Endocrinology Vol. 141, No. 1 366-374
Copyright © 2000 by The Endocrine Society
Dopamine Transporters Participate in the Physiological Regulation of Prolactin1
Jamie E. DeMaria,
György M. Nagy,
Anna A. Lerant2,
Márton I. E. Fekete,
Cathy W. Levenson and
Marc E. Freeman
Department of Biological Science (J.E.D., A.A.L., M.E.F.) and
Department of Nutrition, Food, and Exercise Sciences (C.W.L.), Program
in Neuroscience, Florida State University, Tallahassee, Florida
32306-4340; Neuroendocrine Research Laboratory (G.M.N.), Department of
Human Morphology and Embryology, Semmelweis Medical University,
Budapest, Hungary; and Institute of Experimental Medicine
(M.I.E.F.), Hungarian Academy of Sciences, Budapest, Hungary
Address all correspondence and requests for reprints to: Marc E. Freeman, Ph.D., 208 Biomedical Research Facility, Florida State University, Tallahassee, Florida 32306-4340. E-mail:
freeman{at}neuro.fsu.edu
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Abstract
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Three populations of hypothalamic neuroendocrine dopaminergic (NEDA)
neurons, arising from the arcuate and periventricular nuclei of the
hypothalamus release dopamine (DA) that acts at the pituitary gland to
regulate the secretion of PRL. It is generally accepted that NEDA
neurons lack functional DA transporters (DATs), which are responsible
for uptake of DA from the synaptic cleft into the presynaptic axon
terminal. This study localized DATs to the hypothalamo-pituitary axis
and evaluated the effect of DAT blockade on the hypothalamo-pituitary
regulation of PRL. After 7 days of treatment with cocaine (a
nonspecific amine transporter blocker) or mazindol (a specific DAT
blocker), the relative abundance of PRL messenger RNA (mRNA) in the
anterior lobe (AL) of OVX rats was significantly decreased, whereas the
relative abundance of tyrosine hydroxylase mRNA in the hypothalamus was
significantly increased. The effect of cocaine or mazindol
administration on DA turnover and serum PRL concentration was examined
in estradiol (E2)- treated OVX rats. E2
administration (iv) resulted in a significant increase in serum PRL
within 4 h; however, cocaine or mazindol administration abolished
the E2-induced increase of PRL. Cocaine or mazindol
significantly increased the concentration of DA at the site of the axon
terminals within the median eminence (ME), intermediate lobe (IL) and
neural lobe (NL), indicating blockade of uptake. Because formation of
DOPAC requires uptake of DA, concentrations of DOPAC in the ME, IL and
NL decreased following treatment with either cocaine or mazindol. These
data, together with the presence of immunopositive DAT in the ME,
pituitary stalk, IL, and NL, suggest that a functional DAT system is
present within all three populations of NEDA neurons. Moreover,
similarity between the effects of cocaine and mazindol treatment
indicate that blockade of the DAT, but not other amine transporters, is
responsible for suppression of PRL gene expression and secretion.
Blockade of DATs prevent uptake of DA into NEDA neurons and
consequently increases the amount of DA that diffuses into the portal
vasculature and reaches the AL. These data provide evidence that DATs
play a physiological role in the regulation of DA release from and TH
expression in NEDA neurons and consequently PRL secretion and PRL gene
expression and further support our previous observation that the
regulation of PRL secretion involves all three populations of NEDA
neurons.
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Introduction
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THREE POPULATIONS of neurons
contribute dopamine (DA), the physiological inhibitor of the secretion
of PRL (1), to the pituitary gland to regulate the secretion of PRL
(2). Tuberoinfundibular DAergic (TIDA) neurons arising from the arcuate
nucleus (ARN) terminate in the median eminence (ME) (3) and release DA
that is subsequently delivered through long portal vessels to
lactotrophs in the anterior lobe (AL) of the pituitary gland.
Tuberohypophysial DAergic (THDA) neurons originate in the rostral ARN
and terminate in both the intermediate (IL) and neural (NL) lobes of
the pituitary gland (4). Periventricular-hypophysial DAergic (PHDA)
neurons arise from the periventricular nucleus of the hypothalamus and
terminate exclusively in the IL (5, 6). DA released to the IL and NL is
delivered to the AL through short portal vessels (SPV). Based on their
termination on portal vessels, these are known collectively as
neuroendocrine dopaminergic (NEDA) neurons.
The rate-limiting step of DA synthesis is the formation of
L-3,4-dihydroxyphenylalanine (L-DOPA), from tyrosine catalyzed by
tyrosine hydroxylase (TH). L-DOPA is further metabolized by L-amino
acid decarboxylase into DA. DA is packaged into vesicles and
released through a calcium-mediated vesicle docking process (7). Once
released from the neuron, DA either acts at its target (i.e.
DA receptors) or is taken up into the presynaptic neuron by dopamine
transporters (DATs) and oxidized by mitochondrial monoamine oxidase to
produce 3,4-dihydroxyphenylacetic acid (DOPAC) (8). DA not packaged in
secretory granules is also oxidized to produce DOPAC (9).
The DAT is a 12-transmembrane domain transporter belonging to a larger
family of sodium/chloride-dependent transporters (10). Expression of
DAT is regulated by ovarian steroids both at transcriptional and
posttranslational levels (11, 12). Also, phosphorylation of DAT
proteins has been shown to regulate DA transport activity (13, 14). DAT
messenger RNA (mRNA) has been localized in several regions within the
rat brain, including the arcuate (15, 16, 17) and periventricular nuclei of
the hypothalamus (17, 18). Distribution of DAT mRNA and TH mRNA had
been comparatively mapped throughout the brain (19). Although numerous
immunocytochemical studies were performed on localization of DAT in the
central nervous system, most of them were single label studies (17, 18). Studies detecting both DAT and TH proteins focused mostly on the
mesolimbic and nigrostriatal systems (20, 21, 22). Previous reports
suggested that TIDA and THDA neurons lack functional DAT systems
(23, 24, 25). However, immunodetectable DAT is present in the ME and in its
continuation, the pituitary stalk (26). Moreover, administration of DAT
blockers was shown to inhibit the secretion of PRL and disrupt the
estrous cycle of the rat (27). Relatively little is known about the
distribution and function of DAT in the neuroendocrine DAergic neurons
and in their target areas.
The aim of the present study was to visualize DAT in the cell bodies
and terminal areas of the neuroendocrine DAergic neurons by using
double label immunocytochemistry as well as to elucidate the
contribution of DATs to the hypothalamo-pituitary regulation of
expression and secretion of PRL.
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Materials and Methods
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Identification of dopamine transporter in the
hypothalamo-hypophysial dopaminergic system
Female Sprague Dawley rats were housed two per cage in a
climate-controlled environment under 12 h of illumination (lights
on from 0600 h) with water and rat chow provided ad
libitum. Animals were bilaterally ovariectomized (OVX) under
Halothane anesthesia. Ten days after OVX, rats were killed at
1100–1200 h by an overdose of sodium-pentobarbital and transcardially
perfused with 50 ml ice-cold 0.1 M PBS followed
by 100 ml ice-cold fixative (4% paraformaldehyde in 0.1
M PBS, pH 7.4). After cryoprotection (20%
sucrose-PBS for 24 h), brains and pituitary glands were cut into
20-µm coronal sections with a cryostat (Microm, Carl Zeiss, Walldorf, Germany). Brain sections between
3600–4400 µm post bregma and serial sections of the pituitary gland
were thaw mounted onto gelatin-subbed glass microscope slides.
After rinsing three times in PBS, sections were background-blocked with
10% normal horse serum (in PBS containing 0.4% Triton X-100) for 20
min, then successively incubated at 4 C with antibodies to DAT, TH, and
PRL as described in Table 1
. All
antibodies were diluted in Triton-PBS, containing 2% normal horse
serum (Intergen Co., Purchase, NY). Three 5-min washes (in
PBS) were applied between steps. After immunocytochemistry (ICC), the
sections were rinsed in distilled water and cover slips were fixed with
Gelmount (Fisher Scientific, Atlanta, GA).
Confocal images were obtained with a Carl Zeiss LSM410
laser-scanning microscope equipped with two external Ar/Kr lasers and
an internal He/Ne laser. The excitation and emission spectra of the
fluorescent dyes as well as the characteristics of the lasers and
filters used are described in Table 2.
Sections were scanned under identical acquisition parameters. The
images were acquired with a Plan-Neofluar 63/1.4 objective lens using
the LSM410 invert basic program version 3.8. The same program was used
to assess colocalization of DAT and TH immunoreactivity.
Briefly, a scattergram was derived from the overlaid
red-green-blue (RGB) images (green, DAT;
red, TH). Each pixel was compared with an absolute intensity
scale and plotted according to its intensity in red
(x-axis) and in green (y-axis). The
upper left 25% of the scattergram, containing pixels with high
intensity in both colors, was marked by a constant square and
considered as colocalization.
Effects of short-term DAT blockade on DA turnover (DOPAC/DA ratio)
and PRL secretion
Female Sprague Dawley rats were housed individually in a climate
controlled environment under 12 h of illumination (lights on from 0600
h) with water and rat chow provided ad libitum. Animals were
bilaterally OVX under ether anesthesia. Eight days after OVX, a cannula
was inserted into the jugular vein. The following day, a sample of
venous blood was collected at 0845 h, followed by an iv injection of
water soluble cyclodextrin-encapsulated estradiol
(E2, 20 µg; Sigma, St. Louis, MO).
Sixty minutes later, another blood sample was taken, and an ip
injection of either saline (SAL, control, 200 µl), cocaine (COC, a
nonspecific amine transporter blocker, 10 mg/kg; RBI), or mazindol
(MAZ, a specific DAT blocker, 5 mg/kg; RBI) was administered. Animals
were rapidly decapitated every hour for the next 4 h. Trunk blood
was collected, allowed to clot, centrifuged and serum removed and
stored at -40 C. Brains were removed and the ME, AL, IL, and NL
excised, isolated and stored at -40 C in homogenization buffer as
previously described (2, 28, 29, 30). The concentration of PRL in serum was
measured by RIA (31), which has a sensitivity of 1 ng/ml with inter and
intra assay coefficient of variations 10% and 5%, respectively. The
concentration of DA and DOPAC in the ME, IL, NL, and the concentration
of DA in the AL were measured using HPLC coupled to electrochemical
detection (2, 28, 29, 30). The sensitivity of the assay was 30 pg of DA and
33 pg of DOPAC.
Effects of long-term DAT blockade on PRL and TH gene expression
Female Sprague Dawley rats were housed five per box in a
climate controlled environment under 12 h of illumination (lights
on from 0600 h) with water and rat chow provided ad
libitum. Rats were bilaterally OVX under ether anesthesia. The day
following OVX, rats were divided into three groups and received daily
injections (ip) of either SAL, cocaine (2.5 mg/kg), or mazindol (2.5
mg/kg) for seven days. On the seventh day of injections, rats were
decapitated and the AL and hypothalamus were excised. Total cellular
RNA was immediately extracted using the guanidine-phenol-chloroform
method of Chomczynski and Sacchi (32) and 20 µg separated on a 1%
agarose gel (1 x MOPS, 0.66 M
formaldehyde). Integrity of the RNA was confirmed by UV visualization
of ethidium bromide stained RNA. RNA was transferred overnight by
capillary action to Gene Screen (NEN Life Science Products, Boston, MA) and cross-linked under UV light for 3 min.
Blots were then hybridized overnight (65 C) with
32P-labeled complementary DNA (cDNA) probes
specific for rat PRL mRNA (lactotrophs), or TH mRNA (hypothalamus), and
ß-Actin (control for loading). Probes were labeled using a RadPrime
DNA labeling kit (Promega Corp., Madison, WI) and
32P 
dATP (Amersham Pharmacia Biotech, Piscataway, NJ). Blots were exposed to
Kodak X-OMAT AR film at -80 C. Relative abundance of
bound cDNA probe was determined by computer evaluated densitometry
(Quantity One quantification program, Protein and DNA Imaging, PDI,
Boston, MA) and expressed as a function of ß-actin mRNA
abundance.
Statistical analysis
Mean concentrations of DA, DOPAC, PRL, PRL mRNA, TH mRNA, or
DOPAC/DA ratio were analyzed for each time point using a two-way ANOVA.
Tukey’s honestly significant difference test was used as a posthoc
test for comparisons between saline and cocaine- or mazindol-treated
animals and between cocaine and mazindol-treated animals. Values are
considered significant at P < 0.01.
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Results
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Distribution of immunoreactive DAT in the brain
TH immunoreactive fibers in the caudate putamen contained
DAT labeling that appeared as fine, punctate staining. DAT, colocalized
with TH in nigrostriatal DAergic terminals of the caudate putamen was
used as a positive control (Fig. 1).
Compared with the caudate putamen and the median eminence, only scarce
DAT staining and no colocalization was observed in the
tuberoinfundibular DAergic neurons in the dorsomedial part of the
middle arcuate nucleus adjacent to the third ventricle (Fig. 1). DAT
was abundant in the external layer of the ME (Fig. 1), where TIDA
neurons terminate, and the pituitary stalk (Fig. 1), through which the
THDA and PHDA axons course to reach the pituitary gland. Dense,
punctate DAT staining colocalized with TH immunoreactivity in TIDA axon
terminals (Fig. 1), as well as with the NEDA neuronal axons and
terminals in the pituitary stalk (Fig. 1).
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Figure 1. Distribution of immunoreactive DAT in the caudate
putamen (CPu), dorsomedial part of the arcuate nucleus (DMARN), median
eminence (ME), and pituitary stalk (PS). The first two
columns show confocal images obtained from coronal sections of
the rat brain immuno-labeled for tyrosine hydroxylase (TH,
red) and dopamine transporter (DAT,
green). White quadrangles circumscribe
the area shown of higher magnification in the third and fourth
columns. The third column shows the
red and green images overlayed
(TH/DAT), whereas the fourth column
demonstrates the individual pixels exhibiting the greatest intensity in
both red and green (Colocalization).
Bar, 25 µm.
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Distribution of immunoreactive DAT in the pituitary gland
DAT was present in both fibers and axon terminals of the rostral
NL of the pituitary gland (Fig. 2).
However, both TH and DAT labeling were sparse in the caudal aspect of
the NL (data not shown). Interestingly, some intense DAT labeling
appeared in large axon terminals (Herring bodies), which were not
intensely stained with the TH antibody. This may have been a result of
autofluorescence of large oxytocin or arginine-vasopressin-containing
Herring bodies, or dominance of phosphorylated isoform (Ser40) of TH,
which was not detected by the TH antibody used. A fine network of
dopaminergic fibers was observed in the IL (Fig. 2), and colocalization
of DAT and TH was present along membranes of cells in the IL. Neither
TH nor DAT immunoreactivity was detected in the anterior lobe (Fig. 2),
as the intensity of fluorescence in the anterior lobe cells was lower
than the autofluorescence of red blood cells in the sinusoid
capillaries. However, numerous PRL immunoreactive cells attested to the
success of the immunocytochemical labeling in the AL. In addition, TH
immunoreactive fibers were detected at the interface of the anterior
and intermediate lobes (Fig 2).
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Figure 2. Distribution of immunoreactive DAT in the neural
lobe (NL), intermediate lobe (IL), and anterior lobe (AL) of the
pituitary gland. IL and NL: The first two columns show
confocal images obtained from coronal sections of the rat pituitary
gland immuno-labeled for tyrosine hydroxylase (TH, red)
and dopamine transporter (DAT, green). White
quadrangles circumscribes the area shown of higher
magnification in the third and fourth columns. The
third column shows the red and
green images overlayed (TH/DAT), whereas
the fourth column demonstrates the individual pixels
exhibiting the greatest intensity in both red and
green (Colocalization). AL: The first two
columns show confocal images obtained from coronal sections of
the anterior lobe of the pituitary gland immuno-labeled for tyrosine
hydroxylase (TH, red) and dopamine transporter (DAT,
green). Note the lack of specific TH and DAT label, only
nonspecific background and autofluorescence of red blood cells in the
sinusoid capillaries. The third image shows PRL immunoreactivity of
anterior lobe lactotrophs (blue) in the same section,
whereas the fourth image is the result of the overlay of
all three labels (red, green, and
blue). Bar, 25 µm.
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DAT blockade inhibits E2-induced secretion of PRL
Figure 3 shows the concentration of
PRL in serum following DAT blockade. Before iv injection of
water-soluble E2 (0-time), there was no
significant difference between the concentration of PRL in serum.
Moreover, 60 min after injection of E2 and before
DAT blockade, there was no significant difference in the concentration
of PRL in serum. The concentration of PRL in serum of saline-treated
animals significantly increased 2 h after E2
injection, peaked 3 h after E2 injection,
and returned to low levels by 5 h post E2
injection. However, treatment with either cocaine or mazindol
completely blocked the E2-induced increase
of PRL in serum (Fig. 3).
DAT blockade inhibits uptake of DA in the ME
Figure 4 shows the DOPAC/DA ratio
(A), the concentration of DOPAC (B), and DA (C) in the ME. The turnover
of DA in the ME of E2-treated rats injected with
saline increased concomitant with increased PRL secretion (Fig. 4A).
Moreover, the turnover of DA remained elevated 4–5 h after
E2 injection (Fig. 4A). Cocaine or mazindol
treatment significantly decreased the turnover of DA in the ME, except
at 5 h after E2 injection, when the turnover
of DA in cocaine- and mazindol-treated rats was increased. Cocaine- and
mazindol-treated rats had significantly higher concentrations of DA in
the ME than saline-treated rats 2–4 h after E2
injection (Fig. 4C), but significantly lower DOPAC concentrations (Fig. 4B) 3–4 h after E2 injection.
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Figure 4. Inhibition of DAT alters dopamine turnover in the
median eminence. Concentration of DA (C), DOPAC (B), and the DOPAC/DA
ratio (A) in the ME from estradiol-treated rats following saline
(-•-), cocaine (— —), or mazindol (— —) treatment. Ten days
following ovariectomy rats were injected iv with water-soluble
estradiol at time zero. Sixty minutes following estradiol injection,
either saline, cocaine (10 mg/kg, COC), or mazindol (5 mg/kg, MAZ) was
administered iv. Animals were killed every hour for 4 h following
the drug injection. Each point represents the mean
± SE concentration of PRL from 4 rats/time point/treatment
group. *, P < 0.01 for control vs.
cocaine- or mazindol-treated rats at each time point. a,
P < 0.01 for cocaine-treated vs.
mazindol-treated rats.
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DAT blockade modifies THDA and PHDA neuronal activity and DA
uptake
DA turnover in the IL was significantly depressed following DAT
blockade (Fig. 5A). The concentration of
DOPAC in the IL was significantly decreased after administration of
cocaine or mazindol (Fig. 5B); however, the absolute concentration of
DA in the IL significantly increased 1–3 h after administration of
cocaine or mazindol (Fig. 5C).
DA turnover in the NL was also significantly depressed after
administration of DAT blockers (Fig. 6A).
Similar to the IL, the concentration of DOPAC (Fig. 6B) in the NL
decreased and the concentration of DA (Fig. 6C) in the NL increased
after treatment with either cocaine or mazindol.
DAT blockade increases the concentration of DA in the AL
The concentration of DA in the AL (Fig. 7) was significantly increased following
treatment with cocaine or mazindol.
Long-term DAT blockade alters PRL and TH gene expression
After 7 days of treatment with either cocaine or mazindol, the
relative abundance of PRL mRNA in the AL (Fig. 8A) was decreased, whereas the relative
abundance of TH mRNA in the hypothalamus (Fig. 8B) was increased. There
were no significant differences in the relative abundance of PRL mRNA
(Fig. 8A) or TH mRNA (Fig. 8B) between cocaine and mazindol treated
animals.
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Figure 8. Inhibition of DAT alters gene expression in the
NEDA neurons and in lactotroph. Relative abundance of PRL mRNA in the
AL (A) and TH mRNA in the hypothalamus (B) following long-term
treatment DAT blockers. Rats were injected ip with either saline,
cocaine (10 mg/kg), or mazindol (5 mg/kg) for 6 days following
ovariectomy. Eight days post OVX, rats were decapitated and total RNA
was isolated from the anterior lobe of the pituitary gland and the
hypothalamus. Each bar represents the mean ±
SE concentration of PRL from 5 rats/time point/treatment
group. Inset on A shows a representative Northern of
anterior lobe PRL mRNA and ß-Actin mRNA in saline- and
cocaine-treated conditions. *, P < 0.01 for
control vs. cocaine- or mazindol-treated rats at each
time point.
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Discussion
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DATs belong to a larger class of
Na+/Cl- transporters that
also includes transporters for serotonin (5HT) and norepinephrine (NE)
(10). Different classes of drugs nonselectively inhibit all three
transporters (e.g. cocaine) or specifically inhibit DAT
(e.g. mazindol). Given the clinical significance of cocaine
addiction, the effects of cocaine on central DAergic systems are widely
studied. However, because cocaine nonselectively blocks DAT as well as
NE- and 5HT- transporters, both cocaine and mazindol are used in these
experiments to verify that effects elicited by cocaine are indeed
specific to the DAT. DATs play a role in the regulation of DAergic
neurons in other areas of the brain (10); however, the regulatory role
of DATs on NEDA neurons has not been fully appreciated (23, 24, 25).
Three populations of hypothalamic NEDA neurons contribute to the
regulation of the secretion of PRL (3, 4, 5, 6). While DA released from TIDA
neurons has been considered the primary source of DA inhibiting the
secretion of PRL (1), it is evident that DA released to the IL and NL
also plays a critical role in the regulation of the secretion of PRL
(2). It has been demonstrated that acute treatment with cocaine
inhibits the secretion of PRL (27, 33), alters the activity of TIDA
neurons (33), disrupts the estrous cycle of the rat (27), and increases
the amount of DA released in vitro (34). Previously, using
the rat as a model, other studies have suggested that hypothalamic NEDA
neurons involved in the hypothalamo-pituitary regulation of PRL lack
functional high-affinity DAT (23, 24, 25). Through the experiments
presented here we demonstrate: 1) DAT transporters are present in the
terminal areas of NEDA neurons; 2) Acute DAT blockade inhibits the
secretion of PRL; 3) Acute DAT blockade alters all three populations of
NEDA neurons; and 4) Long-term DAT blockade decreases PRL gene
expression, but increases TH gene expression.
Figures 1
and 2 show positive staining for DAT and colocalization of
DAT and TH in the terminal area of TIDA neurons (external zone of the
ME, Fig. 1), and absence of DAT label in the cell bodies of TIDA
neurons of the dorsomedial part of the arcuate nucleus. Although DAT
mRNA has been detected in great abundance in the dorsomedial part of
the arcuate nucleus (19, 35), DAT immunoreactivity was not found in the
hypothalamic DAergic neurons (A11–15 cell groups) in previous studies
(17, 36). One can hypothesize that while the perikaryon is the site of
translation of DAT mRNA, the DAT protein is being quickly transported
to its destination, the plasma membrane of axon terminals. Thus,
immunodetectable amounts of the mature DAT protein may not be present
in the perikaryon that is expressing DAT.
We also found DAT staining in the pituitary stalk through which fibers
of THDA and PHDA neurons course (Fig. 1) and in the IL and the NL where
these fibers terminate (Fig. 2). Previously, DATs were localized in the
external zone of the ME (26), but not in the IL nor the NL.
Considerable amounts of single labeling (DAT, no TH) can be observed in
the posterior and intermediate lobes of the pituitary gland. Although
this finding may seem controversial, both in situ
hybridization (19) and immunocytochemical studies (36) indicate that
localization of TH and DAT mRNA do not overlap completely in certain
areas. One of the possible explanations may be that the dominant form
of TH present is phosphorylated at ser40, a form which is not
recognized by the antibody used in this study. The lack of DAT in the
AL is not surprising as there are no DAergic nerve terminals in the AL.
However, the presence of PRL in the AL (Fig. 2) confirms that the ICC
is valid. Acute administration of either cocaine or mazindol prevented
the E2-induced increase of PRL in serum (Fig. 3),
indicating that the DAT plays a functional role in the secretion of
PRL. These results are consistent with previous studies demonstrating
that cocaine inhibited the secretion of PRL in rats (27), rhesus
monkeys (37), and humans (38). Moreover, the effects of cocaine and
mazindol were equivalent, suggesting that the mechanism of cocaine’s
action are most likely mediated by DAT and not NE- or 5HT-
transporters.
The dopamine uptake mechanism in the ME, IL and NL is less robust than
in other brain regions (23, 24, 25). However, the fact that administration
of 6-hydroxydopamine (a neurotoxin requiring uptake through
transporters) diminishes the concentration of DA and the presence of
DAergic nerve terminals in the ME, IL, and NL suggests the presence of
DAT in these terminal areas (39, 40, 41, 42, 43, 44).
The DOPAC/DA ratio as been shown to be an effective method for
monitoring the activity of NEDA neurons (9, 45). However, the absolute
concentrations of DA and DOPAC must be presented to reflect the basis
for the ratio. Blockade of DAT inhibits the DA uptake mechanism on NEDA
neurons thus increasing the concentration of DA in the perivascular
space, the consequence of which is enhanced diffusion into the portal
vessels to diminish secretion of PRL from the pituitary gland. Because
uptake of DA is essential for formation of DOPAC (9), the blockade of a
functional uptake mechanism decreases the concentration of DOPAC in the
terminal area. Indeed, it has been demonstrated that DAT blockade
decreases the concentration of DOPAC in DAergic terminals of other
areas of the rat brain (46).
An acute increase in the concentration of DA, while an effective
inhibitor of the secretion of PRL from the AL (1), has no effect on the
relative abundance of PRL mRNA in the AL (47). Conversely, chronic
treatment with DA or DA agonists significantly decreases the relative
abundance of PRL mRNA in the AL (47). DA, acting through G
protein-coupled D2 inhibitory receptors (48, 49),
down regulates adenylate cyclase, inhibiting the formation of cAMP and
blocking the transcription of the PRL gene (50). Blockade of DAT
prevents uptake of DA into the neurons, significantly increasing the
amount of DA available for transport to the AL (Fig. 7). Thus,
long-term treatment with either cocaine or mazindol equally decreases
the relative abundance of PRL mRNA in the AL (Fig. 8A). A decrease in
PRL transcription caused by long-term cocaine use, can lead to the
depression of several physiological processes mediated by PRL, such as
immune function (51), osmoregulation (52, 53), stress response (54),
reproduction (55), and milk production in nursing mothers (56). These
data provide new insights into the potential problems induced by
long-term cocaine use.
Previously, it has been shown that cocaine administration up-regulated
TH activity in the hypothalamus (57, 58). Long-term blockade of DAT, by
either cocaine or mazindol, equally increased the relative abundance of
TH mRNA in the hypothalamus (Fig. 8B). Long-term DAT blockade induces a
decrease in positive immunostaining for TH (59) and an increase in the
activity of TH (58, 60). Though we lack experimental evidence to
explain this result, long-term cocaine exposure may lead to removal of
end-point inhibition of TH activity in neurons, which results in a
compensatory increase in the transcription of TH.
The DAT system in the ME and pituitary gland is less efficient than DAT
in areas of the brain where DA acts as a neurotransmitter rather than a
neurohormone (23, 24, 25). However, this does not diminish the
physiological importance of DAT because DAT knockout mice display
anterior pituitary hypoplasia, decreased PRL mRNA, and decreased PRL
content of the pituitary gland (61). Thus, our results, coupled with
data obtained from DAT knockout mice (61), indicate that the DAT system
plays a prominent role in the development and regulation of PRL in the
pituitary gland.
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Acknowledgments
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The authors wish to thank Dr. Richard Maurer for the PRL cDNA
plasmid, Dr. Karen O’Malley for the tyrosine hydroxylase cDNA plasmid,
Dr. Albert Parlow and the National Pituitary Hormone Program for RIA
supplies, Ms. Mária Mészáros for her technical
assistance, and Leigh Ann Clark for critical review of this
manuscript.
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Footnotes
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1 Supported by NIH Grants DK-43200 (to M.E.F.) and DK-50472 (to C.W.L.)
and OTKA 20916 (to G.M.N.). 
2 Present Address: Department of Anatomy, University of Mississippi
Medical Center, Jackson, Mississippi 39216.
Received August 11, 1999.
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Top
Abstract
Introduction
