Cell Mol Neurobiol (2010) 30:1371–1375
DOI 10.1007/s10571-010-9607-8
COMMENTARY
Commentary on Chapters ‘Clinical and Developmental Aspects’
and ‘Stress Responses of the Adrenal Medulla’
Lee E. Eiden
Received: 15 September 2010 / Accepted: 20 September 2010 / Published online: 19 November 2010
Ó Springer Science+Business Media, LLC (outside the USA) 2010
The division of the contributions to this volume into
Chromogranins, Ion Channels, Secretion Mechanisms,
Clinical and Developmental Aspects, and Stress Responses
of the Adrenal Medulla is somewhat arbitrary: there is
clearly much overlap among these domains of chromaffin
cell research. This commentary covers both Clinical/
Developmental, and Stress Responses together, mainly to
emphasize this overlap, and the translational importance of
these two aspects of the chromaffin cell as a stress
transducer.
Genetics, development and stress were three topics of
particular clinical relevance that were developed in depth
through contributions presented at the 15th International
Symposium for Chromaffin Cell Biology. The genetics of
the chromaffin cell in normal physiology and disease were
highlighted by contributions focusing on markers of chro-
maffin cell origin relevant to cancer including pheochro-
mocytoma, and on catecholaminergic markers for
hypertension and other cardiovascular diseases.
Colon, pancreas, prostate and lung outrank the adrenal
medulla as organs of medical concern in human cancer.
Although pheochromocytoma, or cancer of adrenomedul-
lary origin is rare, it is an important subject of study for
several reasons. First, it arises from a limited and distinct
This is a commentary to articles doi:10.1007/s10571-010-9535-7
10.1007/s10571-010-9567-z, 10.1007/s10571-010-9571-3,
10.1007/s10571-010-9578-9, 10.1007/s10571-010-9575-z,
10.1007/s10571-010-9582-0, 10.1007/s10571-010-9583-z,
10.1007/s10571-010-9592-y, 10.1007/s10571-010-9593-x,
10.1007/s10571-010-9594-9, 10.1007/s10571-010-9600-2,
10.1007/s10571-010-9606-9, 10.1007/s10571-010-9620-y.
L. E. Eiden (&)
NIMH-IRP, Bethesda, MD, USA
e-mail: eidenl@mail.nih.gov
set of cell types, and therefore is an attractive model for
etiology of proliferative disease. Second, pheochromocy-
toma can be either metastatic, malignant or benign, and
therefore can be used to search for markers to make this
clinically crucial diagnosis. Murthy et al. propose car-
boxypeptidase E (CPE), the enkephalin prohormone pro-
cessing enzyme first characterized in bovine chromaffin
granules in 1982 (Hook et al., Nature 295:341–342, 1982),
as such a marker. Meta-analysis of microarray expression
data sets in the Gene Expression Omnibus (GEO) allowed
Murthy and colleagues to identify CPE as a transcript
frequently represented in the transcriptomes of metastatic
cancers of both endocrine and epithelial origin. Thus,
cervical, colorectal, renal, and bone (Ewing sarcoma),
astrocytic, and oligodendroglial tumors or cells express
higher levels of CPE mRNA than their non-cancerous cells
and tissues of origin: in fact most of the corresponding cells
of origin do not have appreciable concentrations of CPE.
Lung, pituitary and pheochromocytoma tissue (i.e. neuro-
endocrine tumors) express higher CPE levels than their
tissues of origin. Most significantly, metastatic pheochro-
mocytoma expresses significantly higher CPE mRNA than
benign. Should CPE prove a reliable prognostic marker for
malignancy in pheochromocytoma and other neuroendo-
crine cancers, and perhaps even in non-endocrine cancers,
its potential for translation to standard clinical practice
would be high.
Thouennon and Anouar report on expression of neuro-
peptides in pheochromocytoma that may be involved in
trophic, proliferative and angiogenic paracrine/autocrine
actions contributing to tumor growth. A strong correlation
between NPY and PACAP expression was documented in
25 pheochromocytomas, and between RDC1, the adreno-
medullin receptor, and VEGF, with angiogenic activity.
These authors review the evidence for concerted expression
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of trophic peptides and their receptors in pheochromocy-
toma leading to autocrine/paracrine regulation of tumor cell
growth. An intriguing possibility is that there is ‘cross
regulation’ of heterologous peptides and their receptors
such that a complex coordinated growth program is initiated
and maintained.
O’Connor and colleagues provide two in-depth exam-
inations of genetic effects of catecholamine-related genes
on renal hypertension and stress-induced blood pressure
regulation. The genetic effects of allelic variation in the
tyrosine hydroxylase (TH) gene reviewed by Rao et al.
trace the linkages from genetic variation to functional
variation, with a face-valid hypothesis for penetrance to
altered phenotype resulting in disease (hypertension). The
genetic effects of allelic variation in the chromogranin A
(CHGA) gene reviewed by Chen et al. are more complex;
exhibited at multiple loci in the CHGA gene, some of
which are functional (affecting transcription of the CHGA
mRNA or processing of the CHGA protein); and compli-
cated in extension to phenotype and disease by the phe-
nomenon of heterosis. TH is the rate-limiting enzyme for
catecholamine production. Therefore the identification of a
tetranucleotide repeat in the first intron of the gene with
two common variants (TCAT6 and TCAT10) that predict
autonomic traits in twins provides a straightforward
opportunity to connect TH allelic variation to blood pres-
sure and blood pressure response variation through varia-
tion in catecholamine production in human individuals.
Indeed TCAT9 is associated with higher plasma norepi-
nephrine levels. However, there is not a simple relationship
between TCAT number (variation is commonly from six to
11 copies) and hemodynamic responses to stress or resis-
tance to cardiovascular disease. A second polymorphic
block exists in the TH promoter region, and affects tran-
scriptional activity, possibly through variable binding of
transcription factors (MEF2, SP1, AP2, EGR1, SRY and
FOXD1 are candidates). Variants with the greatest effects
on TH transcription correspond to individuals with highest
catecholamine levels in vivo. At least one allele (C-824T)
shows augmented secretory response in cell culture mod-
els at nicotinic and PACAP receptors, i.e. to the two
co-transmitters at the splanchnicoadrenomedullary syn-
apse. The linkage between TH allelic variation, TH tran-
scription, catecholamine levels and cardiovascular disease
is not always simple (i.e. allelic variation blocks corre-
sponding to greater TH transcription, corresponding to
higher catecholamine levels, corresponding to hyperten-
sion, corresponding to disease). This is not surprising: the
‘‘area under the curve’’ representing integrated sympatho-
adrenal enhanced catecholamine secretion, and recovery,
following stress may be optimized for homeostasis (or for
disease-associated allostasis) at several points of regula-
tion. Lack of simple correlation between disease and
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Cell Mol Neurobiol (2010) 30:1371–1375
transcription rate of the disease-linked gene is more pro-
nounced in studies of genetic variants in CHGA linked to
hypertension and hypertension-associated kidney disease.
Variants in the CHGA promoter linked to glomerular fil-
tration rate (GFR, an index of kidney function and dys-
function) and in the 3’ untranslated region of the CHGA
gene linked to end-stage renal disease are indeed ‘func-
tional’, i.e. associated with altered gene transcription of
correspondingly mutated reporter genes in cells in culture.
Moreover, CHGA promoter variation also predicts plasma
endothelin-1 levels, and chromogranin A releases this
factor along with angiopoietin-2, and von Willebrand
Factor from cultured endothelial cells. Thus, along with its
role in the biogenesis of catecholamine-containing secre-
tory vesicles, and various direct hormonal effects of CHGA
processed protein fragments (see Commentary of K. Helle
on the Chromogranin chapter of this volume) throughout
the cardiovascular system, chromogranin A also affects the
secretion of key regulatory hormones from the endothelium
of the vasculature itself. The genetic studies reviewed by
Rao et al. and Chen et al. suggest the complexity under-
lying stress transduction by the chromaffin cell of the
adrenal medulla that leads to homeostasis and contributes
to allostatic load throughout the lifespan (see Goldstein).
Importantly, creating tighter association between disease
outcome and precursor or intermediate phenotypes that can
be measured simultaneously and often across time, will
provide a statistical framework for broader integration of
multiple genes and their allelic variants into the mosaic of
human diseases which are progressive, predictable and
therefore preventable.
Zeniou-Meyer and colleagues review the cellular and
molecular biology of Coffin-Lowry, an X-linked skeleto-
muscular developmental and mental retardation syndrome
which is associated with mutations in a single gene,
RPS6KA3, encoding the protein kinase RSK2. An
RSK2-KO mouse model with corresponding deficits in
long-term spatial memory and learning has been found by
this group to show long-term potentiation (LTP) synaptic
deficits in the amygdala. However, it was not possible in
the complex milieu of the brain to clearly identify whether
the role of RKS2 in LTP was pre- or post-synaptic. They
therefore turned to the PC12 cell model to determine how
RSK2 hypofunction might affect secretion. First, using
pharmacological inhibition of RSK kinase function, and
RNA interference in PC12 cells, it was shown that RSK2 is
indeed required for depolarization-induced secretion in
PC12 cells (Zeniou-Meyer et al., Proc. Natl. Acad. Sci.
USA 105:8434, 2008). RSK2 function in exocytosis
appears to be through the phosphorylation of phospholipase
D1 (PLD1). Thus, an important new link in regulation of
secretion has been identified, and it is one in which mal-
function is clearly associated with disease. The next step is
Cell Mol Neurobiol (2010) 30:1371–1375
to show that RSK2’s role in corticoamygdalar synaptic
communication is mediated by the same or a similar
mechanism to that found in PC12 cells. Similarities
between secretory mechanisms in highly accessible model
systems and at difficult to study synapses of the brain is
always welcome news in the context of translational
research, from the standpoint of both identification of drug
targets and high-throughput identification of drug candi-
dates. This is an important advance for understanding and
potentially treating Coffin-Lowry patients. It also adds
another mental retardation syndrome to the list of those,
including Rett’s syndrome, Down syndrome and neurofi-
bromatosis (Ehninger et al., Neuron 60:950, 2008), now
seen essentially as reflecting a potentially correctable
synaptic transmission defect, rather than an irreversibly
misbuilt brain.
Cell model systems are necessary for progress in
translational medicine, whether modeling the behavior of
the identical cell within an intact organ, or a gene within a
cell, or using an accessible cell such as the bovine chro-
maffin cell as a model for other post-mitotic neural cells
which cannot be obtained in sufficient numbers or purity
for biochemical experiments. However, a thorough under-
standing of the human neuroendocrine cell, and the prop-
erties of its surrounding environment in a functioning
organ, is also critical. Three contributions in this section
address the development and properties of the chromaffin
cell within the adrenal gland, and the human chromaffin
cell in particular. Perez-Alvarez and colleagues review the
embryonic origin, development, morphology and physiol-
ogy of the human adrenal medulla. They contrast, in par-
ticular, the stimulus-secretion coupling process in the
human compared to mouse, rat and cow adrenal medulla
with respect to type and composition of nicotinic ACh
receptors, voltage-dependent calcium channel (VDCC)
types, and muscarinic receptors. Alpha-7-subunit-contain-
ing nicotinic receptors are now identified in human chro-
maffin cells. Although it is known that PACAP releases
catecholamines from fetal human adrenal chromaffin cells,
the presence of PACAP as a co-transmitter with ACh in
human adrenal gland is as yet unconfirmed. The 25-year
history of human chromaffin cell autologous transplanta-
tion to the brain for treatment of Parkinson’s disease is
described, and the obstacles to success, including low
survival and dopamine production are reviewed. Interest-
ingly, chromaffin cell transplantation to spinal cord for pain
may be more promising, with therapeutic effect imputed to
enkephalin secretion in situ. It is not clear whether ACh or
PACAP, both relatively abundant in spinal cord, is the
relevant secretagogue for chromaffin cells at that location.
Although adult bovine chromaffin cells are of great value
as a neuroendocrine cell model because they are post-
mitotic, a very small percentage—like adult neurons of the
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CNS—are progenitor cells and can be isolated in culture.
The therapeutic promise of adult human chromaffin cell
progenitors grown as chromospheres in culture is explored
by Ehrhart-Bornstein et al. Ehrhart-Bornstein and col-
leagues point out the advantages of culturing adrenal pro-
genitor cells for autologous transplantation in Parkinson’s
disease including: (i) the potential for expansion of the cell
population, (ii) the expected lack of degeneration in situ
seen with mature chromaffin cells following transplanta-
tion to brain and (iii) the ability to encourage a dopami-
nergic phenotype rather than an adrenergic one during cell
expansion in culture. Characterizing the cell population as
one with growth potential and phenotypic plasticity is an
important first step, and this group reports on the existence
of developmentally specific transcription factors, including
Sox1 and Mash1, in their bovine chromosphere cultures.
Given that bovine chromospheres have increased dopamine
(DA) secretion after treatment with ascorbic and retinoic
acid, the use of similarly treated human chromosphere
cultures in autologous transplantation for treatment of
Parkinson’s disease is an exciting prospect.
The contribution of Guerineau and colleagues is an
important link between developmental aspects of chro-
maffin cell function considered above, and the properties of
the adrenal medulla as a stress transducer, which the
remainder of the contributions to this chapter address
directly. The authors review the developmental and stress-
induced changes in cell–cell communication in the adrenal
medulla. They point out that a developmental switch
occurs in the adrenal medulla such that the newborn gland
features robust intercellular gap junction communication
and relatively weak nicotinic cholinergic synaptic trans-
mission. This is reversed during postnatal maturation of the
cholinergic innervation of the gland, so that weak cell–cell
junctional coupling and strong nicotinic signaling charac-
terizes the adult adrenal medulla. During chronic stress in
the adult animal, however, gap junction-mediated inter-
cellular communication seems to be disinhibited. This
would allow amplification of signal transduction primarily
coming from the splanchnic nerve, in addition to the
increased ACh and PACAP release during increased firing,
through synergy with gap junction propagation of cellular
excitation and subsequent catecholamine secretion. The
involvement of gap-junctional plasticity in the effects of
chronic stress is an area of potentially fruitful further study.
Briefly describing David Goldstein’s review of adrenal
responses to stress (a keynote talk at the 15th ISCCB) at
this point in this commentary somewhat ‘buries the head-
line’ of a major theme of the meeting: the chromaffin cell
as a stress transducer. Goldstein traces the concepts of
sympathoadrenal stress developed by Cannon and of HPA
axis activation as developed by Selye. He points out that a
more modern formulation of these two axes in response to
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stress is that not only do the sympathoadrenal and hypo-
thalamohypophysialadrenocortical (HPA) axes differ in
response to specific stressors, but that the sympathetic and
adrenomedullary responses are often quite different. Thus
metabolic stress—hypoglycemia—preferentially activates
the HPA and adrenal medulla while cold stress (prior to
actual lowering of core temperature) activates sympathetic
outflow preferentially to HPA and adrenomedullary acti-
vation. This updating of earlier (but, as Goldstein points
out, still widespread) notions about the ‘unitary’ or gen-
eralized aspects of stress response is important. It allows a
more detailed accounting of human response to stressors
that are predominantly sympathetic, and those that are
mainly HPA/adrenomedullary. A systems overview of
stress and distress from this more detailed perspective is
presented in this review. This perspective also makes
incorporation of the concept of allostatic load—the ‘wear
and tear’ cost of maintaining homeostasis under conditions
in which significant adaptation is required—more conve-
niently applicable to analysis of how the chromaffin cell
might be involved in adaptation, and maladaptation, to
prolonged stress. In particular, this systems analysis may
help shift focus on stress research from ‘sympathoadrenal’
versus HPA to understanding how the hypothalamus
coordinates, perhaps through the parvocellular paraven-
tricular nucleus and CRH release, the activation of the
adrenal gland as a whole—both cortex and medulla—as a
stress-transducing organ.
Ait-Ali et al. further develop the notion of the adrenal
gland as a stress transducer not only at the immediate level
of enhanced catecholamine secretion, but also increased
production of a broader array of secreted informational
molecules, including cytokines, growth factors and neuro-
peptides, than previously appreciated. Pituitary adenylate
cyclase-activating polypeptide (PACAP) is the splanchni-
coadrenomedullary transmitter that mediates catechol-
amine biosynthesis and secretion from the adrenal medulla
in response to both metabolic and psychogenic stress
(Hamelink et al., Proc. Natl. Acad. Sci. 99:461, 2002;
Stroth et al., Neuroscience 165:1025, 2010). Based on these
findings, Ait-Ali and colleagues (the author of this com-
mentary is a co-author of this contribution) examined by
microarray analysis more than 15,000 transcripts poten-
tially regulated by PACAP in cultured bovine chromaffin
cells. Of the 370 or so up-regulated transcripts, more than
10% encoded informational molecules—secreted proteins
such as cytokines, growth factors, and neuropeptides that
act as first messengers at specific cellular receptors—and
enzymes that mediate their intracellular and extracellular
activation. These findings imply that the stress response is
not limited to the known hormones (catecholamines) and
neuropeptides that the chromaffin cell secretes. Rather, it
engages a transcriptional program that greatly increases the
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Cell Mol Neurobiol (2010) 30:1371–1375
variety and amount of signaling molecules that the adrenal
medulla uses to convey the stress response to other organs.
Wong et al. review the role of HIF1alpha as a tran-
scription factor important in mediating the actions of the
inducible transcription factor Egr1 and the ubiquitous
transcription factor Sp1 on PNMT gene activation. Based
on work primarily in PC12 cells from their own laboratory,
this group has determined that HIF1alpha mRNA is up-
regulated by hypoxia (5% O2 for 24 h) in these cells, as are
Egr1, Sp1 and PNMT. The authors postulate that HIF1al-
pha, activated by hypoxia, then activates the downstream
transcription factors Egr1 and Sp1 to elevate PNMT gene
transcription. Although these experiments were carried out
in cells in culture using reporter gene assays, the PC12 cell
model allowed facile deployment of siRNA for HIF1alpha
to establish the functional order of induction of these fac-
tors by hypoxia, i.e. the dependence of Egr-1 and Sp1
protein elevation, and PNMT transcription, on HIF-1alpha
(Tai et al. J. Neurochem. 109:513, 2009; Tai et al., Brain
Res. doi:10.1016/j.brainres.2010.07.036). These findings
are of great interest in further understanding of chromaffin
cell stress transduction for several reasons. First, these data
reveal HIF-1alpha as an inducible transcription factor in
PC12 cells. As the authors point out, HIF-1alpha regulation
in other cell systems occurs predominantly at post-trans-
lational points of regulation. Second, induction of HIF-
1alpha was observed in rat adrenal medulla after immobi-
lization stress or PACAP treatment in vivo. This implies,
since PACAP has been shown to be a major regulator of
adrenomedullary stress responses in vivo (vide supra) that
HIF-1alpha is a stress transducer in chromaffin cells not
only for hypoxia, but for other metabolic, and for psy-
chogenic stressors. Multiple pathways including protein
kinase A, p38, ERK, PKC, IP3 and Akt (protein kinase B)
appear to be involved in this induction, consistent with the
pleiotropic nature of PACAP signaling in chromaffin cells.
Further analysis of this pathway for PNMT induction in
vivo and in bovine chromaffin cells will likely produce
further insights into the nature of stress transduction by the
adrenal gland, and ways in which it might be modulated in
sustained stress responding leading to disease (see
Goldstein).
The extent of adrenomedullary plasticity during stress is
highlighted further by the report of Tillinger et al., dem-
onstrating that the vesicular monoamine transporter 2
(VMAT2) is induced by immobilization stress in PNMT-
positive chromaffin cells in vivo. Vesicular uptake of cat-
echolamines ismediated by two isoforms of VMAT,
VMAT1 and VMAT2 (Slc18A1 and Slc18A2). Only
VMAT2 is expressed in neurons of the central nervous
system, while VMAT1 is expressed in chromaffin cells,
enterochromaffin cells and SIF cells of all species exam-
ined to date. Interestingly, all cells in both human and
Cell Mol Neurobiol (2010) 30:1371–1375
rhesus macaque express both VMAT1 and VMAT2
(Erickson et al., Proc. Natl. Acad. Sci. USA 93:5166, 1996;
Anlauf et al., J. Histochem. Cytochem. 54:201, 2006),
while rodent chromaffin cells express mainly VMAT1,
with only low levels of VMAT2. Tillinger et al. show that
in the rat, VMAT1 is expressed in all chromaffin cells,
while basal VMAT2 is confined to norepinephrine
(TH?PNMT-) cells and absent from epinephrine (TH?
PNMT?) cells. Upon immobilization stress, VMAT2
mRNA levels are increased in the adrenal medulla, and this
occurs via de novo expression in epinephrine-containing
cells. This induction, which occurs after a single episode of
immobilization stress, provides a mechanism for sustained
plasticity of chromaffin cells. A copious literature now
suggests that altered vesicular transporter expression in
CNS is a physiological regulator of quantal size. It is
instructive that this mechanism may also serve to augment
epinephrine secretion in the adrenal gland responding to
stress. The effects of VMAT2 alteration on quantal size and
overall integrated secretion of epinephrine will be an
interesting question to approach quantitatively and geneti-
cally in the mouse in vivo, if VMAT2 induction after stress
can be demonstrated in that species as well.
Douglas et al. reviewed cytokine interactions with
chromaffin cells guided by the key observation that
‘‘[s]ignaling from the activated immune system is a little
explored but potentially important non-neural adrenome-
dullary regulator.’’ The effects of interferon alpha and
gamma on chromaffin cell tyrosine hydroxylase phos-
phorylation and apoptosis, respectively, of TNF-alpha
through TNFR2 on chromaffin cell gene expression, and
the effects of IL-1 on augmentation of catecholamine
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secretion and biosynthesis are all examples. These effects
are mediated through distinct pathways: ERK and STAT1
for IFN-alpha, NF-kB for TNF-alpha, and ERK, nitric
oxide, guanylate cyclase and NPY as intermediates for IL-1
signaling. How cytokine signaling can be important in
adrenomedullary function and stress transduction is per-
haps most clearly indicated by the reviewed literature on
the anti-inflammatory factor IL-6: in vivo adrenomedullary
catecholamine secretion up-regulates plasma IL-6 con-
centrations, and adrenalectomy reduces stress-induced IL-6
elevation more than 80%. Thus centrally-mediated IL-6
down-regulation could occur via control of splanchnicoa-
drenomedullary output, and the adrenal itself may be a
systemic source of this important cytokine. The isolated
chromaffin cell remains a key model for understanding the
mechanistic details of this signaling as a basis for assessing
efficacy, and potential complications, of new treatments for
major and common threats to human life such as sepsis.
Douglas et al. point out also that cytokine signaling to the
chromaffin cell provides key insights into their cellular
actions on neuroendocrine, in contrast to immune, cells
with direct application to elucidating mechanisms of sig-
naling in sickness behavior mediated through cytokines in
the central nervous system, and the grave CNS side effects
of interferons given as drugs in cancer, and elevated in
aging and during stress.
The contributions to this chapter establish that the
chromaffin cell remains the best simple model of a synapse
in the nervous system in which transduction of a presyn-
aptic signal to a functional, physiologically measurable
output can be visualized in detail, and in which translation
to improved clinical practice can also be clearly envisaged.
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