Journal of Neurochemistry, 2006, 97, 310–333 doi:10.1111/j.1471-4159.2006.03717.

REVIEW The norepinephrine transporter and its regulation

Prashant Mandela* and Gregory A. Ordway 

*Departments of Pharmacology & Toxicology and Psychiatry and Human Behavior, University of
Mississippi Medical Center, Jackson, Mississippi, USA
 Department of Pharmacology, Quillen College of Medicine, East Tennessee State University, Johnson
City, Tennessee, USA

Abstract and thereby its function. The NET is a target of action of a

For many years, the norepinephrine transporter (NET) was
considered a ‘static’ protein that contributed to the termination
of the action of norepinephrine in the synapse of noradren-
ergic neurons. The concept that the NET is dynamically
regulated, adjusting noradrenergic transmission by changing
its function and/or expression, was considered initially in the
mid 1980s. Since that time, a plethora of studies demonstrate
that the NET is regulated by several intracellular and extra-
cellular signaling molecules, and that phosphorylation of the
NET is a major pathway regulating its cell surface expression
number of drugs that are used long-term therapeutically or
abused chronically. This has driven numerous investigations
of how the NET and its function are regulated by long-term
exposure to drugs. While repeated exposure to many drugs
has been shown to affect NET function and expression, the
intracellular mechanisms for these effects remains elusive.
Keywords: amphetamine, antidepressants, cocaine, endo-
cytosis, Na+/ Cl– -dependent transporters, regulation.
J. Neurochem. (2006) 97, 310–333.

Norepinephrine (NE) is the principle chemical messenger

employed in central noradrenergic and peripheral sympa-
thetic synapses. The norepinephrine transporter (NET),
present on the plasma membrane of noradrenergic neurons,
limits the action of NE through reuptake into the cytoplasm.
The NET not only regulates the longevity of NE in the
synapse but also plays a very important role in presynaptic
and postsynaptic homeostasis (Axelrod and Kopin 1969;
Iversen 1971; Xu et al. 2000; Zahniser and Doolen 2001).
Many drugs that bind to the NET have been utilized
therapeutically for the treatment of a number of disorders.
These NET-binding drugs directly alter its function, either by
acting as inhibitors or by competing as a substrate for uptake.
In 1983, the first report appeared that suggested that the NET
could be regulated by drugs (Lee et al. 1983). These authors
demonstrated that repeated exposure to drugs that modulate
noradrenergic transmission altered the expression of the
NET. These initial findings demonstrated that the neurons use
changes in NET expression to adapt to changes in transmis-
sion, and opened the door for studies of the cellular
mechanisms by which the NET is regulated. This paper
reviews research that has contributed to the current under-
standing of the regulation of the NET by numerous factors,
and the cellular and molecular mechanisms by which the
NET is regulated.
NET Structure and Function
The NET belongs to a family of Na+/Cl– dependent transport-
ers that include transporters for serotonin, dopamine, glycine
and GABA (Amara 1992; Masson et al. 1999; Robinson
2002). These transporters share similar amino acid sequences
and presumed protein structures, and ‘pump’ mechanisms.
Electrochemical energy derived from the inward gradient of
Na+ drives the intracellular accumulation of neurotransmitter
via the NET. The function of the NET is selectively blocked by
the Na+/Cl– dependent binding of drugs such as desipramine
Received September 9, 2005; revised manuscript received December 7,
2005; accepted December 12, 2005.
Address correspondence and reprint requests to Dr Gregory A.
Ordway, Department of Pharmacology, Quillen College of Medicine,
East Tennessee State University, PO Box 70577, Johnson City, TN
37614. E-mail: ordway@etsu.edu
Abbreviations used: ANP, atrial natriuretic peptide; bNET, bovine
NET; BNP, brain natriuretic peptide; Br-, bromine; CaM kinase II,
calcium calmodulin kinase II; CNP, C-type natriuretic factor; DMI,
desipramine; hNET, human NET; MLC kinase II, myosin light chain
kinase II; NE, Norepinephrine; NET, norepinephrine transporter; NO,
nitric oxide; PI3 kinase, phosphotidyl-3-OH kinase; PKA, protein kinase
A; PKC, protein kinase C; PMA, phorbol 12-myristate-13-acetate; rNET,
rat NET; SERT, serotonin transporter.

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2006 International Society for Neurochemistry, J. Neurochem. (2006) 97, 310–333

(DMI) and nisoxetine. In fact, the binding properties of these
drugs have been used to identify and characterize the NET, as
well as localize its distribution in the body (Iversen 1971;
Amara and Kuhar 1993; Bönisch and Brüss 1994; Ordway
et al. 1997; Bauman et al. 2000; Blakely and Bauman 2000;
Robinson 2002). Recent evidence indicates that the NET
occurs as a homo-oligomer (Kocabas et al. 2003).
Na+ and Cl– are the two critical ions involved in normal
NET function. Br– (Bromine) is the only other ion that can
partially mimic the function of Cl– (Friedrich and Bönisch
1986; Bönisch and Brüss 1994). Kinetic studies of the
transporter suggest that binding of Na+ and then Cl– is a
prerequisite for the substrate to bind to the transporter
(Bönisch and Brüss 1994; Trendelenburg 1991). NE transport
by NET is electrogenic with a net gain of positive charge
(Trendelenburg 1991). Following the binding of substrate,
both Na+ and Cl– are cotransported along with the substrate
into the cytoplasm. Na+/K+-ATPase, a key ion pump,
provides the driving force for this secondarily active
cotransport by maintaining a Na+ concentration gradient
across the plasma membrane. Na+/Cl– dependent transport of
NE into the cytoplasm also requires a critical balance of
K+ ions across the cell membrane (Harder and Bönisch
1985; Bönisch and Brüss 1994). A negative membrane
potential created by a high extracellular concentration of K+
contributes to the driving force of amine transport (Harder and
Bönisch 1985). Removal of Na+ from the uptake buffer
impairs uptake by the NET. In addition to contributing to NET
function, Na+ can modulate both the affinity and velocity of
NE uptake. Changes in Na+ concentrations in the uptake
buffer results in changes in the transporter Km and Vmax (Role
and Perlman 1983). Further, Na+ demand for NET function
varies depending upon the extracellular concentration of NE
(Role and Perlman 1983). Alterations in membrane Na+/K+-
ATPase activity can directly impact the ability of the NET to
move NE across the membrane. Manipulations leading to a
decrease in Na+/K+-ATPase function are followed by a
decrease in uptake of NE and reversal of transporter function.
Reversal of transporter function can only be detected if
enough substrate is available in the cytoplasm. Drugs that
block ATP synthesis (cyanide) or Na+/K+-ATPase pump
(oubain) simultaneously decrease NET function (Harder and
Bönisch 1985; Bönisch and Brüss 1994).
NET structure and systems regulating its function are
currently under intense investigation because, like the
serotonin transporter (SERT), the NET is a target for
antidepressant and psychostimulant drugs. Analysis of NET
structure reveals critical information regarding the functional
residues and domains responsible for NET function, substrate
binding, drug binding, and regulation. The human NET
(hNET), bovine NET (bNET), and rat NET (rNET) have
been cloned (Pacholczyk et al. 1991; Lingen et al. 1994;
Brüss et al. 1997). The hNET gene is localized at chromo-
some 16q12.2 (Gelernter et al. 1993; Brüss et al. 1993). The

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Regulation of the norepinephrine transporter 311
NET in each species exhibits the same topology, including 12
transmembrane domains and sites for glycosylation. The
NET of each species is similar with respect to binding
affinities for drugs, with some exception (Bönisch and Brüss
1994; Paczkowski et al. 1999). The structural composition of
the hNET, rNET and bNET are discussed here, emphasizing
similarities and differences between the species. It should be
kept in mind that some of these differences could contribute
to species-specific regulation of the NET.
The cloned hNET cDNA is an 1857 base pair open reading
frame, predicting a protein of 617 amino acids with a mass of
approximately 69 kDa (Pacholczyk et al. 1991). When this
cDNA is transfected into cultured cells, the protein that is
expressed demonstrates NE uptake properties and responds
to the drugs known to affect NET function. Similar to other
species, the hNET protein consists of 12–13 hydrophobic
segments each of 18–25 amino acids in length forming the
transmembrane domains. Both amino and carboxy termini of
NET are located in the cytoplasmic side. The extracellular
loop between TM3 and TM4 has three potential N-glycosy-
lation sites. These N-linked carbohydrate additions contrib-
ute to one of the key mechanisms that regulate transporter
expression on the plasma membrane surface, which neces-
sarily impacts the function of the NET. For example, addition
of a carbohydrate moiety has been shown to dramatically
increase uptake (Melikian et al. 1994; Melikian et al. 1996;
Nguyen and Amara 1996). There is a greater likelihood of
glycosylated NET protein being expressed on the cell surface
than the unglycosylated forms or partially glycosylated
forms. Poorly glycosylated forms or unglycosylated forms
are retained in the cytoplasm and are not directed to the
surface (Melikian et al. 1996; Nguyen and Amara 1996).
The difference in cellular location of glycoyslated and
unglycosylated forms, i.e. expressed on the surface and
functional as opposed to intracellular and non-functional,
makes glycosylation sites potential domains for regulation of
NET function. In addition to glycosylation sites, there are
sites for potential phosphorylation by protein kinase C and
casein kinase II within the intracellular part of the transporter.
Comparison of amino-acid sequences and the potential
regulatory sites reveal a close similarity with other trans-
porters including GABA, glycine, SERT, and dopamine
transporters (DAT; Pacholczyk et al. 1991; Amara 1992;
Masson et al. 1999; Robinson 2002).
The rat NET and the bNET contain 617 and 615 amino
acids, respectively. Like the hNET, both rNET and bNET
share structural similarities with other Na+/Cl– neurotrans-
mitter transporters, such as 12 transmembrane domains with
both N- and C- termini on the cytoplasmic face and a large
extracellular loop between TM3 and TM4 (Lingen et al.
1994; Brüss et al. 1997). rNET has two and bNET (like
hNET) has three potential sites for N-glycosylation that are
located in the extracellular loop (Lingen et al. 1994; Brüss
et al. 1997). Analysis of the amino acid sequences reveals
312 P. Mandela and G.A. Ordway
two potential sites for phosphorylation by protein kinase C in
rNET and three in bNET. A potential phosphorylation site at
amino acid 259 is conserved among the 3 species (rat, bovine
and human). Another site for phosphorylation is at amino
acid 579 in the intracellular C-terminus (in rNET and bNET,
but not in hNET) a third phosphorylation site is also in bNET
at the C-terminal (Lingen et al. 1994; Brüss et al. 1997). A
potential casein kinase II phosphorylation site at amino acid
583 near the C-terminus is also conserved among the 3
species (rat, bovine, human). This conserved site for
phosphorylation by casein kinase II among the species
strongly suggests a role of casein kinase in regulating the
transporter function (Brüss et al. 1997) though no such
involvement has yet been reported. Like hNET, bNET and
rNET show characteristic uptake and pharmacological prop-
erties when expressed transiently or stably in other cells
systems. At the nucleotide level, rNET and hNET share 87%
similarity in the coding region, while rNET and bNET share
84% similarity, and hNET and bNET share 90% similarity.
Even the amino acid sequences have very close similarities.
Similarities among species reflect conserved structural fea-
tures that are more conserved than those of two different
catecholamine transporters (e.g. NET and DAT) within the
same species (Brüss et al. 1997).
The cellular trafficking of Na+/Cl– dependent transporters
was once thought to be limited to regulatory events (discussed
below). However, recent studies on these transporters demon-
strate that a dynamic basal transporter recycling occurs
between the plasma membrane and the endosomal compart-
ments that are capable of achieving a rate of 3–5% per minute
(Deken et al. 2003; Loder and Melikian 2003). Recent studies
by Holton et al. have identified a critical region in the C-
terminus of these transporters that plays a vital role in their
constitutive and regulation-dependent endocytosis (Holton
et al. 2005). Residues 584–593 in the C-terminus of the
NET corresponding to amino acid sequence LWERLAYGIT
are a vital component in the NET endocytotic process. This
region of the NET is conserved amongst Na+/Cl– dependent
transporters. Mutations of this sequence block the constitutive
endocytosis of the transporter, and are likely to block its
regulated endocytosis based on findings using a mutated DAT
(Holton et al. 2005).
Alternative splicing has been shown to play a role in
hNET regulation (Pörzgen et al. 1995). The hNET gene
consists of 14 exons, interrupted by 13 introns (Pörzgen et al.
1995). An additional exon (exon 15) has been identified and
can be spliced to exon 13, skipping exon 14. There are two
alternative 5¢-acceptor sites at exon 15 that can lead to two
variants of the alternatively spliced NET containing exon 15.
The shorter and longer forms result in variants with 3 or 18
amino acids, respectively, at the C-terminal end of the NET
(Pörzgen et al. 1998). Transfection of cultured cells with the
long form of the exon 15 splice variant yielded NE uptake
activity and NET cell surface expression that was lower than

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2006 International Society for Neurochemistry, J. Neurochem. (2006) 97, 310–333
the typical hNET (with exon 14), while expression of the short
form of the exon 15 splice variant had no uptake activity and
was not expressed on the cell surface (Kitayama et al. 2001).
The mechanism underlying the reduced surface expression of
the NET may be a result of the inability of the splice variant
hNET to bind to the PDZ-domain containing PICK1 protein
(Distelmaier et al. 2004), a step crucial to cell surface targeting
(see below). Similar findings and the importance of the C-
terminus in targeting of the NET to the plasma membrane has
been demonstrated for the bNET expressed in HEK-293 cells
(Burton et al. 1998).
Regulation by Intracellular Signaling Molecules
The NET is regulated by a number of intracellular signaling
molecules. One common pathway employed by these
molecules is phosphorylation. Several signaling cascades
are considered below and these are summarized in Table 1.
Cyclic AMP and protein kinase A
Considerable research demonstrates a role for cyclic AMP
(cAMP) in regulating the NET and its function. Cyclic AMP
is a key component in the second messenger system linking
upstream G protein-coupled receptors to downstream sign-
aling cascades and is produced by activation of adenylyl
cyclase. A membrane permeable, metabolically stable cAMP
analogue, 8 Br-cAMP, is commonly employed to study
cAMP-mediated events. 8 Br-cAMP induces a concentration-
dependent decrease in uptake of [3H]NE in bovine adrenal
medullary chromaffin cells (Bunn et al. 1992). In contrast,
forskolin, a drug that stimulates production of intracellular
cAMP by directly activating adenylyl cyclase, has a biphasic
effect on NE uptake in chromaffin cells. At low concentra-
tions, forskolin (1.0–3.0 lmol/L) stimulates an increase in
[3H]NE uptake, while at higher concentrations (10–
100 lmol/L) forskolin inhibits uptake and returns [3H]NE
accumulation to basal levels (Bunn et al. 1992). In the same
cell line pertussis toxin, which facilitates an increase in
cAMP by inhibiting Gi, decreases uptake of [3H]NE. The
effect of pertussis toxin on uptake appears to be only partially
accounted for by cAMP actions (Bunn et al. 1992).
Short-term (15 min) exposure of rat PC12 cells to forskolin
or a stable cAMP analogue (but not cGMP) has been shown to
decrease NET activity (Bryan-Lluka et al. 2001). This inhib-
itory effect is rapidly reversed when the drugs are removed
from the uptake assay. The effect of forskolin on NET function
is inhibited by staurosporine, a mixed protein kinase inhibitor
that has no effect on NET function by itself in PC12 cells.
Short-term exposure of PC12 cells to forskolin reduces the
Vmax of the NET. Longer exposure (24 h) of PC12 cells to
forskolin is accompanied by a reduction in NET gene
expression (Bryan-Lluka et al. 2001). While acute studies
(30 min exposures) demonstrate that protein kinase C (PKC)
activation can decrease Vmax and move transporters away from
 Table 1 Intracellular signal regulation of the NET

Messenger Exposure NE Cell Surface NET

System Agent Cell type Time Uptake NET Kinetics Expression mRNA References

PKC bPMA SK-N-SH 30 min fl « Km fl Vmax fl – Apparsundaram et al. (1998a)

2006 The Authors

bPMA COS-7 30 min fl – – – Bönisch et al. (1998)
(trasfected with hNET,
bNET, hNETs259A)
bPMA COS-7 24 h fl « Km fl Vmax – – Bönisch et al. (1998)
(transfected with hNET,
bNET, hNETs259A)
bPMA SK-N-SH-SY5Y 24 h fl « Km fl Vmax fl – Bönisch et al. (1998)
bPMA Placental 30 min fl « Km fl Vmax fl – Jayanthi et al. (2004)
trophoblasts (rat)
bPMA HEK293,LLC-PK1, 30 min fl « Km fl Vmax fl – Apparsundaram et al. (1998b)
(stably transfected)
cAMP/PKA cAMP analogues SK-N-SH-SY5Y 15 min, 24 h « – – – Bönisch et al. (1998)
(8Br-cAMP,db-cAMP)
cAMP anaologues SK-N-SH cells 40 min « – – – Apparsundaram et al. (1998a)
(8-pCT-cAMP,
Rp-8-pCT-cAMp)
8-Br-cAMP Chromaffin cells 25 min fl – – – Bunn et al. (1992)
dbcAMP PC12 cells 15 min, 24 h fl – – fl (24 h) Bryan-Lluka et al. (2001)
dbcAMP COS-7, transfected 15 min › – – – Bryan-Lluka et al. (2001)
with rat NET
dbcAMP COS-7, transfected 24 h › – – – Bryan-Lluka et al. (2001)
with hNET
Forskolin SK-N-SH-SY5Y 15 min, 24 h « – – – Bönisch et al. (1998)
Forskolin Chromaffin cells 25 min Biphasic – – – Bunn et al. (1992)
effect ›fl

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2006 International Society for Neurochemistry, J. Neurochem. (2006) 97, 310–333
Forskolin PC12 cells 15 min fl « Km fl Vmax « – Bryan-Lluka et al. (2001)
Forskolin PC12 cells 24 h fl ? – fl Bryan-Lluka et al. (2001)
Forskolin COS-7 ratNET 15 min « – – – Bryan-Lluka et al. (2001)
Forskolin (100 lM) COS7 ratNET 24 h › – – – Bryan-Lluka et al. (2001)
Forskolin COS7 hNET 24 h › – – – Bryan-Lluka et al. (2001)
Cholera toxin SK-N-SH-SY5Y 15 min, 24 h « – – – Bönisch et al. (1998)
Cholera toxin PC12 24 h fl – – – Bönisch et al. (1998);
Bryan-Lluka et al. (2001)
Pertussis toxin Chromafin cells 90min fl « Km fl Vmax – – Bunn et al. (1992)
cGMP/PKG 8-br-cGMP PC12, SK-N-SH-SY5Y 15 min « – – – Bryan-Lluka et al. (2001)
Regulation of the norepinephrine transporter

CaM Kinase KN93 (inhibitor) SK-N-SH 40 min fl – – – Apparsundaram et al. (1998a)

313
314 P. Mandela and G.A. Ordway

Bauman et al. (2000)

the plasma membrane in SK-N-SH cells (Apparsundaram

Uchida et al. (1998)

Sung et al. (2003)

Sung et al. (2003)

Kaye et al. (1997)
et al. 1998a) (see below), such redistribution is not seen with
cAMP-induced regulation of NE uptake where the Bmax of

et al. (2004)
References
[3H]nisoxetine binding in intact cells does not change (Bryan-

Jayanthi
Lluka et al. 2001). It is noteworthy here that [3H]nisoxetine
binding assays in this latter study were performed in a manner
that limits [3H]nisoxetine binding to surface transporters
(Apparsundaram et al. 1998a; Distelmaier et al. 2004).
mRNA

cAMP-mediated regulation of NET in PC12 cells therefore

NET

may result from a change in the conformation of the transporter

–
–

?
that hinders reorientation of the transporter during the transport
cycle, and thereby decreases the Vmax without effecting the
Cell Surface
Expression

affinity (Km) of the transporter for the substrate and without

changing the level of expression of NET protein on the cell
surface. It is highly unlikely that cAMP has direct effects on the
›
–
–

NET, since the effects of cAMP are cell-line dependent (see

below).
NET Kinetics

Numerous studies demonstrate that cAMP-induced effects

fl Km › Vmax

on NET function are not consistent across cell lines or

species. In contrast to effects in PC12 cells, cAMP analogues
had no effects on NE uptake in human SK-N-SH-SY5Y cells
–
–

(naturally expressing the NET) or COS-7 cells transiently

transfected with rat or human NET cDNA (Bryan-Lluka
Uptake

et al. 2001). It is notable in this regard that PC12 cells and

NE

fl
fl

SK-N-SHSY5Y cells have different origins (rat, human).

Treatment of another human neuroblastoma cell line that
naturally expresses the hNET, SK-N-SH cells, with cAMP
Exposure

10 min
30 min
30 min

analogues also does not affect NE uptake (Apparsundaram

2 days
Time

1h

1h

et al. 1998a). Together, these studies suggest that cAMP-

induced regulation is not a general phenomenon in NET
regulation but is cell line- and species-dependent.
Vas deferens, C. cortex (rat)

Cyclic GMP
Vas deferens (inhibitor)

CAD-hNET, SK-N-SH,
Placental trophoblast

sympathetic neurons

The cGMP analogue, 8-bromo-c-GMP or dbcGMP has no

effect on NET activity in both PC12 and SK-N-SHSY5Y
cells, suggesting that cGMP and cGMP-dependent protein
PC12 and rat

CAD-hNET

PC12 cells

kinases do not play a role in regulating rat and human NET

Cell type

(Bryan-Lluka et al. 2001).

Protein kinase C (PKC)

The involvement of PKC in the regulation of transporter
SNAP (NOx donor)

function and membrane expression is proving to be universal

Extracellular Ca2+
oligonucleotides

among monoamine transporters. There are a number of

Okadaic acid
Okadaic acid

Syntaxin 1A

potential sites for phosphorylation by PKC of the NET in

BONT/C1
antisense
(inhibitor)

several species (see above). Information from these early

Agent

studies has been confirmed by multiple studies demonstrating

that the PKC pathway is the most common intracellular
mechanism through which transporters can be regulated.
Nitric oxide pathway

Studies involving both transfected cell lines and cell lines

Table 1 Continued

Dephosphorylation

Vesicular shuttling

naturally expressing the NET have shown that PKC can

regulate the expression of transporter on the cell surface, and
Messenger

thereby affect NET function.

System

To investigate the role of PKC phosphorylation sites on

Other

NET expression and function, Bönisch et al. (1998) utilized

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COS-7 cells transfected with hNET (stated to have one site
for PKC-induced phosphorylation, see below), bNET (has
three sites for PKC-induced phosphorylation) and a mutant
hNET, hNETS259A. COS-7 cells transfected with
hNETS259 express a NET that has serine 259 replaced with
alanine, thereby removing the putative single site for
phosphorylation by PKC. Acute treatment (10 min) of these
cell lines with phorbol 12-myristate-13-acetate (PMA; PKC
agonist), okadaic acid (protein phosphotase inhibitor) and
staurosporine (PKC antagonist) did not affect NE uptake.
However, longer exposure to PMA resulted in decreased
[3H]NE uptake via NET in all 3 cell lines. The effects of
PMA were inhibited by staurosporine, which had no effect on
uptake on its own. The change in NE uptake following a 24 h
exposure to PMA was due to a reduction in transport velocity
(Vmax) without a change in the affinity (Km) of the
transporter for the substrate. Reduced uptake induced by
PMA exposure in COS-7 cells with all 3 varieties of
transfected NET, as well as SK-N-SH-SY5Y cells (expres-
sing the wild-type hNET), was accompanied by a reduction
of the binding of [3H]nisoxetine to NET on these cells
(Bönisch et al. 1998). Okadaic acid, a protein phosphatase
inhibitor, did not have any effect on PMA-induced decreases
of uptake. Together, these findings demonstrate that NET
function is sensitive to PKC, although direct phosphorylation
of the NET at serine 259 by PKC is not necessary to down-
regulate its function. Furthermore, in contrast to cAMP-
dependent regulation, PKC-dependent regulation of the NET
is irrespective of species and cell line.
Further studies on stable and cell lines, HEK-293, LLC-
PK1, and COS-7 cells, transiently transfected with NET have
confirmed NET internalization after PKC stimulation. PMA
decreases uptake in a time-dependent and dose-dependent
manner. PKC-induced regulation of surface transporter
function appears to be a specialized effect on NET function
in these cell lines because a similar effect is not seen with
other transporter systems (e.g. Na+-dependent leucine trans-
porter) present in the same cell lines. Activation of PKC
results in phosphorylation of NET. Although the NET does
not appear to be regulated by PKC through phosphorylation
at serine 259 (see above; Bönisch and coworkers, 1998), it is
phosphorylated at other sites (Apparsundaram et al. 1998b).
Phosphorylation of the NET is followed by the internalizat-
ion of the transporter into the cytoplasm from the plasma
membrane. Reduced surface expression of NET is the
primary mechanism contributing to PKC- induced decrease
in NET activity (Apparsundaram et al. 1998b).
Muscarinic receptors can signal through Ca2+- and PKC-
dependent mechanisms to modulate NET function (Appars-
undaram et al. 1998a). Stimulation of muscarinic receptors
on SK-N-SH cells with methacholine reduces [3H]NE
uptake. Like direct PKC activation, muscarinic activation
decreases maximum transport velocity (Vmax) secondary to a
relocation of surface transporters to the cytoplasm. NET

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Regulation of the norepinephrine transporter 315
affinity (Km) for the substrate is unaffected by PKC
activation. Muscarinic receptor activation increases intracel-
lular concentrations of Ca2+ (through inositol triphosphate
generation) and diacylglycerol, and both Ca2+ and diacyl-
glycerol activate PKC. PKC activation leads to phosphory-
lation of the NET and decreased surface expression of the
NET. Inhibition of PKC blocks PMA-mediated effects on
NET, but does not completely block muscarinic receptor-
induced effects. Co-treatment of cells with methacholine
plus BAPTA/AM, a Ca2+ chelator, eliminates the effect of
methacholine on NE uptake. These observations suggest a
possible second mechanism, other than Ca2+-dependent PKC
activation, through which intracellular Ca2+ can regulate NE
uptake at the plasma membrane (Apparsundaram et al.
1998a).
Recent studies on NET in placental trophoblasts have
provided a wealth of information regarding PKC-induced
phosphorylation and internalization of NET. A robust
expression of functional endogenous NET has been shown
in rat placental trophoblasts (Jayanthi et al. 2002). As
demonstrated in other cell lines, PMA stimulation of PKC
decreases [3H]NE uptake in a time- dependent and concen-
tration-dependent fashion, producing a robust reduction in
Vmax with no effect on Km. [3H]Nisoxetine binding in whole
cell trophoblasts, as wells as cell surface biotinylation assays,
demonstrate reductions in surface expression of the NET
(Jayanthi et al. 2004). This effect of PMA is mediated by
PKC in a Ca2+-independent manner. Removal of both
intracellular and extracellular Ca2+ did not prevent the effect
of PMA on NET regulation. This observation suggests a role
for Ca2+-independent PKC isoforms in NET regulation
(Jayanthi et al. 2004).
Jayanthi et al. (2004) have further demonstrated that the
NET is directly phosphorylated by PKC activation. Exposure
of rat placental trophoblasts to PMA or okadaic acid (a PP1/
2 A inhibitor) results in phosphorylation of the NET.
Inhibition of clathirin-mediated endocytosis by concanavalin
A or monodansyl cadaverine does not block PMA-induced
down-regulation of NET, suggesting that NET regulation by
PKC occurs via a clathrin-independent pathway. Placental
trophoblasts expressing dominant negative mutants of dyn-
amin 1 or dynamin 2 were also susceptible to PMA-induced
regulation of NET, again indicating that this system does not
play a role in NET endocytosis induced by PKC activation.
In addition, placental trophoblast cells expressing dominant
negative mutants of caveolin 1 or caveolin 3 did not disrupt
PMA-induced internalization of NET. In contrast, the
cholesterol-disrupting agent, filipin, completely blocked
PMA-induced effects on the NET. The inhibitory effect of
filipin appears to be limited to PKC-mediated NET inter-
nalization since other PMA-mediated inhibitions, e.g. taurine
uptake, were unaffected by filipin. These latter observations
provide evidence for a role of lipid rafts in PMA-mediated
NET internalization in rat placental trophoblasts. The authors
316 P. Mandela and G.A. Ordway
also suggest that lipid rafts may function as transition phase
docking sites before NET internalization (Jayanthi et al.
2004).
Taken together, these findings demonstrate that PKC can
regulate NET function by altering the equilibrium between
the plasma membrane and the cytoplasmic fractions of
transporters. PKC-induced reductions in NET function can
function independently of the endocytosis pathway mediated
by dynamin and clathrin, at least in rat placental trophoblasts
(Jayanthi et al. 2004). Recently, Holton et al. (2005) have
demonstrated that an endocytic signal near the C terminus
of DAT, one that is shared by other Na+/Cl– dependent
transporters including the NET, is vital to PKC-driven
regulation as well as constitutive endocytotic cycling of the
DAT. Specific mutations within this sequence block consti-
tutive recycling without affecting PKC-induced regulation,
while other mutations block both constitutive endocytotic
cycling and PKC-induced regulation. Finally, while PKC-
induced reduction in NET function appears to be independ-
ent of species and cell line differences, the mechanisms of
NET sequestration following phosphorylation may exhibit
cell line-dependence. For example, while dynamin mutants
did not effect PMA-induced effects on the NET in rat
trophoblasts, PMA had no effect on NET sequestration in
HEK-293 cells coexpressing both human NET and a K44A
mutant of dynamin 1 (Jayanthi et al. 2004).
Ca2+ and CaM kinase II
Extracellular Ca2+ plays an influential role in NET function.
PC12 cells preincubated with 0.3–10 mM Ca2+ for 10 min
show a marked increase in NE uptake (Uchida et al. 1998),
characterized by an increase in maximum transport velocity
(Vmax) and a decrease in substrate affinity (Km). Despite
removal of Ca2+ (1 mM) from the suspension medium after
the initial exposure, the increase in NE uptake persists,
suggesting a role for an unknown indirect mechanism
through which Ca2+ produces an effect (Uchida et al.
1998). Ca2+-induced alterations of uptake kinetics are also
observed for other monoamine transporters, including the
DAT and SERT, although there are qualitative differences
(Uchikawa et al. 1995; Yura et al. 1996). Regulation of
transporter function by changes in [Ca2+] could result from
changes in the activity of Ca2+-dependent kinases such as
PKC, Ca2+/calmodulin-dependent kinase II (CaM kinase II),
and/or myosin light chain kinase (MLC kinase). However,
PKC inhibitors have no affect on Ca2+-induced increases in
uptake. In contrast, CaM kinase II and MLC kinase inhibitors
robustly inhibit Ca2+-dependent increases in NE uptake.
CaM kinase II could directly phosphorylate the transporter
and regulate its function, or it could work through activating
MLC kinase and subsequent phosphorylation and movement
of the transporter from the cytoplasm to the plasma
membrane. MLC kinase could also be independently contri-

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buting to the increase in uptake, but the key in either case is
phosphorylation. Cells pretreated with 1 mM Ca2+ show a
significant increase in the Bmax of [3H]DMI binding meas-
ured in crude membrane homogenates, raising the possibility
that Ca2+ induces relocation of transporter from cytoplasm to
the plasma membrane (Uchida et al. 1998). Interestingly,
pretreatment of SK-N-SH cells with KN-93, a CAM kinase II
inhibitor, reduces uptake independent of external Ca2+
(Apparsundaram et al. 1998a), and preliminary studies in
our laboratory also are consistent with the idea that basal
CaM kinase II activity is required for normal NET function.
Apparently Ca2+, when applied externally, can influence
transporter function by increasing uptake through CaM
kinase II and MLC kinase rather than decreasing the uptake
by taking the PKC route. Overall, current evidence suggests
that the pathway of activation by Ca2+ when applied
externally is different to the pathway activated when internal
stores of Ca2+ are released.
Protein phosphatases
Protein kinases and phosphatases control protein targeting
and trafficking through phosphorylation and dephosphoryla-
tion processes, respectively. The fine balance of the state of
phosphorylation is orchestrated by these two critical classes
of enzymes and represents a vital regulatory mechanism
of protein function. Okadaic acid (OA) and calyculin A,
inhibitors of protein phosphatase 1/2A trigger SERT phos-
phorylation and reduce transporter activity (Ramamoorthy
et al. 1998). Similar mechanisms have been demonstrated for
the regulation of the NET. Treatment of vas deferens with
OA decreases NE uptake. Immunoprecipitation of the
extracts from the same tissue reveal the presence of NET/
PP2Ac complex. Further treatment of vas deferens with OA
decreases PP2Ac levels in NET immunoprecipitants and no
such effect is noticed in total extracts (Bauman et al. 2000).
Okadaic acid treatment blocks protein phosphatase and thus
inhibits its interaction with NET. The phosphatase–NET
interaction is essential for stabilizing the NET and for surface
expression of the NET.
Recent studies from rat placental trophoblasts also dem-
onstrate a similar mechanism. Okadaic acid reduces NE
uptake via the NET and also increases the phosphorylated
NET fraction (Jayanthi et al. 2004). These findings collec-
tively demonstrate a role for phosphatases in phosphoryla-
tion-dependent regulation of NET. Blockade of phosphatase
function can severely restrict transporter recycling to the
plasma membrane by increasing the phosphorylated fraction
of the NET and leading to a decrease in NE uptake via NET
(Jayanthi et al. 2004).
ATP
ATP is an important molecule in biological systems that
caters to energy demands at the cellular level. Besides its role
as an energy ferrying molecule, ATP mediates a wide variety

of cellular functions by acting through signaling systems.
ATP present in the extracellular compartment is incapable of
penetrating cell/neuronal membranes, it thus exerts its
influence on cellular systems through a wide range of
mediators present externally (Caldwell et al. 1960; Burn-
stock et al. 1997). These mediators include ATP receptors
(purinergic receptors) present on the plasma membrane
(Burnstock et al. 1997; Burnstock 1981) as well as externally
facing enzymes such as ecto-ATPases (Nagy et al. 1986) and
ecto-protein kinases (Ehrlich et al. 1986).
ATP is also present in vesicles loaded with neurotrans-
mitters. The presence of ATP and neurotransmitters in the
same vesicular packet raises the possibility that ATP may
function as a cotransmitter. ATP is stored in the synaptic
vesicles containing NE and is concomitantly released with
NE during a depolarization event (Geffen and Livett 1971). It
is present in the extracellular medium while NE reuptake
is in progress (Cao et al. 1990). Furthermore, ATP can
stimulate NE uptake as shown in rat iris preparations
(Angelakos 1964). ATP can also alter uptake of dopamine
and NE in rat occipital cortex synaptosomes (Cao et al.
1990). These initial findings implicated a role for ATP in NE
uptake.
Further variations in the ionic environment (Ca2+, Mg2+)
have been shown to play a critical role in ATP-induced
regulation of NET (Hardwick et al. 1989). ATP alters NE
uptake in a concentration- and cation-dependent fashion.
ATP increases NE uptake at lower concentrations (0.1 lM)
and decreases NE uptake at higher concentrations (1 lM).
The effects of ATP on altering NE uptake levels are greater in
buffers containing Ca2+ rather than Mg2+. Low concentra-
tions of ATP (0.1 lM) stimulate NE uptake in a Ca2+-
dependent fashion in PC12 cells (Hardwick et al. 1989).
These finding suggest that a Ca2+ rich environment is a
requirement for ATP to exert its influence on NET. A non-
hydrolyzable analog of ATP has no effect on uptake,
suggesting that the actions of ATP and ATPcS are mediated
through an ecto-protein kinase pathway (Hardwick et al.
1989).
Some of the effects of ATP seen, such as the biphasic
effect, were not noticed in another study involving the same
cell line (PC12 cells; Eshleman et al. 1995). In this study,
ATP caused a concentration-dependent increase in uptake of
NE rather than a biphasic effect. The ATP concentrations
ranged from 0.1 to 3 mM. In addition, GTP and UTP were
also effective in stimulating NE uptake. Further, the ATP-
induced increase in NE uptake was sensitive to temperature
and dependent on Na+. The buffer media also played a
pivotal role in dictating ATP’s actions. ATP’s actions were
more profound in bicarbonate-buffered media than in HEPES
or phosphate buffered media. Similar findings were seen in
ATP-induced reduction in the binding of DMI and cocaine to
the transporter. This study suggests an altogether different
route for ATP-induced alterations in transporter function. The

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Regulation of the norepinephrine transporter 317
ecto-kinase pathway could play a role at submicromolar
concentrations but at higher concentrations ATP could
directly interact with the transporter and alter its function
in an allosteric manner or act via the purinergic receptors and
influence transport function via a second messenger system
(Eshleman et al. 1995). The differences between the two
studies mentioned above are likely due to various experi-
mental conditions such as cell culture conditions and buffer
media (Eshleman et al. 1995). These studies provide some
important regulatory mechanisms through which ATP can
work and exert its influence on NE uptake. More work is
needed to understand the role and dynamics of ATP actions
at NE terminals. ATP effects on the NET seem to be of
particular physiological relevance, since ATP is released with
NE from neuronal terminals.
Proteins contributing to cellular NET trafficking
Synaptic proteins
Recent evidence strongly implicates a role of synaptic
proteins in regulating the expression of cell surface proteins
such as transporters (summarized in Table 1). Synaptic
proteins presently known to regulate NET function are the
SNARE (Sung et al. 2003) (soluble N-ethylamide-sensitive
factor attachment protein receptor) proteins and PICK1
(Torres et al. 2001). These studies are of considerable
importance as they focus on the likelihood of NET surface
expression and localization being coupled to NE release.
Syntaxin 1A, a SNARE protein, plays an important role in
the release of neurotransmitters. Recent evidence suggests
that SNARE proteins interact with neurotransmitter trans-
porters (SERT, glycine, GABA) on the plasma membrane
and play an important regulatory role in determining their
function (Quick et al. 1997; Beckman et al. 1998; Geerlings
et al. 2000; Haase et al. 2001). Cells naturally expressing
NET (SK-N-SH) or cells transfected with NET (CAD hNET)
show a decrease in NE uptake following treatment with
antisense oligonucleotides to syntaxin. In addition, treatment
of cells with botulinum toxin C1 (BoNT/C1), which blocks
syntaxin 1A association to the NET, decreases NE uptake by
the NET. Using immunofluorescence, SDS-PAGE, and
immunoblotting methods, colocalization of syntaxin 1A with
the NET has been demonstrated in noradrenergic varicosities.
In addition, SDS-PAGE and immunoblotting assays demon-
strate that syntaxin 1A is physically associated with the NET
(Sung et al. 2003). Since high concentrations of syntaxin 1A
are observed in noradrenergic varicosities, it is logical to
assume that membrane insertion of NET via syntaxin 1A via
a membrane fusion event could localize NET to the site of
NE release, improving the efficiency of NE reuptake. SNAP-
25, another protein involved with neurotransmitter release, is
not associated with NET/syntaxin complexes. Interestingly,
the interaction of NET with syntaxin 1A appears to be
318 P. Mandela and G.A. Ordway
required for protein kinase-induced NET regulation. When
the NET/syntaxin 1A complex is disrupted with botulinum
toxin, PKC-induced NET regulation is abolished (Sung et al.
2003). Sensitivity of both cytoplasmic and plasma membrane
NET/syntaxin 1A complexes to phosphorylation by protein
kinases show that the surface pool of these complexes can be
destabilized, but not the cytoplamsic pool. These studies
reveal mechanisms by which neurotransmitter and transpor-
ter conservation is fine-tuned. Syntaxin, a protein mediating
release-induced insertion of the NET into the plasma
membrane links NET function to the release process.
Dissociation of the syntaxin/NET complex would be expec-
ted to result in a delayed clearance of the transmitter (Sung
et al. 2003). The NET has been shown to be colocalized with
SNAP-25 and synaptophysin at axonal varicosities, also
consistent with the link of NET function to release (Schroeter
et al. 2000). However, NET-immunoreactivity is also found
in intervaricose segments of axons (Schroeter et al. 2000).
Interestingly, DAT immunolabeling in rat striatum has been
shown in plasma membranes of axons and their terminals but
not at the active sites of release, indicating that DAT is not
localized to the immediate area of dopamine release (Hersch
et al. 1997). However, this same study also reported that in
rat substantia nigra, DAT was present close to the active zone
and most likely facilitates rapid uptake of dopamine. From
these findings it has been speculated that transporter
localization to active release zones may be cell type- or
region-dependent.
PDZ domain-containing PICK1 protein
PDZ domain-containing proteins play a role in cell signaling
and membrane trafficking of proteins (see Table 1). PDZ
domains are interacting modules of globular proteins that
play a crucial role in directing proteins to the cell membrane
and organizing proteins to form supramolecular signaling
molecules (Fan and Zhang 2002). The synaptic proteins,
PICK1, are PDZ domain-containing proteins known to
interact with monoamine transporters. The PICK1 family is
demonstrating increasing importance in the targeting and
clustering of receptors and ion channels. The DAT is known
to bind to PICK1, forming clusters and colocalizng with
PICK1 in native dopamine-expressing neurons, as well as in
NET-transfected cell lines. An important prerequisite for
PDZ domain–mediated interactions is a consensus sequence
located in the carboxy terminus of the interacting proteins.
NET has sequence similarities to the DAT and the NET
carboxy terminus has a consensus sequence for a class II
PDZ domain binding, making the NET a likely candidate for
regulation by PICK1. In fact, the carboxy terminus of the
NET has been shown to interact with PICK1 in a yeast two-
hybrid system. PICK 1 interaction with NET is totally
eliminated when the last three residues of the carboxy
terminus are deleted (Torres et al. 2001; Distelmaier et al.
2004).

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HEK-293 cells transfected with cDNAs encoding NET and
PICK1 reveal important information regarding the intimate
association between these two proteins (Torres et al. 2001;
Distelmaier et al. 2004). PICK1 coimmunoprecipitates with
NET regardless of the NET antibody used. When expressed
individually, PICK1 and hNET show different localization
sites: NET is expressed predominantly on the plasma
membrane and PICK1 shows a diffuse intracellular distribu-
tion. Co-expression of both the proteins show a strong
colocalization and formation of clusters similar to that of
DAT association with PICK1. Primary cultures of rat locus
coeruleus neurons also demonstrate an association between
PICK1 and NET (Torres et al. 2001). Although association
and colocalization of NET with PICK1 have been observed,
there is no clear-cut evidence showing that the association is
related to regulation of transporter function or to pharmaco-
logical properties of the NET. Since PICK1 associates with
DAT, PICK1 could regulate NET in a manner similar to DAT.
PICK1 interacts with DAT in a positive manner by enhancing
DAT uptake activity in both mammalian cells and neurons
(Torres et al. 2001). Given the unknown mechanisms
involved in inhibitor-induced regulation of the NET (see
below), it is tempting to speculate that these drugs may work
by disrupting intimate relationships between the NET and
proteins associated with intracellular trafficking.
Regulation of the NET by neurotransmitters and
hormones
NE and other neurotransmitters also play an important role in
regulating the membrane expression of the NET and its
function. Animal and cell culture studies have provided the
evidence for these regulatory pathways. A few neuro-
transmitter regulatory systems are discussed here and are
summarized in Table 1. The regulation of the NET produced
by NE itself is considered in the section below discussing
the effects of substrates.
Acetylcholine
Acetylcholine (ACh) robustly decreases (by 90%) NE
uptake in isolated guinea pig chromaffin cells. ACh alters
Km, without altering the Vmax of the transporter (Role and
Perlman 1983). Recent studies using SK-N-SH cells have
shown that muscarinic receptor agonists can regulate trans-
porter function via a PKC dependent pathway (Apparsunda-
ram et al. 1998a). ACh may work through a similar
mechanism in isolated guinea pig chromaffin cells, although
further studies are required to prove this in these cells.
Nicotine, working through cholinergic nicotinic receptors,
affects NET function. High concentrations of nicotine
(5 mM) reduce uptake in synaptosomes prepared from rat
hippocampus and hypothalamus. In contrast, treatment with
lower concentrations of nicotine produces a small increase in
uptake (Balfour 1973).

Angiotensin
Angiotensin II affects blood pressure through actions on the
sympathetic nervous system, which uses NE as a neurotrans-
mitter. Angiotensin II activates sympathetic pathways by
regulating several of its components, such as the NET and
transcriptional control of the synthesis and release of NE (Lu
et al. 1996; Yang et al. 1996; Yang and Raizada 1999).
Exposure of neuronal cultures (hypothalamus and brain stem)
from Wistar Kyoto (WKY) rats to angiotensin II at concen-
trations as low as 100 nM results in time- and concentration-
dependent increases in NE uptake. Angiotensin II stimulates
increases in NE uptake that reach maximum, i.e. 5-fold above
basal levels, after 30 min of exposure. In contrast to uptake,
angiotensin II induces a gradual increase in NET mRNA levels
starting at 2 h of incubation with angiotensin II, and reaches a
peak (5 · basal levels) after 8 h of incubation (Lu et al. 1996).
The AT1 receptor, but not the AT2 receptor, mediates the
effects of angiotensin II on NET function. The acute (minutes
of exposure) effects of angitensin II are mediated post-
transcriptionally and are not PKC-dependent. In contrast, the
effects of longer exposure (hours) to angiotensin II involve
PKC, cFos and NET gene transcription (Lu et al. 1996).
Brains of spontaneously hypertensive (SH) rats express
higher levels of angiotensin II, NET mRNA, and NE
compared with WKY rats. Neuronal cultures (hypothalamus
and brainstem) show a similar response to angiotensin II
treatment, but the relative increase in NE uptake and NET
mRNA expression is much larger than that observed in WKY
rats. High AT1 receptor gene expression, and parallel
increases in AT1 receptors and AT1 receptor-stimulated
signalling in SH rats (Raizada et al. 1993) provide a possible
explanation for the differences seen between the two strains
of rats following angiotensin II treatment.
As noted above, angiotensin II employs more than one
mechanism to regulate NET function. Furthermore, angio-
tensin II can regulate NET function by affecting NET gene
expression, and in SH rats can do this via multiple pathways.
cFos antisense oligonucleotide treatment considerably limits
the ability of angiotensin II to stimulate NET mRNA
production (Lu et al. 1996). Antisense oligoneucleotide for
MAP kinase attenuates increases in NET gene expression
following chronic treatment with angiotensin II in WKY rat
neuronal cultures. This reduction in NET mRNA expression
is also accompanied by reduction in cfos and TH mRNA
(Yang et al. 1996). The phosphatidylinositol-3-OH kinase
(PI3 kinase) and PKB/Akt pathway is another route through
which NET mRNA production is altered by angiotensin II,
but in neuronal cultures from only SH rats (Yang and
Raizada 1999). These complex findings suggest that angio-
tensin II affects NET function through multiple pathways.
Dysregulation of the NET by angiotensin II likely contributes
to the pathogenesis of hypertension in SH rats, and
implicates a possible role of angiotensin II-mediated regu-
lation of the NET in human hypertension.

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Regulation of the norepinephrine transporter 319
GABA
GABA is an inhibitory neurotransmitter present in the
nervous system. GABA’s role in altering NET uptake activity
has been studied in rat pineal gland. GABA decreases NE
uptake in a concentration-dependent manner with an IC50 of
about 100 lM (Rosenstein et al. 1990). The physiological
relevance of this effect is unknown, since extracellular
GABA has been estimated to occur at low micromolar
concentrations (Timmerman and Westerink 1997; Shin et al.
2002) and to activate chloride conductance at these low
concentrations (Mtchedlishvili and Kapur 2005). GABA’s
effect on NE uptake is mimicked by muscimol, a GABA type
agonist. GABA’s action on NE uptake is inhibited by
bicuculline a GABA antagonist. Bicuculline by itself does
not affect NE uptake. The effects of GABA on NE uptake are
dependent on the concentration of NE used in the uptake
assay. While GABA decreases uptake at low NE concentra-
tions (0.5 lM), GABA increases NE uptake at higher NE
concentrations 5 lM (Rosenstein et al. 1990).
Insulin
Insulin is a key endocrine hormone regulating glucose
homeostasis in the body. Insulin also stimulates catecholam-
ine release from catecholaminergic neurons and can affect
the uptake of NE. The effect of insulin on NET function has
been studied in PC12 and SK-N-SH cells. Acute treatment of
cells with insulin (20 min exposure) decreases NE uptake in
PC12 cells (Figlewicz et al. 1993a). This effect is resistant to
reserpine, a drug that depletes both NE and DA, demonstra-
ting that the insulin-induced reduction in NE uptake is not
related to competition of endogenous NE (released by
insulin) with radiolabelled NE in the uptake assay. PC12
cells treated with IGF-1 (insulin-like growth factor) show an
increase in uptake rather than a decrease, distinguishing
between the 2 major routes through which insulin exerts its
cellular effects (insulin receptors, insulin-like growth recep-
tors). The insulin-induced decrease in uptake is characterized
by a decrease in Vmax, as a result of a decrease in transporter
number on the cell surface (Figlewicz et al. 1993a). Insulin-
dependent decreases in NE uptake may be due to direct
action of insulin at the transporter or through influencing
transporter gene expression. Insulin is known to decrease
NET mRNA expression in locus coeruleus neurons of the rat
(Figlewicz et al. 1993b).
Insulin’s action on the NET is dependent on the cell line in
which it is studied. Exposure of SK-N-SH cells to insulin
increases the uptake of NE. Insulin in this cell line works
through multiple pathways to increase NET function. The
insulin receptor is a membrane bound tyrosine kinase that
when activated phosphorylates tyrosine and other substrates
associated with it. Down stream signaling following insulin
receptor stimulation also includes activation of a lipid kinase,
PI3 kinase. SK-N-SH cells treated with tyrosine kinase and
PI3 kinase inhibitors show a drastic decrease in NE uptake.
320 P. Mandela and G.A. Ordway
This reveals that constitutive tyrosine kinase and PI3 kinase
activity is necessary for basal NE transport activity (Appars-
undaram et al. 2001), at least in this cell line. Tyrosine kinase
and PI3 kinase inhibitors are not known to have any affinity
for NET directly, suggesting mediation through intracellular
sigalling pathways. Furthermore, whole cell binding experi-
ments reveal that these inhibitors decrease surface expression
of the NET. The findings suggest that basal activities of PI3
kinase and tryosine kinase regulate cell surface expression of
the NET (Apparsundaram et al. 2001). In contrast, insulin-
stimulated increases in uptake are independent of changes in
cell surface expression of the transporter (Apparsundaram
et al. 2001), and therefore may involve an insulin-induced
conformational change in the NET that changes its uptake
activity.
Possible roles of ERK (extracellular signal receptor-
activated kinase) and p38 MAPK (p38 mitogen-activated
protein kinase) on insulin-induced regulation of NET
function have also been investigated in SK-N-SH cells
(Apparsundaram et al. 2001). The activity of GLUT 4, a
glucose transporter, is increased via p38 MAPK mechan-
ism, suggesting a possible connection of p38 MAP kinase
with insulin’s action on the NET. Inhibitors of p38 MAP
kinase and ERK enzymes do not affect the basal NE
uptake, but a p38 MAP kinase inhibitor blocks insulin-
dependent increases in uptake. In contrast, ERK inhibition
does not affect insulin’s action on the NET (Apparsundaram
et al. 2001).
The effect of insulin on the NET in SK-N-SH cells is also
mediated, at least in part, through a Ca2+-dependent pathway
(Apparsundaram et al. 2001). Uptake assays performed in
the absence of extracellular Ca2+, or in the presence of
BAPTA/AM (an intracellular Ca2+ chelator) in a Ca2+-
containing buffer, robustly decreases insulin’s influence on
uptake. Thapsigargin, an inhibitor of the sarcolemmal Ca2+
pump that depletes intracellular stores of Ca2+, decreases
insulin’s ability to stimulate NE uptake in SK-N-SH cells.
These findings suggest that the insulin-dependent increase in
uptake is secondary to an insulin-triggered influx of Ca2+
across the plasma membrane. Insulin does not release Ca2+
from intracellular stores, but the Ca2+ requirement for
insulin’s action is met through Ca2+ entry via voltage-
dependent Ca2+ channels. The study by Apparsundaram and
coworkers emphasizes the role of protein phosphatase2A
(PP2A) in NET regulation. Protein phosphatases were shown
to associate with NET (see above) and dictate NET’s
phosphorylation state. This association between NET and
phosphatases is critical for insulin’s actions on NET since
this NET–phosphatase association is vital for normal func-
tion of NET. Further this study also demonstrates that NE
uptake function can be increased without altering NET
plasma membrane expression (Apparsundaram et al. 2001).
Together, these findings demonstrate that regulation of
NET function by insulin involves multiple pathways and NE

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uptake regulation can be independent of changes in plasma
membrane NET expression (Apparsundaram et al. 2001).
Natriuretic peptides (NP)
Polypeptide hormones such as atrial natriuretic factor (ANF)
(de Bold et al. 2001), brain natriuretic peptide (BNP) (Sudoh
et al. 1989) and C-type natriuretic factor (CNP) belong to the
family of natriuretic peptides (NP). These peptides constitute
a major class of cardiac hormones that have strong influences
on endocrine regulation of blood volume and vascular tone.
ANF and BNP are also expressed outside the cardiac system
in a wide variety of sites and are believed to act in an
autocrine or paracrine fashion (Casco et al. 2002). CNP is
also abundantly present in the nervous system and is also
produced by vascular endothelium (Furuya et al. 1990;
Sudoh et al. 1990; Stingo et al. 1992). ANF exerts hypo-
tensive effects on the cardiovascular system via its interac-
tions with catecholamine metabolism and by modulating
sympathetic activity (Vatta et al. 1993). ANF produces a
time-dependent increase, reaching a plateau at 30 min, in NE
uptake via the NET in adrenal medullary slices from male
Wistar rats. ANF induced increases in NE uptake via NET
may result from increasing NET availability at the plasma
membrane or by increasing NET synthesis (Vatta et al.
1993). Both BNP and CNP stimulate NE uptake via NET in
hypothalamic slices from male Wistar rats in both concen-
tration- and time-dependent fashions. The concentration
required to produce an increase of NE uptake is 100-fold
higher for BNP (100 nM) as compared to CNP (1 nM). (Vatta
et al. 1996). Non-neuronal uptake of NE is unaffected by NF
peptides, although a specific effect on NET vs. other Na+-
dependent transporters has not been ruled out. Clearly
natriuretic peptides influence NE uptake, although the
cellular mechanisms mediating these effects are unknown.
Further study of these mechanisms could help in under-
standing the intricate relationship between natriuretic pep-
tides and the cardiovascular system.
Nerve growth factor (NGF)
Exposure of PC12 cells to NGF for 24 h decreases NE
uptake. In contrast, if PC12 cells are grown in serum-free
media and exposed to NGF, NE uptake is slightly increased
after a 24 h exposure to NGF, and then there is robustly
decreased uptake after 96 h of exposure (Ikeda et al. 2001).
In serum-containing medium, NGF exposure decreases
uptake. Kinetic studies of uptake performed after 48 h of
NGF treatment show a decrease in Vmax without altering Km.
In addition to these effects, NGF also decreases NET mRNA
expression in PC12 cells. The decrease in mRNA levels
could contribute to a decrease in availability of new NET
protein thus resulting in the decrease of NE uptake (Ikeda
et al. 2001). In bovine adrenal chromaffin cells, NGF
exposure increases NE uptake and increases NET mRNA
expression (Wakade et al. 1996). Other growth factors like

EGF have also been shown to affect (decrease) NE uptake in
PC12 cells without altering NET mRNA expression (Ikeda
et al. 2001). It is interesting to note that NGF’s effect on NE
uptake and NET mRNA levels varies with presence and
absence of serum (Ikeda et al. 2001). The mechanism by
which NGF affects the NET is poorly understood.
Nitric oxide
Nitric oxide (NO) and its congeners are key signaling
molecules in the cardiovascular system and have a putative
role as neurotransmitters in the nervous system. Regulation
of noradrenergic signaling by NO and its congeners, nitrogen
oxides (NOx), have been studied in PC12 cells and rat
sympathetic neurons in primary culture. PC12 cells treated
with NOx inhibit the uptake of NE. NOx signaling includes
both cGMP-dependent and independent pathways, but
studies of the molecular mechanism of NOx-induced
decreases in NE uptake indicate that the cGMP-independent
pathway is the most likely mediator. The cGMP analogues,
8-BrcGMP and LY-83583, as well as inhibitors of soluble
guanylyl cyclase, do not affect NOx-induced decreases in
uptake (Kaye et al. 1997). It should be noted that besides
cGMP signaling, NO inhibits Na+/K+-ATPase, a key pump in
maintaining the uptake process. Also, NO may exert its
influence through a direct action on the transporter. This view
is supported by findings wherein treatment with 1 mM
cysteine significantly decreases the effect of S-nitroso-
acetylpencillamine (SNAP), a NOx donor, on uptake. This
observation suggests nonenzymatic reactions of NOx with
sulfhydryl groups. The NET has many cysteine residues that
are candidates for trans-nitrosation (Kaye et al. 1997). In
SK-N-SH cells, pretreatment with the nitric oxide synthase
(NOS) inhibitor, l-NMMA, fails to alter methacholine-
induced reductions in uptake. SNAP treatments in SK-N-
SH cells show an increase rather than a decrease in uptake
(Apparsundaram et al. 1998a). Overall, these findings dem-
onstrate that more research is needed to understand molecular
mechanisms of NO-induced regulation of the NET, and to
elucidate its physiological significance.
Ovarian hormones
Estrogen plays a vital role in maintaining homeostatic
conditions in the female reproductive system (Couse and
Korach 1999). Estrogens exert their effect on cell systems in
both a genomic and a non-genomic way (McEwen 1991;
Couse and Korach 1999). Environmental estrogens are
another class of substances that do not share the same
chemical structure or steroidal properties of estrogen, but
have similar actions. One such environmental estrogenic
compound is bisphenol A (Krishnan et al. 1993). Bisphenol
A, 17b-estradiol, and p-nonyl-phenol decrease NE uptake in
bovine adrenal medullary cells following brief exposures
(30 min) (Toyohira et al. 2003). The mechanism by which
these compounds influence NE uptake is not understood.

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Regulation of the norepinephrine transporter 321
However, these compounds change the saturation kinetics of
NE at the NET, although there are differences in the specific
kinetic effects of bisphenol A and 17b-estradiol (Toyohira
et al. 2003). Hence, the effects of these compounds may be
mediated by direct modification of NET activity via modu-
latory binding sites on the transporter, rather than through
regulation of transporter expression or activity via intracell-
ularly mediated pathways.
Regulation by Substrates and Inhibitors Acting at the
NET
The study of the regulation of the NET by substrates and
inhibitors of the NET is a growing area of research because
the NET is a site of action of drugs that are typically
administered chronically. Many drugs can affect the NET
acutely, usually as a result of binding to the NET or
disrupting ionic gradients. The studies reviewed below and
summarized in Table 3 consider the effect of continuous or
repeated exposure of the NET to NET substrates and NET
inhibitors on NET function and expression. Since these drugs
bind the NET, a criterion for discussing the regulatory effects
of these drugs is that the substrate or inhbitor is absent
(except for NE) at the time that function and/or expression is
assessed. The effect of drugs, treatments, or stress, which are
known to modulate NE, on NET expression are also
reviewed below and summarized in Table 4.
Inhibitors
One of the earliest antidepressant drugs was the tricyclic
antidepressant, imipramine. Many early antidepressants,
some of which are still in use today, are variations on this
original structure, including DMI, amitriptyline, and nor-
triptyline. These drugs bind to the NET and block the
reuptake of NE. The pharmacological action of these drugs at
the synapse is rapid, i.e. uptake is inhibited upon binding to
the transporter. However, the therapeutic effect of these drugs
in the treatment of depression requires several weeks of daily
treatment. Thus, while uptake inhibition has been a valuable
index for identifying potential antidepressants, studies
designed to understand the relationship between biology
and therapeutic action have concentrated on the effects of
long-term treatment with antidepressant drugs. Both acute
and chronic exposure to tricyclic antidepressants decrease
uptake of NE, at least in part, by blockade of NET function.
In addition to inhibition of uptake as a result of binding to the
NET, chronic treatment (e.g. 21 days) of rats with DMI
results in a decrease in NET binding sites in specific brain
regions (Bauer and Tejani-Butt 1992; Hebert et al. 2001).
The effect of DMI treatment in vivo on NET expression is not
seen following a single dose of DMI (Bauer and Tejani-Butt
1992). The down-regulation of the NET produced by
repeated DMI administration is accompanied by a reduction
in uptake (Benmansour et al. 2004). In addition, the
322 P. Mandela and G.A. Ordway
structurally dissimilar NET inhibitor, reboxetine, also down-
regulates NET (as measured by radioligand binding) follow-
ing its chronic administration to rats (Gould et al. 2003),
while paroxetine, a SERT inhibitor with low affinity for the
NET, does not affect NET binding or function (Benmansour
et al. 2004). The ability of DMI and reboxetine to down-
regulate the NET, an effect that is functionally similar to
acute blockade of the NET, has raised speculation that this
regulatory effect may contribute to the utility of transporter
inhibitors in the treatment of depression.
In vivo experiments designed to address mechanisms of
DMI-induced regulation of the NET reveal an ambiguous
role of changes in NET gene expression on DMI-induced
changes in the NET. Repeated treatment of rats with
antidepressants for 14 days, but not 3 days, down-regulates
NET-immunoreactivity in the locus coeruleus, as measured
by Western blotting (Zhu et al. 2002). However, NET
mRNA levels have been reported to increase (Szot et al.
1993) or decrease (Zhu et al. 2002) after short-term
treatment of DMI to rats, and increase after 14 days
treatment (Zhu et al. 2002). Hence, changes in NET mRNA
levels in the locus coeruleus following DMI treatment do not
parallel changes in NET binding.
In an effort to elucidate the molecular mechanisms
involved in inhibitor-induced regulation of the NET, the
effect of exposure to NET inhibitors on NET expression and
function has been studied recently in in vitro cell culture
systems. Since the inhibitors themselves affect NET function
acutely, it is important to note that the functional (uptake)
effects of the drugs are measured under conditions that
assume that the drug has been washed from the cells
following the exposure (see below for pitfalls in this
assumption). Exposure (3 days) of both PC12 cells (produce
NE and NET) and 293-hNET cells (HEK cells transfected
with hNET, but do not produce NE) to DMI produces
reductions in uptake capacity, radioligand binding to the
NET, and NET-immunoreactivity (Zhu and Ordway 1997;
Zhu et al. 1998). These effects of DMI appear to be specific
to NET binding antidepressants, since similar exposure of
cells to the selective serotonin uptake inhibitor, citalopram,
does not affect NET binding. Furthermore, exposure to
nisoxetine, a NET inhibitor structurally dissimilar from DMI,
produces effects on the NET very similar to DMI. NET
binding and NET immunoreactivity slowly recovers (over the
course of days) to normal levels following the removal of
DMI. Hence, NET regulation in response to NET inhibitor
exposure in cell culture systems appears to be similar to
NET regulation as induced by prolonged exposure to NET
inhibitors in vivo. It is worth noting however, that if NET
binding by the inhibitor were the only factor contributing to
regulation in vivo during NET inhibitor treatment, then it
becomes difficult to explain why the NET is not regulated
similarly (Bauer and Tejani-Butt 1992) in all brain regions
after chronic NET inhibitor treatment.

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While DMI does not produce effects on NET binding
following a single dose in rats, in vitro studies show an acute
regulation of the transporter by antidepressants. Ordway and
coworkers (Zhu and Ordway 1997; Zhu et al. 1998)
originally reported that short exposures (4 h) to DMI could
down-regulate the NET. However, more recently these
authors (Ordway et al. 2005) have demonstrated that in vitro
studies of DMI’s effects on NET function and binding can be
complicated by the tenacity at which DMI is retained in cell
membranes (Zhu et al. 2004). This property of DMI, and
presumably other lipophilic psychotherapeutic compounds,
creates an obstacle to the study of the acute effects on
regulation of transporter function. In fact, it is speculated that
retention of transporter inhibitors in the brain following
cessation of treatments to rats, could potentially complicate
binding assays (see Zhu et al. 2004 for discussion). This
complication arises because of the membrane retention of
DMI (and nisoxetine) and because these drugs have a very
high affinity for the very protein being studied, i.e. the NET.
Uptake and binding assays are performed in very small
volumes and redistribution of DMI from membranes to the
small volume of the reaction mixture can result in pharma-
cologically relevant concentrations of NET inhibitor in the
assay (Zhu et al. 2004; Ordway et al. 2005). The retention
and redistribution of DMI hampers evaluation of NET by
certain radioligand binding assays and uptake assays, but is
not expected to interfere with immunologically detected NET
using methods such as Western blotting. It is noted, however,
that a lack of an effect of DMI on antibody binding to the
NET has not been specifically demonstrated. Ordway et al.
(2005) recently developed an assay to evaluate binding and
uptake at time points as short as 2 h following DMI
exposure, without the interference of residual drug. Using
these methods, exposure to DMI for 16 h is necessary before
reductions of NET function (uptake) in the absence of DMI
can be measured.
The molecular mechanisms responsible for NET inhibitor-
induced regulation of the NET expression and function are
only beginning to be elucidated. One possible mediator of
this event could be NE itself. Inhibition of NE uptake by
NET inhibitors could result in extracellular actions of NE, i.e.
through receptors for NE that lead to intracellular events
mediating NET down-regulation. However, treatment of
PC12 cells with concentrations of NE as high as 1 mM did
not affect radioligand binding to the NET (Zhu and Ordway
1997). Furthermore, exposure of 293-hNET cells, which
express the NET but do not synthesize NE, to DMI or
nisoxetine down-regulates the NET just as it does in PC12
cells that synthesize NE. Together, these findings strongly
suggest that NE does not play a role in the down-regulation
of the NET produced by NET inhibitor exposure.
Several lines of evidence suggest that down-regulation of
the NET by DMI occurs as a consequence of binding to the
transporter, rather than as a result of secondary effects.

Similar time-dependent and concentration-dependent down-
regulations of the NET are produced by DMI exposure in rat
PC12 cells (Zhu and Ordway 1997), human SK-N-SHSY5Y
cells (Zavosh et al. 1999), and in non-catecholaminergic
293-hNET cells (Zhu et al. 1998). Also, the structurally
dissimilar NET inhibitors, nisoxetine and reboxetine, down-
regulate the NET (Zhu and Ordway 1997; Gould et al.
2003). Although these data suggest that regulation of the
NET by inhibitors is a basic property of inhibitors, it is
emphasized that cocaine, an inhibitor of the NET, does not
down-regulate the NET in cultured cells (Zhu et al. 2000). In
addition, exposure of NET-expressing cells to amphetamine,
a substrate for the NET, also results in down-regulation of the
NET (Zhu et al. 2000). Hence, it can be speculated that
uptake inhibition by itself is not sufficient to initiate the
process of down-regulation. Rather, it seems to be some
specific domain(s) of the NET protein engaging binding to
the ligand that is required for down-regulation. Oligomeri-
zation is another important process that plays a vital role in
NET function (Kocabas et al. 2003) and drug-induced
disruption of oligmerization could conceivably contribute
to disruption of transporter function and to its ultimate down-
regulation.
The possibility that DMI-induced regulation of the NET is
mediated at the level of changes in NET mRNA has been
investigated using cultured cells. In 293 h-hNET cells, DMI
exposure, at times (e.g. 3 days) and concentrations (e.g.
500 nM) that down-regulate NET protein, does not affect
NET mRNA levels (Zhu et al. 1998). However, it is worth
noting that this transfected cell line has a very active
promoter driving NET gene transcription. In the SK-N-
BE(2)M17 cell line, which expresses native hNET, exposure
to DMI for 3 days reduces levels of NET mRNA, whereas
exposure for 14 days increases NET mRNA. Despite the
fluctuation in mRNA levels following DMI exposure, NET
protein levels are down-regulated following 3 and 14 days of
DMI exposure. Similar experiments have been performed in
the NET expressing cell line SK-N-SHSY5Y. Cells treated
with DMI for 1 and 3 days demonstrate a decrease in
radioligand binding to the NET. After 1 day of DMI
exposure, there is no effect on NET mRNA, but a 3 day
exposure decreases NET mRNA. Since a decrease in mRNA
content followed a decrease in protein content, the change in
mRNA content is at best a contributor to reduce NET protein,
but not the initial cause of loss of NET protein.
Cocaine is also a NET inhibitor and a number of studies
using different treatment paradigms have investigated the
effects of cocaine on the NET. Continuous exposure of
293hNET cells to cocaine causes a small reduction in uptake
and no effects on NET protein (Zhu et al. 2000). Studies
examining the effect of repeated treatment of rats with
cocaine have revealed conflicting findings. Prenatal exposure
to cocaine has been reported to increase radioligand binding
to the NET in the placenta (Shearman and Meyer 1999), to

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Regulation of the norepinephrine transporter 323
decrease radioligand binding to the NET in postnatal rat
cardiac tissue (Zhao and Sun 2004), and to have no effect on
NET protein in postnatal rat brain (Shearman and Meyer
1999). Benmansour et al. (1992) and Belej et al. (1996)
reported that repeated cocaine administration to rats does
produce major effects on radioligand binding to the brain
NET, although both increases and decreases have has been
demonstrated in very discrete brain regions (Belej et al.
1996). Chronic self-administration of cocaine in non-human
primates results in an increase in radioligand binding to the
NET in several brain regions (Macey et al. 2003; Beveridge
et al. 2005). In the human brain, as assessed postmortem in
chronic cocaine users, cocaine has been reported to increase
NET protein (Mash et al. 2005). Hence, while studies of the
effects of antidepressant inhibitors of the NET consistently
demonstrate down-regulation of NET protein and/or func-
tion, studies examining chronic cocaine exposure have
yielded incongruent findings. Exposure in vivo to cocaine
appears to increase NET protein. Since this effect does not
occur in cultured cells expressing the NET and lacking
synaptic contacts, the effect of cocaine on the NET in vivo
may be mediated through actions of cocaine other than direct
binding to the NET.
Overall, numerous investigators have demonstrated that
specific NET ligands (e.g. DMI, nisoxetine, amphetamine),
but not all NET inhibitors (e.g. cocaine), down-regulate the
NET. The specific molecular mechanisms responsible for
ligand-induced regulation have not been fully elucidated.
In vivo and in vitro studies demonstrate that the mechanisms
involved in DMI-induced regulation of NET protein are
complex, may be brain region-specific, and are not tightly
associated with NET gene transcription. Preliminary studies
in this laboratory demonstrate that initially, DMI induces a
redistribution of NET away from the plasma membrane
surface (Pan et al. 2002), and that DMI’s effects on the
transporter are unaffected by inhibitors of several protein
kinases and protein phosphatases (Pan et al. 2003). Antide-
pressant-block of the NET antagonizes NET function (reup-
take) and may give the NET a non-functional tag. Unknown
cellular proteins may recognize this inactivity and work
together to internalize the NET protein in order to recycle
new functional NET proteins to the surface. This process
would be expected to take some time and could explain the
lack of immediate recovery following antidepressant expo-
sure. Unfortunately, the intracellular mediators for this
process remain elusive.
Substrates
Studies using 293hNET cells have shown that NET function
(uptake) is reduced as a result of exposure to NE followed by
cell washing. This effect of NE is concentration-dependent
(Zhu and Ordway 1997). NE’s effect on the NET is opposite
to that observed for serotonin and its cognate monoamine
transporter, the SERT. Blakely and coworkers have demon-
324 P. Mandela and G.A. Ordway
strated that SERT regulation is activity dependent, such that
the presence of serotonin increases transporter activity
(Ramamoorthy and Blakely 1999). Recently, Mao et al.
(2004) have demonstrated that the reduction in NET activity
(reduced Vmax) produced by exposure (24 h) of PC12 cells to
NE is likely mediated by oxidative stress pathways (Mao
et al. 2004). NE produces cellular oxidative stress that is
enhanced by copper, and exposure to NE plus copper
increases the magnitude of NE-induced reductions in NET
activity and NET protein. NE appears to reduce NET activity,
at least in part, by decreasing N-glycosylation of the NET, an
effect induced by endoplasmic reticulum stress pathways
induced by NE-derived oxidative metabolites (Mao et al.
2005).
Amphetamine is a psychostimulant that induces neuro-
transmitter release from neuron terminals. It also is a
substrate for the NET. Amphetamine works through intra-
cellular Ca2+ and protein kinase-dependent mechanisms to
reverse transporter function. Amphetamine-mediated neuro-
transmitter release through the NET is attenuated by protein
kinase inhibitors and BAPTA/AM an intracellular calcium
chelator (Kantor and Gnegy 1998; Kantor et al. 2001). As
mentioned above, exposures of cultured NET-expressing
cells to amphetamine results in a down-regulation of NET
and its function (Zhu et al. 2000). The molecular mecha-
nisms responsible for this effect have not been revealed.
Treatments that reduce or elevate endogenous NE regulate
the expression of the NET. These treatments might produce
their effects through actions of the substrate (NE) or through
other unknown direct or indirect effects on the NET. The
earliest study demonstrated that manipulation of NE levels
by administration of reserpine (depletes NE and other
monoamines) or monoamine oxidase inhibitors (elevate
NE) to rats results in reciprocal effects on radioligand
binding to the NET in the cerebral cortex (Lee et al. 1983).
Reserpine treatment reduces radioligand binding to the NET,
whereas monoamine oxidase inhibitor treatment increases
radioligand binding to the NET. Subsequently, others have
shown that depletion of NE in rats by either reserpine
(Cubells et al. 1995) or a-methyl-p-tyrosine treatment (Xiao
et al. 1995) reduces NET mRNA levels in the locus
coeruleus.
Stress induces the release of NE in the brain and chronic
stress can deplete NE. Hence, it is interesting to consider the
effects of stress on NET expression in the context of potential
substrate-mediated effects. Exposure of rats to acute stress
does not affect radioligand binding to the NET (Zafar et al.
1997) or NET gene expression (Sands et al. 2000). In these
studies, NET binding or gene expression was measured
shortly after the stressor. When the NET is measured several
days after acute exposure of mice to a stressor, NET binding
has been found to be increased (Hwang et al. 1999). In
contrast to acute stress, repeated restraint stress decreases
NET binding sites in the brain (Tejani-Butt et al. 1994; Zafar

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2006 International Society for Neurochemistry, J. Neurochem. (2006) 97, 310–333
et al. 1997). Likewise, repeated exposure of rats to cold
stress reduces NE uptake capacity and NET binding sites in
brown adipose tissue (King et al. 1999). An elevation of
NET gene expression has been reported following exposure
of rats to repeated restraint stress, but this effect is not
observed in the locus coeruleus (Rusnak et al. 2001), the
principal source of brain NE. One human postmortem study
measuring binding to the NET reports reduced NET in the
locus coeruleus in subjects with major depression, a stress-
related disorder, as compared to psychiatrically normal
control subjects (Klimek et al. 1997). Together, these studies
suggest that repeated stress decreases NET protein and
function, and these effects are consistent with the effects of
pharmacological depletion of NE on the NET as discussed
above. The mechanisms by which stress regulate the NET
may be related to reduced activity of the transporter in
response to stress-induced reduction in substrate. Alternat-
ively, stress hormones such as CRF and glucocorticoids may
mediate changes in the expression of the NET through as yet
unidentified signaling cascades. Clearly, a considerably
amount of research effort has to be focused on this issue to
understand how stress regulates the NET and whether this
effect is mediated by change in synaptic concentrations of
NE or through other mechanisms.
Regulation of the NET by other drugs
Certain drugs affect NET function through mechanisms that
are largely unknown. The regulatory effect of these drugs on
NET function, following continuous exposure of the drugs to
the NET, have been studied in some cases. These effects are
discussed here and summarized in Table 2.
Anesthetics
Anesthetics both general and local exert their effect by
altering neurotransmission. Ketamine has anesthetic and
analgesic properties, and also has a wide variety of
psychotropic and sympathomimetic effects. Ketamine acts
on numerous targets including glutamatergic, opioidergic,
and monoaminergic systems. The sympathomimetic effects
of ketamine result from its inhibitory actions on NE reup-
take (Taube et al. 1975; Graf et al. 1995). However, the
actions of ketamine on the NET are complex and vary with
the length of time of exposure to ketamine. Acute exposure
of bovine adrenal medullary cells to ketamine (30 min)
decreases NE uptake, characterized by decreased Vmax and
no apparent change in Km (Hara et al. 1998). In stark
contrast, exposure of bovine adrenal medullary cells to
ketamine for longer than 3 h increases NE uptake (Hara et al.
2002; Table 2). The effects of the longer exposure are
accompanied by a change in the Vmax without changes in
Km. The effects of ketamine are modestly attenuated in the
presence of the RNA polymerase inhibitor, actinomycin D,
and blocked in the presence of the ribosomal protein

2006 The Authors
Table 2 Regulation of the NET by neurotransmitters, hormones, and drugs

Messenger Exposure Agent Cell Surface

System Cell type Time NE Uptake NET Kinetics Expression NET mRNA References

2+
Methacholine SK-N-SH cells 30min Ca , PKC fl « Km fl Vmax fl – Apparsundaram et al.
(1998a)
Insulin SK-N-SH cells 60 min PI3 Kinase › « Km › Vmax « – Apparsundaram et al.
(2001)
PC 12 cells (reserpine treated) 20 min – fl ? fl – Figlewicz et al. (1993a)
Rat locus coeruleus – – – – – fl Figlewicz et al. (1993b)
Angiotensin II Primary culture of 30 min, MAP kinase, › – – › only with Lu et al. (1996);
WKY rat neurons 4h cfos 4 h treatment Yang and Raizada (1998)
Primary culture of 30 min, MAP kinase, › – – › only with Lu et al. (1996);
SH rat neurons 4h cfos 4 h treatment
ATP PC12 cells 5 min ? › « Km › Vmax – – Eshleman et al. (1995)
PC12 cells 3 min – › (0.1 lM) – – – Hardwick et al. (1989)
only in Ca
KRH buffer,
fl (1 lM)
ATPcS PC12 cells 30 min – › « Km › Vmax – – Hardwick et al. (1989)
pre-treatment
ANF Adrenal medullary slices (Wistar rats) 60 min – › – – – Vatta et al. (1993)
BNP, CNP Hypothalamus slices (Wistar rats) 60 min – › – – – Vatta et al. (1996)

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Ketamine Bovine adrenal medullary cells 12 h – › « Km › Vmax › NP › Hara et al. (2002)
Propofol Bovine adrenal medullary cells 6–24 h – › – › NP › Hara et al. (2000)
Clozapine Bovine adrenal medullary cells 2–168 h – fl (2–48 h) fl Vmax (6 h) fl NP (6 h) › (12–96 h) Yoshimura et al. (2001)
› (72–168 h) › Vmax (96 h) › (96 h)
Regulation of the norepinephrine transporter
325
 Table 3 Regulation of the NET by substrates and inhibitors of the NET

Messenger NET protein (NP)*

Cell type or Time period of system or Cell Surface
Agent Species exposure involved NE uptake NET Kinetics Expression (CSE) NET mRNA References

Amphetamine 293hNET cells 3 days – fl « Km fl Vmax fl NP « Zhu et al. (2000)

326 P. Mandela and G.A. Ordway

Citalopram PC12 cells, 3 days – – – « NP – Zhu and Ordway (1997)

293-hNET Zhu et al. (1998)
Cocaine Rat, pregnant 3 days – – – › NP (placenta) « – Shearman and Meyer (1999)
dams NP (fetal brain)
Rat, pregnant Perinatal – fl (myocardial) – « NP (myocardial) – Zhao and Sun (2004)
dams
Rat 10 days – – – « NP (brain) Benmansour et al. (1992)
Rat 4 week – – – ›, fl, « NPdepending – Belej et al. (1996)

2006 The Authors

on brain region
Non-human 100 days – – – › NP (brain) – Macey et al. (2003)
primate Beveridge et al. (2005)
Human, Chronic – – – › NP (brain) – Mash et al. (2005)
postmortem
293hNET cells 3 days – fl (small change) « Km « Vmax « NP « Zhu et al. (2000)
Desipramine Rat 1 day, – – – fl (brain; 21 days) – Bauer and Tejani-Butt (1992)
21 days i.p. Hebert et al. (2001)
Rat 3 week, 6 week – fl – fl (brain) « Benmansour et al. (2004)
PC12 and 3 days – fl – fl NP – Zhu and Ordway (1997)
293hNET cells 1–21 days – fl (3 days) – fl (‡ 2 days) NP « Zhu et al. (1998)
PC12 cells 4 h)3 days – fl (‡ 16 h) – fl (‡ 3 days) NP – Ordway et al. (2005)
293-hNET fl ((‡ 16 h) CSE
Nisoxetine PC12 3 days – – – fl NP – Zhu and Ordway (1997)
Norepinephrine PC12 3 days – – – « – Zhu and Ordway (1997)
PC12 24 h Free radicals fl « Km fl Vmax fl NP fl CSE « Mao et al. (2004) Mao
et al. (2005)
Paroxetine Rat 3 week, 6 week – « – « – Gould et al. (2003)
Benmansour et al. (2004)
Reboxetine Rat 3 week, 6 week – – – fl – Gould et al. (2003)

*NET protein measured by binding of [3H]nisoxetine or with NET antibody using crude membrane homogenates.

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2006 The Authors
Table 4 Regulation of the NET by other drug treatments, stress, or stress-related disease

NET protein (NP)* or

Cell type or Time period of NET Cell Surface
Agent/Illness Action Species exposure NE uptake Kinetics Expression (CSE) NET mRNA References

Iproniazid MAO inhibitor Rat 18 days fl** › NP (brain – Lee et al. 1983;
Pargyline MAO inhibitor Rat 10, 18 days fl** – › NP (brain) – Lee et al. (1983)
Reserpine Monoamine Rat 1–10 days fl – fl (brain) – Lee et al. (1983)
depletion Rat 1 day – – – fl (brain and Cubells et al. (1995)
adrenal)
a-methyl-p-tyrosine Catecholamine Rat 1, 3, 7 days – – – fl 3, 7 days Xiao et al. (1995)
depeletion (brain)
Restraint Stress Rat 30 min – – – « Sands et al. (2000)
Rat 2 h/day for – – fl 7 days (brain) – Zafar et al. (1997)
1 or 7 days
Mice 2h › NP (brain) – Hwang et al. (1999)
Rat 21 days, including – – fl (brain) – Tejani-Butt et al. (1994)
other stressors
Rat 2 h/day for 41 days – – – › (isolated Rusnak et al. (2001)
changes)
Cold Stress Rat 2–7 days fl 7 days – fl NP 3–7 days – King et al. (1999)
(adipose)
Major depression Stress-related Human – – – fl (brain) – Klimek et al. (1997)
Citalopram Serotonin PC12 and, 3 days – – « NP – Zhu and Ordway (1997)

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uptake inhibitor 293-hNET cells Zhu et al. (1998)
Paroxetine Serotonin Rat 3 week, 6 week « – « NP – Gould et al. (2003)
uptake inhibitor Benmansour et al. (2004)

*NET protein measured by binding of [3H]nisoxetine or with NET antibody using crude membrane homogenates.
**These drugs have modest acute inhibitory effects on uptake, and were considered by the authors to be present in the uptake assay (Lee et al. 1983).
Regulation of the norepinephrine transporter
327
328 P. Mandela and G.A. Ordway
synthesis inhibitor cycloheximide. Following a 24 h incuba-
tion with ketamine, NET mRNA expression increases (Hara
et al. 2002). The effect of ketamine is not stereo-selective,
that is both (+) ketamine and (–) ketamine have similar
effects on NE uptake (Hara et al. 2002). Although protein
synthesis clearly plays a role, the mechanism by which
ketamine affects transporter function is largely unknown. It is
intriguing to consider the possibility that ketamine may be
affecting the function of the norepinephrine transporter
through its actions at the NMDA receptor.
Other anesthetics
Other anesthetics such as propofol, thiamylal and diazepam
inhibit NE uptake in a concentration-dependent manner in
bovine adrenal medullary cells. Propofol demonstrates the
highest potency followed by diazepam and thiamylal. In
addition, propofol exhibits a biphasic effect on NE uptake
depending on the times of exposure (Hara et al. 2000;
Table 2). These anesthetics exhibit similar properties to those
of ketamine in altering NET expression and uptake kinetics.
Further these findings carry some pharmacological import-
ance as it has been demonstrated that propofol and thiamylal
can further increase epinephrine-induced arrhythmias, an
effect likely to be mediated via the inhibition of NET
function. Furthermore, some of the antinociceptive actions of
these anesthetics may be mediated by NE secondary to NET
inhibition (Hara et al. 2000).
Clozapine
Clozapine is an atypical antipsychotic that has mixed actions
on NE uptake. Clozapine has relatively low affinity for the
NET, i.e. in the micromolar range (Fernandez et al. 2005).
Exposure of bovine adrenal medullary cells to clozapine has
biphasic actions on the NET, decreasing NET function
initially followed by increasing NET function. Both phases
of clozapine’s action on the NET require continuous
exposure and appear to be regulatory in nature, rather than
as a result of simply acute binding of clozapine to the
transporter. Both phases of action of clozapine are accom-
panied by correlative changes in NET protein, although only
the longer clozapine-induced increase in NET function is
accompanied by both increases in NET protein and NET
mRNA (Yoshimura et al. 2001). The unusual actions of
clozapine on NET function require further study, particularly
with regard to whether the actions of clozapine on the NET
contribute to its unique actions in the treatment of schizo-
phrenia and whether NET effects extend to other atypical
antipsychotic drugs.
Summary
The NET plays a major role in modulating transmission at
noradrenergic synapses. The function of the NET can be
altered by numerous drugs that bind directly to the transpor-

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2006 International Society for Neurochemistry, J. Neurochem. (2006) 97, 310–333
ter and either inhibit its function, essentially as an antagonist,
or alter its function by acting as a competitive substrate.
Besides these functional changes in the NET as a result of
binding to the NET, drugs, hormones, and other transmitters
can regulate the function of the NET through interactions
with other receptors or binding sites, either by activation of
known second messenger systems, or by activating or
inhibiting other signaling pathways that are currently
unknown. In addition, repeated or continuous exposure of
the NET to substrates or inhibitors regulates the physical
expression of the NET on the cell surface, through as yet
unknown cellular mechanisms.
Although drugs that directly manipulate the function of the
NET have been used for over a half century, it is only within
the past 10–15 years that considerable advances have been
made in understanding how the NET functions at the
molecular level and how its function is regulated by repeated
or continuous exposure to drugs or hormones. The key
development in studying this transporter was its cloning by
Pacholczyk et al. in 1991. Since then, cellular transfection
studies of the NET across species have paved way for many
important studies yielding information about multiple cellu-
lar systems that participate in regulating the transporters.
NET function is regulated by a wide variety of drugs,
neurotransmitters, peptides, psychostimulants, temperature,
ionic environment, nucleotides and various pathological
conditions. Across the species, phosphorylation is the most
significant intracellular event that dictates NET expression
and function, although the number of potential sites for
phosphorylation varies from one species to another. Second
messenger systems and subsequent intracellular signaling
cascades, particularly but not limited to PKC, are intracel-
lular triggers that regulate NET surface expression and
function. Besides working through second messenger sys-
tems, it is interesting to consider the potential for allosteric
inhibition of the NET. For example, insulin increases NE
uptake in SK-N-SH cells without altering the membrane
expression of NET (Apparsundaram et al. 2001). Though
allosteric mechanisms have been proposed by various
investigators, it is not known how allosterism operates at
the molecular level, i.e. how agents bring about conforma-
tional or topographic changes to increase or decrease NET
function. Another secondary mechanism of regulating NET
function is through altering Na+/K+-ATPase activity or by
otherwise disturbing the ionic gradient required for NET
function. In addition, altering the oligomerization of the NET
represents another potential regulatory pathway.
Clearly, most studies of the regulation of NET have
utilized in vitro systems to identify cellular mechanisms. In
order for the field to move forward, more research is needed
to try to understand how regulation of the transporter affects
noradrenergic signaling in vivo, and to delineate the physio-
logical consequences of these effects. Given the cell-line
dependence of many cellular mechanisms involved in

regulation, it is important to identify which mechanisms are
relevant to the in vivo situation, and to also consider the
possibility of species-specificity. Recent studies demonstra-
ting that the NET is predominately located in the noradren-
ergic terminal cytoplasm, rather in the plasma membrane, in
certain brain regions (Miner et al. 2003) emphasizes the
dearth of understanding of the physiological significance of
NET regulation in vivo. Elucidation of cellular pathways for
NET regulation in vivo will move the field one step closer to
developing novel drugs capable of modulating NET activity.
These types of drugs could have unique activities for the
treatment of CNS and PNS disorders, such as depression,
attention deficit, cardiovascular, or other disorders where
modulation of noradrenergic transmission can yield thera-
peutic effects.
Acknowledgements
This work was supported in part by the National Institute of Mental
Health (MH58211 and MH02031).
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