0013-7227/08/$15.

00/0 Endocrinology 149(4):1470 –1479

Printed in U.S.A. Copyright © 2008 by The Endocrine Society
doi: 10.1210/en.2007-0980

Dopamine Inhibits Basal Prolactin Release in Pituitary

Lactotrophs through Pertussis Toxin-Sensitive and -
Insensitive Signaling Pathways
Arturo E. Gonzalez-Iglesias,* Takayo Murano,* Shuo Li, Melanija Tomić, and Stanko S. Stojilkovic
Section on Cellular Signaling, Program in Developmental Neuroscience, National Institute of Child Health and Human
Development (NICHD), National Institutes of Health, Bethesda, Maryland 20892

Dopamine D2 receptors signal through the pertussis toxin effect on voltage-gated Ca2ⴙ channel gating is sufficient to
(PTX)-sensitive Gi/o and PTX-insensitive Gz proteins, as well as inhibit spontaneous Ca2ⴙ influx. However, agonist-induced
through a G protein-independent, ␤-arrestin/glycogen syn- inhibition of PRL release was only partially relieved in PTX-
thase kinase-3-dependent pathway. Activation of these recep- treated cells, indicating that dopamine receptors also inhibit
tors in pituitary lactotrophs leads to inhibition of prolactin exocytosis downstream of voltage-gated Ca2ⴙ influx. The PTX-
(PRL) release. It has been suggested that this inhibition oc- insensitive step in agonist-induced inhibition of PRL release
curs through the Gi/o-␣ protein-mediated inhibition of cAMP was not affected by the addition of wortmannin, an inhibitor
production and/or Gi/o-␤␥ dimer-mediated activation of in- of phosphatidylinositol 3-kinase, and lithium, an inhibitor of
ward rectifier Kⴙ channels and inhibition of voltage-gated glycogen synthase kinase-3, but was attenuated in the pres-
Ca2ⴙ channels. Here we show that the dopamine agonist-in- ence of phorbol 12-myristate 13-acetate, which inhibits Gz sig-
duced inhibition of spontaneous Ca2ⴙ influx and release of naling pathway in a protein kinase C-dependent manner.
prestored PRL was preserved when cAMP levels were ele- Thus, dopamine inhibits basal PRL release by blocking volt-
vated by forskolin treatment. We further observed that dopa- age-gated Ca2ⴙ influx through the PTX-sensitive signaling
mine agonists inhibited both spontaneous and depolariza- pathway and by desensitizing Ca2ⴙ secretion coupling
tion-induced Ca2ⴙ influx in untreated but not in PTX-treated through the PTX-insensitive and protein kinase C-sensitive
cells. This inhibition was also observed in cells with blocked signaling pathway. (Endocrinology 149: 1470 –1479, 2008)
inward rectifier Kⴙ channels, suggesting that the dopamine

D OPAMINE SECRETED FROM hypophyseal hypotha-

lamic neurons is a principal inhibitory regulator of
prolactin (PRL) release by pituitary lactotrophs (1, 2). In low
pamine-induced inhibition of PRL release is also affected by
PTX treatment (3, 10, 11), suggesting that this signaling path-
way is involved in control of secretion. Two intracellular
concentrations, dopamine also stimulates PRL release (3). By messengers affected by activation of PTX-sensitive pathways
using the radioligand binding assay, it was shown in the late in pituitary cells, Ca2⫹ and cAMP, play major roles in con-
1970s that the dopamine D2 subtype of receptors mediates trolling the fusion of secretory vesicles with the plasma mem-
the tonic inhibitory control of hypothalamic dopamine on brane to release hormones in endocrine cells (12). Electro-
PRL release in these cells (4). Later investigations showed physiological experiments revealed that spontaneous action
that two subtypes of D2 receptors, termed D2S and D2L, are potential-dependent fluctuations in intracellular calcium
generated by alternative splicing in lactotrophs and other cell concentration ([Ca2⫹]i) account for high basal PRL release
types (5, 6). Lactotrophs may express a different ratio of these (13). Removal of extracellular Ca2⫹ abolished spontaneous
two subtype receptors, depending on the level of gonadal firing of action potentials and reduced basal PRL release to
steroids (7). Consistent with these findings, the knockout D2 the levels observed during the application of dopamine ago-
mice showed chronic hyperprolactinemia, pituitary hyper-
nists (13, 14). Consistent with these observations, activation
plasia, and a moderate decrease in MSH content (8). In ad-
of dopamine receptors was found to inhibit voltage-gated
dition to lactotrophs, dopamine D2 receptors have been iden-
Ca2⫹ influx (VGCI). At the present time, it is not clear
tified in the intermediate lobe of the pituitary gland (4).
The pituitary dopamine receptors are functionally associ- whether inhibition of voltage-gated T-type and L-type
ated with pertussis toxin (PTX)-sensitive G proteins (9). Do- Ca2⫹ (Cav) channels (15–17) and/or activation of K⫹ cur-
rents (18 –21) accounts for such inhibition. In pituitary
cells, dopamine also inhibits adenylyl cyclase (AC) activity
First Published Online December 20, 2007
* A.E.G.-I. and T.M. contributed equally to this work.
in a PTX-sensitive manner (10, 22), which could contribute
Abbreviations: AC, Adenylyl cyclase; [Ca2⫹]i, intracellular calcium to inhibition of PRL release (10, 11). Such an action could
concentration; Cav, voltage-gated Ca2⫹; GSK, glycogen synthase kinase; be coupled to the effects of cAMP and its kinase on the
Kir, inward rectifier K⫹; PI3, phosphoinositide 3; PMA, phorbol 12- exocytotic pathway (23) and/or inhibition of Ca2⫹ signal-
myristate 13-acetate; PRL, prolactin; PTX, pertussis toxin; VGCI, voltage-
gated Ca2⫹ influx; Vm, membrane potential.
ing pathway (24). However, the relevance of cAMP in
dopamine actions on PRL release was questioned by the
Endocrinology is published monthly by The Endocrine Society (http://
www.endo-society.org), the foremost professional society serving the finding that dopamine inhibits PRL secretion in cells with
endocrine community. activated ACs by forskolin (25).

1470

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 October 2015. at 00:42 For personal use only. No other uses without permission. . All rights reserved.
Gonzalez-Iglesias et al. • PTX-Insensitive Inhibition of PRL Release Endocrinology, April 2008, 149(4):1470 –1479 1471

Two additional transduction mechanisms have also M199 medium containing Hanks’ balanced solution, 0.1% BSA, 25 mm
been reported for D2 dopamine receptors in target tissues, HEPES, and antibiotics before being treated at 37 C with wortmannin
(Sigma Chemical Co., St. Louis, MO) for 10 min only or first treated with
but their significance in dopamine-induced control of PRL 1 ␮m wortmannin for 10 min and then treated with 3 mm lithium
release has not been addressed. First, dopamine D2S and chloride for an additional 10 min.
D2L receptors couple to the same extent to the PTX-sen-
sitive Gi/o protein and to the PTX-insensitive Gz proteins PRL and cAMP measurements
in vitro (26) and in vivo (27). Other subtypes of dopamine
Pituitary cells (1 million per well) were plated in 24-well plates in
receptors also couple to Gz proteins (26, 28). It has been serum-containing M199 and incubated overnight at 37 C under 5%
shown that the Gz signaling pathway plays a major role in CO2-air and saturated humidity. Before experiments, cells were washed
endothelin-A receptor-dependent inhibition of basal PRL with serum-free medium and stimulated at 37 C under 5% CO2-air and
release (29), raising the possibility that dopamine D2 re- saturated humidity for 120 min if not otherwise stated. Hormone se-
cretion was also monitored using cell column perifusion experiments.
ceptor may also use the same pathway, at least in part. Briefly, 1.5 ⫻ 107 cells were incubated with preswollen cytodex-1 beads
Second, the D2 type of dopamine receptors also exert their in 60-mm petri dishes for 18 h. The beads were then transferred to 0.5-ml
actions independently of G proteins by promoting the chambers and perifused with Hanks’ M199 containing 25 mm HEPES,
formation of a signaling protein complex composed of 0.1% BSA, and penicillin (100 U/ml)/streptomycin (100 ␮g/ml) for 2.5 h
␤-arrestin, Akt, and protein phosphatase-2A (30). In chro- at a flow rate of 0.8 ml/min and at 37 C to establish stable basal secretion.
PRL measurements, perifusion, and static culture experiments were
maffin cells, Akt-induced phosphorylation of cysteine always conducted with medium containing no phosphodiesterase in-
string protein plays a role in late stages of exocytosis (31). hibitors, whereas for cAMP measurements, experiments were done in
Akt also regulates the PRL promoter activity (32), whereas cells perifused without (Figs. 2, 4, and 5) and with 1 mm 3-isobutyl-1-
the contribution of this signaling pathway on dopamine- methylxanthine (Table 1). Fractions were collected in 1-min intervals and
later assayed for PRL and cAMP contents using RIA. Primary antibody
controlled PRL synthesis and release has not been studied. and standard for PRL assay were purchased from the National Pituitary
In that respect, it is important to stress that the PTX sen- Agency and Dr. A. F. Parlow (Harbor-UCLA Medical Center, Torrance,
sitivity of dopamine-induced inhibition of PRL release CA). cAMP was determined using specific antiserum provided by Albert
was originally evaluated with cells in static cultures dur- Baukal (NICHD, Bethesda, MD). [125I]PRL and [125I]cAMP were pur-
ing 2- to 24-h treatments (3, 11, 33), which do not dissociate chased from PerkinElmer Life Sciences (Boston, MA).
effects of this signaling pathway on exocytosis from those
Single-cell calcium measurements
mediated by synthesis of hormone.
Here we reexamined the mechanism by which dopa- For [Ca2⫹]i measurements, cells were incubated in Hanks’ M199,
mine inhibits PRL secretion in vitro. In our experiments, supplemented with 2 ␮m fura-2 AM (Molecular Probes, Eugene OR) at
37 C for 60 min. Coverslips with cells were then washed and mounted
the contribution of de novo synthesis on PRL release was on the stage of an Axiovert 135 microscope (Carl Zeiss, Oberkochen,
minimized by performing Ca2⫹, cAMP, and PRL measure- Germany) attached to the Attofluor Digital Fluorescence Microscopy
ments in perifused pituitary cells during the first 30 min System (Atto Instruments, Rockville, MD). Cells were examined under
of agonist application. The results of these investigations a ⫻40 oil immersion objective during exposure to alternating 340- and
revealed that dopamine inhibits PRL release by blocking 380-nm light beams, and the intensity of light emission at 520 nm was
measured. The ratio of light intensities, F340/F380, which reflects changes
VGCI in a PTX-sensitive manner, as well as by desensi- in [Ca2⫹]i, was followed in several single cells simultaneously at the rate
tizing calcium-secretion coupling in a PTX-insensitive of one point per second.
manner. Such a dual action of dopamine through multiple
intracellular signaling pathways provides an effective Simultaneous recording of [Ca2⫹]i and membrane
mechanism for control of exocytosis. The redundancy of potential (Vm)
pathways not only reinforces the blockade of basal PRL Pituitary cells were incubated for 15 min at 37 C in phenol red-free
release but also provides a mechanism for controlling PRL medium 199 containing Hanks’ salts, 20 mm sodium bicarbonate, 20 mm
release and simultaneously using calcium signaling path- HEPES, and 0.5 ␮m indo-1 AM (Molecular Probes). The Vm and bulk
ways by other receptors. [Ca2⫹]i was simultaneously monitored using a Nikon photon counter
system as previously described (13). The Vm and bulk [Ca2⫹]i were
captured simultaneously at rate of 5 kHz using a PC equipped with a
Materials and Methods Digidata 1200 A/D interface in conjunction with Clampex 8 (Molecular
Cell cultures Devices, Sunnyvale, CA). The [Ca2⫹]i was calibrated in vivo according
to Kao (35).
Experiments were performed on anterior pituitary cells from normal
postpubertal female Sprague Dawley rats obtained from Taconic Farm Western blot analysis
(Germantown, MD). Pituitary cells were dispersed and cultured as
mixed cells or enriched lactotrophs in medium 199 containing Earle’s Total proteins were isolated with M-PER mammalian protein extrac-
salts, sodium bicarbonate, 10% heat-inactivated horse serum, penicillin tion regents (Pierce, Rockford, IL), and protein concentration was de-
(100 U/ml), and streptomycin (100 ␮g/ml). A two-stage Percoll dis- termined using a BCA kit (Pierce) according to the manufacturer’s pro-
continuous density gradient procedure was used to obtain enriched tocol. Total proteins were separated on 12.5% SDS-PAGE and
lactotrophs, and their further identification in single-cell studies was transferred onto polyvinylidene difluoride membrane. The membrane
done by the addition of dopamine agonists and TRH (34). Experiments was blocked with Tris-buffered saline with Tween 20 buffer [10 mm
were done 24 – 48 h after dispersion. When indicated, cultured pituitary Tris-Cl (pH 7.5), 150 mm NaCl, and 0.1% Tween 20] containing 5% BSA
cells were treated overnight with 250 ng/ml PTX before experiments. fraction V and then incubated with diluted polyclonal phospho-Akt
Human embryonic kidney (HEK) 293 cells were cultured in DMEM antibodies (1:2000; Cell Signaling, Boston, MA) overnight. After that,
(Invitrogen, Carlsbad, CA), supplemented with 5% heat-inactivated fetal positive signals of individual blots were visualized by incubating the
bovine serum, according to standard protocols. The HEK cells were membrane with peroxidase-conjugated goat antirabbit secondary anti-
seeded at the concentration of 1.5 million/35-mm dish and cultured 24 h body (1:10,000; KPL, Gaithersburg, MD) and subsequent treatment with
before any treatment. Before experiment, cells were washed twice with SuperSignal West Pico Luminol/Enhanced solution (Pierce). The total

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 October 2015. at 00:42 For personal use only. No other uses without permission. . All rights reserved.
1472 Endocrinology, April 2008, 149(4):1470 –1479
Akt protein was detected by reprobing the membranes with a polyclonal
Akt antibody (1:2000; Cell Signaling).
Calculations
PRL release by perifused pituitary cells, [Ca2⫹]i, and Vm oscillations
are shown as representative traces. Secretory data for cAMP and PRL are
also summarized as means ⫾ sem values from at least three independent
experiments (Tables 1 and 2). The static culture results shown are
means ⫾ sem values of sextuplicate incubations in one of at least three
similar experiments, each giving the same statistical conclusions (Fig. 1),
or from pooled data from several experiments (Tables 1 and 2). Signif-
icant differences with P ⬍ 0.01 were determined by Student’s t test.
Results
Dopamine inhibits release of prestored PRL
To clarify the extent to which the prestored PRL contrib-
utes to dopamine-controlled hormone release, static cultures
of pituitary cells were treated with 50 ␮g/ml cycloheximide,
a protein synthesis inhibitor. As shown in Fig. 1A, basal PRL
FIG. 1. Dependence of rapid dopamine-induced inhibition of prolac-
tin (PRL) release on prestored hormone. A and C, Time course of
cycloheximide effects on basal PRL release in pituitary cells in static
culture (A) and perifused pituitary cells (C). B and D, Dopamine
(DA)-induced inhibition of PRL release in cells in static culture (B)
and perifused pituitary cells (D). E, Dose- and time-dependent effects
of dopamine on PRL release in perifused pituitary cells. In C and D,
samples were collected every minute, and in E, samples were collected
every 3 min. *, P ⬍ 0.01 between pairs.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 October 2015. at 00:42 For personal use only. No other uses without permission. . All rights reserved.
Gonzalez-Iglesias et al. • PTX-Insensitive Inhibition of PRL Release
release was not obviously affected by cycloheximide treat-
ment during the first 2 h incubation but was significantly
attenuated at longer incubation periods. In parallel experi-
ments, 1 ␮m dopamine added to cells in static cultures ef-
fectively stopped PRL release 30 min after application and
kept it low for 8 h (Fig. 1B). Effects of cycloheximide and
dopamine on PRL release were also evaluated in perifused
pituitary cells loaded in temperature-controlled chambers.
Figure 1C shows that the obvious inhibitory effect of cyclo-
heximide on basal PRL release was observed during treat-
ments longer than 60 min. In contrast, dopamine-induced
inhibition of basal PRL release in perifused pituitary cells
occurs rapidly, within 6 –7 min of application, and the wash-
out of agonist was accompanied by the full recovery of basal
secretion (Fig. 1D). Dopamine was fully effective in 1 and 0.5
␮m concentrations, whereas at lower concentration, only par-
tial inhibition was observed (Fig. E). These results indicate
that dopamine affects the fusion of prestored secretory
vesicles and that de novo synthesis of hormone plays a
minor role during shorter-term application of agonist. In
other experiments, we focused on the effects of dopamine
and its agonists on signaling and secretion using using 1
␮m concentration to reach full inhibition in treatments not
longer than 30 min.
Dopamine agonists inhibit basal AC activity and
spontaneous VGCI
We showed previously a strong correlation between extracel-
lular and intracellular cAMP levels in both perifused and pituitary
cells in static cultures, indicating that the measurement of extra-
cellular cAMP concentration is sufficient to evaluate the status of
AC activity (24). To study the dependence of agonist-induced
inhibition of PRL release on cAMP production, we measured PRL
and cAMP contents in the same samples obtained from perifusion
experiments. Figure 2 illustrates such an experiment. Like dopa-
mine (Fig. 1D), the dopamine agonists bromocriptine (Fig. 2A) and
apomorphine (Fig. 2C) also induced a rapid inhibition of PRL
release, but the recovery of secretion after their washouts was
delayed when compared with dopamine washout. In parallel to
changes in the rate of PRL release, both agonists also inhibited
cAMP release, which was sustained after their removal (Fig. 2, B
and D). This finding could indicate that dopamine-induced down-
regulation of basal AC activity accounts for or contributes to in-
hibition of PRL release.
Simultaneous measurements of electrical activity and in-
tracellular calcium in perforated cells revealed that apomor-
phine promptly abolished spontaneous firing of action po-
tentials and accompanied Ca2⫹ transients (Fig. 3B). TRH also
inhibited spontaneous electrical activity but transiently, fol-
lowed by sustained depolarization of cells and increase in the
frequency of spiking. Consistent with the Ca2⫹-mobilizing
action of this agonist and the consequent activation of the SK
type of Ca2⫹-controlled K⫹ channels (36), the transient hy-
perpolarization coincided with the spike response in [Ca2⫹]i,
whereas the sustained spiking coincided with the plateau
rise in [Ca2⫹]i (Fig. 3A). The inhibitory effect of apomorphine
(Fig. 3C) and bromocriptine (Fig. 3D) on spontaneous Ca2⫹
transients was also observed in intact cells loaded with
fura-2. In these experiments, TRH was applied at the end of
Gonzalez-Iglesias et al. • PTX-Insensitive Inhibition of PRL Release Endocrinology, April 2008, 149(4):1470 –1479 1473

FIG. 2. Parallelism in the actions of dopamine agonists on PRL and cAMP

release in perifused pituitary cells. A and B, Effects of bromocriptine
(Bromo) on PRL (A) and cAMP (B) release. C and D, Effects of apomorphine
(Apo) on PRL (C) and cAMP (D) release. In this and following figures, PRL
and cAMP contents were determined from the same samples, and cells were
perifused with medium without phosphodiesterase inhibitors added. Gray
areas indicate duration of dopamine agonist application.

recording to identify lactotrophs. Parallelism in the actions of

dopamine and its agonists on spontaneous Ca2⫹ transients
and basal PRL release is consistent with the role of VGCI in
basal PRL secretion (13).

Dopamine inhibits VGCI and PRL release in cells with

elevated cAMP production
In other experiments, basal AC activity was elevated by
perifusion of cells with forskolin (Fig. 4, A and C). Measure-
ments of PRL content in the same samples revealed that the
increase in cAMP production was accompanied by stimula-
tion of PRL release (Fig. 4, B and D). Forskolin treatment also
elevated [Ca2⫹]i in single lactotrophs (Fig. 4E). Application
of bromocriptine and apomorphine attenuated AC activity in FIG. 3. Dopamine agonists inhibit spontaneous electrical activity and
the presence of forskolin, but the residual cAMP production calcium transients in single cells. A and B, Simultaneous measurements
was severalfold higher than in untreated cells (Fig. 4, A and of electrical activity and calcium signals in perforated cells with acti-
vated TRH (A) and dopamine (B) receptors. Horizontal black bars in-
C; basal ⫽ 82 ⫾ 7, forskolin ⫽ 1377 ⫾ 203, and forskolin plus dicate duration of TRH and apomorphine applications. C and D, Inhib-
apomorphine ⫽ 416 ⫾ 33 pmol/min; (n ⫽ 4). At such ele- itory effect of apomorphine (C) and bromocriptine (D) on spontaneous
vated cAMP levels, both agonists inhibited PRL release (Fig. calcium transients in intact cells. For this and the following figures, TRH
4, B and D) in a manner highly comparable to that observed was applied at the end of recording to identify lactotrophs. Agonists were
present in incubation medium from the moment of their application
in controls (Fig. 2, A and C). Apomorphine also abolished (indicated by arrows) to the end of recording.
stimulatory effect of forskolin on Ca2⫹ transients in a ma-
jority of TRH-responsive cells (Fig. 4E). In the absence of
apomorphine, there was a sustain elevation in cAMP pro- Dopamine inhibits Ca2⫹ transients and PRL release in cells
duction, VGCI, and PRL release in forskolin-treated cells (24). with inhibited inward rectifier K⫹ (Kir) channels
These results indicate that inhibition of VGCI alone is suf- The role of dopamine in activation of Kir channels in pituitary
ficient to block rapid PRL release. lactotrophs is well established (19, 20). Extracellular Cs⫹ in 2–5

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 October 2015. at 00:42 For personal use only. No other uses without permission. . All rights reserved.
1474 Endocrinology, April 2008, 149(4):1470 –1479
FIG. 4. Dopamine agonists inhibit calcium transients and PRL re-
lease in cells with elevated cAMP levels. A and B, Bromocriptine-
induced inhibition of cAMP (A) and PRL (B) release in cells treated
with forskolin. C and D, Apomorphine-induced inhibition of cAMP (C)
and PRL (D) release in forskolin-treated cells. Notice that during
bromocriptine and apomorphine applications, cAMP levels were sev-
eralfold higher in forskolin-treated cells compared with untreated
cells. E, Inhibition of forskolin-induced calcium transients by apo-
morphine, which was added in medium containing forskolin. Apo,
Apomorphine; Bromo, bromocriptine.
mm concentrations fully blocks Kir channels in these cells (37).
The addition of 5 mm Cs⫹ had a minor stimulatory effect on
basal PRL release but did not affect bromocriptine-induced (Fig.
5A) and apomorphine-induced (Fig. 5C) inhibition of PRL re-
lease. In the presence of Cs⫹, agonist-induced inhibition of AC
activity was also preserved (Fig. 5, B and D). In agreement with
previously published data in GH3 cells (38), Cs⫹ elevated
[Ca2⫹]i in single lactotrophs. Furthermore, in the presence of
Cs⫹, apomorphine inhibited spontaneous Ca2⫹ transients in all
TRH-responsive cells (Fig. 5E). The same effects were also ob-
served in the presence of 2 mm Cs⫹ (data not shown). Dopam-
ine-induced inhibition of PRL release was also observed in cells
bathed in the presence of 1 mm Ba2⫹, a blocker of Kir and
Ca2⫹-conrolled K⫹ channels (38). Basal PRL release in control
cells was 87 ⫾ 4.3 ng/min and in the presence of 1 mm Ba2⫹ was
96 ⫾ 4.8 ng/min. In the presence of 1 ␮m dopamine, perifused
pituitary cells released 8.2 ⫾ 1.3 ng/min in the absence of Ba2⫹
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 October 2015. at 00:42 For personal use only. No other uses without permission. . All rights reserved.
Gonzalez-Iglesias et al. • PTX-Insensitive Inhibition of PRL Release
FIG. 5. Parallelism in the actions of dopamine agonists on basal PRL
release, adenylyl cyclase activity, and calcium transients in cells with
blocked inward rectifier potassium channels by extracellular Cs⫹. A
and B, Bromocriptine-induced inhibition of basal PRL (A) and cAMP
(B) release in cells with blocked inward rectifier potassium channels
by Cs⫹. C and D, Apomorphine-induced inhibition of PRL (C) and
cAMP (D) release in cesium-treated cells. E, Apomorphine-induced
inhibition of Ca2⫹ transients in cells bathed in 5 mM Cs⫹-containing
medium. Notice increase in [Ca2⫹]i after the addition of Cs⫹. Apo,
Apomorphine; Bromo, bromocriptine.
and 9.1 ⫾ 1.1 ng/min in the presence of Ba2⫹. These results
suggest that dopamine can inhibit spontaneous VGCI and basal
PRL release independently of the status of Kir channels.
Dopamine inhibits basal PRL release downstream of VGCI
In additional experiments, we analyzed the relevance of the
coupling of dopamine receptors to Gi/o signaling pathways for
PRL release. To do this, cells were treated with 250 ng/ml PTX
overnight. In such treated cells, agonist-induced inhibition of
basal AC activity was dramatically reduced but not completely
abolished (Table 1). The inhibitory effect of apomorphine and
bromocriptine on spontaneous Ca2⫹ transients was lost in PTX-
treated cells (Fig. 6, A and B). However, the same PTX treatment
could exert only a partial loss of inhibitory effects of bromocrip-
tine and apomorphine on PRL release (Fig. 6, C and D, and
Table 2). To examine the possibility that the PTX dose used in
Gonzalez-Iglesias et al. • PTX-Insensitive Inhibition of PRL Release
TABLE 1. Effects of dopamine agonists on basal cAMP release in
perifused pituitary cells
cAMP release (pmol/min)
Treatment
Without PTX
Basal 143 ⫾ 5 (10)
Agonist-stimulated 54 ⫾ 3 (10)a
To amplify the cAMP response, cells were perifused with medium
containing 1 mM 3-isobutyl-1-methylxanthine. Data shown are
mean ⫾ SEM values; numbers in parentheses indicate the number of
experiments per treatment. In each experiment, samples were col-
lected every minute, and individual values were generated from 10
measurements, starting 10 min after the application of 1 ␮M agonist
(bromocriptine or apomorphine) and 1 ␮M forskolin.
a
P ⬍ 0.01 vs. basal.
b
P ⬍ 0.01 vs. without PTX.
these experiments was not sufficient to fully block Gi/o signal-
ing pathway, we performed dose-dependent studies. As shown
in Fig. 6E, no further reduction in effectiveness of dopamine
was observed in cells in static cultures treated with PTX in 0.5
and 1 ␮g/ml concentration. These findings are in full accord
with the literature (1, 4) and indicate that PTX treatment was
fully effective in our experiments when used in 250 ng/ml
concentration.
To further tests the hypothesis that dopamine inhibits
rapid PRL release in cells downstream of Ca2⫹ influx in a Gi/o
protein-independent manner, we stimulated VGCI and PRL
release by depolarizing cells with 25 mm KCl. As shown in
Fig. 7A, depolarization-induced Ca2⫹ influx was reduced by
FIG. 6. Characterization of PTX treatment on dopamine agonist-induced
inhibition of [Ca2⫹]i and PRL release. A and B, The lack of effects of apo-
morphine (A) and bromocriptine (B) on spontaneous Ca2⫹ transients in
lactotrophs treated with PTX. C and D, PTX-sensitive and -insensitive
components of apomorphine-induced (C) and bromocriptine-induced (D)
inhibition of basal PRL release in perifused pituitary cells. Mean ⫾ SEM
values are shown in Table 2. E, Dose-dependent effects of PTX on basal and
dopamine (DA)-induced inhibition of PRL release in static cultures of pi-
tuitary cells. In all experiments, cells were treated with PTX overnight. In
A–D, cells were treated with 250 ng/ml PTX.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 October 2015. at 00:42 For personal use only. No other uses without permission. . All rights reserved.
Endocrinology, April 2008, 149(4):1470 –1479 1475
TABLE 2. Effects of dopamine agonists on basal PRL release in
perifused pituitary cells
PRL release (ng/min)
Treatment
With PTX Without PTX With PTX
137 ⫾ 4 (10) Basal 86 ⫾ 4 (23) 93 ⫾ 10 (9)
117 ⫾ 5 (10)a,b Apomorphine (1 ␮M) 7 ⫾ 1 (9)a 24 ⫾ 3 (3)a,b
Bromocriptine (1 ␮M) 8 ⫾ 2 (8)a 26 ⫾ 2 (3)a,b
Dopamine (1 ␮M) 7 ⫾ 2 (6)a 23 ⫾ 3 (3)a,b
Cells were perifused with medium without 3-isobutyl-1-methylx-
anthine. Data shown are mean ⫾ SEM values; numbers in parentheses
indicate the number of experiments per treatment. In each experi-
ment, samples were collected every minute, and individual values
were generated from 10 measurements, starting 10 min after the
application of agonist.
a
P ⬍ 0.01 vs. basal.
b
P ⬍ 0.01 vs. without PTX.
apomorphine in controls but not in 31 of 33 PTX-treated
TRH-responsive cells. Although [Ca2⫹]i levels were elevated
in PTX-treated cells during the sustained depolarization,
apomorphine effectively blocked PRL release below the basal
levels (Fig. 7B), confirming the hypothesis that dopamine can
also inhibit PRL release in a Ca2⫹-independent manner.
Effects of wortmannin, lithium, and phorbol 12-myristate 13-
acetate (PMA) on dopamine-induced inhibition of PRL release
In general, dopamine could inhibit PRL release through G
protein-dependent and G protein-independent, ␤-arrestin-
dependent pathways, the latter being discovered recently by
Caron’s group (30). The emerging components of this path-
way include Akt (a member of serine/threonine kinase fam-
ily), protein phosphatase-2A, and glycogen synthase kinase
(GSK) (39). Via its pleckstrin homology domain, Akt is reg-
ulated by the phosphoinositide 3 (PI3)-kinase (40), which is
effectively blocked by wortmannin. However, dopamine-
induced inhibition of basal PRL release was not affected by
wortmannin (Fig. 8C). Lithium antagonizes dopamine-de-
pendent effects mediated by Akt/GSK-3 signaling cascade
(41). Yet, extracellular Li⫹ did not affect dopamine agonist-
induced inhibition of PRL release in cells without (Fig. 8, A
and C) and with (Fig. 8B) inhibited Gi/o signaling pathway.
To test the efficacy of wortmannin and Li⫹ treatments to
inhibit Akt phosphorylation, we used HEK293 cells that
show constitutively high levels of Akt phosphorylation on
Ser473 and Thr308. In these cells, wortmannin inhibited
phosphorylation of Akt in a dose-dependent manner (Fig. 9,
left panel). On the other hand, Li⫹ stimulated Akt phosphor-
ylation (Fig. 9, center panel), which is consistent with obser-
vations in other cell types (41). Akt is also present in pituitary
cells, but we could not observe the presence of phosphory-
lating forms of this protein, suggesting that in our experi-
mental conditions, dopamine cannot signal through the Akt/
␤-arrestin signaling complex by dephosphorylating Akt on
Thr308.
In other experiments, cells were stimulated with phorbol
ester PMA. Such a treatment is known to silence the Gz
signaling pathway through protein kinase C-dependent
phosphorylation of the Gz␣ subunits (42, 43). As shown in
Fig. 8D, the inhibitory effect of dopamine on PRL secretion
was dramatically reduced in PMA-treated cells with opera-
1476 Endocrinology, April 2008, 149(4):1470 –1479 Gonzalez-Iglesias et al. • PTX-Insensitive Inhibition of PRL Release

FIG. 7. Effects of apomorphine on high potassium-induced calcium

influx and PRL release in control and PTX-treated lactotrophs. A,
Mean values of KCl-induced [Ca2⫹]i response in PTX-treated (black)
and – untreated (gray) cells. Numbers above traces indicate the num-
ber of TRH-responsive cells, in which such pattern was observed. B,
Apomorphine (Apo)-induced inhibition of high KCl-stimulated PRL
release in perifused pituitary cells.

tive Gi/o signaling pathway. Dopamine-induced inhibition of

secretion was further reduced in PTX- plus PMA-treated cells
(Fig. 8D). These results are consistent with the possibility that
Gz protein-dependent signaling pathway accounts for the
PTX-insensitive component in dopamine-induced rapid in-
hibition of PRL release.
FIG. 8. Dopamine-induced inhibition of basal PRL release in cells
Discussion with inhibited PI3-kinase and Akt/␤-arrestin signaling pathways. A
The fusion of secretory vesicles with the plasma membrane and B, The lack of effect of 3 mM Li⫹, an inhibitor of GSK-3, on
bromocriptine (Bromo)-induced inhibition of basal PRL release in
is termed regulated exocytosis and leads to release of hor- cells without (A) and with (B) blocked Gi/o signaling pathway (250
mones from endocrine cells and neurotransmitters from neu- ng/ml PTX, overnight). Traces shown are representative from three
rons (12). Exocytosis is mediated by complex protein ma- similar experiments. C, The lack of effects of 1 ␮M wortmannin (WT),
chinery, which is conserved in organisms ranging from yeast an inhibitor of PI3-kinase, and 3 mM Li⫹, on dopamine (DA)-induced
inhibition of basal PRL release. Traces shown are pooled data from
to mammals and includes docking, ATP-dependent priming five independent experiments. D, Effects of acute treatment with 100
and fusion of vesicle membranes through interactions of nM PMA on 1 ␮M dopamine (DA)-induced inhibition of PRL release in
proteins and their modulations by intracellular messengers cells with and without blocked Gi/o signaling pathway. PMA was
(44 – 46). Calcium is the primary intracellular signaling mol- applied 30 min before and during application of dopamine. Traces
ecule controlling hormone release. An increase in [Ca2⫹]i is shown are representative from three similar experiments.
required for two steps in regulated exocytosis: the priming
lated by TRH, pituitary adenylate cyclase-activating peptide,
of secretory vesicles, which occurs on the time-scale of tens
vasopressin-oxytocin, galanin, and angiotensin II receptors
of seconds, and triggering the fusion, which occurs within
(1). The parallelism we observed in the actions of dopamine
1–2 sec (12). Both the depolarization-driven Ca2⫹ entry (47)
on cAMP/Ca2⫹ signaling and secretion is in full agreement
and Ca2⫹ mobilization from intracellular stores (48) can trig-
with the literature, describing inhibition of AC (10, 22) and
ger the fusion of primed secretory vesicles. Other signaling
pathways, including cAMP/protein kinase A (23, 49), diac-
ylglycerol/protein kinase C (50 –52), and phosphatidylino-
sitol bisphosphate/PI3-kinase (46, 53), also contribute to the
control of exocytosis.
Lactotrophs exhibit spontaneous firing of action poten-
tials, and the associated VGCI is sufficient to maintain PRL
release at high and steady levels in cells in the population for
many hours (13). Such secretion is termed intrinsic, sponta-
neous, or basal and in vivo can be achieved by removing the
FIG. 9. Wortmannin and lithium sensitivity of Akt phosphorylation.
hypothalamo-pituitary connection or by transplantation of Western blot showing the relative levels of total and phospho-Akt (Ser-
pituitary glands under the kidney capsule (2). Slow sponta- 473 and Thr-308) in extracts from HEK293 and pituitary cells. A phos-
neous secretion from single lactotrophs could be monitored pho-independent antibody was used as a loading control. HEK293 cells
with confocal microscopy using FM4-64-loaded cells (54, 55). were treated with wortmannin (WT) for 10 min only (left panel) or first
with 1 ␮M wortmannin for 10 min and then with 3 mM Li⫹ for an
In vivo and in vitro, basal PRL release is modulated by nu- additional 10 min (center panel). Pituitary cells were treated with 1 ␮M
merous G protein-coupled receptors; it is inhibited by do- wortmannin for 10 min, followed by treatment with 1 ␮M dopamine (DA)
pamine, somatostatin, and endothelin receptors and stimu- or 100 ng/ml epidermal growth factor (EGF) for 5 min.

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 October 2015. at 00:42 For personal use only. No other uses without permission. . All rights reserved.
Gonzalez-Iglesias et al. • PTX-Insensitive Inhibition of PRL Release Endocrinology, April 2008, 149(4):1470 –1479 1477

Cav channels (15, 56) and activation of Kir channels, which in basal AC activity. Furthermore, the heterotrimeric Gz pro-
turn inhibits VGCI (20). These findings suggest that basal teins provide a pathway through which endothelin receptors
PRL release could reflect changes in intracellular concentra- can stop PRL release for a prolonged period of time. Two
tions of cAMP and Ca2⫹. lines of evidence supported this conclusion. First, down-
Here we confirmed the PTX-sensitive coupling of dopa- regulating the Gz␣ expression led to abolition of the inhib-
mine receptors to ACs as well as the PTX-sensitive inhibition itory phase in PRL release. Second, in experiments with
of VGCI, suggesting the major role of Gi/o signaling pathway phorbol esters, which silence the Gz signaling pathway
in control of these two intracellular messengers. Our results through protein kinase C-dependent phosphorylation of the
indicate that dopamine agonists can inhibit PRL secretion not Gz␣ subunits (42, 43), the inhibitory action of endothelin-1
only when the coupling of these receptors to AC is attenuated was attenuated (29). In parallel to that, the PTX-insensitive
but also when the activity of this enzyme is elevated. We dopaminergic inhibition of PRL release was also remarkably
further show that Kir channels contribute to the control of attenuated in cells treated with phorbol esters. These results
basal [Ca2⫹]i and PRL release, a finding consistent with ob- are consistent with a hypothesis that dopamine inhibits PRL
servations in GH3 cells (38), but that dopamine-induced in- release through the Gz signaling pathway, but additional
hibition of spontaneous VGCI was preserved in cells bathed work is required to demonstrate it.
in the presence of blockers of these channels. Thus, dopam- The PTX-insensitive inhibition of PRL release by dopa-
ine-induced inhibition of the Cav channel gating alone is mine could reflect desensitization of Ca2⫹-secretion cou-
sufficient to block spontaneous Ca2⫹ influx. Finally, we show pling, as indicated by the ability of dopamine to inhibit PRL
that dopamine receptors can inhibit PRL release indepen- release in PTX-treated cells, which showed no inhibition of
dently of the status of VGCI, at least in part. This was ex- spontaneous electrical activity. In the case of endothelin re-
perimentally achieved by PTX treatment, which effectively ceptors, both arms of Gz proteins, ␣ and ␤␥, were suggested
blocked dopamine-induced inhibition of spontaneous and to contribute to inhibition of PRL release downstream of
depolarization-induced VGCI but was unable to abolish do- VGCI (57). The contribution of Gz␣-mediated attenuation of
pamine-induced inhibition of PRL release at elevated [Ca2⫹]i AC activity in pituitary cells appeared not to be sufficient in
levels. dopamine-induced rapid inhibition of PRL release, because
At the present time, we do not know by which mechanism such inhibition was preserved in forskolin-treated cells.
dopamine inhibits PRL release in cells with blocked PTX- Thus, liberation of Gz␤␥ may represent the primary factor in
sensitive signaling pathway. In addition to Gi/o signaling dopamine-induced inhibition of Ca2⫹-secretion coupling.
pathway, dopamine receptors also signal through a recently Such a conclusion is in accordance with the finding in other
discovered signaling complex composed of Akt, protein cell types about the role of G␤␥ subunits in control of se-
phosphatase-2A, and ␤-arrestin (30). Formation of this com- cretion independently of their actions on AC, phospholipase
plex leads to inactivation of Akt, mediated by protein phos- C-␤2, and several tyrosine kinases (58). Recent experiments
phatase-2A-induced dephosphorylation of Thr-308, and ac- by Martin’s group in permeabilized PC12 cells also demon-
tivation of GSK-3 (39). PI3-kinase affects Akt via its pleckstrin strated that G protein ␤␥ directly regulates soluble N-eth-
homology domain (40). In our hands, however, wortmannin ylmaleimide-sensitive factor attachment protein receptor
did not influence dopamine-induced inhibition of PRL re- protein fusion machinery to modulate secretory granule exo-
lease. In the mouse striatum, dopamine-stimulated dephos- cytosis. This action occurs rapidly and affects ATP-primed
phorylation of Akt leads to a reduction in kinase activity and secretory vesicles (59).
activation of GSK-3, and Li⫹ antagonizes dopamine-depen- The presence of the PTX-insensitive step in dopamine ac-
dent effects mediated by Akt/GSK-3 signaling cascade (41). tion on PRL release does not argue against the relevance of
In perifused pituitary cells with and without blocked Gi/o AC, Kir channels, and Cav channels in this process in vivo but
signaling pathway, we observed no effects of extracellular demonstrates that pharmacological exclusion of a single
Li⫹ on dopamine- and bromocriptine-induced inhibition of pathway does not provide a valid experimental model for a
PRL release. Furthermore, we were unable to observe the system simultaneously controlled by multiple pathways. In
presence of phosphorylated forms of Akt in resting cells. vitro, dopamine-mediated control of VGCI is more than suf-
These observations argue against the role of ␤-arrestin-Akt ficient to block PRL release, as is shown in experiments with
signaling pathway in rapid inhibition of PRL release by do- removal of extracellular Ca2⫹ (13, 14). The triple control of
pamine, but more work is required to clarify the potential VGCI by inhibiting AC and Cav and activating Kir channels,
role of this G protein-independent signaling pathway on D2 combined with the mechanism for desensitization of Ca2⫹-
dopamine receptor-mediated regulation of PRL synthesis secretion coupling, not only secures inhibition of PRL release
and sustained release. In that respect, it is of relevance to but also provides the possibility for maintaining such inhi-
mention that Akt regulates PRL promoter activity (32) and bition when cells are exposed to multiple agonists. For ex-
that the novel signaling pathway of dopamine receptors is ample, the Gz signaling pathway probably contributes to the
operative during prolonged stimulation of the D2 class of control of PRL release by dopamine when the electrical ac-
receptors (41). tivity in lactotrophs is stimulated by other factors.
Anterior pituitary cells also express Gz␣ (29), and the cou- In conclusion, here we show that dopamine receptors ac-
pling of dopamine receptors (including the D2 subtype) with tivate multiple signaling pathways in lactotrophs and that
these proteins is well established (26 –28). Consistent with such redundancy reinforces the blockade of basal PRL re-
this finding, we observed the presence of PTX-sensitive and lease (Fig. 10). Our results indicate that dopamine rapidly
-insensitive components in dopamine-induced inhibition of reduced basal cAMP production, a second messenger in-

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 October 2015. at 00:42 For personal use only. No other uses without permission. . All rights reserved.
1478 Endocrinology, April 2008, 149(4):1470 –1479
FIG. 10. Activation of multiple signaling pathways by dopamine-2
receptors in lactotrophs. Arrows indicate stimulatory actions, and
circles indicate inhibitory actions.
volved in both synthesis and release of PRL as well as in
control of VGCI. Independently of the status of AC activity,
however, these receptors inhibited spontaneous firing of ac-
tion potentials and the associated VGCI in lactotrophs. Such
inhibition was also observed in the presence of Cs⫹ and Ba2⫹
in concentrations sufficient to completely inhibit Kir chan-
nels, suggesting that inhibition of Cav channels per se was
enough to stop spontaneous Ca2⫹ influx. Dopamine-induced
inhibition of AC and electrical activity required the coupling
of receptors to PTX-sensitive Gi/o proteins. In contrast, the
coupling of dopamine receptors to both the PTX-sensitive
and -insensitive class of G proteins and/or signaling by other
pathways was required for full inhibition of AC activity and
PRL release. More studies are required to clarify the mech-
anism by which dopamine inhibits PRL secretion down-
stream of VGCI.
Acknowledgments
We are thankful to Dr. Fredrick Van Goor for electrophysiological
measurements and Dr. Tamas Balla for help with Akt experiments.
Received July 18, 2007. Accepted December 10, 2007.
Address all correspondence and requests for reprints to: Dr. Stanko
Stojilkovic, National Institute of Child Health and Human Development,
Building 49, Room 6A-36, 49 Convent Drive, Bethesda, Maryland 20892-
4510. E-mail: stankos@helix.nih.gov.
This work was Supported by the Intramural Research Program of the
NICHD, National Institutes of Health.
Disclosure Statement: A.E.G.-I., T.M., S.L., M.T., and S.S.S. have noth-
ing to declare.
References
1. Freeman ME, Kanyicska B, Lerant A, Nagy G 2000 Prolactin: structure, func-
tion, and regulation of secretion. Physiol Rev 80:1523–1631
2. Ben-Jonathan N, Hnasko R 2001 Dopamine as a prolactin (PRL) inhibitor.
Endocr Rev 22:724 –763
3. Burris TP, Nguyen DN, Smith SG, Freeman ME 1992 The stimulatory and
inhibitory effects of dopamine on prolactin secretion involve different G-
proteins. Endocrinology 130:926 –932
4. Missale C, Nash SR, Robinson SW, Jaber M, Caron MG 1998 Dopamine
receptors: from structure to function. Physiol Rev 78:189 –225
5. Giros B, Sokoloff P, Martres MP, Riou JF, Emorine LJ, Schwartz JC 1989
Alternative splicing directs the expression of two D2 dopamine receptor iso-
forms. Nature 342:923–926
6. Monsma Jr FJ, McVittie LD, Gerfen CR, Mahan LC, Sibley DR 1989 Multiple
D2 dopamine receptors produced by alternative RNA splicing. Nature 342:
926 –929
7. Kukstas LA, Domec C, Bascles L, Bonnet J, Verrier D, Israel JM, Vincent JD
1991 Different expression of the two dopaminergic D2 receptors, D2415 and
D2444, in two types of lactotroph each characterized by their response to
dopamine, and modification of expression by sex steroids. Endocrinology
129:1101–1103
8. Kelly MA, Rubinstein M, Asa SL, Zhang G, Saez C, Bunzow JR, Allen RG,
Hnasko R, Ben-Jonathan N, Grandy DK, Low MJ 1997 Pituitary lactotroph
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 October 2015. at 00:42 For personal use only. No other uses without permission. . All rights reserved.
Gonzalez-Iglesias et al. • PTX-Insensitive Inhibition of PRL Release
hyperplasia and chronic hyperprolactinemia in dopamine D2 receptor-defi-
cient mice. Neuron 19:103–113
9. Senogles SE, Benovic JL, Amlaiky N, Unson C, Milligan G, Vinitsky R,
Spiegel AM, Caron MG 1987 The D2-dopamine receptor of anterior pituitary
is functionally associated with a pertussis toxin-sensitive guanine nucleotide
binding protein. J Biol Chem 262:4860 – 4867
10. Enjalbert A, Bockaert J 1983 Pharmacological characterization of the D2 do-
pamine receptor negatively coupled with adenylate cyclase in rat anterior
pituitary. Mol Pharmacol 23:576 –584
11. Cronin MJ, Myers GA, MacLeod RM, Hewlett EL 1983 Pertussis toxin un-
couples dopamine agonist inhibition of prolactin release. Am J Physiol 244:
E499 –E504
12. Martin TF 2003 Tuning exocytosis for speed: fast and slow modes. Biochim
Biophys Acta 1641:157–165
13. Van Goor F, Zivadinovic D, Martinez-Fuentes AJ, Stojilkovic SS 2001 De-
pendence of pituitary hormone secretion on the pattern of spontaneous volt-
age-gated calcium influx. Cell type-specific action potential secretion coupling.
J Biol Chem 276:33840 –33846
14. Lamberts SW, Macleod RM 1990 Regulation of prolactin secretion at the level
of the lactotroph. Physiol Rev 70:279 –318
15. Lledo PM, Legendre P, Israel JM, Vincent JD 1990 Dopamine inhibits two
characterized voltage-dependent calcium currents in identified rat lactotroph
cells. Endocrinology 127:990 –1001
16. Lledo PM, Homburger V, Bockaert J, Vincent JD 1992 Differential G protein-
mediated coupling of D2 dopamine receptors to K⫹ and Ca2⫹ currents in rat
anterior pituitary cells. Neuron 8:455– 463
17. Seabrook GR, Knowles M, Brown N, Myers J, Sinclair H, Patel S, Freedman
SB, McAllister G 1994 Pharmacology of high-threshold calcium currents in
GH4C1 pituitary cells and their regulation by activation of human D2 and D4
dopamine receptors. Br J Pharmacol 112:728 –734
18. Israel JM, Kirk C, Vincent JD 1987 Electrophysiological responses to dopa-
mine of rat hypophysial cells in lactotroph-enriched primary cultures. J Physiol
390:1–22
19. Gregerson KA, Flagg TP, O’Neill TJ, Anderson M, Lauring O, Horel JS,
Welling PA 2001 Identification of G protein-coupled, inward rectifier potas-
sium channel gene products from the rat anterior pituitary gland. Endocri-
nology 142:2820 –2832
20. Einhorn LC, Gregerson KA, Oxford GS 1991 D2 dopamine receptor activation
of potassium channels in identified rat lactotrophs: whole-cell and single-
channel recording. J Neurosci 11:3727–3737
21. Lledo PM, Vernier P, Vincent JD, Mason WT, Zorec R 1994 New approaches
in the study of stimulus-secretion coupling in anterior pituitary cells. Ann NY
Acad Sci 710:301–318
22. McDonald WM, Sibley DR, Kilpatrick BF, Caron MG 1984 Dopaminergic
inhibition of adenylate cyclase correlates with high affinity agonist binding to
anterior pituitary D2 dopamine receptors. Mol Cell Endocrinol 36:201–209
23. Nagy G, Reim K, Matti U, Brose N, Binz T, Rettig J, Neher E, Sorensen JB
2004 Regulation of releasable vesicle pool sizes by protein kinase A-dependent
phosphorylation of SNAP-25. Neuron 41:417– 429
24. Gonzalez-Iglesias AE, Jiang Y, Tomic M, Kretschmannova K, Andric SA,
Zemkova H, Stojilkovic SS 2006 Dependence of electrical activity and calcium
influx-controlled prolactin release on adenylyl cyclase signaling pathway in
pituitary lactotrophs. Mol Endocrinol 20:2231–2246
25. Boyd RS, Ray KP, Wallis M 1988 Actions of pertussis toxin on the inhibitory
effects of dopamine and somatostatin on prolactin and growth hormone re-
lease from ovine anterior pituitary cells. J Mol Endocrinol 1:179 –186
26. Obadiah J, Avidor-Reiss T, Fishburn CS, Carmon S, Bayewitch M, Vogel Z,
Fuchs S, Levavi-Sivan B 1999 Adenylyl cyclase interaction with the D2 do-
pamine receptor family; differential coupling to Gi, Gz, and Gs. Cell Mol
Neurobiol 19:653– 664
27. Leck KJ, Blaha CD, Matthaei KI, Forster GL, Holgate J, Hendry IA 2006 Gz
proteins are functionally coupled to dopamine D2-like receptors in vivo. Neu-
ropharmacology 51:597– 605
28. Sidhu A, Kimura K, Uh M, White BH, Patel S 1998 Multiple coupling of
human D5 dopamine receptors to guanine nucleotide binding proteins Gs and
Gz. J Neurochem 70:2459 –2467
29. Andric SA, Zivadinovic D, Gonzalez-Iglesias AE, Lachowicz A, Tomic M,
Stojilkovic SS 2005 Endothelin-induced, long lasting, and Ca2⫹ influx-inde-
pendent blockade of intrinsic secretion in pituitary cells by Gz subunits. J Biol
Chem 280:26896 –26903
30. Beaulieu JM, Sotnikova TD, Marion S, Lefkowitz RJ, Gainetdinov RR,
Caron MG 2005 An Akt/␤-arrestin 2/PP2A signaling complex mediates do-
paminergic neurotransmission and behavior. Cell 122:261–273
31. Evans GJ, Barclay JW, Prescott GR, Jo SR, Burgoyne RD, Birnbaum MJ,
Morgan A 2006 Protein kinase B/Akt is a novel cysteine string protein kinase
that regulates exocytosis release kinetics and quantal size. J Biol Chem 281:
1564 –1572
32. Hayakawa J, Ohmichi M, Tasaka K, Kanda Y, Adachi K, Nishio Y, Hisamoto
K, Mabuchi S, Hinuma S, Murata Y 2002 Regulation of the PRL promoter by
Akt through cAMP response element binding protein. Endocrinology 143:
13–22
33. Enjalbert A, Musset F, Chenard C, Priam M, Kordon C, Heisler S 1988
Gonzalez-Iglesias et al. • PTX-Insensitive Inhibition of PRL Release
Dopamine inhibits prolactin secretion stimulated by the calcium channel ag-
onist Bay-K-8644 through a pertussis toxin-sensitive G protein in anterior
pituitary cells. Endocrinology 123:406 – 412
34. Koshimizu TA, Tomic M, Wong AO, Zivadinovic D, Stojilkovic SS 2000
Characterization of purinergic receptors and receptor-channels expressed in
anterior pituitary cells. Endocrinology 141:4091– 4099
35. Kao JP 1994 Practical aspects of measuring [Ca2⫹] with fluorescent indicators.
Methods Cell Biol 40:155–181
36. Ashworth R, Hinkle PM 1996 Thyrotropin-releasing hormone-induced intra-
cellular calcium responses in individual rat lactotrophs and thyrotrophs. En-
docrinology 137:5205–5212
37. Tomic M, Van Goor F, He ML, Zivadinovic D, Stojilkovic SS 2002 Ca2⫹-
mobilizing endothelin-A receptors inhibit voltage-gated Ca2⫹ influx through
Gi/o signaling pathway in pituitary lactotrophs. Mol Pharmacol 61:1329 –1339
38. Charles AC, Piros ET, Evans CJ, Hales TG 1999 L-type Ca2⫹ channels and K⫹
channels specifically modulate the frequency and amplitude of spontaneous
Ca2⫹ oscillations and have distinct roles in prolactin release in GH3 cells. J Biol
Chem 274:7508 –7515
39. Bibb JA 2005 Decoding dopamine signaling. Cell 122:153–155
40. Burgering BM, Coffer PJ 1995 Protein kinase B (c-Akt) in phosphatidylino-
sitol-3-OH kinase signal transduction. Nature 376:599 – 602
41. Beaulieu JM, Sotnikova TD, Yao WD, Kockeritz L, Woodgett JR, Gainet-
dinov RR, Caron MG 2004 Lithium antagonizes dopamine-dependent be-
haviors mediated by an AKT/glycogen synthase kinase 3 signaling cascade.
Proc Natl Acad Sci USA 101:5099 –5104
42. Fields TA, Casey PJ 1995 Phosphorylation of Gz␣ by protein kinase C blocks
interaction with the ␤␥ complex. J Biol Chem 270:23119 –23125
43. Wang J, Frost JA, Cobb MH, Ross EM 1999 Reciprocal signaling between
heterotrimeric G proteins and the p21-stimulated protein kinase. J Biol Chem
274:31641–31647
44. Rettig J, Neher E 2002 Emerging roles of presynaptic proteins in Ca⫹⫹-
triggered exocytosis. Science 298:781–785
45. An SJ, Almers W 2004 Tracking SNARE complex formation in live endocrine
cells. Science 306:1042–1046
46. Di Paolo G, Moskowitz HS, Gipson K, Wenk MR, Voronov S, Obayashi M,
Flavell R, Fitzsimonds RM, Ryan TA, De Camilli P 2004 Impaired
PtdIns(4,5)P2 synthesis in nerve terminals produces defects in synaptic vesicle
trafficking. Nature 431:415– 422
Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the
endocrine community.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 October 2015. at 00:42 For personal use only. No other uses without permission. . All rights reserved.
Endocrinology, April 2008, 149(4):1470 –1479 1479
47. Felmy F, Neher E, Schneggenburger R 2003 The timing of phasic transmitter
release is Ca2⫹-dependent and lacks a direct influence of presynaptic mem-
brane potential. Proc Natl Acad Sci USA 100:15200 –15205
48. Tse A, Tse FW, Almers W, Hille B 1993 Rhythmic exocytosis stimulated by
GnRH-induced calcium oscillations in rat gonadotropes. Science 260:82– 84
49. Sakaba T, Neher E 2003 Direct modulation of synaptic vesicle priming by
GABA(B) receptor activation at a glutamatergic synapse. Nature 424:775–778
50. Gillis KD, Mossner R, Neher E 1996 Protein kinase C enhances exocytosis
from chromaffin cells by increasing the size of the readily releasable pool of
secretory granules. Neuron 16:1209 –1220
51. Yang Y, Udayasankar S, Dunning J, Chen P, Gillis KD 2002 A highly Ca2⫹-
sensitive pool of vesicles is regulated by protein kinase C in adrenal chromaffin
cells. Proc Natl Acad Sci USA 99:17060 –17065
52. Zhu H, Hille B, Xu T 2002 Sensitization of regulated exocytosis by protein
kinase C. Proc Natl Acad Sci USA 99:17055–17059
53. Grishanin RN, Kowalchyk JA, Klenchin VA, Ann K, Earles CA, Chapman
ER, Gerona RR, Martin TF 2004 CAPS acts at a prefusion step in dense-core
vesicle exocytosis as a PIP2 binding protein. Neuron 43:551–562
54. Stenovec M, Kreft M, Poberaj I, Betz WJ, Zorec R 2004 Slow spontaneous
secretion from single large dense-core vesicles monitored in neuroendocrine
cells. FASEB J 18:1270 –1272
55. Vardjan N, Stenovec M, Jorgacevski J, Kreft M, Zorec R 2007 Subnanometer
fusion pores in spontaneous exocytosis of peptidergic vesicles. J Neurosci
27:4737– 4746
56. Malgaroli A, Vallar L, Elahi FR, Pozzan T, Spada A, Meldolesi J 1987 Do-
pamine inhibits cytosolic Ca2⫹ increases in rat lactotroph cells. Evidence of a
dual mechanism of action. J Biol Chem 262:13920 –13927
57. Bertram R, Tabak J, Toporikova N, Freeman ME 2006 Endothelin action on
pituitary lactotrophs: one receptor, many GTP-binding proteins. Sci STKE
2006:er2
58. Blackmer T, Larsen EC, Takahashi M, Martin TF, Alford S, Hamm HE 2001
G protein ␤␥ subunit-mediated presynaptic inhibition: regulation of exocytotic
fusion downstream of Ca2⫹ entry. Science 292:293–297
59. Blackmer T, Larsen EC, Bartleson C, Kowalchyk JA, Yoon EJ, Preininger AM,
Alford S, Hamm HE, Martin TF 2005 G protein ␤␥ directly regulates SNARE
protein fusion machinery for secretory granule exocytosis. Nat Neurosci
8:421– 425