CCK 2019

Cholecystokinin (CCK) Regulation of Pancreatic
Acinar Cells: Physiological Actions and Signal

Transduction Mechanisms
John A. Williams*1
ABSTRACT
Pancreatic acinar cells synthesize and secrete about 20 digestive enzymes and ancillary proteins
with the processes that match the supply of these enzymes to their need in digestion being regu-
lated by a number of hormones (CCK, secretin and insulin), neurotransmitters (acetylcholine and
VIP) and growth factors (EGF and IGF). Of these regulators, one of the most important and best
studied is the gastrointestinal hormone, cholecystokinin (CCK). Furthermore, the acinar cell has
become a model for seven transmembrane, heterotrimeric G protein coupled receptors to regulate
multiple processes by distinct signal transduction cascades. In this review, we briefly describe the
chemistry and physiology of CCK and then consider the major physiological effects of CCK on
pancreatic acinar cells. The majority of the review is devoted to the physiologic signaling path-
ways activated by CCK receptors and heterotrimeric G proteins and the functions they affect. The
pathways covered include the traditional second messenger pathways PLC-IP3-Ca2+ , DAG-PKC,
and AC-cAMP-PKA/EPAC that primarily relate to secretion. Then there are the protein-protein in-
teraction pathways Akt-mTOR-S6K, the three major MAPK pathways (ERK, JNK, and p38 MAPK),
and Ca2+ -calcineurin-NFAT pathways that primarily regulate non-secretory processes including
biosynthesis and growth, and several miscellaneous pathways that include the Rho family small
G proteins, PKD, FAK, and Src that may regulate both secretory and nonsecretory processes but
are not as well understood. © 2019 American Physiological Society. Compr Physiol 9:535-564,
2019.
Didactic Synopsis r Other signaling proteins that can be activated by CCK

include PKD, Src, and FAK.
Major teaching points
r Cholecystokinin (CCK) is a peptide hormone and paracrine
regulator produced in unique intestinal enteroendocrine Introduction: Cholecystokinin as a
cells and specific neurons in the brain.
r The major targets for CCK regulation in the gastrointestinal Hormone and Paracrine Regulator
tract are the pancreatic acinar cell, which is stimulated to Cholecystokinin (CCK) is a hormone and paracrine regula-
secrete, gallbladder smooth muscle which contracts and tor primarily originating in specialized endocrine cells of the
gastric smooth muscle which is relaxed to inhibit gastric intestinal mucosa, which acts through the blood on a num-
emptying. ber of target organs, which are involved in the digestion and
r The actions of CCK on pancreatic acinar cells are mediated metabolism of food (243). Its first action to be discovered,
from which its name arises, is the contraction of gallbladder
by CCK type1 receptors, heterotrimeric G proteins and smooth muscle (129) which along with the relaxation of the
signal transduction pathways. Sphincter of Oddi propels bile stored in the gallbladder into
r Acinar cell digestive enzyme secretion is mediated by the the small intestine where it participates in fat digestion. Its
traditional second messenger pathways centered on Ca2+ , second action to be discovered was to stimulate the secre-
cAMP, and diacylglycerol—PKC. tion of pancreatic digestive enzymes, which travel through
r Distinct pathways regulating pancreatic adaptive growth * Correspondence to jawillms@umich.edu
and gene expression are mediated by mTOR, the three 1 Departmentsof Molecular & Integrative Physiology and Internal
MAPK pathways centered on ERK, JNK, and p38MAPK Medicine (Gastroenterology), University of Michigan, Ann Arbor,
as well as the calcineurin—NFAT. Michigan, USA
r Small G proteins Rho and Rac are activated by CCK and Published online, April 2019 (comprehensivephysiology.com)
DOI: 10.1002/cphy.c180014
affect both secretory and nonsecretory processes.
Copyright © American Physiological Society.
Volume 9, April 2019 535

CCK Regulation of Pancreatic Acinar Cells Comprehensive Physiology
the pancreatic ducts to the intestine (112). This led to the the lumen of the small intestine. These cells are stimulated
alternative name of pancreozymin. After purification estab- directly by the products of fat and protein digestion. In many
lished the identity of the peptides producing the two actions, species but most prominently in rodents, an additional mech-
it was known for a period as CCK-PZ. It is now referred anism is the feedback from luminal protease, which con-
to as CCK based on the prior discovery of this action. It is trols secretion of an intestinal derived CCK-releasing peptide.
also one of several gastrointestinal hormones that inhibit gas- I-cells have been isolated through fluorescence activated cell
tric emptying through effects on gastric smooth muscle thus sorting and their secretion studied in vitro. As an adjunct
allowing time for digestion in the small intestine. CCK also method, STC-1 cells, a transformed intestinal cell line pro-
acts on beta cells of the endocrine pancreas although it is not ducing CCK has been used for the study of the control of
considered to be a classical “incretin.” Peripheral CCK has CCK secretion. Released CCK can enter capillaries and travel
effects on the CNS as a short-term satiety factor (293) and through the blood to reach target tissues or act in a paracrine
can synergize with leptin (60). Whether this is a hormonal manner to stimulate CCK receptors on vagal afferent nerve
or neural action is unclear. Overall, its actions are directed endings. I cells are most numerous in the duodenum and
at promoting digestion in the small intestine. As a paracrine decrease in number progressively in the distal small intestine.
regulator, CCK acts on vagal afferent nerve endings in the CCK is present in plasma at low concentrations during
intestinal submucosa and initiates a long neural reflex with fasting due both to a low rate of secretion and rapid degra-
vagal efferents stimulating the exocrine pancreas. This neural dation by endothelial protease. A major problem in CCK
action may also influence beta cell secretion and satiety. Of research has been the difficulty in measuring CCK because
the actions of CCK, those on pancreatic acinar cells have been of its low plasma levels and overlap in structure with gas-
studied in the most detail, in part because of the availability of trin that exists in plasma at higher concentrations. Several
readily prepared isolated cell or acinar systems. CCK is also sensitive and specific radioimunoassays and a bioassay for
synthesized in the brain making it a brain/gut peptide but that CCK have been developed (38, 171, 242). Basal CCK levels
location will not be covered in detail. There are also reports are as low as 0.5 to 1 pmol/L and increase to 5 to 15 pmol/L
of small amounts of CCK mRNA in other tissues. after a meal (14,118,169,170). Generally, multiple molecular
The gene for CCK includes three exons making up about forms are present and contribute to plasma CCK, although
7 kilobases and in humans is located on chromosome 3 (168). the larger forms such as CCK58 are more abundant in dog
The mature CCK mRNA is about 750 bases and codes for a and rat (74, 241) and CCK33 in humans (245). In response
115 amino acid precursor that is processed by specific pro- to a meal the increase in plasma CCK is transient, returning
hormone convertase enzymes in gut and brain to peptides toward baseline as the meal passes down the intestine. Plasma
containing the carboxyl terminal 58, 39, 33, or 8 amino acids CCK is also degraded by neutral endopeptidase on capillary
(67, 243). With better techniques to prevent proteolysis, the endothelial cells and the plasma half-life is only 1.5 to 5 min
larger forms especially CCK 58 appear to be the most abun- with CCK58 and CCK33 having a longer half-life than CCK8
dant in plasma (168). All active forms have essential post- (121, 132, 137).
translational modifications that include sulfation of the tyro-
sine residue 7 amino acids in from the C-terminal and an
amidated C terminal processed from glycine. All forms of
CCK containing the sulfated tyrosine act on the same recep- Actions of Cholecystokinin on the
tor (the CCK1R also known as the CCK-AR) although there Exocrine Pancreas
are modest differences in potency due to affinity for the recep-
tor and half-life in plasma. CCK is also synthesized in brain Stimulation of digestive enzyme secretion
where the primary processed form is CCK8. In the brain, it by acinar cells
is not clear if CCK is a neurotransmitter with rapid actions Digestive enzymes are synthesized in the acinar cell by ribo-
or a neuromodulator (68). In any case, these actions are dis- somes on the endoplasmic reticulum (ER), translocated into
tinct from its satiety actions. CCK is also related to the hor- the lumen of the ER where they follow the canonical secre-
mone gastrin in its C-terminal sequence where the terminal tory pathway to the Golgi apparatus and are packaged into
tetrapeptides are identical, although the two are encoded by large secretory granules, the zymogen granule (ZG) named
separate genes and exert most of their biological effects on because of their content of proteases synthesized as inactive
separate receptors, with gastrin acting on CCK2R. Both the precursors or zymogens. Zymogen granules are localized to
two hormones and the two receptors evolved from common the apical pole of acinar cells and the major pathway for secre-
precursors during vertebrate evolution (244, 330). tion involves their fusion with the apical plasma membrane
CCK is synthesized in and released from a specific type and the release of their content by exocytosis.
of enteroendocrine cell termed I-cells (168). The name comes The action of CCK to stimulate digestive enzyme secre-
from the intermediate size of their secretory granules when tion from pancreatic acinar cells has been studied extensively
viewed by electron microscopy, which are concentrated in both in vivo and in vitro (169). Early studies of partially
their basolateral pole. As with other enteroendocrine cells, purified intestinal extracts containing CCK showed a power-
I-cells have receptors on their apical pole which contacts ful action to stimulate enzyme secretion. Subsequent studies
536 Volume 9, April 2019

Comprehensive Physiology CCK Regulation of Pancreatic Acinar Cells
showed that purified CCK could increase pancreatic secretion Isolated or dispersed pancreatic cells were first described by
when plasma levels were similar to those seen after a meal Amsterdam & Jamieson (4) and isolated acini a few years
(14). Moreover, CCK receptor antagonists loxiglumide and latter (226,346). Isolated acini prepared by collagenase diges-
devazepide decreased pancreatic secretion induced by a meal tion have become the standard preparation to study CCK
by 40% to 60% (31,117,283). Because pancreatic acinar cells action in vitro as they are stable with maintained junctional
had been shown to possess CCK receptors, early work pre- complexes, retain polarity, and release digestive enzymes in
sumed CCK to act through the blood as a GI hormone on response to physiological concentrations of secretagogues for
the acinar cell. However, in rats and humans, the cholinergic several hours (343). They have been used extensively for lig-
blockers atropine and hexamethonium blocked the response to and binding which defined the presence of specific receptors
low doses of CCK including concentrations similar to those on acinar cells. Competitive binding studies along with amy-
seen after a meal (1, 219, 297). Similarly, cutting of vagal lase secretion have defined the binding and signal transduction
afferent nerves also blocked the effect of CCK (165). Of note, characteristics for CCK and other secretagogue receptors. Of
higher concentrations of CCK still acted on the pancreas in the most importance, these studies led to the identification of two
presence of atropine. Other experiments support the action of synergistic signaling mechanisms that work through Ca2+
CCK via the vagal nerve. CCK receptors have been identified and cAMP (348). These pathways potentiate each other with
on vagal afferent nerves (375) and CCK activates electrical regard to digestive enzyme secretion (133, 169, 348).
firing of vagal afferents that can be recorded in the nodose
ganglion (165). Recently I cells of the intestine have been
shown to possess processes that project from the basal pole Stimulation of acinar cell and pancreatic growth
into the submucosa in the neighborhood of enteric neurons Regulation of pancreatic size occurs through genetic and envi-
(36). Overall, these studies show that physiological levels of ronmental influences (especially food intake) and is directed
CCK act predominately through the vagal system to stimu- at ensuring that the organ can carry out its function (342). The
late digestive enzyme secretion but that higher concentrations pancreatic growth that occurs pre- and postnatally is largely
such as those that stimulate pancreatic growth can act directly independent of hormones such as CCK and secretin in rats and
on the acinar cells (358). Both types of action are mediated mice (153, 270, 378). Adaptive growth allows the pancreas to
by CCK1 receptors (also known as CCK-A receptors). increase or decrease its supply of digestive enzymes and is
In vivo studies of pancreatic enzyme secretion have led primarily under the control of CCK and dietary amino acids
to the concept of feedback loops acting to maintain the con- (342). An increase in food intake such as occurs in preg-
centration of trypsin and thereby digestion in the upper small nancy, lactation, and cold exposure leads to an increase in
intestine. Exogenous trypsin inhibitor in the small intestine pancreatic size in rodents mediated by CCK and amino acids
stimulates pancreatic enzyme secretion (98). Furthermore, (110, 148, 186). The converse of increased food intake is star-
diversion of bile-pancreatic juice also stimulates enzyme vation and leads to a decrease in size of the body and compo-
secretion and this can be blocked by duodenal infusion of nent organs but with pancreatic weight decreasing more than
trypsin. Feedback regulation of pancreatic enzyme secretion other organs. Moreover, pancreatic protein decreased more
is dependent on protease secretion and mediated by CCK. than DNA consistent with cellular atrophy (199). That reduc-
The presence of feedback in humans has been difficult to tion of food in the GI tract is important for this atrophy is
establish as it requires broader protease suppression as by the shown by the results of parenteral feeding in rats where the
Bowman-Birk soybean trypsin inhibitor (172). Trypsin and pancreas atrophy (13, 78). Interestingly, CCK administration
other proteases suppress further CCK secretion by cleaving reduces pancreatic atrophy induced by TPN (78).
CCK releasing factors; conversely, protein and most potently A large number of studies have shown that exogenous
trypsin inhibitor bind trypsin allowing CCK releasing pep- CCK can stimulate pancreatic growth in rats, mice and ham-
tides to stimulate more CCK release (167). In addition to the sters (82, 209, 229, 295). There is a parallel increase in wet
intestinal CCK-releasing factors, pancreatic juice contains a weight, protein, and usually DNA indicating cellular hyper-
distinct peptide known as monitor peptide (also known as plasia with normal cell size. At least in the early stage of such
pancreatic secretory trypsin inhibitor-1) that interacts with growth there is increased synthesis of DNA (296). Primary
receptors on I cells to stimulate CCK release (359). Monitor cultures of acinar and duct cells have also shown that CCK
peptide provides positive feedback from the pancreas to CCK can stimulate DNA synthesis (175,176). Endogenous CCK is
release. Thus, high protein diet induces monitor peptide syn- secreted in response to feeding trypsin inhibitor, which blocks
thesis and secretion that acts to maintain CCK release. For normal feedback of trypsin on CCK secretion and induces
further details of these feedback systems see Liddle (169). pancreatic growth, which can be blocked by gene deletion of
Considerable support for CCK in stimulating enzyme CCK or CCK1R (275,309). Rodent and hamster pancreas can
secretion and increased mechanistic understanding of this pro- double in size in a week and will return to the original size
cess has come from in vitro studies of CCK-stimulated pancre- when trypsin inhibitor is withdrawn from the chow (46).
atic digestive enzyme secretion. Early studies used cut pieces Feeding a high protein diet is also well established to
of pancreas or micro-dissected pancreatic lobules, but both induce pancreatic growth. This is partly mediated by CCK
have problems due to damage and inadequate oxygenation. as shown by the inhibition by CCK antagonists (97, 198).

However, the growth effect is also mediated by amino acids (7,124,136) we will mainly cover here how CCK and secretin
as it can occur in a mouse without CCK (47). In this case, the activate duct cell secretion and potentiate each other. The
growth was due to cellular hypertrophy rather than hyperpla- major transporter promoting entry of HCO3 − across the
sia. The converse occurs in mice fed a protein free diet, which duct basolateral membrane is the Na+ -HCO3 − cotransporter
leads to pancreatic acinar cell atrophy (44). NBCe1B that is activated by PKA phosphorylation. Exit of
Stimulation of acinar cell growth involves a number of HCO3 − and Cl− across the duct apical membrane into the
anabolic events both to provide energy and to synthesize more lumen is by the CFTR anion channel and the SLC26A anion
structural protein, lipids, and nucleic acids. While these pro- exchanger, which promotes HCO3 − efflux in exchange for
cesses are similar to those in other cell types, the regulation Cl− uptake. In some species, a Ca2+ activated Cl− channel
by CCK is not yet fully understood. Regulation of protein exists in duct luminal membrane as well as in acinar cell lumi-
synthesis is covered later in the section on mTOR signaling. nal membrane. IRBIT (IP3 receptor binding protein) binds
to the IP3 receptor and inhibits Ca2+ release. Phosphoryla-
tion of IP3 R by PKA decreases the affinity for IRBIT and
Stimulation or potentiation of ductal secretion increases the affinity for IP3 . IRBIT is then released and can
The secretion of bicarbonate rich pancreatic juice serves to bind to CFTR, SLC26, and NBCe1B to promote their func-
solubilize digestive enzymes secreted by acinar cells and par- tion (136). Knockout of IRBIT blocks the synergism between
ticipates in the neutralization of gastric acid in the duode- cAMP/PKA and the Ca2+ signaling pathway.
num; this secretion is primarily controlled by the GI hormone
secretin and the vagal nerve (36, 40, 136). Secretin and some
neurotransmitters such as vasoactive intestinal polypeptide
(VIP) bind to receptors, which activate adenylate cyclase (AC) Mechanism of Action of CCK on
which produces cyclic AMP. Vagal nerve endings in some Pancreatic Acinar Cells
species contain abundant VIP but in most animals, the main
vagal efferent neurotransmitters are acetylcholine and GRP Receptors and transmembrane signal transduction
(gastrin releasing peptide) which act through activating PLC, CCK receptors are 7-transmembrane domain, G-protein cou-
IP3 , and increasing intracellular Ca2+ . Other hormones and pled receptors that are located on the plasma membrane of
neurotransmitters including CCK that act through intracellu- CCK regulated cells. Two distinct CCK receptors bind CCK
lar Ca2+ will stimulate ductal secretion but in the course of a with high affinity and were originally termed CCKA (alimen-
meal their main effect is to potentiate the action of secretin. tary) and CCKB (brain) but now are known as CCK1R and
The relative importance of CCK in stimulating pancre- CCK2R (73). CCK1R is highly selective for CCK with a sul-
atic fluid secretion is very species dependent. In rats and fated tyrosine residue seven amino acids from the C terminal
mice, exogenous CCK stimulates a high volume of pancreatic and has a much lower affinity for the related hormone gastrin.
juice with a low bicarbonate concentration (40-70 mmol/L) It is found in the pancreas, gallbladder smooth muscle, stom-
(66,286,305). In humans, cats, and guinea pigs, which secrete ach, and on certain neurons particularly vagal afferent nerve
bicarbonate rich pancreatic juice (130-140 mmol/L) CCK has endings of multiple species. However, the presence of CCK1R
a much smaller effect. While there is some evidence that on human pancreatic acinar cells is controversial with differ-
acinar cells can secrete a chloride rich fluid most of the pan- ent results reported (135, 166, 195, 202). It now seems likely
creatic juice is of ductal origin and secretion from isolated that human CCK1R is susceptible to collagenase used to iso-
ducts reproduces the above pattern of bicarbonate secretion. late acini as a strong response was seen in slices not exposed
In rodents at least, ducts as well as acinar cells possess CCK to protease (166). CCK2R binds both CCK and gastrin simi-
receptors (264, 305) and isolated pancreatic ducts of rodents larly and does not require a sulfated tyrosine in the ligand. It
can respond to CCK indicating a direct action as well as stim- is present in the stomach and widely distributed in the brain
ulating a vagal-vagal pathway. In humans, CCK alone has and is sometimes referred to as the gastrin receptor. Following
at most a modest effect on pancreatic fluid and bicarbonate cDNA cloning the primary amino acid sequence predicts both
secretion but will potentiate the action of secretin (364). The CCK receptors have seven transmembrane helices (based on
CCK antagonist, loxiglumide at a dose that blocked the effect hydropathy plots) connected by extracellular and intracellular
of exogenous CCK inhibited bicarbonate secretion into the loops with an extracellular amino terminal and intracellular
duodenum by only 25% (283) and other studies have found carboxyl terminal (62, 337). While this is often illustrated in
no significant effect. In guinea pig isolated pancreatic duct, a two dimensions (Fig. 1) the transmembrane domains actu-
good model for high bicarbonate secretion, CCK stimulated ally form a heptahelical bundle which is similar to rhodopsin
fluid and bicarbonate secretion with the effect blocked by the (191). The mature receptor possesses an interchain disulfide
CCK antagonist, devazepide (304). Thus, whether duct cells linkage, is glycosylated on the amino terminal and an extra-
secrete a high or low bicarbonate is dependent on the molecu- cellular loop, and is phosphorylated on Ser and Thr residues
lar machinery present rather than the stimulating agonist (7). in the third intracellular loop and the carboxyl terminal fol-
Because a number of good reviews exist on the mech- lowing agonist stimulation. This phosphorylation which is
anisms by which duct cells secrete bicarbonate rich fluid carried out by PKC and a G protein coupled receptor kinase

Figure 1 Two-dimensional structure of CCK1R showing membrane spanning topology and

sites of posttranslational modification. The amino terminal is outside the cell membrane and the
carboxy terminal is inside. Sites of glycosylation are indicated by a Y and sites of phosphory-
lation by P in a circle. There is a site of palmitoylation in the carboxyl terminal tail indicated
by two Cs anchored to the membrane. The natural CCK peptide is indicated as a brown oval
docked to the receptor. Reproduced, with permission, from reference (62).
plays a role in receptor desensitization and possibly internal- Whether they fit into the CCK binding pocket or bind to a sep-
ization. The carboxyl terminal also possesses two cysteines, arate site is not always clear. At least one allosteric binding site
which are palmitoylated and anchor the C-terminal to the has been identified on the CCK1R for small molecule agonists
membrane (63). and antagonists (34, 111). Two examples of CCK1R antag-
Considerable effort has been expended to determine how onists that have been important for physiological research
CCK interacts with receptor with many publications too are the benzodiazepines such as devazepide (also known as
numerous to include. Techniques used have included, recep- L-364,718), the most commonly used CCK1R antagonist and
tor mutagenesis, cross-linking of ligand to receptors, NMR, JMV-180, a peptide analog of CCK8, which exerts some but
fluorescence and photoaffinity labeling of receptor residues not all of the effects of CCK. A yet incompletely understood
within a calibrated distance by a probe containing a ligand in phenomenon is that CCK1R molecules bind CCK in two affin-
which a specific amino acid contains a photoactivatable ligand ity states, high and low affinities with different signal trans-
(59, 191). These have been combined with three dimensional duction mechanisms assigned to each state. JMV-180 acts as
receptor models. Two distinct receptor models continue to be an agonist at high affinity receptors and an antagonist at low
considered. In one, the entire CCK active peptide (usually affinity receptors (63, 183). Another class of CCK receptor
7-9 amino acids containing the pharmacophore) sits at the antagonists are derivatives of proglumide, such as loxiglu-
surface of the lipid bilayer (Fig. 1) with the amino terminal mide, which are highly specific for CCK1R. Recent studies
over the first transmembrane segment of the receptor (191). In have also shown that the CCK1R is affected by the lipid com-
the other, the carboxy terminal of the CCK peptide dips into position of the plasma membrane as first shown for choles-
the helical bundle (116). A variety of small molecule antago- terol but now known to also be affected by specific phospho-
nists and a few agonists for the CCK1R have been developed. lipids that can change in metabolic disease (63). Specifically,

Figure 2 CCK1R signals through multiple G proteins to activate different signaling cascades.
Whether β-Arrestin and Gβ/γ play a role in pancreatic acinar cells is unclear (368).
elevated membrane cholesterol reduces sensitivity to CCK relevant literature and will focus on initial observations and
and this is prominent in obesity and diabetes (61). reviews related to the pathway. For other aspects of intra-
The activated CCK1R transduces its actions through dif- cellular Ca2+ regulation and stimulus secretion coupling see
ferent heterotrimeric G proteins, located at the cytoplasmic (228, 348, 368).
face of the plasma membrane, which are activated by differ-
ent regions of the receptor. The third cytoplasmic loop is the
main activation region of the receptor, although the second
intracellular loop is believed to activate Gαs. When the recep- PLC and IP3 Production
tor activates a heterotrimeric G protein, the α subunit binds This pathway begins with activation of the phosphoinositide-
GTP and separates from the β/γ complex. Most actions are specific phospholipase C (PLC) which cleaves
mediated by the activated α subunits. There are 16 α-, 5 β-, and phosphatidylinositol-4,5-bisphosphate (PIP2 ) in the plasma
12 γ-subunits in the human genome. The four major classes membrane to generate the second messengers inositol 1,4,5-
of heterotrimeric G proteins are Gs , Gq/11 , G12/13 , and Gi/o . G trisphosphate (IP3 ) and diacylglycerol (DAG) (17). DAG will
protein α subunits that mediate Ca2+ signaling and digestive be considered later as part of the PKC pathway. There are 13
enzyme secretion include αq , α11 , and α14 (348); these have isoforms of PLC all of which can act on phosphoinositides
been identified in acinar cells and microinjection of a blocking (99). They have different tissue distributions and activators.
antibody inhibits Ca2+ signaling (369). The signaling path- Four PLCβ isoforms exist and are the main forms activated
ways activated by specific G proteins are shown in Figure 2. by G protein coupled receptors through αq/11, βγ, and Rho
Heterotrimeric G proteins go through the standard G protein family small GTPases whereas PLCγ forms are activated by
cycle with the receptor acting as a GEF and RGS proteins or growth factors which activate receptors with tyrosine kinase
in some cases phospholipase C (PLC) acting as GAPs. Some activity (203). CCK activates the PLC β isoforms, which are
such as Gq can activate more than one downstream effector also known to be activated by purified Gαq (294, 310). Initial
(174). The G proteins are also the target of bacterial toxins western blotting identified PLC-β1, -β3, -γ1, and -δ1 on rat
such as cholera, pertussis, and pasturella toxins. For more pancreatic acinar membranes (232). Inhibition of PLC-β1
details, see reference (348). by specific antibody inhibited CCK activation of PLC while
antibody to PLC-β3 was more effective in blocking carbachol
stimulation. A more recent study showed that all four PLCβ
PLC - IP3 - Ca2+ pathway and two PLCγ isoforms were present in rat acini (145).
This is the major pathway stimulating digestive enzyme secre- Interestingly, several isoforms (-β1, -β2, and -γ1) have been
tion and also affects many other functions. Because it has been localized over ZG in mouse pancreas (336) Multiple PLC
the focus of much work, we will not be able to cite all the isoforms have also been localized to pancreatic islets.

Following stimulation with CCK and other Gq coupled 200

agonists, IP3 is rapidly formed. In rat pancreatic acini labeled
with [3 H]-inositol, formation of [3 H]-IP3 can be measured
in as little as 5 s and probably occurs faster (255). A non-
[Ca2+]i (nmol/L)
150
radioactive mass assay based on competitive binding to the IP3
receptor showed a larger increase in response to CCK stim-
ulation (184). While these studies were all carried out with
maximal agonist concentrations, physiological concentrations 100
of CCK and other calcium mobilizing secretagogues have not 10 pmol/L CCK
shown an effect on IP3 formation. It is not clear whether low –Ca
concentrations of agonists work through a separate second 50
messenger such as nicotinic acid adenine dinucleotide phos- 0 5 10 15 20 25
phate (NADDP) that induces localized Ca2+ release through Time (min)
two pore channels (360, 368) or that this reflects technical
Figure 3 Ca2+ oscillations induced by pmol/L concentrations of
issues with the assay of small amounts of IP3 since IP3 recep- CCK that are initially independent of extracellular Ca2+ . Ca2+ was
tor blockers inhibit this signaling (315, 369). There is also removed and EGTA added 1 min before CCK was added. Reproduced,
considerable literature on other chemical derivatives of IP3 with permission, from reference (323).
with additional phosphate groups, none of these have been
shown to exert significant effects on calcium signaling. [Ca2+ ] showed that stimulation by a maximal concentration of
That IP3 can release Ca2+ from intracellular stores was CCK, similar to acetylcholine (ACh) and bombesin, induced
first shown in permeabilized pancreatic acinar cells by Streb, a rapid increase in [Ca2+ ] from a basal value of around
Irvine, Berridge, and Schulz in 1983 (300). These studies led 100 nmol/L to a peak value of 500 nmol/L to 1 μmol/L. This
to the identification of the endoplasmic reticulum (ER) as the peak then declined to a plateau level of 75 to 100 nmol/L above
source of released, Ca2+ and confirmed earlier work in intact the basal level that was maintained as long as secretagogue
acini that in response to CCK and acetylcholine, Ca2+ was was present. The initial peak was independent of extracellular
released from the ER but not ZG or mitochondria (69). Fol- Ca2+ and is due to mobilization of intracellular stored Ca2+ in
lowing the identification of the IP3 receptor (84), all three of response to IP3 and possibly other messengers. By contrast,
its isoforms, IP3 R1, IP3 R2, and IP3 R3 have been shown to the plateau phase is dependent on extracellular Ca2+ and due
be present in pancreatic acinar cells and concentrated imme- to Ca2+ influx (228, 348).
diately underneath the apical and lateral membrane domains In contrast to the response to maximal concentrations
near the web of actin fibers (158,204,370). Studies where IP3 of secretagogues, lower physiological concentrations induce
was released from a caged precursor in various areas of mouse Ca2+ oscillations (Fig. 3). In response to 1 to 30 pmol/L CCK,
acinar cell showed that the apical region was most sensitive to regular Ca2+ oscillations rise off either the basal level or a
IP3 (81). The major forms of IP3 receptors in acinar cells are slightly elevated plateau to a peak of 200 to 500 pmol/L with
R2 and R3, which together make up 90% and genetic dele- a frequency of 1 to 3 per minute (323, 371). The height of the
tion of these two isoforms blocks Ca2+ release and digestive oscillations increases with CCK concentration while the fre-
enzyme secretion (85). quency is fairly constant. Ca2+ oscillations in acinar cells can
IP3 receptors are homo- or heterotetramers with each probe mimicked by activators of heterotrimeric G proteins and
tein subunit having a binding site for IP3 near the N-terminus, by introduction IP3 (218). They are believed to be the result
which extends into the cytoplasm while the C-terminus of of cycles of Ca2+ release and reuptake with the latter being
the four subunits together form a transmembrane gated pore. ATP dependent. Oscillating levels of IP3 are not necessary
Further complexities are the result of the fact that receptors but may occur. Ca2+ oscillations run down in the absence of
can be assembled from the three different isoforms and that extracellular Ca2+ as some Ca2+ is pumped out of the cell
the binding affinity and channel properties can be altered by by the plasma membrane calcium ATPase (PMCA) with each
posttranslational modification especially phosphorylation and oscillatory peak (27, 312).
by binding Ca2+ , ATP and other modulators (37). Physiolog- Spatial properties of Ca2+ signaling have been elucidated
ical concentrations of CCK but not acetylcholine result in a as a result of high-resolution digital imaging that allows
PKA-dependent phosphorylation of IP3 R (299), which may resolution at the subcellular level. This has included use
underlie the difference in Ca2+ signaling initiated by these of advanced imaging techniques including confocal, mul-
two agonists. tiphoton and total internal reflectance fluorescence (TIRF)
microscopy. Early imaging studies with maximal stimula-
tion showed a Ca2+ increase in the apical region of the
Ca2+ Signaling
cell that spread as a wave toward the cell’s basal pole
Temporal and spatial properties Studies using ratio- (94, 138, 205, 299, 320). This wave travels at 5 to 40 μm/s and
metric fluorescence imaging of the Ca2+ probe fura-2 in sin- is affected by ryanodine receptors, PKC, and other regulatory
gle pancreatic acinar cells to estimate the global intracellular signals. At more physiological concentrations, Ca2+ signals

20 pmol/L CCK
(A)
700
[Ca2+]i (nmol/L)
500
300
100
100 s
(B)
Brightfield CCK (20 pmpl/L)
c b
900
a Basal 8 sec 9.5 11 12.5
[Ca2+]i (nmol/L)
450
100 15 18 21 24 45 46.5
40
48 51 52.5 57 63 8/
Figure 4 CCK initiates Ca2+ waves that travel around an acini. (A) Ca2+ signaling shown for a single cell was typical
of all cells in an acinar cluster; however, they did not occur synchronously. (B) Pseudocolor image of Ca2+ concentration
shows that superfusion with 20 pmol/L CCK initiates a signal in one cell that then propagates in each cell around the
cluster. After a basal period, a new cycle is initiated in the same cell. Reproduced, with permission, from reference (372).
are also initiated in the apical region but do not always spread Ca2± influx across the plasma membrane Ca2+ influx
(314). The apical site is also most sensitive when release of across the plasma membrane is required to maintain Ca2+ sig-
caged IP3 is carried out in different positions and has been naling and secretion in acinar cells. This influx mechanism
referred to as the trigger zone (368). Low concentrations of is not voltage sensitive but can be blocked with lanthanum
ACh induce repetitive local Ca2+ spikes in this region. It is and is sensitive to extracellular pH (322, 323). The influx
also the portion of the cell with the highest concentration pathway is initiated by depletion of intracellular Ca2+ stores
of secretagogue receptors, IP3 channels, and Ca2+ pumps and is referred to as the store operated Ca2+ entry mecha-
(139, 158, 287). One striking aspect of this cellular polariza- nism (SOCE). It can be demonstrated by using thapsigargin
tion is that stimuli applied to the basal membrane by a patch or another SERCA inhibitor to deplete Ca2+ stores in the
pipette can “tunnel” through the cell to increase Ca2+ in the absence of extracellular Ca2+ followed by the readdition of
apical region (8). The initiation of Ca2+ spikes in the trig- Ca2+ to the medium, which enters the cell independent of
ger zone is functionally coupled to exocytosis of zymogen receptor activation. The ER Ca2+ sensor has been identified
granules and activation of Cl− channels, which maintain fluid as stromal interaction molecule 1 (Stim 1) (173). Stim 1 is
secretion in rodent acini. an integral ER membrane protein with an intraluminal Ca2+
Another aspect of Ca2+ signaling in pancreatic acini binding domain. Following intraluminal Ca2+ depletion, Stim
results from the cells being coupled to their neighbors by gap 1 molecules aggregate and move closer to the PM where they
junctions. These allow passage of small intracellular messen- interact with the Ca2+ channel Orai1. Both Stim1 and Orai1
gers such as IP3 and in some cases Ca2+ itself. While maximal are expressed in mouse pancreatic acinar cells (177) and Orai1
stimulation leads to junctional closure, physiological stimula- inhibitors have been proposed as potential tools for pancre-
tion induces a wave of Ca2+ elevation traveling through cells atitis therapy (91). In exocrine cells, there is also evidence
around the acini (298, 372). Each cycle of Ca2+ increases is that a transient receptor potential channel (Trp C) can also
induced in the same cell that can be considered a pacemaker contribute as an influx channel to SOCE. Trp C3 and C6 are
(Fig. 4). The function of these Ca2+ waves is not fully estab- expressed in acinar cells and TrpC3 KO mice show reduced
lished but it should allow the most sensitive cell to drive the CCK stimulated Ca2+ influx and amylase secretion (147).
entire acinus. This is supported by the fact that isolated acini Finally, another Ca2+ entry mechanism is that activated by
are more sensitive to secretagogue induced amylase secretion arachidonic acid, the ARC channel that may also be respon-
than are isolated acinar cells. sible for Ca2+ influx (190, 313).

For consideration of the cellular mechanisms bringing

about clearance of Ca2+ from the cytoplasm after stimulation,
see (368).
Ca2± -induced physiological events That an increase

in Ca2+ can induce a number of physiological events has
been shown by the use of Ca2+ ionophores such as A23187
and inhibitors of the SERCA ATPase such as thapsigargin
both of which will increase intracellular Ca2+ without acti-
vating earlier events in the signaling pathway. An alterna-
tive approach has been to permeabilize acinar cells with pore
forming proteins or electric shock such that intracellular pro-
teins are maintained and [Ca2+ ] is set by Ca2+ buffers in the
medium (348). Permeabilization usually works for a short
time as important molecules will leak out of the cell. Increas-
ing Ca2+ alters a number of functions including enzyme secre-
tion, fluid secretion, energy metabolism, and mitogenesis.
This regulation is often complex and involves direct effects
of Ca2+ on proteins and more commonly by Ca2+ binding to
a regulatory protein of which the most common is calmod-
ulin (CaM) (41). One class of such proteins are the Ca2+
activated protein kinases. In contrast to a single broadly dis-
tributed cAMP-activated kinase, there are a number of distinct Figure 5 Intracellular Ca2+ signaling in pancreatic acinar cells is ini-
Ca2+ -activated calmodulin-dependent kinases. Some such as tiated by the binding of Acetylcholine to Muscarinic M3 receptors (M3R)
and by Cholecystokinin (CCK) to CCK receptors, predominately the
myosin light chain kinase (MLCK) and phosphorylase kinase CCK1R in mice and rats. Both receptors are classical seven transmem-
have single substrate proteins, while CaM kinase II (CaMKII) brane domain receptors coupled to guanine nucleotide-binding proteins
is multifunctional and can phosphorylate large numbers of (G proteins). Activation of both receptors leads to stimulation of Gαq
and increased activity of phospholipase C-β (PLC) which cleaves the
proteins (187). Five Ca2+ activated kinases have been identi- membrane phospholipid phosphatidylinositol,4,5,-bisphosphate (PIP2)
fied in exocrine pancreas including MLCK, CaMKI, CaMKII, into diacylglycerol and inositol 1,4,5-trisphosphate (IP3). IP3 diffuses
CaMKIII, and CaMKIV (348). Of these, MLCK has been through the cytoplasm and interacts with InsP3 receptors (IP3R), largely
type-2 and -3 present predominantly on the apical endoplasmic reticu-
related to secretion through its action on actin-myosin inter- lum (ER) resulting in Ca2+ release into the cytoplasm. Ca2+ release from
actions under the apical plasma membrane. CaMKIII phos- IP3R acts a co-agonist to increase further IP3R activity and also acts on
phorylates and inhibits elongation factor 2 and is responsible Ryanodine receptors (RyR) to induce Ca2+ release. Depletion of Ca2+
within the ER results in Ca2 + influx from the extracellular space. The ER
in part for high concentrations of intracellular Ca2+ inhibiting Ca2+ sensor has been identified as stromal interaction molecule-1 (Stim-
protein synthesis. While CaMKII has been most studied and 1). Following ER depletion, Ca2+ is released from an EF hand present in
its properties allow it to integrate signals from Ca2+ oscilla- a domain of Stim-1 within the ER lumen and this results in aggregation
of several Stim-1 molecules. Aggregation of Stim-1 is sufficient to gate
tions, its importance in secretion and other pancreatic func- plasma membrane Ca2+ channels and leads to Ca2+ influx. Good can-
tions is unclear. The Ca2+ -activated phosphatase calcineurin didates for the channel pore are the proteins Orai-1 and TRPC3. CCK
or PP2B is also activated by Ca2+ -calmodulin and is covered receptor stimulation also stimulates ADP-ribosyl cyclase activity resulting
in the formation of two additional Ca2+ mobilizing second messengers;
in the Calcineurin - NFAT pathway. Intracellular Ca2+ signal- nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic-ADP
ing and effectors as related to digestive enzyme secretion is ribose (cADPr). The particular cyclase responsible has not been defined
diagramed in Figure 5. in pancreas but good candidates include CD38 and CD157. cADPr is
generally thought to act on RyR, while the target of NAADP is currently
CCK stimulation also increases the rate of ATP production the subject of intense research. Candidates include the RyR and Two
2 to 2.5 fold to counter the increased use of ATP during secre- Pore Channel (TPC). In addition to the ER, Ca2+ can also be released
tion. This results in increased cytosolic and intramitochondrial from a store, which accumulates Ca2+ in a manner dependent on a
proton gradient- known as the “acidic store.” This pool likely represents
ATP through both glycolytic and oxidative metabolism (335). the endolysosomal compartment. This pool has been reported to be
This effect of CCK is mediated by Ca2+ as it can be mimicked responsive to IP3, cADPr and NAADP and may represent the store ini-
with ionomycin or thapsigargin to bypass receptors as well tially released following receptor stimulation. Ca2+ is removed from the
cytoplasm by the concerted action of SERCA (ER Ca2+ -ATPase), PMCA
as by the other Ca2+ mobilizing secretagogues. As a general (PM Ca2+ -ATPase), and the action of MCU (mitochondrial uniporter).
phenomenon in Ca2+ regulation, elevated cytoplasmic Ca2+ Figure and legend reproduced, with permission, from reference (368).
is taken up by mitochondria into the matrix and enhances the
activity of the dehydrogenases of the Krebs cycle (185). This
has been validated in pancreatic acinar cells where NAD(P)H glycolysis and oxidative phosphorylation and supplies ATP
produced by acinar cells oscillates along with the increase in to the secretory machinery (334). The converse of cytosolic
Ca2+ (333). This leads to an increase in ATP production with- Ca2+ stimulating ATP production is that a fall in ATP inhibits
out change in the mitochondrial potential that is due to both Ca2+ signaling (334). Finally, although outside the scope of

this review, prolonged high [Ca2+ ] as occurs in experimental (AKAPs) are involved (196). Conventional PKCs bind Ca2+
pancreatitis can lead to mitochondrial damage with opening of and target to the plasma membrane where they bind to PIP2
the mitochondrial transition pore and cell death by apoptosis while novel PKCs which lack a C2 domain target to DAG rich
or necrosis. endomembrane domains particularly the Golgi. Novel PKCs
have a longer period of activation due to a longer increase in
DAG before it is metabolized by DAG kinase.
DAG-PKC pathway Study of the action of PKC in pancreatic as well as
Diacylglycerol (DAG) is an established second messenger other cells was initially facilitated by the use of cell per-
produced by phospholipase action on membrane phospholipid meant phorbol esters, which mimic the action of DAG but are
with its main messenger action being to activate PKC (protein more potent. Phorbol esters were initially discovered as plant
kinase C (18, 210). The initial production of DAG is from derived tumor promoters present in Croton oil. They contain
the cleavage of phosphatidylinositol-4,5-bis-phosphate (PIP2 ) a phorbol moiety esterified to two fatty acids leading to com-
by a phosphoinositide specific phospholipase C that yields pounds such as PMA (phorbol myristate acetate) also known
IP3 and DAG. However, while IP3 is produced as a rapid as TPA being the most potent (353). Phorbol ester can increase
initial peak, DAG levels continue to rise over many minutes the amount of membrane bound conventional and novel PKC
in response to CCK stimulation of pancreatic acinar cells without a rise in free Ca2+ (151). The other early tool to study
(184,222). Using a mass assay for DAG an initial peak can be PKC action were small molecule general PKC inhibitors with
detected in several seconds that occurs simultaneously with the bisindolylmaleimides being most prominent. Latter devel-
the initial peak in IP3 (184). These initial peaks are only seen opment established inhibitors with some degree of specificity
at high concentrations of CCK, are not produced by JMV-180, for groups such as conventional PKCs or individual PKCs
and are presumed generated by the low affinity state of the although the latter are not perfectly specific and some such as
CCK receptor. The latter increase in DAG is believed to come rottlerin, a presumptive PKCδ inhibitor, have been discredited
from the hydrolysis of other phospholipids particularly PC (353). More specific inhibitors of individual forms have been
(phosphatidylcholine). CCK, CCh, and bombesin all activate targeted at RACKS or have been dominant negative PKCs,
the hydrolysis of PC (184,256). Since phorbol esters and Ca2+ which have been targeted using viral vectors (161).
ionophore can also cause PC hydrolysis in acinar cells there In the 1980s phorbol esters were shown to partially
may also be control by secondary events that follow the initial stimulate amylase release from isolated acini, bring about
PIP2 hydrolysis. the translocation and activation of PKC activity and the
The main target for diacylglycerol are certain members of phosphorylation of specific substrates (179, 212, 351, 352).
the PKC (protein kinase C) family. PKC has a kinase domain Moreover, the combination of calcium ionophore and phor-
related to PKA and PKB but distinct regulatory domains that bol ester reproduced the stimulation by CCK or carba-
target the kinase and regulate its activity along with an autoin- chol, which is also known to induce membrane transloca-
hibitory domain and phosphorylation events (5, 207). The tion of PKC (28, 128). Conversely, down regulation of PKC
PKC family contains 10 members from 9 genes divided into by prolonged incubation with phorbol ester or incubation
conventional PKCs (α, βI, βII, and γ) which require both Ca2+ with PKC inhibitors such as GF109203X reduced amylase
and DAG, novel PKCs (δ, ε, η, and θ) which require DAG but secretion induced by CCK or carbachol by 40% to 50%
not Ca2+ , and atypical PKCs (ζ and λ) which require neither (3, 161, 189, 301). While most studies with amylase secretion
Ca2+ or DAG but interact with specific proteins especially have concluded that phorbol esters alone can stimulate exo-
in establishing cell polarity. The primary structure of PKC cytosis, a more recent study quantitating exocytotic images
includes an amino terminal regulatory region, which contains showed that phorbol esters could not stimulate exocytosis
C1 domains, which bind DAG, C2 domains that bind Ca2+ without a rise in Ca2+ but increased the number of granules
and a pseudosubstrate domain, which binds to and inhibits able to undergo exocytosis (157).
the catalytic domain. The carboxyl terminal kinase region is While initial studies measured PKC activity through a
separated by a hinge domain that also contains regulatory kinase assay, with the cloning and development of specific
phosphorylation sites (207). In contrast to most other kinases, antibodies to different PKC isoforms the emphases shifted to
phosphorylation in constitutive, occurs shortly after synthesis determining the localization and function of specific PKC iso-
and maintains PKC in the cytoplasm in a primed but inactive forms. Four PKC isoforms have been identified in pancreatic
folded configuration in which the pseudosubstrate domain acinar cells from rodents (α, δ, ε, and ζ) (12,145,146,161,249)
binds to and inhibits the catalytic domain and protects the with additional isoforms present in human pancreas some
kinase from degradation. Activation requires targeting to a of which are islet specific (80). Both broad-spectrum PKC
membrane followed by opening and exposure of the catalytic inhibitors and downregulation with phorbol ester will act on
domain (5, 207). Some PKC isoforms have additional control PKC α, δ, and ε. To relate individual isoforms to a biological
mechanisms such as Tyr phosphorylation of PKC-δ. Mem- function more specific inhibitors such a cell permeant peptides
brane targeting of active PKC involves interaction with a tar- blocking binding to RACKs and dominant negative kinases
geting protein, most often a RACK (Receptor for Activated C introduced by viral vectors or antisense oligonucleotides have
Kinase) although in some cases A kinase anchoring proteins been used. By these techniques, PKCα has been linked to

EGF CCK, ACh

Gq PIP2 DAG
PIP 2
PC
P LCγ PI-PLC-β
P
P PC-PLC
P
P
DAG + IP3 IP3 P-Chol
ER Protein-protein
Ca2+ interactions
Classical Novel Unconventional
PKC PKCα PKCδ PKCζ

isoforms PKCβ
PKγ
PKCε
PKCη
PKCλ
PKCθ
Figure 6 Activation of PKC isoforms in pancreatic acinar cells. CCK and ACh activate PLCβ
and EGF activates PLCγ all of which lead to production of diacylglycerol (DAG) from PIP2 . CCK
also induces DAG formation from phosphatidylcholine (PC). Ca2+ and DAG together activate the
classical isoforms PKC α, β, and γ; DAG activates the novel isoforms PKC δ, ε, η, and θ; while
unconventional isoforms PKC ζ and λ do not require Ca2+ or DAG. Reproduced, with permission,
from Pancreapedia, DAG–PKC Signaling Pathway, 2013.
cell proliferation (376), S6K activation (374) and amylase MAPK pathways
secretion (376). In differentiated acinar cells PKCα has been
Mitogen activated protein kinases (MAPKs) are serine-
related to proliferation in monolayer culture (Guo, Sans, and
threonine directed protein kinases that were originally iden-
Williams, unpublished data) and basolateral zymogen granule
tified as mitogen-activated. Now they are known to be acti-
exocytosis in response to alcohol and CCK or carbachol stim-
vated by diverse stimuli including cytokines, growth factors,
ulation (42, 43). The novel PKCs δ and ε have been related to
hormones, and stresses. MAPK cascades are composed of
a number of functions with some overlap. PKCδ is required
three protein kinases acting in a series generically termed
for amylase and insulin secretion, chemokine synthesis by
MAP3K, MAP2K, and MAPK, which sequentially activate
acinar cells, NF-κB activation and cerulein-induced zymo-
the downstream component. The pathways (Fig. 7) are named
gen activation (161,236,276,316,325) although a latter paper
for the central kinase that affects cell function, ERK, JNK, p38
disputed the effect on acinar cell amylase secretion (317).
MAPK, and ERK5. The ERK pathway is activated by growth
PKCε is required for zymogen activation, NF-κB activation,
factors, mitogens, hormones and some neurotransmitters that
ethanol sensitization of NF-κB activation, and ERK activation
bind to tyrosine kinase and G protein coupled receptors (235).
(162,231,276,316). Activation of PKC isoforms by DAG and
The JNK and p38 MAPK pathways are most often activated by
Ca2+ in pancreatic acinar cells is diagramed in Figure 6.
While PKC activity has now been related to a number
of actions in pancreatic acinar cells, only a few molecular
targets have been identified. In the case of the role of PKCα to
stimulate basolateral exocytosis, the target is Munc18c which
when phosphorylated dissociates from Syntaxin-4 allowing
formation of a SNARE complex between zymogen granules
and the basolateral membrane (42, 43). Whether a similar
phosphorylation of Munc18b on the apical membrane occurs
in physiological secretion is unclear. In the activation of NF-
κB, PKC phosphorylates the IκB inhibitory subunit leading
to its degradation such that the active component can move
into the nucleus (3). Finally, in the case of ERK activation,
PKCε phosphorylates cRaf-1 (162). Other known PKC targets
include the MARCKS protein (277) and PKD where PKC
phosphorylates the activation loop (247). For more details
and possible actions of PKC in pancreatic duct and stellate Figure 7 Parallel nature of MAPK cascades in pancreas. Modified
cells, see (3). and updated, with permission, from reference (281).

cytokines and cell stress. At many levels of the three pathways formation (52,55,70,71,236). By contrast, human acini show
there are multiple forms such as ERK1 and ERK2 (ERK1/2) an increase in pERK in response to cholinergic agonist but
and JNK1/2/3 (235). These multiple species are more closely not CCK although this was ascribed to the absence of CCK
related and in some cases have the same actions. For the receptors (135). ERK1/2 is also activated by EGF, HGF, IGF-
ERK pathway, Human ERK1 and ERK2 are 84% identical 1, and other growth factors, which activate Tyr kinase con-
and all known stimuli activate both forms (30). By contrast, taining receptors (6, 52, 339). Most mechanistic studies have
the p38 Map Kinase has four forms (α, β, γ, δ) which may utilized CCK and EGF as they induce the most robust acti-
have different regulation and actions. MAPK cascade compo- vation. Both of these agonists activate ERK1/2 within min-
nents are all inactivated by phosphatases including pSer/Thr utes in vitro with the response sustained for at least an hour.
phosphatases such as pp2A, Tyr phosphatases or in the case CCK effects are seen at 3 or 10 pmol/L, which is slightly
of the MAPKs themselves, by dual specificity phosphatases higher than that required to mobilize Ca2+ or stimulate diges-
known as DUSPS (or map kinase phosphates, MKPs) that tive enzyme secretion. TGF-β has also been shown to acti-
dephosphorylate both Ser/Thr and Tyr (126, 141). There are vate ERK1/2 in pancreatic acini with the effect mediated by
ten catalytically active DUSPS arranged in three families by Smad4 (288). In vivo, there is little change in phospho ERK
their nuclear or cytosolic localization (33). between fasting and refeeding chow but phospho ERK shows
a large increase after refeeding chow with trypsin inhibitor
that increases plasma CCK to around 10 pmol/L and induces
ERK Pathway
adaptive pancreatic growth (103, 308, 309). In another type
The ERK pathway is activated by growth factors, mitogens, of growth, p42 and p44 MAPK were shown to be activated
hormones and some neurotransmitters that bind to tyrosine during pancreatic regeneration following partial pancreatec-
kinase and G protein coupled receptors. For the ERK path- tomy (197).
way, the three kinases in the cascade are Raf, MEK and ERK ERK appears to be activated in pancreatic acinar cells by
(Fig. 7). The MAPK pathways are often organized by scaf- the canonical pathway of RAF-MEK-ERK as RafA, RafB,
folding proteins (150, 152, 238). For ERK the best-studied and c-Raf1 as well as MEK1 and MEK2 are all present and
scaffold is KSR1 (Kinase Suppressor of Ras-1) which binds activated by CCK and EGF (70, 71) (Fig. 8). EGF activates
all three members of the ERK kinase cascade. The MAPK this pathway by activating Ras and is not blocked by inhibit-
cascades all have multiple actions in both the nucleus and ing Protein Kinase C (PKC) (52). Whether CCK activates Ras
cytoplasm. In the nucleus, ERK and its downstream effec- is unclear with different reports indicating activation (56, 71)
tors such as p90 Ribosomal S6 Kinase (RSK) phosphorylate or lack of activation (52). The two studies showing activa-
ternary complex factors such as ELK-1 and thereby stimulate tion used high concentrations of CCK. In addition, the effects
transcription of early response genes such as Fos and Egr1 of CCK to activate ERK were blocked by a PKC inhibitor
and are important in initiating mitogenesis (251, 363). In the but not by dominant negative Ras (50, 208). It appears that
cytoplasm, ERK and another downstream kinase, MAPK- the CCK1 receptor and receptors for other agonists that acti-
interacting protein kinase (MNK-1) phosphorylate specific vate Gq primarily activate PKC via Ca2+ and diacylglycerol
translational factors including eIF4E and eIF4G as well as (DAG) and thereby activate the ERK pathway and the ERK
cPLA2 (cytoplasmic phospholipase A2). ERK also localizes mediator RSK (22). In some other cell types, a pertussis toxin
to other organelles including endosomes, caveolae, Golgi, and (PTx) sensitive G protein is involved in ERK activation. In
cytoskeleton (362). AR42J cells derived from a rat pancreatic tumor, PTx par-
tially inhibited ERK activation in response to CCK, EGF, and
Activation of ERK pathway in pancreatic acinar cells phorbol ester (231). However, this was shown to be due to
ERK activation is usually monitored by following the dual disinhibition of adenylyl cyclase signaling. Gastrin or CCK2
phosphorylation of the Thr and Tyr residues in the Thr-Glu- receptors have also been shown to be able to activate ERK
Tyr activation sequence brought about by MEK as there are (50, 57). In contrast to rodent pancreatic acini, in AR42J cells
a number of good phosphospecific antibodies directed at this the action of CCK to induce ERK activation was mediated by
epitope. It can also be shown by phosphorylation of myelin the CCK2 receptor, the tyrosine kinase Yes and transactivation
basic protein either in a test tube or by an in gel technique fol- of the EGFR (230). This transactivation is known to occur in
lowing gel electrophoresis and renaturization. Both Western some cells but in rat pancreatic acinar cells no CCK-induced
blots and the in gel kinase procedure reveal the two forms of EGFR activation was observed (54). Another mechanism for
ERK at approximately 44 and 42 kDa; in fact, the molecules ERK activation in some cells is through G protein coupled
were originally referred to as p42 and p44 MAPK with p42 receptors kinases that phosphorylate the receptor, which then
being what is now referred to as ERK2 and p44 now being binds β-arrestins, which recruit other signaling molecules and
ERK1. Using isolated rat or mouse pancreatic acini in vitro, mediate the prolonged activation of ERK (192). However, a
ERK1/2 is activated by CCK, bombesin, substance P, and β-arrestin mechanism has not been described for pancreatic
carbachol, all of which activate G protein coupled receptors acini or the CCK1R.
coupled to Gq and calcium mobilization, but not by secretin There is only a little information on the inactivation of
or VIP which activate receptors coupled to Gs and cAMP ERK in acinar cells. In one study, pancreatic DUSP mRNA

Figure 8 Activation of ERK signaling pathway by CCK and EGF in pancreatic acinar cells. CCK1R sig-
nals through PKC and the activation by phosphorylation of Raf isoforms. Raf sequentially phosphorylates
and activates MEK1/2 and ERK1/2. By contrast, EGFR autophosphorylates and then binds Shc, Grb2 and
SOS with the latter being a GEF for Ras; active Ras binds and activates Raf and the kinase cascade ending
in ERK. Activated ERK phosphorylates cytoplasmic substrates and downstream kinases, which phosphory-
lates transcription factors thereby activating expression of specific genes. Reproduced, with permission, from
reference (345).
levels were very low but DUSP-5, -6, and -10 were induced by mTOR complex 1 (TORC1). However, most of the cellular
caerulein hyperstimulation such that they could be considered responses of ERK signaling cascade are related to prolifer-
early response genes (122). ation, differentiation, angiogenesis, survival, or metastasis.
Many of the experimental studies have used MEK inhibitors
Actions of CCK mediated by ERK in pancreatic aci- as MEK is the only known activator of ERK1/2. The first MEK
nar cells ERK1/2 as a protein kinase has broad substrate inhibitor was PD98059, which is a flavone and highly insol-
specificity for Ser or Thr residues upstream from a Pro residue uble in water (72). Another MEK inhibitor U0126 was dis-
and can phosphorylate a large number of proteins with 659 covered shortly thereafter and is slightly more potent. These
target sites listed in a recent compendium (326). There are inhibitors have been used for in vitro studies of ERK sig-
also 296 reported ERK-interacting proteins whose function naling, but were not very useful for in vivo studies due to
may thereby be influenced by ERK (331). These include a poor solubility and short half-life. This led to the devel-
number of molecules known to be important in pancreatic opment of PD325901 and later Trametinib (GSK1120212),
function including Stim1 which regulates Ca2+ entry chan- which are longer acting and effective experimentally in vivo
nels (233) and Raptor (32) which promotes activation of the (284).

In pancreas, as in other cell types, most studies of ERK at the plasma membrane (327). Overactivation of JNK can
action have focused on growth, proliferation and regenera- lead to apotosis in part by phosphorylation of Bcl2 family
tion as ERK1/2 is recognized as a master regulator of the cell members. The three levels of kinase activation are facilitated
cycle focused on the G1 to S phase transition (188). To study by scaffolding proteins such as the JNK interacting proteins,
adaptive pancreatic growth mediated by CCK, Holtz et al. JIP1 and JIP2 (341). Similar to ERKS, JNKs are inactivated
fed mice trypsin inhibitor and showed that both PD325901 by tyrosine, serine/threonine, and dual specific phosphatases
and Trametinib blocked acinar cell mitogenesis and pancre- such as DUSP-7 (349).
atic growth (123). The drugs were effective when fed orally Only a few reports have evaluated the regulation of JNKs
by gavage or mixed into food and a single bolus dose was in the pancreas using either an in gel kinase assay with c-
effective for at least 12 hours. Moreover, the drugs had no Jun as substrate or active site dual phosphorylation Western
effect on other signaling pathways including mTOR, JNK, blots (347). Using isolated rat acini JNK was activated by
and STAT3. Cell cycle proteins including cyclin D1, D3 and CCK with lesser activation by carbachol and bombesin and
E as well as PCNA and BrdU incorporation into DNA were at higher concentrations than required to activate ERK. A
inhibited. In vitro, inhibiting ERK with U0126 or PD98059 longer stimulation time was also required (51). VIP, TPA,
blocked proliferation of acinar cell monolayer cultures (104). and increasing intracellular Ca2+ failed to activate JNK. A
ERK activation has also been shown to play a role in similarly high concentration of CCK delivered by IV infu-
cytokine production by pancreatic acinar cells. In isolated sion was required to activate JNK in vivo and the concen-
mouse acinar cells, PD98059 decreased production of MCP- tration used induced pancreatitis (96). Bombesin had a much
1, MCP1α and MIP-2 induced by Substance P (236) and in rat lower response and induces little or no pancreatitis. Because
pancreatic fragments, PD98059 reduced production of TNF- of these observations, it may be that JNK activation is a
α and IL-1β induced by cerulein (265). The stimulation of result of ER stress rather than a signal transduction cascade.
cytokine production in both cases involved AP-1 transcrip- Blocking the ER stress response with chemical chaperones
tion factor that is also blocked by inhibitors of JNK. EGR-1 or anti-inflammatory agents reduced JNK activation by high
is another early response gene whose expression in AR42J concentrations of CCK (180, 181, 355). Arguing for a more
cells was shown to be blocked by ERK inhibitor PD98059 or physiological role, JNK activation was observed prolonged
overexpression of DUSP-1 (MKP-1) (140). MEK inhibition trypsin inhibitor feeding to mice which induced endogenous
by either targeted shRNA or Trametanib has also been shown CCK release to a plasma concentration of about 10 pmol/L and
to reduce inflammatory cytokines in cerulein-induced chronic which induces pancreatic growth (308). In monolayer culture
pancreatitis (108). Some of this activation of MAPKs may be of mouse pancreatic acini, JNK was required to increase AP-1
mediated by reactive oxygen species as hydrogen peroxide activity and thymidine incorporation (104).
and menadione strongly activated all three MAPKs and the
activation by CCK was reduced by antioxidants (49).
P38 MAPK
The p38MAPKs are a third family of MAPK that respond to
JNK Pathway
stress and various extracellular signals. They include four iso-
The c-Jun NH2-terminal kinases (JNK) also known as stress- forms, -α, -β, -γ, and -δ coded for by different genes with dif-
activated protein kinase (SAPK) are MAPKs that possess ferent expression patterns. P38α is ubiquitously expressed and
the dual phosphorylation motif Thr-Gly-Tyr (58, 235). They mediates most actions referred to as p38MAPK. Pyridinyl-
are activated by treatment of cells with cytokines and by imidazole inhibitors such as SB203580 which are commonly
different forms of environmental stress. There are three forms used to demonstrate p38 action block the α and β form but not
of JNK (-1,-2, and -3) that are coded by different genes with the other two (48). P38γ and δ, which are about 60% iden-
JNK-1 and JNK-2 expressed widely and JNK-3 limited to tical to p38α are now known to perform specific functions
specific tissues such as brain and testis. The major substrate in different tissues (76). This discussion will focus on p38α.
for JNKs is c-Jun that is phosphorylated on Ser-63 and Ser The canonical path of activation involves dual phosphoryla-
73, which causes increased transcriptional activity as part of tion on the Thr-Gly-Tyr sequence present in the activation
an AP-1 complex. Other related AP-1 components such as loop by MKK3, MKK4, and MKK6 (48, 235). MKK3 and
JunB, JunD, and ATF2 are also phosphorylated by JNK while MKK6 are highly specific for p38MAPK but the JNK activa-
the other major AP-1 component c-Fos is phosphorylated by tor MKK4 can also activate p38α. Several MAP3Ks activate
ERK. JNKs are activated by dual phosphorylation by MKK-4 the MAP2Ks including ASK1, TAK1, MLK3 and MEKK3
and MKK-7 sometimes known as SEK by analogy to MEK and MEKK4. Inactivation by phosphatases is similar to that
(Fig. 7). MKK-4 can also activate p38 MAPK and is primarily of ERK and JNK with dual specific phosphatases, MKP1,
activated by stress while MKK-7 is more specific and may 4, 5, and 7 most important. Scaffold proteins may also be
mediate activation by cytokines (58). Multiple MKKK can involved. P38α is localized to both the cytoplasm and nucleus
activate the JNK pathway including MEKK proteins, mixed and while most of its actions are mediated by its kinase activity
lineage protein kinases, TAK1, and the ASK group (87, 340). or that of downstream kinases, it also has kinase independent
Some of these are activated by ER stress rather than events functions (48). Kinases activated by p38MAPK are involved

in gene expression especially MSK1 and MSK2 (mitogen are shared. However, TORC1 uniquely contains the scaffold-
and stress-activated kinase). MAPK-activated protein kinase ing protein Raptor (Regulatory associated protein of mam-
(MAPKAPK) also known as MKs include MK2 which plays malian target of rapamycin) (109) and PRAS40 (Proline rich
a role in actin remodeling by phosphorylation of Hsp27 (heat Akt substrate of 40 kDa) (329), whereas TORC2 contains
shock protein of 27 kDa) (86). Additionally, the MAP kinase among other components Rictor (rapamycin-insensitive com-
interacting kinases Mnk1 and Mnk2 are activated by ERKs panion of rapamycin) (154). Both raptor and PRAS40 are
and P38MAPK and regulate protein synthesis by phosphory- inhibitory proteins; phosphorylation blocks this inhibition.
lating eIF4E (35). PRAS40 represents an essential component for insulin acti-
The presence of p38 MAPK in rat pancreatic acini and vation of TORC1. Raptor is an essential component and its
its activation by CCK stimulation was first demonstrated by genetic deletion leads to loss of TORC1 activity (15).
Schafer et al. (53, 280). CCK, carbachol and bombesin as Much less is known as to the functions of TORC2. It
well as UV-B irradiation and sorbitol-induced osmotic stress is a regulator of the actin cytoskeleton in both yeast and
activated P38 MAPK as quantitated by immunoprecipitating mammalian cells (377). More recent studies have shown it
p38 and then carrying out a kinase assay with GST-ATF2 to be active in phosphorylating various protein kinases espe-
as substrate; VIP which increases cellular cAMP had no cially Akt.
effect. The p38α substrate MAPKAP kinase-2 was also iden- Only TORC1 is acutely sensitive to rapamycin, which
tified by immunoprecipitation and shown to phosphorylate a inhibits some, but not all, of TORC1 functions (234). This
peptide from Hsp27. The kinase activity of p38 MAPK and inhibition requires FKBP-12 (FK506 binding protein of
MAPKAPK-2 could be blocked by the inhibitor SB203580 12 kDa). Much is known about the function of TORC1 which
indicating that the p38 MAPK activity almost certainly is mediates the growth promoting effects including protein syn-
p38α. Little is known of the upstream components of the thesis, lipid synthesis, inhibition of autophagy, ribosome and
p38MAPK signal cascade in acini and the primary down- lysosome biogenesis and energy metabolism. The effects on
stream target that has been studied is Hsp27. CCK stimulated protein synthesis are mediated by activation of S6K1 (S6
phosphorylation on two sites of Hsp27 so the unphospho- Kinase-1), which phosphorylates ribosomal protein S6 and
rylated, monophosphorylated and dephosphorylated forms activates initiation and elongation translation factors, the latter
could be separated by isoelectric focusing or 2D gel elec- through elongation factor 2 kinase (234). In addition, TORC1
trophoresis (100). This was mediated in vivo by MAPKAPK- phosphorylates 4E-BP1, a binding protein that, upon phos-
2 as shown by its absence in MAPKAPK-2 gene deleted mice phorylation, releases the initiation factor eIF4E which acts
(319). CCK stimulation of acini increased the amount of fil- as a mRNA cap binding protein (332). The overall rate of
amentous actin and this effect was blocked with SB203580 protein synthesis also depends on the number of ribosomes,
(280). CCK was also shown to have similar effects on Hsp27 and TORC1 also enhances the synthesis of ribosomal proteins
phosphorylation in CHO cells transfected with the CCK1 and RNA (234). TORC1 promotes lipid synthesis by activat-
receptor (279). The older literature on stress kinases and heat ing SREBP (sterol responsive element binding protein) (278).
shock proteins in pancreas is reviewed in (281). MAPKAPK- The action of TORC1 to inhibit autophagy is mediated by
2 also is involved in upregulation of TNF-α and IL-6 while phosphorylation of ULK1 (unc-51 like autophagy activating
p38α downregulates expression of BAD, a proapoptotic pro- Kinase-1) and Atg13 (autophagy related 13) which blocks the
tein (223). formation of the phagosome (285). Recently, TORC1 has also
been shown to regulate the abundance of proteasomes; when
TORC1 is inhibited, the ability of proteasomes to degrade
mTOR pathway protein increases (253).
The mTOR signaling pathway is a nutrient sensing mecha- A variety of upstream signals regulate TORC1 including
nism coupled to mTOR, the mammalian or mechanistic target growth factors (and some hormones that stimulate growth of
of rapamycin (79, 154, 282). Rapamycin is named after the their target cells), amino acids, oxygen levels, and glucose
island Rapa Nui (Easter Island) from whose soil it was first (Fig. 9). Growth factors and insulin reflect the fed state of the
isolated and has broad antiproliferative and immunosuppre- organism and promote anabolic processes. Binding of insulin
sive properties (278). Genetic screens in the early 1990’s in to its receptor activates PI3K and Akt that are upstream of
yeast identified two genes TOR1 and TOR2 that mediated TORC1, Akt phosphorylates and inhibits TSC1/2 the tuberous
the effects of rapamycin. Biochemical studies then led to the sclerosus complex which acts as a tumor suppressor by acting
identification of the single mammalian form (26,258). mTOR as a GAP for Rheb (Ras homolog enriched in brain), a GTPase
is an atypical protein kinase related to phosphoinositide 3- which is one of the major activators of TORC1. Akt also
kinase family although it is a Ser/Thr-targeted kinase and not phosphorylates PRAS40 and thereby relieves its inhibition
a lipid kinase. It is a large protein of about 2500 amino acids of mTOR in TORC1. TORC1 limits its own activation by
with multiple domains including a C terminal kinase domain the negative feedback of S6K1 on the early steps in PI3K
and a FKBP-rapamycin binding (FRB) domain (79). activation especially by insulin (285).
mTOR is a component of two distinct complexes, TORC1 Another major signal regulating TORC1 activity is the
and TORC2; each contains other proteins, some of which abundance of amino acids, which tells the organism to

CCK Insulin pathway’s downstream mediators S6 and 4E-BP1. Phos-

phospecific antibodies are widely available for S6 and its
upstream kinase, S6K1 that is activated by TORC1. 4E-BP1
resolves into multiple bands on Western blots with the higher
PI3K
band being most highly phosphorylated. Such measurements
showed that in isolated rodent pancreatic acini CCK and sim-
AMP Hypoxia
ilarly acting secretagogues (bombesin, carbachol) activated
Akt/PKB
S6K1 (23), increased phosphorylation of S6, 4E-BP1, and
AMPK EF2K (elongation factor 2 Kinase) (23, 24, 274, 303) and
Amino acids that these effects were blocked by rapamycin, the TORC1
TSC complex inhibitor. Moreover, rapamycin blocked the increase in pro-
(TSC1/TSC2)
Rag tein synthesis stimulated by CCK in isolated acini (24). These
complex Translocation studies showed that the primary cell type involved in TORC1
RHEB
activation in the exocrine pancreas is the acinar cell and this
Activation has been reinforced in vivo where CCK injection increased
Lysosome mTORC1
phosphorylation of S6 and 4E-BP1 as well as the phosphory-
S6K
lation of eIF4E and the formation of the eIF4F initiation com-
plex (25). Elevating endogenous CCK by feeding the trypsin
4E-BP1
SREBP1 ULK1
inhibitor camostat or by diverting bile pancreatic juice also led
Mitogenesis to similar effects (45, 113). As discussed earlier, amino acids
Protein activate the TORC1 pathway and leucine and other branched
Lipid Autophagy synthesis chain amino acids activate protein synthesis and the TORC1
biosynthesis pathway both in pancreas in vivo and in isolated pancreatic
acini (114, 271, 321). S6 and 4E-BP1 signaling in the pan-
Figure 9 General and simplified diagram of mTORC1 pathway set creas are also affected by insulin and diabetes (225,268,302).
in pancreatic acinar cell, where the pathway is regulated by CCK and
insulin. Red arrows indicate activation; Black arrows indicate inhibi-
TORC1 signaling is not required for secretion of digestive
tion; Green arrow indicates translocation. Biological processes regu- enzymes (23) but is required for protein synthesis and adap-
lated by mTORC1 are shown at the bottom of the figure, along with the tive growth (24, 45, 47).
key proteins mediating the effect. Reproduced, with permission, from
reference (273).
In addition to studies of acinar cells, the TORC 1 path-
way appears to play a role in activated pancreatic stellate cells
where it mediates effects of insulin to enhance collagen syn-
undergo anabolic activity (10, 134). Conversely, the absence thesis and fibrosis (361). These in vitro effects of insulin were
of adequate amino acids is a stress, which leads to the shut- blocked by TOR inhibitors rapamycin and KU63794. TORC1
ting down of biosynthetic pathways and the induction of also plays a role in the endocrine pancreas where it is involved
autophagy. The major amino acids sensed are the branched in islet development, beta cell growth and insulin processing
chain amino acids, especially leucine, as well as arginine and secretion (11, 21, 75, 164, 289).
and glutamine. Amino acid sensing involves recruitment of The importance of the TORC1 pathway in the exocrine
TORC1 from cytoplasm to lysosomes where it interacts with pancreatic response to feeding is shown by the activation of
proteins including the Rag GTPases (143), a protein com- the downstream components when mice fasted overnight are
plex termed the ragulator (266), amino acid transporters (93), refed (269). In this study, protein synthesis also increased
and the lysosomal vacuolar ATPase (64). In the presence of with feeding without a change in mRNA levels for digestive
amino acids, Rheb that is also localized on the lysosome, enzymes, indicating the importance of translational control
is activated and in turn activates TORC1. Furthermore, amino primarily by the TORC1 pathway in synthesis of new diges-
acids are necessary for almost all other mechanisms activating tive enzymes after secretion. Similar effects have also been
TORC1. Low oxygen or low glucose levels prevent TORC1 seen in neonatal pigs (90). TORC1 was also shown to play a
signaling through AMP Kinase and reduce the activity of the role in the hypertrophic response to feeding a high protein diet
proteins REDD and BMIP3 (285, 365). DNA damage also and this was independent of CCK (47). Conversely, pancreatic
inhibits TORC1. As a result of this network of interactions, atrophy was seen along with to a loss of TORC1 signaling
growth and other anabolic activities can only take place in the when mice were fed a protein free diet (44). These in vivo
presence of a supporting milieu. responses involve multiple hormones including CCK and
insulin and nutrients acting directly, particularly amino acids.
Actions of CCK mediated by mTOR signaling in
pancreatic cells Calcineurin-NFAT pathway
mTOR signaling in pancreas was first recognized, and is Calcineurin (Cn), a unique calcium/calmodulin activated
most commonly followed, through phosphorylation of the serine/threonine phosphatase is a calcium effector usually

requiring prolonged Ca2+ elevation, which both alters gene by additional phosphorylation by GSK-3. This leads to rapid
expression and has cellular effectors leading to immediate export from the nucleus preventing brief Ca2+ signals from
responses (163). Much of the gene regulation is mediated initiating gene expression (239, 354).
by NFAT (nuclear factor of activated T cells). Other cal-
cineurin substrates or binding partners are the K+ channel
Actions of CCK mediated by calcineurin
TRESK, KSR2 (kinase suppressor of Ras 2), AKAP79 (A-
in acinar cells
kinase anchoring protein 79), the transcription factors MEF2A
and Crz1 and the binding protein, Rcan1 (regulator of cal- A Ca2+ /CaM dependent protein phosphatase has been demon-
cineurin 1). Calcineurin is specifically inhibited by immuno- strated in mouse and rat pancreatic acinar cytosol (29, 101).
suppressant drugs such as cyclosporine A and FK506. Two-dimensional gel electrophoresis of P32 labeled proteins
Calcineurin is a heterodimer made up of the two subunits showed a limited set dephosphorylated by CCK stimula-
CnA and CnB that are noncovalently attached. There are three tion (350). One of these was heat stable, had an approxi-
isoforms of CnA: CnAα, CnAβ, and CnAγ while CnB has mate molecular mass of 24 kDa and its dephosphorylation
two isoforms: CnB1 and CnB2. CnAγ and CnB2 are primarily was blocked by treatment with cyclosporine A (102). This
found in testis while the other isoforms are broadly distributed. protein termed CRHSP-24 was sequenced, had its phospho-
CnA is an approximately 60-kDa protein and contains the rylation sites identified, and its crystal structure determined
catalytic domain, a binding domain for CnB, a calmodulin (102, 125, 159). It appears to be an RNA binding protein that
(CaM) binding domain and a autoinhibitory domain (149,200, participates in the stress response but its main significance to
257). The regulatory subunit CnB is approximately 19 kDa in date is as a readout of calcineurin activation in acinar cells.
size and contains four Ca2+ binding EF hands two of high A second action of this pathway involved calcineurin
affinity and two with lower affinity. Complete activation of activation, NFAT dephosphorylation, and pancreatic adap-
calcineurin involves binding of Ca2+ to the low affinity sites tive growth. Tashiro et al. (308, 309) used the feeding of
on CnB followed by binding of Ca2+/ CaM to CnA (163). This the trypsin inhibitor camostat to increase plasma CCK and
leads to a conformational change such that the autoinhibitory stimulate pancreatic adaptive growth in mice. Other organs
domain moves out of the catalytic groove. This activation were not affected and the response was absent in CCK gene
occurs in the range of Ca2+ concentrations from 100 nmol/L deleted mice. Both cyclosporine A (CsA) and FK506 blocked
to several μmol/L. this growth. The amount of Cn and its activity were increased
A major target of calcineurin is the NFAT family of tran- by the elevated CCK. CsA and FK506 did not affect the
scription factors (239). Of the five members, four are regu- increase in plasma CCK. Several other signal transduction
lated by calcineurin (NFAT 1, 2, 3, 4) and are also known as pathways were also affected by calcineurin inhibitors (308).
NFAT c2, c1, c4, and c3, respectively. They evolved at the CCK both in vitro and in vivo released by trypsin inhibitor
time of the origin of vertebrates and contain in addition to the feeding induced NFAT translocation to the nucleus of aci-
amino terminal Cn regulated domain, a DNA binding domain nar cells and that this correlated with activation of a NFAT
related to the Rel family of transcription factors (354). The luciferase reporter (106). NFATc1, c2, and c3 were present
regulatory domain possesses 13 phosphorylation sites which and all translocated. The effect of CCK was blocked by the
are dephosphorylated by Cn. Dephosphorylation exposes a calcineurin inhibitors CsA, FK506 and overexpression of the
nuclear localization signal (NLS) and the NFAT then moves calcineurin inhibitory protein, CAIN. Utilizing the model
into the nucleus. The Rel like domain alone binds weakly to of trypsin inhibitor feeding to induce pancreatic growth, 39
DNA but binds cooperatively with other factors such as AP1, genes were identified as having their expression increased in
GATA, and MEF2 (193, 354). NFAT signaling was initially a manner blocked by FK506 (105), Most of these genes were
identified in T cells where it connects the T cell antigen recep- predicted to be NFAT regulated and ChiP assay showed bind-
tor (TCR) to expression of the IL-2 gene. With the advent of ing of NFATc1 induced by CCK. The most highly induced
NFAT gene deletion it has been discovered that NFATs play genes were Rcan1 (regulator of calcineurin 1 also known as
a role in the development of the nervous system, the heart, DSCR, Down Syndrome Critical Region 1), FGF21, Kel, and
lungs, skeletal muscle, blood vessels and pancreatic islets as Socs3. A mouse line acutely overexpressing Rcan1 in aci-
well as postnatal cardiac hypertrophy (354). Most individ- nar cells showed inhibition of calcineurin-NFAT signaling
ual genes that are activated by NFATS are proinflamatory or and pancreatic growth (105). Thus, Rcan1 can be considered
proangiogenic. However, one of the most strongly activated a physiological feedback inhibitor. Interestingly other feed-
genes is Rcan1 also known as Dscr1 designated because it back inhibitor proteins such as Rgs2 were also induced. Rcan1
resides in the Downs syndrome critical region of chromosome overexpression along with aging has been shown to promote
21. Rcan1 strongly inhibits Cn mediated NFAT signaling by diabetes (227). The activation of the Ca2+ -calcineurin-NFAT
inhibiting the binding of Cn to NFAT and its enzymatic activ- pathway in pancreatic acinar cells is diagramed in Figure 10.
ity. Calcineurin action depends on docking to target proteins Other actions of CCK that may involve calcineurin are
through a PxIxIT motif originally identified in NFAT. the synthesis and secretion of digestive enzymes. Calcineurin
NFAT signaling ends when the protein is phosphorylated inhibitors, FK506 and CsA dose dependently inhibited the
by dual specificity tyrosine kinase 1 (Dyrk1) or PKA followed stimulation of amino acid incorporation into protein of

Figure 10 Activation of the Ca2+ -calcineurin-NFAT signaling pathway by CCK and cholinergic ago-
nists in pancreatic acinar cells. The initial steps in the pathway involve receptor-mediated elevation of
intracellular Ca2+ . Ca2+ binds to calmodulin (CaM) and the complex to the Ca2 + activated phosphatase
calcineurin. Calcineurin can dephosphorylate a number of substrates but the most generally important are
NFATS which when dephosphorylated move into the nucleus and bind alone or together with coactiva-
tors such as AP-1 to activate specific genes including FGF21, Socs3, Rgs2, Hbegf and Rcan1 with the
latter feeding back to inhibit calcineurin. Reproduced, with permission, from the Pancreapedia, Calcium–
Calcineurin–NFAT Signaling Pathway, 2012.
isolated rat pancreatic acini (272). In this study, FK506 unpublished data). Thus, whether secretion of digestive
blocked the phosphorylation of 4EBP-1 and reduced the for- enzymes requires calcineurin is unclear.
mation of the eIF4F complex but did not affect the phospho- Similar studies of calcineurin NFAT signaling have been
rylation of ribosomal protein S6 or eIF4E. This study needs carried out in islet beta cells and this pathway has been shown
confirmation and more detailed mechanistic understanding. to play a role in development, adaptive growth and insulin
The effect of calcineurin inhibitors on secretion of diges- synthesis (65,95,115,155).The effects of FK506 on beta cells
tive enzymes has also been studied. Early work showed that is also of interest because of its use as an immunosuppressive
CsA and FK506 partially reduced stimulated amylase release agent following pancreas transplantation.
from rat pancreatic lobules and isolated acini (101, 119, 338).
However, relative potency of the inhibitors differed and did
not always follow their ability to inhibit Cn activity. More- Rho and Rac pathways
over, CsA at μmol/L concentrations affects the mitochondrial CCK is known to mediate effects through small G proteins of
permeability pore and effects on secretion were seen at 1 the Ras superfamily (344). Rho family proteins include about
to 400 μmol/L CsA. Latter studies have not seen effects of 20 proteins in mammalian cells and are best known as regu-
FK506 on secretion in rat or mouse acini (127) (Williams JA, lators of the actin cytoskeleton but also can affect other cell

processes including gene expression, proliferation, intracellu- These include WASP (Wiskott-Aldrich syndrome protein) and
lar trafficking, cell polarity, and cell migration (77, 120, 201). related proteins which help stimulate actin filament formation,
The most highly conserved and best studied Rho family mem- formins such as mammalian diaphanous 1 (mDia1) which act
bers are RhoA, Rac1, and Cdc42, all of which have well as both initiating and elongation factors and the actin depoly-
known actions on the actin cytoskeleton (211). The study merizing proteins ADF/cofilin (92, 107). Cofilin is inhib-
of Rho family G proteins in secretion has been carried out ited by LIM Kinase induced phosphorylation and LIMK is
largely because of the importance of the cytoskeleton and activated by the Rho effector ROCK (Rho Kinase). Thus
particular actin in the secretory process. These studies have RhoA signaling increases actin polymerization and decreases
included platelets (9) and neutrophils as well as pancreatic depolymerization. ROCK also phosphorylates MLCK and
exocrine (344) and endocrine cells. Rho family members thereby promotes actin-myosin interaction. In the terminal
are posttranslationally modified by addition of farnesyl or steps in secretion zymogen granules must pass through an
geranylgeranyl lipid chains to cysteine residues at the car- actin network under the apical plasma membrane and follow-
boxyl terminal. Thus, they tend to associate with cellular ing exocytosis actin polymerizes as a coating around the secre-
membranes. tory granule, a process requiring active Rho (131, 206, 328).
Classically, Rho family members are activated by extra- Finally, other effectors such as SRF (serum response factor)
cellular soluble molecules, cell-cell interactions, and mechan- mediate effects on gene expression and proliferation.
ical stress (107). Activation of the Rho molecules is by bind-
ing of GTP in place of GDP, which is facilitated by GEFs
Rho Proteins in the pancreas
(guanine nucleotide exchange factors). Inactivation of the
G protein is by a GAP (GTP-ase activating protein) which Studies of Rho family G proteins in the exocrine pancreas
enhances the rate of GTP hydrolysis. A third class of regula- have been limited to RhoA, Rac1, and Cdc42. Using pull down
tors are the GDIs (guanine nucleotide dissociation inhibitors) assays to monitor the G protein in the active, GTP liganded
which can extract G proteins from membranes and sequester state, CCK was shown to increase active RhoA and Rac1 but
the inactive G proteins in the cytoplasm. In addition to these not Cdc42 in isolated rodent pancreatic acini (20). Both RhoA
three proteins, Rho GTPases are regulated by posttranslational and Rac1 were observed to translocate within the cell upon
modifications including phosphorylation, ubiquitination, and CCK stimulation. Moreover, expression of dominant negative
sumoylation (120). Rho and Rac inhibited CCK stimulated amylase secretion
The first identified mammalian Rho GEF was isolated from isolated acini. We will therefore consider further what is
from B-lymphoma cells and was designated as Dbl. Most known of the role of RhoA (Fig. 11) and Rac1 in pancreatic
Rho GEFs contain a region of homology to Dbl. Adjacent to acinar cells. There is evidence, however, for Cdc42 playing a
the Dbl domain is a PH or pleckstrin homology domain which role in pancreatic islet beta cells.
binds to phosphoinositides and localizes the Rho GEF to The earliest work studying the importance of RhoA for
plasma membranes. The 70 Dbl containing GEFs also contain pancreatic enzyme secretion involved botulinum C3 exotoxin.
other structural domains, which determine which G protein is This toxin inactivates RhoA through ADP ribosylation at Asp
their target. Some are specific to a single protein such as RhoA 41 (2). Although it is difficult to get the protein into intact
while others such as Vav and Tiam can act on multiple Rho cells, Rosada et al. were able to incubate isolated rat acini
family members including Rac1 (252). Some Rho GEFs are with C3 toxin and ADP-ribosylate RhoA and reduce CCK-
activated by phosphorylation or protein-protein interactions; stimulated secretion (250). Kiehne et al. used digitonin per-
others can also act as scaffolding proteins. Heterotrimeric G meabilized acini to allow entry of C3 toxin and showed that
proteins that are activated by G protein coupled membrane it blocked both the basolateral blebbing and Tyr phosphory-
receptors act on specific Rho GEFs including p115-Rho GEF, lation of p125FAK (142). CCK also was shown to induce
Larg, and pdz-Rho GEF all of which contain a RGS domain the translocation of RhoA in rat pancreatic acini (214). Using
(83, 252). Most of these GEFs are activated by Gα12/13 and NIH3T3 cells containing CCK1 receptors, CCK was shown to
in some cases by Gαq/11 (83). About 11 other Rho GEFs lack activate Rho, induce actin containing stress fibers, and acti-
the Dbl domain but are related to DOCK1 and possess a dock vate a SRE luciferase reporter (156). The effects of CCK
homology region (DHR). were mimicked by adenoviral expression of constitutively
Rho GAPS contain a 150 amino acid RhoGAP domain. active RhoV14 and blocked by expression of dominant neg-
About 80 Rho GAPS exist in humans and many are regulated ative RhoN19, which lock the protein into the GTP or GDP
by post-translational modifications, especially phosphoryla- liganded structure respectively. Other studies indicated that
tion (120,311). The number of Rho GAPS exceeds the number the most likely heterotrimeric G protein mediating the effect
of Rho G proteins with some showing tissue specific expres- of CCK to activate Rho was Gα12/13 (156).
sion. Similar to GEFs Rho GAPS have multiple structural Isolated mouse acini have been studied using adenoviral
domains and some act on a single GTPase while most act on vectors to introduce modified G proteins or the active region
multiple Rho proteins. of C3 toxin. Dominant negative RhoA and Rac1 decreased
The best studied Rho family G protein effectors are CCK stimulated amylase secretion with either DN RhoA
involved in regulation of the actin cytoskeleton (130). and Rac1 or C3 toxin plus DN Rac1 being additive (20).

Figure 11 Rho Activation pathway by CCK and ACh. The CCK1 and muscarinic M3 receptors activate Gα12/13, which
activate a Rho GEF of which several present in the pancreas are shown. Rho with bound GTP can activate mDia1 or ROCK (Rho
kinase) thereby regulating the actin cytoskeleton or activate SRF (serum response factor) and activate gene expression. GTP
bound Rho is inactivated by a Rho GAP of which several present in pancreas are shown. Botulinum exotoxin can permanently
inactivate Rho. Reproduced, with permission, from Pancreapedia, Galpha12/13-Rho Signaling Pathway, 2010.
The same proteins decreased amylase release stimulated by acini by adenoviral vectors only Gα13 activated RhoA while
cholinergic agonist but did not affect the increase in intracel- both Gαq and Gα13 activated Rac1 and the combination of
lular Ca2+ . Conversely, introduction of constitutively active Gαq and Gα13 strongly activated Rac1 (260). G12/13 acti-
RhoA or Rac1 had no effect on basal secretion but led to an vates specific Rho GEFs that possess a RGS domain (Regula-
approximate doubling of CCK-stimulated amylase secretion. tor of G protein signaling). Two of these proteins, p115 Rho
Active or inhibitory Rho and Rac also induced changes in the GEF and LARG have been identified in acinar cells (344).
actin cytoskeleton as visualized by phalloidin staining (19). Rho A activation as well as stimulation of amylase secretion
That these effects were mediated by the actin cytoskeleton could be blocked by expression of a p115-RGS domain (260).
was shown using jasplakinoid, an actin polymerizing toxin The blebbing induced by active Gα13 could be blocked with a
which induced basolateral blebs, while latrunculin, which ROCK inhibitor. Further evidence that Gαq was not involved
binds and sequesters actin monomers prevented the morpho- in Rho activation was shown using the expression of RGS2,
logical changes induced by CCK (19). Actin has also been which abolished CCK-induced Ca2+ mobilization but did not
visualized by introducting Life Act – GFP that revealed short affect Rho activation. RhoA has also been shown to partic-
actin cable perpendicular to the apical membrane induced ipate in a complex with Vav2 and Src whose formation is
by mDia1, a downstream mediator of RhoA. Granules were induced by CCK stimulation (144).
shown to move along these cables to reach the apical mem- Rac1 activation could be inhibited with both RGS2 and
brane (92). p115-RGS. Thus, both Gαq and Gα13 are necessary to medi-
Only a little is known as to which GEFs activate RhoA ate CCK activation of Rac1 and this action was also shown to
and Rac1 or which effector molecule they utilize in pancreatic be phospholipase C independent (260). Rac1 participated in
acini. Both Gα12/13 and Gαq proteins activate Rho and Rac CCK-induced amylase secretion and this action was mediated
GEFs in different cell types (252). When constitutively active by PAK2 (J. Lim & J. A. Williams, unpublished data) (215).
α subunits of Gq , Gs , and G13 were introduced into mouse The activation of PAK2 was mediated by the adapter protein

Beta-PIX. Blockage of PAK2 expression or activity inhibited is the isoform responsible for stimulating enzyme secretion
amylase secretion. in mouse pancreas (262). cAMP acts through binding to the
Less is known of the GAPs and GDIs that also regulate regulatory subunits of PKA which leads to dissociation and
Rho and Rac in acinar cells. There are three forms of GDI activation of the catalytic subunits. Two isoforms of PKA
and RhoGDI1 and RhoGDI3 are present in cytosol where with differing regulatory subunits exist and both have been
they bind RhoA and Rac1 (263). CCK stimulated PKCα can described in pancreas. An alternate cAMP target is EPAC
phosphorylate RhoGDI1 on Ser96 and thereby release RhoA (exchange protein directly activated by cAMP). There are
and Rac1. Rho GAPs play a role in inactivating Rho A and two EPAC isoforms that upon binding cAMP act as GEFs
Rac1. There are almost no studies of GAPS in acini although for the small G protein Rap1. EPAC1 is present in acini in
p50RhoGAP, p122RhoGAP, p190RhoGAP-B, and RICS have the ZG area while EPAC2 is present in islets (261). Rap1,
been observed by mass spectrometry (248) or by PCR which is also activated by CCK stimulation acting through
(E. Sabbatini & J. A. Williams, unpublished data). a calcium and DAG activated GEF, plays a necessary role in
pancreatic digestive enzyme secretion, which can be inhibited
by overexpressing a RAP GAP (261).
Adenylyl Cyclase-cAMP-Protein Kinase A pathway The CCK1 receptor has the ability to increase AC and
The adenylyl cyclase (AC)-cAMP-protein kinase A (PKA) the concentration of cAMP in transfected cell models such
pathway was one of the first signal transduction cascades as HEK and CHO cells with the first intracellular loop of the
worked out in mammalian cells. It plays a role in the pancreas receptor involved in signaling through Gαs to AC (357, 373).
primarily in duct and islet cells but may have a supporting role The CCK2 receptor does not couple to Gαs but can follow-
in acinar cells (Fig. 12). It is activated by secretin, VIP and ing conversion of serine 82 to asparagine in the first intra-
GLP-1 all of which act on G-protein coupled receptors. There cellular loop (356). High concentrations of CCK have been
are 9 different transmembrane AC isoforms divided into four shown to increase adenylyl cyclase activity in homogenates
groups and one soluble form (259). AC3, -4, -6, -7, and -9 have of guinea pig pancreatic acini (292). In both guinea pig and
been shown to be expressed in mouse pancreas (262). AC6 rat acini in the presence of the phosphodiesterase inhibitor
Figure 12 Activation of cAMP pathway in pancreatic acinar and duct cells. Secretin, VIP and CCK bind to their receptors
which activate adenylyl cyclase (AC) through Gαs while Somatostatin acts on SomstostatinR2 to activate Gαi which inhibits
AC. Cholera toxin activates Gαs while forskolin directly activates AC and Pertussis toxin inhibits Gαi. AC produces cyclic
AMP (cAMP) which is broken down by phosphodiesterase (PDE) which can be inhibited by isobutylmethylxanthine (IBMX).
cAMP activates PKA which can phosphorylate a number of proteins including CREB, CFTR and the IP3 receptor. cAMP also
activates EPAC1 which acts as a GEF to activate the small G protein Rap1 present on zymogen granules. Reproduced, with
permission, from Pancreapedia, cAMP Signaling Pathway, 2012.

IBMX, high concentrations of CCK (10-100 nmol/L) moder- involved in the initial phosphorylation and there is evidence
ately increased cAMP levels (89, 182, 246) and PKA activ- for Rho and Src also being involved (88,220). The function of
ity (182). A lower concentration of 0.5 nmol/L CCK without this signaling pathway physiologically is unclear although it
IBMX increased PKA without affecting cAMP levels (182). has been suggested to mediate activation of the MAP Kinase
Most studies using more physiological levels of CCK have pathway (306). It likely mediates the dissociation of the actin
not shown effects on cAMP signaling and no physiological cytoskeleton that occurs with supramaximal stimulation and
actions of CCK have been linked to this pathway. Rather, participates in overstimulation pancreatitis.
cAMP signaling induced by secretin or VIP acts to potentiate
amylase secretion induced by CCK through Ca2+ signaling
Src family kinases
(7, 259, 348).
c-Src is the prototypic member of a family of cytosolic nonre-
ceptor tyrosine kinases and homologous to viral V-Src. There
Other signaling molecules or pathways activated are 11 Src family kinases, which are about 60 kDa in size and
by CCK possess an amino terminal myristolation site followed by 4
PKD SH (Src homology) domains, a hinge region and a carboxyl
terminal catalytic domain (290). Src activation is related to
Protein kinase D was originally classified as an atypical PKC phosphorylation at tyrosine 416, which holds the kinase in
but is now known to be a distinct seine/threonine kinase fam- the open configuration. When Src is activated SH2 and SH3
ily. The PKD family contains three isoforms, PKD1, PKD2, domains are exposed which can bind substrates including
and PKD3 and each possesses an amino terminal regulatory paxillin, cortactin, and p130 Crk.
and a carboxyl terminal kinase domain, which has homol- Src family members identified in pancreatic acinar cells
ogy to myosin light chain kinase (254). In unstimulated cells, include c-Src, Yes, Lyn and possibly Fyn; Src, Yes, and
PKD has a low catalytic activity as a result of autoinhibi- Lyn are activated by high concentrations of CCK and other
tion by an amino terminal domain. Treatment with phorbol PLC coupled acinar cell secretagogues as shown by kinase
esters induces rapid activation that can be blocked with a activity on exogenous substrates or autophosphorylation
PKC inhibitor and involves phosphorylation by PKC on an (178, 213, 221, 237, 267). Immunolocalization shows concen-
activation loop. PKD is believed to play a role in cell pro- tration under the apical membrane in association with fila-
liferation, polarity, inflammation, membrane trafficking and mentous actin in the resting state and translocation to the
angiogenesis (254). cytoplasm after stimulation (178, 291). How Src proteins
In pancreatic acini, PKD1 and PKD3 are activated by are activated is not fully understood but involves Ca2+
CCK and PKD1 activation has been shown to involve PKCδ influx through store operated calcium channels (240, 324).
(16, 39). PKD isoforms have been associated with amy- It has been proposed that a Ca2+ activated tyrosine kinase is
lase secretion, NF-κB activation, intracellular trypsin activa- involved which phosphorylates Src but the identity of such a
tion, inflammation, and experimental pancreatitis (318, 366). kinase is not clear.
Recently, small molecule inhibitors of PKD that do not affect Src family members have been proposed to regulate var-
PKC have been developed. These inhibitors block cerulein ious biological functions in acinar cells including enzyme
activation of NF-κB, activation of trypsinogen and the sever- secretion (178, 213), store mediated calcium entry (240),
ity of experimental pancreatitis (318, 367). However, they endocytosis (224), changes in actin localization and acinar
have not yet been tested on models of pancreatitis other than cell blebbing (178, 291), activation of PKC-delta (307) and
cerulein-induced. chemokine production in response to substance P (237). Most
of these are induced by supraphysiological levels of secreta-
gogues and the primary evidence is based on the use of the
FAK tyrosine phosphorylation
chemical inhibitors PP1 and PP2, which may have additional
CCK is known to induce the tyrosine phosphorylation of a effects. Thus, the role of Src in these processes requires further
number of proteins in pancreatic acinar cells and two were work. Some of the high dose effects of CCK on Src are prob-
identified as the focal adhesion kinase, p125 FAK and the ably related to cerulein induced pancreatitis in which actin
proline rich kinase 2, Pyk2 often referred to as paxillin. FAK redistribution is an important component. A recent in vitro
is a cytosolic protein known to be associated with focal adhe- study of rat acini showed that Src kinases inhibited apoptosis
sions and paxillin is associated with the cytoskeleton. In rat and promoted necrosis but did not affect physiological secre-
pancreatic acini, this tyrosine phosphorylation is mediated by tion (217). The Src inhibitors PP2 and Dasatinib have also
physiological concentrations of CCK although the maximum been shown to reduce the severity of caeulein-induced pan-
effect requires supermaximal concentrations (88, 160). It is creatitis in mice in vivo (194,237). A recent study showed that
often taken that FAK is activated first and then phosphorylates Src kinases affected a number of other signaling pathways that
paxillin although evidence for this is not clear. There are six may also be involved in caerulein-induced pancreatitis (216).
Tyr phosphorylation sites in FAK and several are conserved in To our knowledge, the role of Src kinases in other models of
Pyk2. Both PLC dependent and independent mechanisms are pancreatitis has not yet been explored. Gene deletion of Src

kinases is often lethal but acinar cell specific deletion could pathways in rat pancreatic acini. Biochim Biophys Acta 1643: 37-46,
be a useful approach. 2003.
7. Argent BE, Gray MA, MSteward MC, Case RM. Cell physiology of
pancreatic ducts. In: Physiol of the Gastrointest Tract, edited by Johnson
LR. Boston: Academic Press, 2012, pp. 1399-1423.
8. Ashby MC, Camello-Almaraz C, Gerasimenko OV, Petersen OH,
Tepikin AV. Long distance communication between muscarinic recep-
Summary and Conclusions tors and Ca2+ release channels revealed by carbachol uncaging in
cell-attached patch pipette. J Biol Chem 278: 20860-20864, 2003.
The pancreatic acinar cell is specialized to synthesize and 9. Aslan JE, McCarty OJ. Rho GTPases in platelet function. J Thromb
Haemost 11: 35-46, 2013.
secrete digestive enzymes that perform luminal digestion of 10. Bar-Peled L, Sabatini DM. Regulation of mTORC1 by amino acids.
nutrients in the small intestine that is necessary for nutri- Trends Cell Biol 24: 400-406, 2014.
11. Bartolome A, Guillen C. Role of the mammalian target of rapamycin
ent assimilation. While early work focused on the control of (mTOR) complexes in pancreatic beta-cell mass regulation. Vitam
secretion, more recent work has shown the presence of most of Horm 95: 425-469, 2014.
12. Bastani B, Yang L, Baldassare JJ, Pollo DA, Gardner JD. Cellular
the major intracellular signaling pathways in acinar cells and distribution of isoforms of protein kinase C (PKC) in pancreatic acini.
some have been identified in duct or stellate cells. Many have Biochim Biophys Acta 1269: 307-315, 1995.
13. Baumler MD, Nelson DW, Ney DM, Groblewski GE. Loss of exocrine
been tied to other acinar cell functions such as protein synthe- pancreatic stimulation during parenteral feeding suppresses digestive
sis (translation), adaptive organ growth and the regulation of enzyme expression and induces Hsp70 expression. Am J Physiol Gas-
trointest Liver Physiol 292: G857-G866, 2007.
gene expression. In some cases, it is not yet clear whether this 14. Beglinger C, Fried M, Whitehouse I, Jansen JB, Lamers CB, Gyr K.
regulation is physiologic, pathophysiologic or possesses yet Pancreatic enzyme response to a liquid meal and to hormonal stim-
ulation. Correlation with plasma secretin and cholecystokinin levels.
unclear regulatory functions. One major point is clear; CCK J Clin Invest 75: 1471-1476, 1985.
acting through the CCK1R can regulate multiple functions of 15. Bentzinger CF, Romanino K, Cloetta D, Lin S, Mascarenhas JB, Oliveri
F, Xia J, Casanova E, Costa CF, Brink M, Zorzato F, Hall MN, Ruegg
acinar cells but acting through different signaling pathways. MA. Skeletal muscle-specific ablation of raptor, but not of rictor, causes
Thus while Ca2+ is the major regulator of exocytotic secre- metabolic changes and results in muscle dystrophy. Cell Metab 8: 411-
424, 2008.
tion, the mTORC1, MAPK, and calcineurin-NFAT pathways 16. Berna MJ, Hoffmann KM, Tapia JA, Thill M, Pace A, Mantey SA,
regulate protein synthesis and growth. At present interest is Jensen RT. CCK causes PKD1 activation in pancreatic acini by sig-
naling through PKC-delta and PKC-independent pathways. Biochim
centered on the role of these pathways in diseases such as Biophys Acta 1773: 483-501, 2007.
pancreatitis and pancreatic cancer. However, this understand- 17. Berridge MJ. Rapid accumulation of inositol trisphosphate reveals that
agonists hydrolyse polyphosphoinositides instead of phosphatidylinos-
ing will be developed based on understanding of the role of itol. Biochem J 212: 849-858, 1983.
signaling pathways in physiological regulation. 18. Berridge MJ. Inositol trisphosphate and diacylglycerol: Two interacting
second messengers. Annu Rev Biochem 56: 159-193, 1987.
19. Bi Y, Page SL, Williams JA. Rho and Rac promote acinar morphological
changes, actin reorganization, and amylase secretion. Am J Physiol
Gastrointest Liver Physiol 289: G561-G570, 2005.
Acknowledgements 20. Bi Y, Williams JA. A role for Rho and Rac in secretagogue-induced
amylase release by pancreatic acini. Am J Physiol Cell Physiol 289:
C22-C32, 2005.
I would like to thank all the students, fellows, and researchers 21. Blandino-Rosano M, Barbaresso R, Jimenez-Palomares M, Bozadjieva
N, Werneck-de-Castro JP, Hatanaka M, Mirmira RG, Sonenberg N,
over the last 45 years who contributed directly or indirectly to Liu M, Ruegg MA, Hall MN, Bernal-Mizrachi E. Loss of mTORC1
the research from my laboratory that is reviewed here as well signalling impairs beta-cell homeostasis and insulin processing. Nat
Commun 8: 16014, 2017.
as other research that is not covered. I would especially thank 22. Bragado MJ, Dabrowski A, Groblewski GE, Williams JA. CCK acti-
David Yule, Rodger Liddle, M. Dolors Sans, and Eugenia vates p90rsk in rat pancreatic acini through protein kinase C. Am J
Physiol 272: G401-G407, 1997.
Sabbatini who made figures and read and edited the review 23. Bragado MJ, Groblewski GE, Williams JA. p70s6k is activated by CCK
as it was in preparation. Juliana Lam provided support for in rat pancreatic acini. Am J Physiol 273: C101-C109, 1997.
24. Bragado MJ, Groblewski GE, Williams JA. Regulation of protein
the preparation of the manuscript. I also thank the NIH for synthesis by cholecystokinin in rat pancreatic acini involves PHAS-
funding my research over this period. I and the p70 S6 kinase pathway. Gastroenterology 115: 733-742,
1998.
25. Bragado MJ, Tashiro M, Williams JA. Regulation of the initiation of
pancreatic digestive enzyme protein synthesis by cholecystokinin in rat
pancreas in vivo. Gastroenterology 119: 1731-1739, 2000.
References 26. Brown EJ, Albers MW, Shin TB, Ichikawa K, Keith CT, Lane
WS, Schreiber SL. A mammalian protein targeted by G1-arresting
rapamycin-receptor complex. Nature 369: 756-758, 1994.
1. Adler G, Beglinger C, Braun U, Reinshagen M, Koop I, Schafmayer A, 27. Bruce JIE. Metabolic regulation of the PMCA: Role in cell death and
Rovati L, Arnold R. Interaction of the cholinergic system and cholecys- survival. Cell Calcium 69: 28-36, 2018.
tokinin in the regulation of endogenous and exogenous stimulation of 28. Bruzzone R, Regazzi R, Wollheim CB. Caerulein causes translocation
pancreatic secretion in humans. Gastroenterology 100: 537-543, 1991. of protein kinase C in rat acini without increasing cytosolic free Ca2+.
2. Aktories K, Hall A. Botulinum ADP-ribosyltransferase C3: A new tool Am J Physiol 255: G33-G39, 1988.
to study low molecular weight GTP-binding proteins. Trends Pharma- 29. Burnham DB. Characterization of Ca2+-activated protein phosphatase
col Sci 10: 415-418, 1989. activity in exocrine pancreas. Biochem J 231: 335-341, 1985.
3. Alexandre M, Thrower EC. PKC. Pancreapedia: Exocrine Pancreas 30. Busca R, Pouyssegur J, Lenormand P. ERK1 and ERK2 map kinases:
Knowledge Base, DOI: 10.3998/panc.2012.13, 2012. Specific roles or functional redundancy? Front Cell Dev Biol 4: 53,
4. Amsterdam A, Jamieson JD. Structural and functional characterization 2016.
of isolated pancreatic exocrine cells. Proc Natl Acad Sci U S A 69: 31. Cantor P, Olsen O, Gertz BJ, Gjorup I, Worning H. Inhibition of
3028-3032, 1972. cholecystokinin-stimulated pancreaticobiliary output in man by the
5. Antal CE, Newton AC. Tuning the signalling output of protein kinase cholecystokinin receptor antagonist MK-329. Scand J Gastroenterol
C. Biochem Soc Trans 42: 1477-1483, 2014. 26: 627-637, 1991.
6. Aparicio IM, Garcia-Marin LJ, Andreolotti AG, Bodega G, Jensen RT, 32. Carriere A, Romeo Y, Acosta-Jaquez HA, Moreau J, Bonneil E,
Bragado MJ. Hepatocyte growth factor activates several transduction Thibault P, Fingar DC, Roux PP. ERK1/2 phosphorylate Raptor to

promote Ras-dependent activation of mTOR complex 1 (mTORC1). 57. Daulhac L, Kowalski-Chauvel A, Pradayrol L, Vaysse N, Seva C.
J Biol Chem 286: 567-577, 2011. Src-family tyrosine kinases in activation of ERK-1 and p85/p110-
33. Caunt CJ, Keyse SM. Dual-specificity MAP kinase phosphatases phosphatidylinositol 3-kinase by G/CCKB receptors. J Biol Chem 274:
(MKPs): Shaping the outcome of MAP kinase signalling. FEBS J 280: 20657-20663, 1999.
489-504, 2013. 58. Davis RJ. Signal transduction by the JNK group of MAP kinases. Cell
34. Cawston EE, Lam PC, Harikumar KG, Dong M, Ball AM, Augustine 103: 239-252, 2000.
ML, Akgun E, Portoghese PS, Orry A, Abagyan R, Sexton PM, 59. Dawson ES, Henne RM, Miller LJ, Lybrand TP. Molecular models for
Miller LJ. Molecular basis for binding and subtype selectivity of cholecystokinin-A receptor. Pharmacology & Toxicology 91: 290-296,
1,4-benzodiazepine antagonist ligands of the cholecystokinin receptor. 2002.
J Biol Chem 287: 18618-18635, 2012. 60. de Lartigue G, Barbier de la Serre C, Espero E, Lee J, Raybould HE.
35. Cendrowski J, Lobo VJ, Sendler M, Salas A, Kuhn JP, Molero X, Leptin resistance in vagal afferent neurons inhibits cholecystokinin
Fukunaga R, Mayerle J, Lerch MM, Real FX. Mnk1 is a novel aci- signaling and satiation in diet induced obese rats. PLoS One 7: e32967,
nar cell-specific kinase required for exocrine pancreatic secretion and 2012.
response to pancreatitis in mice. Gut 64: 937-947, 2015. 61. Desai AJ, Dong M, Langlais BT, Dueck AC, Miller LJ. Cholecystokinin
36. Chandra R, Samsa LA, Vigna SR, Liddle RA. Pseudopod-like basal responsiveness varies across the population dependent on metabolic
cell processes in intestinal cholecystokinin cells. Cell Tissue Res 341: phenotype. Am J Clin Nutr 106: 447-456, 2017.
289-297, 2010. 62. Desai AJ, Miller LJ. Cholecystokinin type 1 receptor. Pancreapedia:
37. Chandrasekhar R, Yule DI, Wang L. Inositol 1,4,5-trisphosphate recep- Exocrine Pancreas Knowledge Base, DOI: 10.3998/panc.2013.9, 2013.
tors (InsP3R). Pancreapedia: Exocrine Pancreas Knowledge Base, 63. Desai AJ, Miller LJ. Changes in the plasma membrane in metabolic
DOI: 10.3998/panc.2017.07, 2017. disease: Impact of the membrane environment on G protein-coupled
38. Chang TM, Chey WY. Radioimmunoassay of cholecystokinin. Dig Dis receptor structure and function. Br J Pharmacol 175: 4009-4025,
Sci 28: 456-468, 1983. 2017.
39. Chen LA, Li J, Silva SR, Jackson LN, Zhou Y, Watanabe H, Ives KL, 64. Dibble CC, Manning BD. Signal integration by mTORC1 coordinates
Hellmich MR, Evers BM. PKD3 is the predominant protein kinase D nutrient input with biosynthetic output. Nat Cell Biol 15: 555-564,
isoform in mouse exocrine pancreas and promotes hormone-induced 2013.
amylase secretion. J Biol Chem 284: 2459-2471, 2009. 65. Dirice E, Walpita D, Vetere A, Meier BC, Kahraman S, Hu J, Dancik V,
40. Chey WY, Chang T-M. Secretin, 100 years later. Journal of Gastroen- Burns SM, Gilbert TJ, Olson DE, Clemons PA, Kulkarni RN, Wagner
terology 38: 1025-1035, 2003. BK. Inhibition of DYRK1A stimulates human beta-cell proliferation.
41. Chin D, Means AR. Calmodulin: A prototypical calcium sensor. Trends Diabetes 65: 1660-1671, 2016.
Cell Biol 10: 322-328, 2000. 66. Dockray GJ. The action of scretin, cholecystokinin-pancreozymin and
42. Cosen-Binker LI, Lam PP, Binker MG, Gaisano HY. Alcohol-induced caerulein on pancreatic secretion in the rat. J Physiol 225: 679-692,
protein kinase Calpha phosphorylation of Munc18c in carbachol- 1972.
stimulated acini causes basolateral exocytosis. Gastroenterology 132: 67. Dockray GJ. Gastrointestinal hormones: Gastrin, cholecystokinin,
1527-1545, 2007. somatostatin, and ghrelin. In: Physiology of the Gastrointestinal Tract
43. Cosen-Binker LI, Lam PP, Binker MG, Reeve J, Pandol S, Gaisano HY. (Fourth Edition). Ed. Johnson LR, Burlington: Academic Press, 2006,
Alcohol/cholecystokinin-evoked pancreatic acinar basolateral exocyto- pp. 91-120.
sis is mediated by protein kinase C alpha phosphorylation of Munc18c. 68. Dockray GJ. Cholecystokinin and gut-brain signalling. Regul Pept 155:
J Biol Chem 282: 13047-13058, 2007. 6-10, 2009.
44. Crozier SJ, D’Alecy LG, Ernst SA, Ginsburg LE, Williams JA. Molec- 69. Dormer RL, Williams JA. Secretagogue-induced changes in subcellular
ular mechanisms of pancreatic dysfunction induced by protein malnu- Ca2+ distribution in isolated pancreatic acini. Am J Physiol 240: G130-
trition. Gastroenterology 137: 1093-1101, 1101 e1091-1093, 2009. G140, 1981.
45. Crozier SJ, Sans MD, Guo L, D’Alecy LG, Williams JA. Activation 70. Duan RD, Williams JA. Cholecystokinin rapidly activates mitogen-
of the mTOR signalling pathway is required for pancreatic growth in activated protein kinase in rat pancreatic acini. Am J Physiol 267:
protease-inhibitor-fed mice. J Physiol 573: 775-786, 2006. G401-G408, 1994.
46. Crozier SJ, Sans MD, Lang CH, D’Alecy LG, Ernst SA, Williams JA. 71. Duan RD, Zheng CF, Guan KL, Williams JA. Activation of MAP kinase
CCK-induced pancreatic growth is not limited by mitogenic capacity kinase (MEK) and Ras by cholecystokinin in rat pancreatic acini. Am J
in mice. Am J Physiol Gastrointest Liver Physiol 294: G1148-1157, Physiol 268: G1060-G1065, 1995.
2008. 72. Dudley DT, Pang L, Decker SJ, Bridges AJ, Saltiel AR. A synthetic
47. Crozier SJ, Sans MD, Wang JY, Lentz SI, Ernst SA, Williams JA. inhibitor of the mitogen-activated protein kinase cascade. Proc Natl
CCK-independent mTORC1 activation during dietary protein-induced Acad Sci U S A 92: 7686-7689, 1995.
exocrine pancreas growth. Am J Physiol Gastrointest Liver Physiol 299: 73. Dufresne M, Seva C, Fourmy D. Cholecystokinin and gastrin receptors.
G1154-G1163, 2010. Physiol Rev 86: 805-847, 2006.
48. Cuadrado A, Nebreda AR. Mechanisms and functions of p38 MAPK 74. Eberlein GA, Eysselein VE, Hesse WH, Goebell H, Schaefer M, Reeve
signalling. Biochem J 429: 403-417, 2010. JR, Jr. Detection of cholecystokinin-58 in human blood by inhibition
49. Dabrowski A, Boguslowicz C, Dabrowska M, Tribillo I, Gabryelewicz of degradation. Am J Physiol 253: G477-G482, 1987.
A. Reactive oxygen species activate mitogen-activated protein kinases 75. Elghazi L, Blandino-Rosano M, Alejandro E, Cras-Meneur C, Bernal-
in pancreatic acinar cells. Pancreas 21: 376-384, 2000. Mizrachi E. Role of nutrients and mTOR signaling in the regulation of
50. Dabrowski A, Detjen KM, Logsdon CD, Williams JA. Stimulation of pancreatic progenitors development. Mol Metab 6: 560-573, 2017.
both CCK-A and CCK-B receptors activates MAP kinases in AR42J 76. Escos A, Risco A, Alsina-Beauchamp D, Cuenda A. p38gamma and
and receptor-transfected CHO cells. Digestion 58: 361-367, 1997. p38delta mitogen activated protein kinases (MAPKs), new stars in the
51. Dabrowski A, Grady T, Logsdon CD, Williams JA. Jun kinases are MAPK galaxy. Front Cell Dev Biol 4: 31, 2016.
rapidly activated by cholecystokinin in rat pancreas both in vitro and in 77. Etienne-Manneville S, Hall A. Rho GTPases in cell biology. Nature
vivo. J Biol Chem 271: 5686-5690, 1996. 420: 629-635, 2002.
52. Dabrowski A, Groblewski GE, Schafer C, Guan KL, Williams JA. 78. Fan BG, Axelson J, Sternby B, Rehfeld J, Ihse I, Ekelund M. Total
Cholecystokinin and EGF activate a MAPK cascade by different mech- parenteral nutrition affects the tropic effect of cholecystokinin on the
anisms in rat pancreatic acinar cells. Am J Physiol 273: C1472-C1479, exocrine pancreas. Scand J Gastroenterol 32: 380-386, 1997.
1997. 79. Fingar DC, Blenis J. Target of rapamycin (TOR): An integrator of
53. Dabrowski A, Tribillo I, Dabrowska MI, Wereszczynska- nutrient and growth factor signals and coordinator of cell growth and
Siemiatkowska U, Gabryelewicz A. Activation of mitogen-activated cell cycle progression. Oncogene 23: 3151-3171, 2004.
protein kinases in different models of pancreatic acinar cell damage. Z 80. Fleming AK, Storz P. Protein kinase C isoforms in the normal pancreas
Gastroenterol 38: 469-481, 2000. and in pancreatic disease. Cell Signal 40: 1-9, 2017.
54. Dabrowski A, VanderKuur JA, Carter-Su C, Williams JA. Cholecys- 81. Fogarty KE, Kidd JF, Tuft DA, Thorn P. Mechanisms underlying InsP3-
tokinin stimulates formation of shc-grb2 complex in rat pancreatic evoked global Ca2+ signals in mouse pancreatic acinar cells. J Physiol
acinar cells through a protein kinase C-dependent mechanism. J Biol 526 Pt 3: 515-526, 2000.
Chem 271: 27125-27129, 1996. 82. Folsch UR, Winckler K, Wormsley KG. Influence of repeated adminis-
55. Daniluk J, Dabrowski A. The effect of concomitant stimulation with tration of cholecystokinin and secretin on the pancreas of the rat. Scand
cholecystokinin and epidermal growth factor on extracellular signal- J Gastroenterol 13: 663-671, 1978.
regulated kinase (ERK) activity in pancreatic acinar cells. J Physiol 83. Fukuhara S, Chikumi H, Gutkind JS. RGS-containing RhoGEFs: The
Pharmacol 58: 441-453, 2007. missing link between transforming G proteins and Rho? Oncogene 20:
56. Daniluk J, Liu Y, Deng D, Chu J, Huang H, Gaiser S, Cruz-Monserrate 1661-1668, 2001.
Z, Wang H, Ji B, Logsdon CD. An NF-kappaB pathway-mediated 84. Furuichi T, Yoshikawa S, Miyawaki A, Wada K, Maeda N, Mikoshiba
positive feedback loop amplifies Ras activity to pathological levels in K. Primary structure and functional expression of the inositol 1,4,5-
mice. J Clin Invest 122: 1519-1528, 2012. trisphosphate-binding protein P400. Nature 342: 32-38, 1989.

85. Futatsugi A, Nakamura T, Yamada MK, Ebisui E, Nakamura K, Uchida 109. Hara K, Maruki Y, Long X, Yoshino K, Oshiro N, Hidayat S, Tokunaga
K, Kitaguchi T, Takahashi-Iwanaga H, Noda T, Aruga J, Mikoshiba K. C, Avruch J, Yonezawa K. Raptor, a binding partner of target of
IP3 receptor types 2 and 3 mediate exocrine secretion underlying energy rapamycin (TOR), mediates TOR action. Cell 110: 177-189, 2002.
metabolism. Science 309: 2232-2234, 2005. 110. Harada E, Kanno T. Progressive enhancement in the secretory functions
86. Gaestel M. MAPKAP kinases-MKs-two’s company, three’s a crowd. of the digestive system of the rat in the course of cold acclimation.
Nat Rev Mol Cell Biol 7: 120-130, 2006. J Physiol 260: 629-645, 1976.
87. Gallo KA, Johnson GL. Mixed-lineage kinase control of JNK and p38 111. Harikumar KG, Cawston EE, Lam PC, Patil A, Orry A, Henke BR,
MAPK pathways. Nat Rev Mol Cell Biol 3: 663-672, 2002. Abagyan R, Christopoulos A, Sexton PM, Miller LJ. Molecular basis
88. Garcia LJ, Rosado JA, Gonzalez A, Jensen RT. Cholecystokinin- for benzodiazepine agonist action at the type 1 cholecystokinin receptor.
stimulated tyrosine phosphorylation of p125FAK and paxillin is medi- J Biol Chem 288: 21082-21095, 2013.
ated by phospholipase C-dependent and -independent mechanisms and 112. Harper AA, Raper HS. Pancreozymin, a stimulant of the secretion of
requires the integrity of the actin cytoskeleton and participation of pancreatic enzymes in extracts of the small intestine. J Physiol 102:
p21rho. Biochem J 327(Pt 2): 461-472, 1997. 115-125, 1943.
89. Gardner JD, Sutliff VE, Walker MD, Jensen RT. Effects of inhibitors 113. Hashi M, Yoshizawa F, Onozuka E, Ogata M, Hara H. Adaptive changes
of cyclic nucleotide phosphodiesterase on actions of cholecystokinin, in translation initiation activities for rat pancreatic protein synthesis
bombesin, and carbachol on pancreatic acini. Am J Physiol 245: G676- with feeding of a high-protein diet. J Nutr Biochem 16: 507-512, 2005.
G680, 1983. 114. Hashimoto N, Hara H. Dietary amino acids promote pancreatic protease
90. Gazzaneo MC, Orellana RA, Suryawan A, Tuckow AP, Kimball SR, synthesis at the translation stage in rats. J Nutr 133: 3052-3057, 2003.
Wilson FA, Nguyen HV, Torrazza RM, Fiorotto ML, Davis TA. Differ- 115. Heit JJ, Apelqvist AA, Gu X, Winslow MM, Neilson JR, Crabtree GR,
ential regulation of protein synthesis and mTOR signaling in skeletal Kim SK. Calcineurin/NFAT signalling regulates pancreatic beta-cell
muscle and visceral tissues of neonatal pigs after a meal. Pediatr Res growth and function. Nature 443: 345-349, 2006.
70: 253-260, 2011. 116. Henin J, Maigret B, Tarek M, Escrieut C, Fourmy D, Chipot C. Probing
91. Gerasimenko JV, Gryshchenko O, Ferdek PE, Stapleton E, Hebert a model of a GPCR/ligand complex in an explicit membrane environ-
TO, Bychkova S, Peng S, Begg M, Gerasimenko OV, Petersen OH. ment: The human cholecystokinin-1 receptor. Biophys J 90: 1232-1240,
Ca2+ release-activated Ca2+ channel blockade as a potential tool in 2006.
antipancreatitis therapy. Proc Natl Acad Sci U S A 110: 13186-13191, 117. Hildebrand P, Beglinger C, Gyr K, Jansen JB, Rovati LC, Zuercher M,
2013. Lamers CB, Setnikar I, Stalder GA. Effects of a cholecystokinin recep-
92. Geron E, Schejter ED, Shilo BZ. Directing exocrine secretory vesicles tor antagonist on intestinal phase of pancreatic and biliary responses in
to the apical membrane by actin cables generated by the formin mDia1. man. J Clin Invest 85: 640-646, 1990.
Proc Natl Acad Sci U S A 110: 10652-10657, 2013. 118. Himeno S, Tarui S, Kanayama S, Kuroshima T, Shinomura Y, Hayashi
93. Goberdhan DC, Wilson C, Harris AL. Amino acid sensing by mTORC1: C, Tateishi K, Imagawa K, Hashimura E, Hamaoka T. Plasma chole-
Intracellular transporters mark the spot. Cell Metab 23: 580-589, 2016. cystokinin responses after ingestion of liquid meal and intraduodenal
94. Gonzalez A, Schmid A, Sternfeld L, Krause E, Salido GM, Schulz infusion of fat, amino acids, or hydrochloric acid in man: Analysis with
I. Cholecystokinin-evoked Ca(2+) waves in isolated mouse pancreatic region specific radioimmunoassay. Am J Gastroenterol 78: 703-707,
acinar cells are modulated by activation of cytosolic phospholipase 1983.
A(2), phospholipase D, and protein kinase C. Biochem Biophys Res 119. Hocker M, Waschulewski IH, Kern HF, Domagk KA, Schwarzhoff R,
Commun 261: 726-733, 1999. Folsch UR, Schmidt WE. Cyclosporine A inhibits protein-kinase-C-
95. Goodyer WR, Gu X, Liu Y, Bottino R, Crabtree GR, Kim SK. Neonatal mediated amylase release from isolated rat pancreatic acini. Digestion
beta cell development in mice and humans is regulated by calcineurin/ 55: 380-388, 1994.
NFAT. Dev Cell 23: 21-34, 2012. 120. Hodge RG, Ridley AJ. Regulating Rho GTPases and their regulators.
96. Grady T, Dabrowski A, Williams JA, Logsdon CD. Stress-activated Nat Rev Mol Cell Biol 17: 496-510, 2016.
protein kinase activation is the earliest direct correlate to the induc- 121. Hoffmann P, Eberlein GA, Reeve JR, Jr., Bunte RH, Grandt D, Goebell
tion of secretagogue-induced pancreatitis in rats. Biochem Biophys Res H, Eysselein VE. Comparison of clearance and metabolism of infused
Commun 227: 1-7, 1996. cholecystokinins 8 and 58 in dogs. Gastroenterology 105: 1732-1736,
97. Green GM, Levan VH, Liddle RA. Plasma cholecystokinin and pan- 1993.
creatic growth during adaptation to dietary protein. Am J Physiol 251: 122. Hofken T, Keller N, Fleischer F, Goke B, Wagner AC. Map kinase
G70-G74, 1986. phosphatases (MKP’s) are early responsive genes during induction of
98. Green GM, Lyman RL. Feedback regulation of pancreatic enzyme cerulein hyperstimulation pancreatitis. Biochem Biophys Res Commun
secretion as a mechanism for trypsin inhibitor-induced hypersecretion 276: 680-685, 2000.
in rats. Proc Soc Exp Biol Med 140: 6-12, 1972. 123. Holtz BJ, Lodewyk KB, Sebolt-Leopold JS, Ernst SA, Williams JA.
99. Gresset A, Sondek J, Harden TK. The phospholipase C isozymes and ERK activation is required for CCK-mediated pancreatic adaptive
their regulation. Subcell Biochem 58: 61-94, 2012. growth in mice. Am J Physiol Gastrointest Liver Physiol 307: G700-
100. Groblewski GE, Grady T, Mehta N, Lambert H, Logsdon CD, Landry G710, 2014.
J, Williams JA. Cholecystokinin stimulates heat shock protein 27 phos- 124. Hong JH, Park S, Shcheynikov N, Muallem S. Mechanism and syn-
phorylation in rat pancreas both in vivo and in vitro. Gastroenterology ergism in epithelial fluid and electrolyte secretion. Pflugers Arch 466:
112: 1354-1361, 1997. 1487-1499, 2014.
101. Groblewski GE, Wagner AC, Williams JA. Cyclosporin A inhibits 125. Hou H, Wang F, Zhang W, Wang D, Li X, Bartlam M, Yao X, Rao Z.
Ca2+/calmodulin-dependent protein phosphatase and secretion in pan- Structure-functional analyses of CRHSP-24 plasticity and dynamics in
creatic acinar cells. J Biol Chem 269: 15111-15117, 1994. oxidative stress response. J Biol Chem 286: 9623-9635, 2011.
102. Groblewski GE, Yoshida M, Bragado MJ, Ernst SA, Leykam J, 126. Huang CY, Tan TH. DUSPs, to MAP kinases and beyond. Cell Biosci
Williams JA. Purification and characterization of a novel physiolog- 2: 24, 2012.
ical substrate for calcineurin in mammalian cells. J Biol Chem 273: 127. Husain SZ, Grant WM, Gorelick FS, Nathanson MH, Shah AU.
22738-22744, 1998. Caerulein-induced intracellular pancreatic zymogen activation is
103. Guo L, Sans MD, Gurda GT, Lee SH, Ernst SA, Williams JA. Induction dependent on calcineurin. Am J Physiol Gastrointest Liver Physiol
of early response genes in trypsin inhibitor-induced pancreatic growth. 292: G1594-G1599, 2007.
Am J Physiol Gastrointest Liver Physiol 292: G667-G677, 2007. 128. Ishizuka T, Ito Y, Kajita K, Miura K, Nagao S, Nagata K, Nozawa Y.
104. Guo L, Sans MD, Hou Y, Ernst SA, Williams JA. c-Jun/AP-1 is required Redistribution of protein kinase C in pancreatic acinar cells stimulated
for CCK-induced pancreatic acinar cell dedifferentiation and DNA syn- with caerulein or carbachol. Biochem Biophys Res Commun 144: 551-
thesis in vitro. Am J Physiol Gastrointest Liver Physiol 302: G1381- 559, 1987.
G1396, 2012. 129. Ivy AC. A hormone mechanism for gall-bladder contraction & evacu-
105. Gurda GT, Crozier SJ, Ji B, Ernst SA, Logsdon CD, Rothermel ation. Amer J Physiology 86: 599-613, 1928.
BA, Williams JA. Regulator of calcineurin 1 controls growth plas- 130. Jaffe AB, Hall A. Rho GTPases: Biochemistry and biology. Annu Rev
ticity of adult pancreas. Gastroenterology 139: 609-619, 619 e601-606, Cell Dev Biol 21: 247-269, 2005.
2010. 131. Jang Y, Soekmadji C, Mitchell JM, Thomas WG, Thorn P. Real-time
106. Gurda GT, Guo L, Lee SH, Molkentin JD, Williams JA. Cholecys- measurement of F-actin remodelling during exocytosis using Lifeact-
tokinin activates pancreatic calcineurin-NFAT signaling in vitro and in EGFP transgenic animals. PLoS One 7: e39815, 2012.
vivo. Mol Biol Cell 19: 198-206, 2008. 132. Jebbink MC, Jansen JB, Masclee AA, Lamers CB. Lack of effect of
107. Haga RB, Ridley AJ. Rho GTPases: Regulation and roles in cancer cell the specific cholecystokinin receptor antagonist loxiglumide on chole-
biology. Small GTPases 7: 207-221, 2016. cystokinin clearance from plasma in man. Br J Clin Pharmacol 29:
108. Halbrook CJ, Wen HJ, Ruggeri JM, Takeuchi KK, Zhang Y, di Magliano 770-773, 1990.
MP, Crawford HC. Mitogen-activated protein kinase kinase activity 133. Jensen RT, Wank SA, Rowley WH, Sato S, Gardner JD. Interaction of
maintains acinar-to-ductal metaplasia and is required for organ regen- CCK with pancreatic acinar cells. Trends Pharmacol Sci 10: 418-423,
eration in pancreatitis. Cell Mol Gastroenterol Hepatol 3: 99-118, 2017. 1989.

134. Jewell JL, Russell RC, Guan KL. Amino acid signalling upstream of 161. Li C, Chen X, Williams JA. Regulation of CCK-induced amylase
mTOR. Nat Rev Mol Cell Biol 14: 133-139, 2013. release by PKC-delta in rat pancreatic acinar cells. Am J Physiol Gas-
135. Ji B, Bi Y, Simeone D, Mortensen RM, Logsdon CD. Human pancreatic trointest Liver Physiol 287: G764-G771, 2004.
acinar cells lack functional responses to cholecystokinin and gastrin. 162. Li C, Williams JA. Regulation of CCK-induced ERK1/2 activation
Gastroenterology 121: 1380-1390, 2001. by PKC epsilon in rat pancreatic acinar cells. AIMS Molecular Sci 4:
136. Jun I, Lee MG, Muallem S. Molecular mechanisms of pancreatic bicar- 463-477, 2017.
bonate secretion. Pancreapedia 2017. 163. Li H, Rao A, Hogan PG. Interaction of calcineurin with substrates and
137. Kanayama S, Himeno S, Kurokawa M, Shinomura Y, Kuroshima T, targeting proteins. Trends Cell Biol 21: 91-103, 2011.
Okuno M, Tsuji K, Higashimoto Y, Ikei N, Hashimura E, et al. Marked 164. Li W, Zhang H, Nie A, Ni Q, Li F, Ning G, Li X, Gu Y, Wang Q.
prolongation in disappearance half-time of plasma cholecystokinin- mTORC1 pathway mediates beta cell compensatory proliferation in 60
octapeptide in patients with hepatic cirrhosis. Am J Gastroenterol 80: % partial-pancreatectomy mice. Endocrine 53: 117-128, 2016.
557-560, 1985. 165. Li Y, Owyang C. Vagal afferent pathway mediates physiological action
138. Kasai H, Augustine GJ. Cytosolic Ca2+ gradients triggering unidirec- of cholecystokinin on pancreatic enzyme secretion. J Clin Invest 92:
tional fluid secretion from exocrine pancreas. Nature 348: 735-738, 418-424, 1993.
1990. 166. Liang T, Dolai S, Xie L, Winter E, Orabi AI, Karimian N, Cosen-
139. Kasai H, Li YX, Miyashita Y. Subcellular distribution of Ca2+ release Binker LI, Huang YC, Thorn P, Cattral MS, Gaisano HY. Ex vivo
channels underlying Ca2+ waves and oscillations in exocrine pancreas. human pancreatic slice preparations offer a valuable model for studying
Cell 74: 669-677, 1993. pancreatic exocrine biology. J Biol Chem 292: 5957-5969, 2017.
140. Kaufmann A, Rossler OG, Thiel G. Expression of the transcription fac- 167. Liddle RA. Regulation of cholecystokinin secretion by intraluminal
tor Egr-1 in pancreatic acinar cells following stimulation of cholecys- releasing factors. Am J Physiol 269: G319-G327, 1995.
tokinin or Galphaq-coupled designer receptors. Cell Physiol Biochem 168. Liddle RA. Cholecystokinin cells. Annu Rev Physiol 59: 221-242, 1997.
33: 1411-1425, 2014. 169. Liddle RA. Regulation of Pancreatic Secretion. In: Physiology of the
141. Kidger AM, Keyse SM. The regulation of oncogenic Ras/ERK sig- Gastrointestinal Tract (Fifth Edition), edited by Johnson LR, Ghishan
nalling by dual-specificity mitogen activated protein kinase phos- FK, Kaunitz JD, Merchant JL, Said HM, Wood JD. Boston: Academic
phatases (MKPs). Semin Cell Dev Biol 50: 125-132, 2016. Press, 2012, pp. 1425-1460.
142. Kiehne K, Herzig KH, Folsch UR. CCK-stimulated changes in pan- 170. Liddle RA, Goldfine ID, Rosen MS, Taplitz RA, Williams JA. Chole-
creatic acinar morphology are mediated by rho. Digestion 65: 47-55, cystokinin bioactivity in human plasma. Molecular forms, responses to
2002. feeding, and relationship to gallbladder contraction. J Clin Invest 75:
143. Kim J, Guan KL. Amino acid signaling in TOR activation. Annu Rev 1144-1152, 1985.
Biochem 80: 1001-1032, 2011. 171. Liddle RA, Goldfine ID, Williams JA. Bioassay of plasma cholecys-
144. Kim M, Nozu F, Kusama K, Imawari M. Cholecystokinin stimulates tokinin in rats: Effects of food, trypsin inhibitor, and alcohol. Gastroen-
the recruitment of the Src-RhoA-phosphoinositide 3-kinase pathway by terology 87: 542-549, 1984.
Vav-2 downstream of G(alpha13) in pancreatic acini. Biochem Biophys 172. Liener IE, Goodale RL, Deshmukh A, Satterberg TL, Ward G, DiPietro
Res Commun 339: 271-276, 2006. CM, Bankey PE, Borner JW. Effect of a trypsin inhibitor from soy-
145. Kim MJ, Lee KH, Min DS, Yoon SH, Hahn SJ, Kim MS, Jo YH. beans (Bowman-Birk) on the secretory activity of the human pancreas.
Distributional patterns of phospholipase C isozymes in rat pancreas. Gastroenterology 94: 419-427, 1988.
Pancreas 22: 47-52, 2001. 173. Liou J, Kim ML, Heo WD, Jones JT, Myers JW, Ferrell JE, Jr., Meyer
146. Kim MJ, Lee YS, Lee KH, Min DS, Yoon SH, Hahn SJ, Kim MS, T. STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered
Jo YH. Site-specific localization of protein kinase C isoforms in rat Ca2+ influx. Curr Biol 15: 1235-1241, 2005.
pancreas. Pancreatology 1: 36-42, 2001. 174. Litosch I. Decoding Galphaq signaling. Life Sci 152: 99-106, 2016.
147. Kim MS, Hong JH, Li Q, Shin DM, Abramowitz J, Birnbaumer L, 175. Logsdon CD. Stimulation of pancreatic acinar cell growth by CCK,
Muallem S. Deletion of TRPC3 in mice reduces store-operated Ca2+ epidermal growth factor, and insulin in vitro. Am J Physiol 251: G487-
influx and the severity of acute pancreatitis. Gastroenterology 137: G494, 1986.
1509-1517, 2009. 176. Logsdon CD, Williams JA. Pancreatic acinar cells in monolayer culture:
148. Kim YS, Kim WJ, Kim HK, Hong SS. Effect of cold and hot environ- Direct trophic effects of caerulein in vitro. Am J Physiol 250: G440-
ments on the exocrine pancreas of rats. Yonsei Med J 11: 1-9, 1970. G447, 1986.
149. Klee CB, Ren H, Wang X. Regulation of the calmodulin-stimulated 177. Lur G, Haynes LP, Prior IA, Gerasimenko OV, Feske S, Petersen
protein phosphatase, calcineurin. J Biol Chem 273: 13367-13370, OH, Burgoyne RD, Tepikin AV. Ribosome-free terminals of rough
1998. ER allow formation of STIM1 puncta and segregation of STIM1 from
150. Kolch W. Coordinating ERK/MAPK signalling through scaffolds and IP(3) receptors. Curr Biol 19: 1648-1653, 2009.
inhibitors. Nat Rev Mol Cell Biol 6: 827-837, 2005. 178. Lynch G, Kohler S, Leser J, Beil M, Garcia-Marin LJ, Lutz MP.
151. Kraft AS, Anderson WB. Phorbol esters increase the amount of Ca2+, The tyrosine kinase Yes regulates actin structure and secretion dur-
phospholipid-dependent protein kinase associated with plasma mem- ing pancreatic acinar cell damage in rats. Pflugers Arch 447: 445-451,
brane. Nature 301: 621-623, 1983. 2004.
152. Krishna M, Narang H. The complexity of mitogen-activated protein 179. Machado-de Domenech E, Soling HD. Effects of stimulation of mus-
kinases (MAPKs) made simple. Cell Mol Life Sci 65: 3525-3544, 2008. carinic and of beta-catecholamine receptors on the intracellular distri-
153. Lacourse KA, Swanberg LJ, Gillespie PJ, Rehfeld JF, Saunders TL, bution of protein kinase C in guinea pig exocrine glands. Biochem J
Samuelson LC. Pancreatic function in CCK-deficient mice: Adaptation 242: 749-754, 1987.
to dietary protein does not require CCK. Am J Physiol Gastrointest 180. Malo A, Kruger B, Goke B, Kubisch CH. 4-Phenylbutyric acid reduces
Liver Physiol 276: G1302-G1309, 1999. endoplasmic reticulum stress, trypsin activation, and acinar cell apop-
154. Laplante M, Sabatini DM. mTOR signaling in growth control and dis- tosis while increasing secretion in rat pancreatic acini. Pancreas 42:
ease. Cell 149: 274-293, 2012. 92-101, 2013.
155. Lawrence MC, Bhatt HS, Watterson JM, Easom RA. Regulation of 181. Malo A, Kruger B, Seyhun E, Schafer C, Hoffmann RT, Goke B,
insulin gene transcription by a Ca(2+)-responsive pathway involving Kubisch CH. Tauroursodeoxycholic acid reduces endoplasmic reticu-
calcineurin and nuclear factor of activated T cells. Mol Endocrinol 15: lum stress, trypsin activation, and acinar cell apoptosis while increasing
1758-1767, 2001. secretion in rat pancreatic acini. Am J Physiol Gastrointest Liver Physiol
156. Le Page SL, Bi Y, Williams JA. CCK-A receptor activates RhoA 299: G877-G886, 2010.
through G alpha 12/13 in NIH3T3 cells. Am J Physiol Cell Physiol 182. Marino CR, Leach SD, Schaefer JF, Miller LJ, Gorelick FS. Charac-
285: C1197-C1206, 2003. terization of cAMP-dependent protein kinase activation by CCK in rat
157. Lee M, Chung S, Uhm DY, Park MK. Regulation of zymogen gran- pancreas. FEBS Lett 316: 48-52, 1993.
ule exocytosis by Ca2+, cAMP, and PKC in pancreatic acinar cells. 183. Matozaki T, Goke B, Tsunoda Y, Rodriguez M, Martinez J, Williams
Biochem Biophys Res Commun 334: 1241-1247, 2005. JA. Two functionally distinct cholecystokinin receptors show different
158. Lee MG, Xu X, Zeng W, Diaz J, Kuo TH, Wuytack F, Racymaekers modes of action on Ca2+ mobilization and phospholipid hydrolysis
L, Muallem S. Polarized expression of Ca2+ pumps in pancreatic in isolated rat pancreatic acini. Studies using a new cholecystokinin
and salivary gland cells. Role in initiation and propagation of [Ca2+]i analog, JMV-180. J Biol Chem 265: 6247-6254, 1990.
waves. J Biol Chem 272: 15771-15776, 1997. 184. Matozaki T, Williams JA. Multiple sources of 1,2-diacylglycerol in iso-
159. Lee S, Wishart MJ, Williams JA. Identification of calcineurin regulated lated rat pancreatic acini stimulated by cholecystokinin. Involvement of
phosphorylation sites on CRHSP-24. Biochem Biophys Res Commun phosphatidylinositol bisphosphate and phosphatidylcholine hydrolysis.
385: 413-417, 2009. J Biol Chem 264: 14729-14734, 1989.
160. Leser J, Luhrs H, Beil MF, Adler G, Lutz MP. Cholecystokinin-induced 185. McCormack JG, Halestrap AP, Denton RM. Role of calcium ions in
redistribution of paxillin in rat pancreatic acinar cells. Biochem Biophys regulation of mammalian intramitochondrial metabolism. Physiol Rev
Res Commun 254: 400-405, 1999. 70: 391-425, 1990.

186. McLaughlin CL, Baile CA, Peikin SR. Hyperphagia during lactation: 212. Noguchi M, Adachi H, Gardner JD, Jensen RT. Calcium-activated,
Satiety response to CCK and growth of the pancreas. Am J Physiol 244: phospholipid-dependent protein kinase in pancreatic acinar cells. Am J
E61-E65, 1983. Physiol Gastrointest Liver Physiol 248: G692-G701, 1985.
187. Means AR. Regulatory cascades involving calmodulin-dependent pro- 213. Nozu F, Owyang C, Tsunoda Y. Involvement of phosphoinositide 3-
tein kinases. Mol Endocrinol 14: 4-13, 2000. kinase and its association with pp60src in cholecystokinin-stimulated
188. Meloche S, Pouyssegur J. The ERK1/2 mitogen-activated protein pancreatic acinar cells. Eur J Cell Biol 79: 803-809, 2000.
kinase pathway as a master regulator of the G1- to S-phase transition. 214. Nozu F, Tsunoda Y, Ibitayo AI, Bitar KN, Owyang C. Involvement
Oncogene 26: 3227-3239, 2007. of RhoA and its interaction with protein kinase C and Src in CCK-
189. Merritt JE, Rubin RP. Pancreatic amylase secretion and cytoplas- stimulated pancreatic acini. Am J Physiol Gastrointest Liver Physiol
mic free calcium. Effects of ionomycin, phorbol dibutyrate and dia- 276: G915-G923, 1999.
cylglycerols alone and in combination. Biochem J 230: 151-159, 215. Nuche-Berenguer B, Jensen RT. Gastrointestinal hormones/
1985. neurotransmitters and growth factors can activate P21 activated kinase
190. Mignen O, Thompson JL, Yule DI, Shuttleworth TJ. Agonist activation 2 in pancreatic acinar cells by novel mechanisms. Biochim Biophys
of arachidonate-regulated Ca2+-selective (ARC) channels in murine Acta 1853: 2371-2382, 2015.
parotid and pancreatic acinar cells. J Physiol 564: 791-801, 2005. 216. Nuche-Berenguer B, Moreno P, Jensen RT. Elucidation of the roles of
191. Miller LJ, Gao F. Structural basis of cholecystokinin receptor binding the Src kinases in pancreatic acinar cell signaling. J Cell Biochem 116:
and regulation. Pharmacol Ther 119: 83-95, 2008. 22-36, 2015.
192. Miller WE, Lefkowitz RJ. Expanding roles for beta-arrestins as scaf- 217. Nuche-Berenguer B, Ramos-Alvarez I, Jensen RT. Src kinases play a
folds and adapters in GPCR signaling and trafficking. Curr Opin Cell novel dual role in acute pancreatitis affecting severity but no role in
Biol 13: 139-145, 2001. stimulated enzyme secretion. Am J Physiol Gastrointest Liver Physiol
193. Minami T. Calcineurin-NFAT activation and DSCR-1 auto-inhibitory 310: G1015-G1027, 2016.
loop: How is homoeostasis regulated? J Biochem 155: 217-226, 218. Osipchuk YV, Wakui M, Yule DI, Gallacher DV, Petersen OH.
2014. Cytoplasmic Ca2+ oscillations evoked by receptor stimulation, G-
194. Mishra V, Cline R, Noel P, Karlsson J, Baty CJ, Orlichenko L, Patel K, protein activation, internal application of inositol trisphosphate or
Trivedi RN, Husain SZ, Acharya C, Durgampudi C, Stolz DB, Navina Ca2+: Simultaneous microfluorimetry and Ca2+ dependent Cl- cur-
S, Singh VP. Src dependent pancreatic acinar injury can be initiated rent recording in single pancreatic acinar cells. EMBO J 9: 697-704,
independent of an increase in cytosolic calcium. PLoS One 8: e66471, 1990.
2013. 219. Owyang C, Logsdon CD. New insights into neurohormonal regulation
195. Miyasaka K, Shinozaki H, Jimi A, Funakoshi A. Amylase secretion of pancreatic secretion. Gastroenterology 127: 957-969, 2004.
from dispersed human pancreatic acini: Neither cholecystokinin a nor 220. Pace A, Garcia-Marin LJ, Tapia JA, Bragado MJ, Jensen RT. Phos-
cholecystokinin B receptors mediate amylase secretion in vitro. Pan- phospecific site tyrosine phosphorylation of p125FAK and proline-rich
creas 25: 161-165, 2002. kinase 2 is differentially regulated by cholecystokinin receptor type A
196. Mochly-Rosen D. Localization of protein kinases by anchoring pro- activation in pancreatic acini. J Biol Chem 278: 19008-19016, 2003.
teins: A theme in signal transduction. Science 268: 247-251, 1995. 221. Pace A, Tapia JA, Garcia-Marin LJ, Jensen RT. The Src family kinase,
197. Morisset J, Aliaga JC, Calvo EL, Bourassa J, Rivard N. Expression and Lyn, is activated in pancreatic acinar cells by gastrointestinal hor-
modulation of p42/p44 MAPKs and cell cycle regulatory proteins in rat mones/neurotransmitters and growth factors which stimulate its associ-
pancreas regeneration. Am J Physiol Gastrointest Liver Physiol 277: ation with numerous other signaling molecules. Biochim Biophys Acta
G953-G959, 1999. 1763: 356-365, 2006.
198. Morisset J, Guan D, Jurkowska G, Rivard N, Green GM. Endoge- 222. Pandol SJ, Schoeffield MS. 1,2-Diacylglycerol, protein kinase C, and
nous cholecystokinin, the major factor responsible for dietary protein- pancreatic enzyme secretion. J Biol Chem 261: 4438-4444, 1986.
induced pancreatic growth. Pancreas 7: 522-529, 1992. 223. Park AM, Kudo M, Hagiwara S, Tabuchi M, Watanabe T, Munakata
199. Morisset JA, Webster PD. Effects of fasting and feeding on protein H, Sakurai T. p38MAPK suppresses chronic pancreatitis by regulating
synthesis by the rat pancreas. J Clin Invest 51: 1-8, 1972. HSP27 and BAD expression. Free Radic Biol Med 52: 2284-2291,
200. Muili KA, Jin S, Orabi AI, Eisses JF, Javed TA, Le T, Bottino R, 2012.
Jayaraman T, Husain SZ. Pancreatic acinar cell nuclear factor kappaB 224. Parker EM, Zaman MM, Freedman SD. GP2, a GPI-anchored pro-
activation because of bile acid exposure is dependent on calcineurin. tein in the apical plasma membrane of the pancreatic acinar cell, co-
J Biol Chem 288: 21065-21073, 2013. immunoprecipitates with src kinases and caveolin. Pancreas 21: 219-
201. Murali A, Rajalingam K. Small Rho GTPases in the control of cell 225, 2000.
shape and mobility. Cell Mol Life Sci 71: 1703-1721, 2014. 225. Patel R, Atherton P, Wackerhage H, Singh J. Signaling proteins asso-
202. Murphy JA, Criddle DN, Sherwood M, Chvanov M, Mukherjee R, ciated with diabetic-induced exocrine pancreatic insufficiency in rats.
McLaughlin E, Booth D, Gerasimenko JV, Raraty MG, Ghaneh P, Ann N Y Acad Sci 1084: 490-502, 2006.
Neoptolemos JP, Gerasimenko OV, Tepikin AV, Green GM, Reeve JR, 226. Peikin SR, Rottman AJ, Batzri S, Gardner JD. Kinetics of amylase
Jr., Petersen OH, Sutton R. Direct activation of cytosolic Ca2+ signaling release by dispersed acini prepared from guinea pig pancreas. Am J
and enzyme secretion by cholecystokinin in human pancreatic acinar Physiol 235: E743-E749, 1978.
cells. Gastroenterology 135: 632-641, 2008. 227. Peiris H, Raghupathi R, Jessup CF, Zanin MP, Mohanasundaram D,
203. Nakamura Y, Fukami K. Regulation and physiological functions of Mackenzie KD, Chataway T, Clarke JN, Brealey J, Coates PT, Pritchard
mammalian phospholipase C. J Biochem 161: 315-321, 2017. MA, Keating DJ. Increased expression of the glucose-responsive gene,
204. Nathanson MH, Fallon MB, Padfield PJ, Maranto AR. Localization RCAN1, causes hypoinsulinemia, beta-cell dysfunction, and diabetes.
of the type 3 inositol 1,4,5-trisphosphate receptor in the Ca2+ wave Endocrinology 153: 5212-5221, 2012.
trigger zone of pancreatic acinar cells. J Biol Chem 269: 4693-4696, 228. Petersen OH, Tepikin AV. Polarized calcium signaling in exocrine gland
1994. cells. Annu Rev Physiol 70: 273-299, 2008.
205. Nathanson MH, Padfield PJ, O’Sullivan AJ, Burgstahler AD, Jamieson 229. Pfeiffer CJ, Chernenko GA, Kohli Y, Barrowman JA. Trophic effects
JD. Mechanism of Ca2+ wave propagation in pancreatic acinar cells. of cholecystokinin octapeptide on the pancreas of the Syrian hamster.
J Biol Chem 267: 18118-18121, 1992. Can J Physiol Pharmacol 60: 358-362, 1982.
206. Nemoto T, Kojima T, Oshima A, Bito H, Kasai H. Stabilization of exo- 230. Piiper A, Elez R, You SJ, Kronenberger B, Loitsch S, Roche S,
cytosis by dynamic F-actin coating of zymogen granules in pancreatic Zeuzem S. Cholecystokinin stimulates extracellular signal-regulated
acini. J Biol Chem 279: 37544-37550, 2004. kinase through activation of the epidermal growth factor receptor, Yes,
207. Newton AC. Regulation of the ABC kinases by phosphorylation: Pro- and protein kinase C. Signal amplification at the level of Raf by activa-
tein kinase C as a paradigm. Biochem J 370: 361-371, 2003. tion of protein kinase Cepsilon. J Biol Chem 278: 7065-7072, 2003.
208. Nicke B, Tseng MJ, Fenrich M, Logsdon CD. Adenovirus-mediated 231. Piiper A, Gebhardt R, Kronenberger B, Giannini CD, Elez R, Zeuzem S.
gene transfer of RasN17 inhibits specific CCK actions on pancreatic Pertussis toxin inhibits cholecystokinin- and epidermal growth factor-
acinar cells. Am J Physiol Gastrointest Liver Physiol 276: G499-G506, induced mitogen-activated protein kinase activation by disinhibition of
1999. the cAMP signaling pathway and inhibition of c-Raf-1. Mol Pharmacol
209. Niederau C, Liddle RA, Williams JA, Grendell JH. Pancreatic growth: 58: 608-613, 2000.
Interaction of exogenous cholecystokinin, a protease inhibitor, and a 232. Piiper A, Stryjek-Kaminska D, Klengel R, Zeuzem S. CCK, carbachol,
cholecystokinin receptor antagonist in mice. Gut 28(Suppl): 63-69, and bombesin activate distinct PLC-beta isoenzymes via Gq/11 in rat
1987. pancreatic acinar membranes. Am J Physiol Gastrointest Liver Physiol
210. Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and 272: G135-G140, 1997.
activation of protein kinase C. Science 258: 607-614, 1992. 233. Pozo-Guisado E, Campbell DG, Deak M, Alvarez-Barrientos A,
211. Nobes CD, Hall A. Rho, rac, and cdc42 GTPases regulate the assembly Morrice NA, Alvarez IS, Alessi DR, Martin-Romero FJ. Phosphoryla-
of multimolecular focal complexes associated with actin stress fibers, tion of STIM1 at ERK1/2 target sites modulates store-operated calcium
lamellipodia, and filopodia. Cell 81: 53-62, 1995. entry. J Cell Sci 123: 3084-3093, 2010.

234. Proud CG. Control of the translational machinery by amino acids. Am 261. Sabbatini ME, Chen X, Ernst SA, Williams JA. Rap1 activation plays
J Clin Nutr 99: 231S-236S, 2014. a regulatory role in pancreatic amylase secretion. J Biol Chem 283:
235. Raman M, Chen W, Cobb MH. Differential regulation and properties 23884-23894, 2008.
of MAPKs. Oncogene 26: 3100-3112, 2007. 262. Sabbatini ME, D’Alecy L, Lentz SI, Tang T, Williams JA. Adenylyl
236. Ramnath RD, Sun J, Adhikari S, Bhatia M. Effect of mitogen- cyclase 6 mediates the action of cyclic AMP-dependent secretagogues
activated protein kinases on chemokine synthesis induced by substance in mouse pancreatic exocrine cells via protein kinase A pathway acti-
P in mouse pancreatic acinar cells. J Cell Mol Med 11: 1326-1341, vation. J Physiol 591: 3693-3707, 2013.
2007. 263. Sabbatini ME, Williams JA. Cholecystokinin-mediated RhoGDI phos-
237. Ramnath RD, Sun J, Bhatia M. Involvement of SRC family kinases in phorylation via PKCalpha promotes both RhoA and Rac1 signaling.
substance P-induced chemokine production in mouse pancreatic acinar PLoS One 8: e66029, 2013.
cells and its significance in acute pancreatitis. J Pharmacol Exp Ther 264. Sakamoto C, Goldfine ID, Roach E, Williams JA. Localization of sat-
329: 418-428, 2009. urable CCK binding sites in rat pancreatic islets by light and electron
238. Ramos JW. The regulation of extracellular signal-regulated kinase microscope autoradiography. Diabetes 34: 390-394, 1985.
(ERK) in mammalian cells. Int J Biochem Cell Biol 40: 2707-2719, 265. Samuel I, Zaheer A, Fisher RA. In vitro evidence for role of ERK, p38,
2008. and JNK in exocrine pancreatic cytokine production. J Gastrointest
239. Rao A, Luo C, Hogan PG. Transcription factors of the NFAT family: Surg 10: 1376-1383, 2006.
Regulation and function. Annu Rev Immunol 15: 707-747, 1997. 266. Sancak Y, Bar-Peled L, Zoncu R, Markhard AL, Nada S, Sabatini DM.
240. Redondo PC, Lajas AI, Salido GM, Gonzalez A, Rosado JA, Pariente Ragulator-Rag complex targets mTORC1 to the lysosomal surface and
JA. Evidence for secretion-like coupling involving pp60src in the activa- is necessary for its activation by amino acids. Cell 141: 290-303, 2010.
tion and maintenance of store-mediated Ca2+ entry in mouse pancreatic 267. Sancho V, Nuche-Berenguer B, Jensen RT. The Src kinase Yes
acinar cells. Biochem J 370: 255-263, 2003. is activated in pancreatic acinar cells by gastrointestinal hormones/
241. Reeve JR, Jr., Green GM, Chew P, Eysselein VE, Keire DA. CCK-58 neurotransmitters, but not pancreatic growth factors, which stimulate
is the only detectable endocrine form of cholecystokinin in rat. Am J its association with numerous other signaling molecules. Biochim Bio-
Physiol Gastrointest Liver Physiol 285: G255-G265, 2003. phys Acta 1823: 1285-1294, 2012.
242. Rehfeld JF. Accurate measurement of cholecystokinin in plasma. Clin 268. Sans M, Amin R, Vogel N, D’Alecy L, Kahn R, Williams J. Spe-
Chem 44: 991-1001, 1998. cific deletion of insulin receptors on pancreatic acinar cells defines
243. Rehfeld JF. Cholecystokinin-from local gut hormone to ubiquitous mes- the insulin-acinar axis: Implications for pancreatic insufficiency in dia-
senger. Front Endocrinol (Lausanne) 8: 47, 2017. betes. Gastroenterology 140: S-156, 2011.
244. Rehfeld JF. Four basic characteristics of the gastrin-cholecystokinin 269. Sans MD, Lee SH, D’Alecy LG, Williams JA. Feeding activates protein
system. Am J Physiol Gastrointest Liver Physiol 240: G255-G266, synthesis in mouse pancreas at the translational level without increase
1981. in mRNA. Am J Physiol Gastrointest Liver Physiol 287: G667-G675,
245. Rehfeld JF, Sun G, Christensen T, Hillingso JG. The predominant 2004.
cholecystokinin in human plasma and intestine is cholecystokinin-33. 270. Sans MD, Sabbatini ME, Ernst SA, D’Alecy LG, Nishijima I, Williams
J Clin Endocrinol Metab 86: 251-258, 2001. JA. Secretin is not necessary for exocrine pancreatic development and
246. Renckens BA, van Emst-de Vries SE, de Pont JJ, Bonting SL. Rat growth in mice. Am J Physiol Gastrointest Liver Physiol 301: G791-
pancreas adenylate cyclase: VII. Effect of extracellular calcium on G798, 2011.
pancreozymin-induced cyclic AMP formation. Biochim Biophys Acta 271. Sans MD, Tashiro M, Vogel NL, Kimball SR, D’Alecy LG, Williams
630: 511-518, 1980. JA. Leucine activates pancreatic translational machinery in rats and
247. Rey O, Reeve JR, Jr., Zhukova E, Sinnett-Smith J, Rozengurt E. G mice through mTOR independently of CCK and insulin. J Nutr 136:
protein-coupled receptor-mediated phosphorylation of the activation 1792-1799, 2006.
loop of protein kinase D: Dependence on plasma membrane translo- 272. Sans MD, Williams JA. Calcineurin is required for translational control
cation and protein kinase Cepsilon. J Biol Chem 279: 34361-34372, of protein synthesis in rat pancreatic acini. Am J Physiol Cell Physiol
2004. 287: C310-C319, 2004.
248. Rindler MJ, Xu CF, Gumper I, Smith NN, Neubert TA. Proteomic 273. Sans MD, Williams JA. The mTOR signaling pathway and regulation
analysis of pancreatic zymogen granules: Identification of new granule of pancreatic function. Pancreapedia 2017.
proteins. J Proteome Res 6: 2978-2992, 2007. 274. Sans MD, Xie Q, Williams JA. Regulation of translation elongation and
249. Rodriguez-Martin E, Boyano-Adanez MC, Bodega G, Martin M, phosphorylation of eEF2 in rat pancreatic acini. Biochem Biophys Res
Hernandez C, Quin Y, Vadillo M, Arilla-Ferreiro E. Redistribution Commun 319: 144-151, 2004.
of protein kinase C isoforms in rat pancreatic acini during lactation and 275. Sato N, Suzuki S, Kanai S, Ohta M, Jimi A, Noda T, Takiguchi S,
weaning. FEBS Lett 445: 356-360, 1999. Funakoshi A, Miyasaka K. Different effects of oral administration of
250. Rosado JA, Salido GM, Jensen RT, Garcia LJ. Are tyrosine phospho- synthetic trypsin inhibitor on the pancreas between cholecystokinin-A
rylation of p125(FAK) and paxillin or the small GTP binding protein, receptor gene knockout mice and wild type mice. Jpn J Pharmacol 89:
rho, needed for CCK-stimulated pancreatic amylase secretion? Biochim 290-295, 2002.
Biophys Acta 1404: 412-426, 1998. 276. Satoh A, Gukovskaya AS, Nieto JM, Cheng JH, Gukovsky I, Reeve
251. Roskoski R, Jr. ERK1/2 MAP kinases: Structure, function, and regula- JR, Jr., Shimosegawa T, Pandol SJ. PKC-delta and -epsilon regulate
tion. Pharmacol Res 66: 105-143, 2012. NF-kappaB activation induced by cholecystokinin and TNF-alpha in
252. Rossman KL, Der CJ, Sondek J. GEF means go: Turning on RHO pancreatic acinar cells. Am J Physiol Gastrointest Liver Physiol 287:
GTPases with guanine nucleotide-exchange factors. Nat Rev Mol Cell G582-G591, 2004.
Biol 6: 167-180, 2005. 277. Satoh K, Narita T, Katsumata-Kato O, Sugiya H, Seo Y. Involvement
253. Rousseau A, Bertolotti A. An evolutionarily conserved pathway con- of myristoylated alanine-rich C kinase substrate phosphorylation and
trols proteasome homeostasis. Nature 536: 184-189, 2016. translocation in cholecystokinin-induced amylase release in rat pancre-
254. Rozengurt E. Protein kinase D signaling: Multiple biological functions atic acini. Am J Physiol Gastrointest Liver Physiol 310: G399-G409,
in health and disease. Physiology (Bethesda) 26: 23-33, 2011. 2016.
255. Rubin RP, Godfrey PP, Chapman DA, Putney JW, Jr. Secretagogue- 278. Saxton RA, Sabatini DM. mTOR signaling in growth, metabolism, and
induced formation of inositol phosphates in rat exocrine pancreas. disease. Cell 169: 361-371, 2017.
Implications for a messenger role for inositol trisphosphate. Biochem J 279. Schafer C, Clapp P, Welsh MJ, Benndorf R, Williams JA. HSP27
219: 655-659, 1984. expression regulates CCK-induced changes of the actin cytoskeleton
256. Rubin RP, Hundley TR, Adolf MA. Regulation of diacylglycerol lev- in CHO-CCK-A cells. Am J Physiol 277: C1032-C1043, 1999.
els in carbachol-stimulated pancreatic acinar cells: Relationship to the 280. Schafer C, Ross SE, Bragado MJ, Groblewski GE, Ernst SA, Williams
breakdown of phosphatidylcholine and metabolism to phosphatidic JA. A role for the p38 mitogen-activated protein kinase/Hsp 27 pathway
acid. Biochim Biophys Acta 1133: 127-132, 1992. in cholecystokinin-induced changes in the actin cytoskeleton in rat
257. Rusnak F, Mertz P. Calcineurin: Form and function. Physiol Rev 80: pancreatic acini. J Biol Chem 273: 24173-24180, 1998.
1483-1521, 2000. 281. Schafer C, Williams JA. Stress kinases and heat shock proteins in the
258. Sabatini DM, Erdjument-Bromage H, Lui M, Tempst P, Snyder SH. pancreas: Possible roles in normal function and disease. J Gastroenterol
RAFT1: A mammalian protein that binds to FKBP12 in a rapamycin- 35: 1-9, 2000.
dependent fashion and is homologous to yeast TORs. Cell 78: 35-43, 282. Schmelzle T, Hall MN. TOR, a central controller of cell growth. Cell
1994. 103: 253-262, 2000.
259. Sabbatini ME. Cyclic nucleotides as mediators of acinar and ductal 283. Schwarzendrube J, Niederau M, Luthen R, Niederau C. Effects of
function. Pancreapedia: Exocrine Pancreas Knowledge Base, DOI: cholecystokinin-receptor blockade on pancreatic and biliary function
10.3998/panc.2016.2, 2016. in healthy volunteers. Gastroenterology 100: 1683-1690, 1991.
260. Sabbatini ME, Bi Y, Ji B, Ernst SA, Williams JA. CCK activates RhoA 284. Sebolt-Leopold JS, Bridges AJ. Road to PD0325901 and beyond: The
and Rac1 differentially through Galpha13 and Galphaq in mouse pan- MEK Inhibitor Quest. In: Kinase Inhibitor Drugs. Hoboken, NJ: John
creatic acini. Am J Physiol Cell Physiol 298: C592-C601, 2010. Wiley & Sons, Inc., 2009, pp. 203-227.

285. Sengupta S, Peterson TR, Sabatini DM. Regulation of the mTOR com- 310. Taylor SJ, Chae HZ, Rhee SG, Exton JH. Activation of the beta 1
plex 1 pathway by nutrients, growth factors, and stress. Mol Cell 40: isozyme of phospholipase C by alpha subunits of the Gq class of G
310-322, 2010. proteins. Nature 350: 516-518, 1991.
286. Sewell WA, Young JA. Secretion of electrolytes by the pancreas of the 311. Tcherkezian J, Lamarche-Vane N. Current knowledge of the large
anaestetized rat. J Physiol 252: 379-396, 1975. RhoGAP family of proteins. Biol Cell 99: 67-86, 2007.
287. Shin DM, Luo X, Wilkie TM, Miller LJ, Peck AB, Humphreys-Beher 312. Tepikin AV, Voronina SG, Gallacher DV, Petersen OH. Pulsatile Ca2+
MG, Muallem S. Polarized expression of G protein-coupled receptors extrusion from single pancreatic acinar cells during receptor-activated
and an all-or-none discharge of Ca2+ pools at initiation sites of [Ca2+]i cytosolic Ca2+ spiking. J Biol Chem 267: 14073-14076, 1992.
waves in polarized exocrine cells. J Biol Chem 276: 44146-44156, 313. Thompson JL, Shuttleworth TJ. Molecular basis of activation of the
2001. arachidonate-regulated Ca2+ (ARC) channel, a store-independent Orai
288. Simeone DM, Zhang L, Graziano K, Nicke B, Pham T, Schaefer C, channel, by plasma membrane STIM1. J Physiol 591: 3507-3523,
Logsdon CD. Smad4 mediates activation of mitogen-activated protein 2013.
kinases by TGF-beta in pancreatic acinar cells. Am J Physiol Cell 314. Thorn P, Lawrie AM, Smith PM, Gallacher DV, Petersen OH. Local and
Physiol 281: C311-C319, 2001. global cytosolic Ca2+ oscillations in exocrine cells evoked by agonists
289. Sinagoga KL, Stone WJ, Schiesser JV, Schweitzer JI, Sampson L, and inositol trisphosphate. Cell 74: 661-668, 1993.
Zheng Y, Wells JM. Distinct roles for the mTOR pathway in postnatal 315. Thorn P, Petersen OH. Calcium oscillations in pancreatic acinar cells,
morphogenesis, maturation and function of pancreatic islets. Develop- evoked by the cholecystokinin analogue JMV-180, depend on functional
ment 144: 2402-2414, 2017. inositol 1,4,5-trisphosphate receptors. J Biol Chem 268: 23219-23221,
290. Singh VP. Src: Regulation and function in the exocrine pancreas. 1993.
Pancreapedia: Exocrine Pancreas Knowledge Base, DOI: 10.3998/ 316. Thrower EC, Osgood S, Shugrue CA, Kolodecik TR, Chaudhuri AM,
panc.2012.5, 2012. Reeve JR, Jr., Pandol SJ, Gorelick FS. The novel protein kinase C iso-
291. Singh VP, McNiven MA. Src-mediated cortactin phosphorylation reg- forms -delta and -epsilon modulate caerulein-induced zymogen activa-
ulates actin localization and injurious blebbing in acinar cells. Mol Biol tion in pancreatic acinar cells. Am J Physiol Gastrointest Liver Physiol
Cell 19: 2339-2347, 2008. 294: G1344-G1353, 2008.
292. Sjodin L, Gardner JD. Effect of cholecystokinin variant (CCK39) on 317. Thrower EC, Wang J, Cheriyan S, Lugea A, Kolodecik TR, Yuan J,
dispersed acinar cells from guinea pig pancreas. Gastroenterology 73: Reeve JR, Jr., Gorelick FS, Pandol SJ. Protein kinase C delta-mediated
1015-1018, 1977. processes in cholecystokinin-8-stimulated pancreatic acini. Pancreas
293. Smith GP, Gibbs J. Satiating effect of cholecystokinin. Ann N Y Acad 38: 930-935, 2009.
Sci 713: 236-241, 1994. 318. Thrower EC, Yuan J, Usmani A, Liu Y, Jones C, Minervini SN,
294. Smrcka AV, Sternweis PC. Regulation of purified subtypes of Alexandre M, Pandol SJ, Guha S. A novel protein kinase D inhibitor
phosphatidylinositol-specific phospholipase C beta by G protein alpha attenuates early events of experimental pancreatitis in isolated rat acini.
and beta gamma subunits. J Biol Chem 268: 9667-9674, 1993. Am J Physiol Gastrointest Liver Physiol 300: G120-G129, 2011.
295. Solomon TE, Petersen H, Elashoff J, Grossman MI. Interaction of 319. Tietz AB, Malo A, Diebold J, Kotlyarov A, Herbst A, Kolligs FT,
caerulein and secretin on pancreatic size and composition in rat. Am J Brandt-Nedelev B, Halangk W, Gaestel M, Goke B, Schafer C. Gene
Physiol 235: E714-E719, 1978. deletion of MK2 inhibits TNF-alpha and IL-6 and protects against
296. Solomon TE, Vanier M, Morisset J. Cell site and time course of DNA cerulein-induced pancreatitis. Am J Physiol Gastrointest Liver Physiol
synthesis in pancreas after caerulein and secretin. Am J Physiol Gas- 290: G1298-G1306, 2006.
trointest Liver Physiol 245: G99-G105, 1983. 320. Toescu EC, Lawrie AM, Petersen OH, Gallacher DV. Spatial and
297. Soudah HC, Lu Y, Hasler WL, Owyang C. Cholecystokinin at phys- temporal distribution of agonist-evoked cytoplasmic Ca2+ signals in
iological levels evokes pancreatic enzyme secretion via a cholinergic exocrine acinar cells analysed by digital image microscopy. EMBO J
pathway. Am J Physiol Gastrointest Liver Physiol 263: G102-G107, 11: 1623-1629, 1992.
1992. 321. Torrazza RM, Suryawan A, Gazzaneo MC, Orellana RA, Frank JW,
298. Stauffer PL, Zhao H, Luby-Phelps K, Moss RL, Star RA, Muallem Nguyen HV, Fiorotto ML, El-Kadi S, Davis TA. Leucine supplementa-
S. Gap junction communication modulates [Ca2+]i oscillations and tion of a low-protein meal increases skeletal muscle and visceral tissue
enzyme secretion in pancreatic acini. J Biol Chem 268: 19769-19775, protein synthesis in neonatal pigs by stimulating mTOR-dependent
1993. translation initiation. J Nutr 140: 2145-2152, 2010.
299. Straub SV, Giovannucci DR, Yule DI. Calcium wave propagation 322. Tsunoda Y, Stuenkel EL, Williams JA. Characterization of sustained
in pancreatic acinar cells: Functional interaction of inositol 1,4,5- [Ca2+]i increase in pancreatic acinar cells and its relation to amylase
trisphosphate receptors, ryanodine receptors, and mitochondria. J Gen secretion. Am J Physiol Gastrointest Liver Physiol 259: G792-G801,
Physiol 116: 547-560, 2000. 1990.
300. Streb H, Irvine RF, Berridge MJ, Schulz I. Release of Ca2+ from a non- 323. Tsunoda Y, Stuenkel EL, Williams JA. Oscillatory mode of calcium
mitochondrial intracellular store in pancreatic acinar cells by inositol- signaling in rat pancreatic acinar cells. Am J Physiol Cell Physiol 258:
1,4,5-trisphosphate. Nature 306: 67-69, 1983. C147-C155, 1990.
301. Sung CK, Hootman SR, Stuenkel EL, Kuroiwa C, Williams JA. Down- 324. Tsunoda Y, Yoshida H, Africa L, Steil GJ, Owyang C. Src kinase path-
regulation of protein kinase C in guinea pig pancreatic acini: Effects on ways in extracellular Ca(2+)-dependent pancreatic enzyme secretion.
secretion. Am J Physiol Gastrointest Liver Physiol 254: G242-G248, Biochem Biophys Res Commun 227: 876-884, 1996.
1988. 325. Uchida T, Iwashita N, Ohara-Imaizumi M, Ogihara T, Nagai S, Choi
302. Sung CK, Williams JA. Insulin and ribosomal protein S6 kinase in rat JB, Tamura Y, Tada N, Kawamori R, Nakayama KI, Nagamatsu S,
pancreatic acini. Diabetes 38: 544-549, 1989. Watada H. Protein kinase Cdelta plays a non-redundant role in insulin
303. Sung CK, Williams JA. Cholecystokinin stimulates a specific ribosomal secretion in pancreatic beta cells. J Biol Chem 282: 2707-2716, 2007.
S6 kinase in rat pancreatic acini. Pancreas 5: 668-676, 1990. 326. Unal EB, Uhlitz F, Bluthgen N. A compendium of ERK targets. FEBS
304. Szalmay G, Varga G, Kajiyama F, Yang XS, Lang TF, Case RM, Lett 591: 2607-2615, 2017.
Steward MC. Bicarbonate and fluid secretion evoked by cholecys- 327. Urano F, Wang X, Bertolotti A, Zhang Y, Chung P, Harding HP, Ron
tokinin, bombesin and acetylcholine in isolated guinea-pig pancreatic D. Coupling of stress in the ER to activation of JNK protein kinases by
ducts. J Physiol 535: 795-807, 2001. transmembrane protein kinase IRE1. Science 287: 664-666, 2000.
305. Takiguchi S, Suzuki S, Sato Y, Kanai S, Miyasaka K, Jimi A, Shinozaki 328. Valentijn JA, Valentijn K, Pastore LM, Jamieson JD. Actin coating
H, Takata Y, Funakoshi A, Kono A, Minowa O, Kobayashi T, Noda of secretory granules during regulated exocytosis correlates with the
T. Role of CCK-A receptor for pancreatic function in mice: A study in release of rab3D. Proc Natl Acad Sci U S A 97: 1091-1095, 2000.
CCK-A receptor knockout mice. Pancreas 24: 276-283, 2002. 329. Vander Haar E, Lee SI, Bandhakavi S, Griffin TJ, Kim DH. Insulin
306. Tapia JA, Ferris HA, Jensen RT, Garcia LJ. Cholecystokinin activates signalling to mTOR mediated by the Akt/PKB substrate PRAS40. Nat
PYK2/CAKbeta by a phospholipase C-dependent mechanism and its Cell Biol 9: 316-323, 2007.
association with the mitogen-activated protein kinase signaling pathway 330. Vigna SR, Thorndyke MC, Williams JA. Evidence for a common evo-
in pancreatic acinar cells. J Biol Chem 274: 31261-31271, 1999. lutionary origin of brain and pancreas cholecystokinin receptors. Proc
307. Tapia JA, Garcia-Marin LJ, Jensen RT. Cholecystokinin-stimulated pro- Natl Acad Sci U S A 83: 4355-4359, 1986.
tein kinase C-delta kinase activation, tyrosine phosphorylation, and 331. von Kriegsheim A, Baiocchi D, Birtwistle M, Sumpton D, Bienvenut W,
translocation are mediated by Src tyrosine kinases in pancreatic acinar Morrice N, Yamada K, Lamond A, Kalna G, Orton R, Gilbert D, Kolch
cells. J Biol Chem 278: 35220-35230, 2003. W. Cell fate decisions are specified by the dynamic ERK interactome.
308. Tashiro M, Dabrowski A, Guo L, Sans MD, Williams JA. Calcineurin- Nat Cell Biol 11: 1458-1464, 2009.
dependent and calcineurin-independent signal transduction pathways 332. von Manteuffel SR, Dennis PB, Pullen N, Gingras AC, Sonenberg N,
activated as part of pancreatic growth. Pancreas 32: 314-320, 2006. Thomas G. The insulin-induced signalling pathway leading to S6 and
309. Tashiro M, Samuelson LC, Liddle RA, Williams JA. Calcineurin medi- initiation factor 4E binding protein 1 phosphorylation bifurcates at a
ates pancreatic growth in protease inhibitor-treated mice. Am J Physiol rapamycin-sensitive point immediately upstream of p70s6k. Mol Cell
Gastrointest Liver Physiol 286: G784-G790, 2004. Biol 17: 5426-5436, 1997.

333. Voronina S, Sukhomlin T, Johnson PR, Erdemli G, Petersen OH, 357. Wu V, Yang M, McRoberts JA, Ren J, Seensalu R, Zeng N, Dagrag
Tepikin A. Correlation of NADH and Ca2+ signals in mouse pancreatic M, Birnbaumer M, Walsh JH. First intracellular loop of the human
acinar cells. J Physiol 539: 41-52, 2002. cholecystokinin-A receptor is essential for cyclic AMP signaling in
334. Voronina S, Tepikin A. Mitochondrial calcium in the life and death of transfected HEK-293 cells. J Biol Chem 272: 9037-9042, 1997.
exocrine secretory cells. Cell Calcium 52: 86-92, 2012. 358. Yamamoto M, Otani M, Jia DM, Fukumitsu K, Yoshikawa H, Akiyama
335. Voronina SG, Barrow SL, Simpson AW, Gerasimenko OV, da Silva T, Otsuki M. Differential mechanism and site of action of CCK on the
Xavier G, Rutter GA, Petersen OH, Tepikin AV. Dynamic changes pancreatic secretion and growth in rats. Am J Physiol Gastrointest Liver
in cytosolic and mitochondrial ATP levels in pancreatic acinar cells. Physiol 285: G681-G687, 2003.
Gastroenterology 138: 1976-1987, 2010. 359. Yamanishi R, Kotera J, Fushiki T, Soneda T, Iwanaga T, Sugimoto E.
336. Wang S, Lukinius A, Zhou Y, Stalberg P, Gobl A, Oberg K, Skogseid B. Characteristic and localization of the monitor peptide receptor. Biosci
Subcellular distribution of phospholipase C isoforms in rodent pancreas Biotechnol Biochem 57: 1153-1156, 1993.
and gastric mucosa. Endocrinology 141: 2589-2593, 2000. 360. Yamasaki M, Thomas JM, Churchill GC, Garnham C, Lewis AM,
337. Wank SA. G protein-coupled receptors in gastrointestinal physiology. I. Cancela JM, Patel S, Galione A. Role of NAADP and cADPR in the
CCK receptors: An exemplary family. Am J Physiol Gastrointest Liver induction and maintenance of agonist-evoked Ca2+ spiking in mouse
Physiol 274: G607-G613, 1998. pancreatic acinar cells. Curr Biol 15: 874-878, 2005.
338. Waschulewski IH, Hall DV, Kern HF, Edwardson JM. Effects of the 361. Yang J, Waldron RT, Su HY, Moro A, Chang HH, Eibl G, Ferreri K,
immunosuppressants cyclosporin A and FK 506 on exocytosis in the Kandeel FR, Lugea A, Li L, Pandol SJ. Insulin promotes prolifera-
rat exocrine pancreas in vitro. Br J Pharmacol 108: 892-900, 1993. tion and fibrosing responses in activated pancreatic stellate cells. Am J
339. Watanabe H, Saito H, Rychahou PG, Uchida T, Evers BM. Aging is Physiol Gastrointest Liver Physiol 311: G675-G687, 2016.
associated with decreased pancreatic acinar cell regeneration and phos- 362. Yao Z, Seger R. The ERK signaling cascade–views from different
phatidylinositol 3-kinase/Akt activation. Gastroenterology 128: 1391- subcellular compartments. Biofactors 35: 407-416, 2009.
1404, 2005. 363. Yoon S, Seger R. The extracellular signal-regulated kinase: Multiple
340. Weston CR, Davis RJ. The JNK signal transduction pathway. Curr substrates regulate diverse cellular functions. Growth Factors 24: 21-
Opin Genet Dev 12: 14-21, 2002. 44, 2006.
341. Whitmarsh AJ, Cavanagh J, Tournier C, Yasuda J, Davis RJ. A mam- 364. You CH, Rominger JM, Chey WY. Potentiation effect of
malian scaffold complex that selectively mediates MAP kinase activa- cholecystokinin-octapeptide on pancreatic bicarbonate secretion stim-
tion. Science 281: 1671-1674, 1998. ulated by a physiologic dose of secretin in humans. Gastroenterology
342. Williams JA. Regulation of normal and adaptive pancreatic growth. 85: 40-45, 1983.
Pancreapedia: Exocrine Pancreas Knowledge Base, DOI: 10.3998/ 365. Yuan HX, Xiong Y, Guan KL. Nutrient sensing, metabolism, and cell
panc.2017.2, 2017. growth control. Mol Cell 49: 379-387, 2013.
343. Williams JA. Isolation of rodent pancreatic acinar cells and acini by 366. Yuan J, Lugea A, Zheng L, Gukovsky I, Edderkaoui M, Rozengurt E,
collagenase digestion. Pancreapedia; Exocrine Pancreas Knowledge Pandol SJ. Protein kinase D1 mediates NF-kappaB activation induced
Base, DOI: 10.3998/panc.2010.18, 2010. by cholecystokinin and cholinergic signaling in pancreatic acinar cells.
344. Williams JA, Chen X, Sabbatini ME. Small G proteins as key regulators Am J Physiol Gastrointest Liver Physiol 295: G1190-G1201, 2008.
of pancreatic digestive enzyme secretion. Am J Physiol Endocrinol 367. Yuan J, Tan T, Geng M, Tan G, Chheda C, Pandol SJ. Novel small
Metab 296: E405-E414, 2009. molecule inhibitors of protein kinase D suppress NF-kappaB activation
345. Williams JA, Holtz BJ. ERK activation and its role in pancreatic acinar and attenuate the severity of rat cerulein pancreatitis. Front Physiol 8:
cell function. Pancreapedia: Exocrine Pancreas Knowledge Base, DOI: 1014, 2017.
10,3998/panc.2017.6, 2017. 368. Yule DI. Ca2+ signaling in pancreatic acinar cells. Pancreapedia:
346. Williams JA, Korc M, Dormer RL. Action of secretagogues on a new Exocrine Pancreas Knowledge Base, DOI: 10.3998/panc.2015.24,
preparation of functionally intact, isolated pancreatic acini. Am J Phys- 2015.
iol 235: 517-524, 1978. 369. Yule DI, Baker CW, Williams JA. Calcium signaling in rat pancreatic
347. Williams JA, Sans MD, Tashiro M, Schafer C, Bragado MJ, Dabrowski acinar cells: A role for Galphaq, Galpha11, and Galpha14. Am J Physiol
A. Cholecystokinin activates a variety of intracellular signal transduc- Gastrointest Liver Physiol 276: G271-G279, 1999.
tion mechanisms in rodent pancreatic acinar cells. Pharmacol Toxicol 370. Yule DI, Ernst SA, Ohnishi H, Wojcikiewicz RJ. Evidence that zymo-
91: 297-303, 2002. gen granules are not a physiologically relevant calcium pool. Defining
348. Williams JA, Yule DI. Stimulus-secretion coupling in pancreatic acinar the distribution of inositol 1,4,5-trisphosphate receptors in pancreatic
cells In: Physiology of the Gastrointestinal Tract (Fifth Edition), edited acinar cells. J Biol Chem 272: 9093-9098, 1997.
by Johnson LR, Ghishan FK, Merchant JL, Said HM, and Wood JD. 371. Yule DI, Lawrie AM, Gallacher DV. Acetylcholine and cholecystokinin
Boston: Academic Press, 2012, pp. 1361-1398. induce different patterns of oscillating calcium signals in pancreatic
349. Willoughby EA, Perkins GR, Collins MK, Whitmarsh AJ. The JNK- acinar cells. Cell Calcium 12: 145-151, 1991.
interacting protein-1 scaffold protein targets MAPK phosphatase-7 to 372. Yule DI, Stuenkel E, Williams JA. Intercellular calcium waves in rat
dephosphorylate JNK. J Biol Chem 278: 10731-10736, 2003. pancreatic acini: Mechanism of transmission. Am J Physiol Cell Physiol
350. Wishart MJ, Groblewski G, Goke BJ, Wagner AC, Williams JA. Sec- 271: C1285-C1294, 1996.
retagogue regulation of pancreatic acinar cell protein phosphorylation 373. Yule DI, Tseng MJ, Williams JA, Logdson CD. A cloned CCK-A
shown by large-scale 2D-PAGE. Am J Physiol Gastrointest Liver Phys- receptor transduces multiple signals in response to full and partial
iol 267: G676-G686, 1994. agonists. Am J Physiol Gastrointest Liver Physiol 265: G999-G1004,
351. Wooten MW, Wrenn RW. Phorbol ester induces intracellular translo- 1993.
cation of phospholipid/Ca2+-dependent protein kinase and stimulates 374. Yutsudo Y, Kido Y, Okabayashi Y, Matsumoto M, Ogawa W, Ohba
amylase secretion in isolated pancreatic acini. FEBS Lett 171: 183-186, M, Kuroki T, Kasuga M. Protein kinase Calpha is implicated in
1984. cholecystokinin-induced activation of 70-kd S6 kinase in AR42J cells.
352. Wooten MW, Wrenn RW. Redistribution of phospholipid/calcium- Pancreas 30: 50-53, 2005.
dependent protein kinase and altered phosphorylation of its soluble 375. Zarbin MA, Wamsley JK, Innis RB, Kuhar MJ. Cholecystokinin recep-
and particulate substrate proteins in phorbol ester-treated rat pancreatic tors: Presence and axonal flow in the rat vagus nerve. Life Sci 29:
acini. Cancer Res 45: 3912-3917, 1985. 697-705, 1981.
353. Wu-Zhang AX, Newton AC. Protein kinase C pharmacology: Refining 376. Zhang X, Wen J, Aletta JM, Rubin RP. Inhibition of expression of PKC-
the toolbox. Biochem J 452: 195-209, 2013. alpha by antisense mRNA is associated with diminished cell growth
354. Wu H, Peisley A, Graef IA, Crabtree GR. NFAT signaling and the and inhibition of amylase secretion by AR4-2J cells. Exp Cell Res 233:
invention of vertebrates. Trends Cell Biol 17: 251-260, 2007. 225-231, 1997.
355. Wu L, Cai B, Zheng S, Liu X, Cai H, Li H. Effect of emodin on 377. Zoncu R, Efeyan A, Sabatini DM. mTOR: From growth signal integra-
endoplasmic reticulum stress in rats with severe acute pancreatitis. tion to cancer, diabetes and ageing. Nat Rev Mol Cell Biol 12: 21-35,
Inflammation 36: 1020-1029, 2013. 2011.
356. Wu SV, Yang M, Avedian D, Birnbaumer M, Walsh JH. Single amino 378. Zucker KA, Adrian TE, Bilchik AJ, Modlin IM. Effects of the CCK
acid substitution of serine82 to asparagine in first intracellular loop receptor antagonist L364,718 on pancreatic growth in adult and devel-
of human cholecystokinin (CCK)-B receptor confers full cyclic AMP oping animals. Am J Physiol Gastrointest Liver Physiol 257: G511-
responses to CCK and gastrin. Mol Pharmacol 55: 795-803, 1999. G516, 1989.

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Cholecystokinin (CCK) Regulation of Pancreatic

Acinar Cells: Physiological Actions and Signal

Didactic Synopsis r Other signaling proteins that can be activated by CCK

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Figure 1 Two-dimensional structure of CCK1R showing membrane spanning topology and

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Following stimulation with CCK and other Gq coupled 200

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For consideration of the cellular mechanisms bringing

Ca2± -induced physiological events That an increase

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EGF CCK, ACh

Classical Novel Unconventional

PKC PKCα PKCδ PKCζ

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CCK Insulin pathway’s downstream mediators S6 and 4E-BP1. Phos-

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