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Endocrinology, doi:10.1210/en.2007-0980
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*12-O-TETRADECANOYLPHORBOL-13-ACETATE
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*DOPAMINE
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*LITHIUM, ELEMENTAL
Endocrinology Vol. 149, No. 4 1470-1479
Copyright © 2008 by The Endocrine Society

Dopamine Inhibits Basal Prolactin Release in Pituitary Lactotrophs through Pertussis Toxin-Sensitive and -Insensitive Signaling Pathways

Arturo E. Gonzalez-Iglesias1, Takayo Murano1, Shuo Li, Melanija Tomic 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

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{at}helix.nih.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Dopamine D2 receptors signal through the pertussis toxin (PTX)-sensitive Gi/o and PTX-insensitive Gz proteins, as well as through a G protein-independent, β-arrestin/glycogen synthase kinase-3-dependent pathway. Activation of these receptors in pituitary lactotrophs leads to inhibition of prolactin (PRL) release. It has been suggested that this inhibition occurs through the Gi/o-{alpha} protein-mediated inhibition of cAMP production and/or Gi/o-β{gamma} dimer-mediated activation of inward rectifier K+ channels and inhibition of voltage-gated Ca2+ channels. Here we show that the dopamine agonist-induced inhibition of spontaneous Ca2+ influx and release of prestored PRL was preserved when cAMP levels were elevated by forskolin treatment. We further observed that dopamine agonists inhibited both spontaneous and depolarization-induced Ca2+ influx in untreated but not in PTX-treated cells. This inhibition was also observed in cells with blocked inward rectifier K+ channels, suggesting that the dopamine effect on voltage-gated Ca2+ channel gating is sufficient to inhibit spontaneous Ca2+ influx. However, agonist-induced inhibition of PRL release was only partially relieved in PTX-treated cells, indicating that dopamine receptors also inhibit exocytosis downstream of voltage-gated Ca2+ influx. The PTX-insensitive step in agonist-induced inhibition of PRL release was not affected by the addition of wortmannin, an inhibitor of phosphatidylinositol 3-kinase, and lithium, an inhibitor of glycogen synthase kinase-3, but was attenuated in the presence of phorbol 12-myristate 13-acetate, which inhibits Gz signaling pathway in a protein kinase C-dependent manner. Thus, dopamine inhibits basal PRL release by blocking voltage-gated Ca2+ influx through the PTX-sensitive signaling pathway and by desensitizing Ca2+ secretion coupling through the PTX-insensitive and protein kinase C-sensitive signaling pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DOPAMINE SECRETED FROM hypophyseal hypothalamic neurons is a principal inhibitory regulator of prolactin (PRL) release by pituitary lactotrophs (1, 2). In low concentrations, dopamine also stimulates PRL release (3). By using the radioligand binding assay, it was shown in the late 1970s that the dopamine D2 subtype of receptors mediates the tonic inhibitory control of hypothalamic dopamine on PRL release in these cells (4). Later investigations showed that two subtypes of D2 receptors, termed D2S and D2L, are generated by alternative splicing in lactotrophs and other cell types (5, 6). Lactotrophs may express a different ratio of these two subtype receptors, depending on the level of gonadal steroids (7). Consistent with these findings, the knockout D2 mice showed chronic hyperprolactinemia, pituitary hyperplasia, and a moderate decrease in MSH content (8). In addition to lactotrophs, dopamine D2 receptors have been identified in the intermediate lobe of the pituitary gland (4).

The pituitary dopamine receptors are functionally associated with pertussis toxin (PTX)-sensitive G proteins (9). Dopamine-induced inhibition of PRL release is also affected by PTX treatment (3, 10, 11), suggesting that this signaling pathway is involved in control of secretion. Two intracellular messengers affected by activation of PTX-sensitive pathways in pituitary cells, Ca2+ and cAMP, play major roles in controlling the fusion of secretory vesicles with the plasma membrane to release hormones in endocrine cells (12). Electrophysiological experiments revealed that spontaneous action potential-dependent fluctuations in intracellular calcium concentration ([Ca2+]i) account for high basal PRL release (13). Removal of extracellular Ca2+ abolished spontaneous firing of action potentials and reduced basal PRL release to the levels observed during the application of dopamine agonists (13, 14). Consistent with these observations, activation of dopamine receptors was found to inhibit voltage-gated Ca2+ influx (VGCI). At the present time, it is not clear whether inhibition of voltage-gated T-type and L-type Ca2+ (Cav) channels (15, 16, 17) and/or activation of K+ currents (18, 19, 20, 21) accounts for such inhibition. In pituitary cells, dopamine also inhibits adenylyl cyclase (AC) activity in a PTX-sensitive manner (10, 22), which could contribute to inhibition of PRL release (10, 11). Such an action could be coupled to the effects of cAMP and its kinase on the exocytotic pathway (23) and/or inhibition of Ca2+ signaling pathway (24). However, the relevance of cAMP in dopamine actions on PRL release was questioned by the finding that dopamine inhibits PRL secretion in cells with activated ACs by forskolin (25).

Two additional transduction mechanisms have also been reported for D2 dopamine receptors in target tissues, but their significance in dopamine-induced control of PRL release has not been addressed. First, dopamine D2S and D2L receptors couple to the same extent to the PTX-sensitive Gi/o protein and to the PTX-insensitive Gz proteins in vitro (26) and in vivo (27). Other subtypes of dopamine receptors also couple to Gz proteins (26, 28). It has been shown that the Gz signaling pathway plays a major role in endothelin-A receptor-dependent inhibition of basal PRL release (29), raising the possibility that dopamine D2 receptor may also use the same pathway, at least in part. Second, the D2 type of dopamine receptors also exert their actions independently of G proteins by promoting the formation of a signaling protein complex composed of β-arrestin, Akt, and protein phosphatase-2A (30). In chromaffin cells, Akt-induced phosphorylation of cysteine string protein plays a role in late stages of exocytosis (31). Akt also regulates the PRL promoter activity (32), whereas the contribution of this signaling pathway on dopamine-controlled PRL synthesis and release has not been studied. In that respect, it is important to stress that the PTX sensitivity of dopamine-induced inhibition of PRL release was originally evaluated with cells in static cultures during 2- to 24-h treatments (3, 11, 33), which do not dissociate effects of this signaling pathway on exocytosis from those mediated by synthesis of hormone.

Here we reexamined the mechanism by which dopamine inhibits PRL secretion in vitro. In our experiments, the contribution of de novo synthesis on PRL release was minimized by performing Ca2+, cAMP, and PRL measurements in perifused pituitary cells during the first 30 min of agonist application. The results of these investigations revealed that dopamine inhibits PRL release by blocking VGCI in a PTX-sensitive manner, as well as by desensitizing calcium-secretion coupling in a PTX-insensitive manner. Such a dual action of dopamine through multiple intracellular signaling pathways provides an effective mechanism for control of exocytosis. The redundancy of pathways not only reinforces the blockade of basal PRL release but also provides a mechanism for controlling PRL release and simultaneously using calcium signaling pathways by other receptors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures
Experiments were performed on anterior pituitary cells from normal postpubertal female Sprague Dawley rats obtained from Taconic Farm (Germantown, MD). Pituitary cells were dispersed and cultured as mixed cells or enriched lactotrophs in medium 199 containing Earle’s salts, sodium bicarbonate, 10% heat-inactivated horse serum, penicillin (100 U/ml), and streptomycin (100 µg/ml). A two-stage Percoll discontinuous density gradient procedure was used to obtain enriched lactotrophs, and their further identification in single-cell studies was done by the addition of dopamine agonists and TRH (34). Experiments were done 24–48 h after dispersion. When indicated, cultured pituitary cells were treated overnight with 250 ng/ml PTX before experiments. Human embryonic kidney (HEK) 293 cells were cultured in DMEM (Invitrogen, Carlsbad, CA), supplemented with 5% heat-inactivated fetal bovine serum, according to standard protocols. The HEK cells were seeded at the concentration of 1.5 million/35-mm dish and cultured 24 h before any treatment. Before experiment, cells were washed twice with M199 medium containing Hanks’ balanced solution, 0.1% BSA, 25 mM 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 1 µM wortmannin for 10 min and then treated with 3 mM lithium chloride for an additional 10 min.

PRL and cAMP measurements
Pituitary cells (1 million per well) were plated in 24-well plates in serum-containing M199 and incubated overnight at 37 C under 5% CO2-air and saturated humidity. Before experiments, cells were washed with serum-free medium and stimulated at 37 C under 5% CO2-air and saturated humidity for 120 min if not otherwise stated. Hormone secretion was also monitored using cell column perifusion experiments. Briefly, 1.5 x 107 cells were incubated with preswollen cytodex-1 beads in 60-mm petri dishes for 18 h. The beads were then transferred to 0.5-ml chambers and perifused with Hanks’ M199 containing 25 mM HEPES, 0.1% BSA, and penicillin (100 U/ml)/streptomycin (100 µg/ml) for 2.5 h 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 always conducted with medium containing no phosphodiesterase inhibitors, whereas for cAMP measurements, experiments were done in cells perifused without (Figs. 2Go, 4Go, and 5Go) and with 1 mM 3-isobutyl-1-methylxanthine (Table 1Go). Fractions were collected in 1-min intervals and later assayed for PRL and cAMP contents using RIA. Primary antibody and standard for PRL assay were purchased from the National Pituitary Agency and Dr. A. F. Parlow (Harbor-UCLA Medical Center, Torrance, CA). cAMP was determined using specific antiserum provided by Albert Baukal (NICHD, Bethesda, MD). [125I]PRL and [125I]cAMP were purchased from PerkinElmer Life Sciences (Boston, MA).


Figure 2
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  		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.

 

Figure 4
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  		FIG. 4. Dopamine agonists inhibit calcium transients and PRL release 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 severalfold higher in forskolin-treated cells compared with untreated cells. E, Inhibition of forskolin-induced calcium transients by apomorphine, which was added in medium containing forskolin. Apo, Apomorphine; Bromo, bromocriptine.

 

Figure 5
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  		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.

 

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  		TABLE 1. Effects of dopamine agonists on basal cAMP release in perifused pituitary cells

 
Single-cell calcium measurements
For [Ca2+]i measurements, cells were incubated in Hanks’ M199, supplemented with 2 µM fura-2 AM (Molecular Probes, Eugene OR) at 37 C for 60 min. Coverslips with cells were then washed and mounted on the stage of an Axiovert 135 microscope (Carl Zeiss, Oberkochen, Germany) attached to the Attofluor Digital Fluorescence Microscopy System (Atto Instruments, Rockville, MD). Cells were examined under a x40 oil immersion objective during exposure to alternating 340- and 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 in [Ca2+]i, was followed in several single cells simultaneously at the rate of one point per second.

Simultaneous recording of [Ca2+]i and membrane potential (Vm)
Pituitary cells were incubated for 15 min at 37 C in phenol red-free medium 199 containing Hanks’ salts, 20 mM sodium bicarbonate, 20 mM HEPES, and 0.5 µM indo-1 AM (Molecular Probes). The Vm and bulk [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 Digidata 1200 A/D interface in conjunction with Clampex 8 (Molecular Devices, Sunnyvale, CA). The [Ca2+]i was calibrated in vivo according to Kao (35).

Western blot analysis
Total proteins were isolated with M-PER mammalian protein extraction regents (Pierce, Rockford, IL), and protein concentration was determined using a BCA kit (Pierce) according to the manufacturer’s protocol. Total proteins were separated on 12.5% SDS-PAGE and transferred onto polyvinylidene difluoride membrane. The membrane was blocked with Tris-buffered saline with Tween 20 buffer [10 mM Tris-Cl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20] containing 5% BSA fraction V and then incubated with diluted polyclonal phospho-Akt antibodies (1:2000; Cell Signaling, Boston, MA) overnight. After that, positive signals of individual blots were visualized by incubating the membrane with peroxidase-conjugated goat antirabbit secondary antibody (1:10,000; KPL, Gaithersburg, MD) and subsequent treatment with SuperSignal West Pico Luminol/Enhanced solution (Pierce). The total 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 1Go and 2Go). 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. 1Go), or from pooled data from several experiments (Tables 1Go and 2Go). Significant differences with P < 0.01 were determined by Student’s t test.


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  		TABLE 2. Effects of dopamine agonists on basal PRL release in perifused pituitary cells

 

Figure 1
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  		FIG. 1. Dependence of rapid dopamine-induced inhibition of prolactin (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.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dopamine inhibits release of prestored PRL
To clarify the extent to which the prestored PRL contributes to dopamine-controlled hormone release, static cultures of pituitary cells were treated with 50 µg/ml cycloheximide, a protein synthesis inhibitor. As shown in Fig. 1AGo, basal PRL release was not obviously affected by cycloheximide treatment during the first 2 h incubation but was significantly attenuated at longer incubation periods. In parallel experiments, 1 µM dopamine added to cells in static cultures effectively stopped PRL release 30 min after application and kept it low for 8 h (Fig. 1BGo). Effects of cycloheximide and dopamine on PRL release were also evaluated in perifused pituitary cells loaded in temperature-controlled chambers. Figure 1CGo shows that the obvious inhibitory effect of cycloheximide on basal PRL release was observed during treatments 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 washout of agonist was accompanied by the full recovery of basal secretion (Fig. 1DGo). Dopamine was fully effective in 1 and 0.5 µM concentrations, whereas at lower concentration, only partial 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 µM 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 extracellular and intracellular cAMP levels in both perifused and pituitary cells in static cultures, indicating that the measurement of extracellular 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 2Go illustrates such an experiment. Like dopamine (Fig. 1DGo), the dopamine agonists bromocriptine (Fig. 2AGo) and apomorphine (Fig. 2CGo) 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. 2Go, B and D). This finding could indicate that dopamine-induced down-regulation of basal AC activity accounts for or contributes to inhibition of PRL release.

Simultaneous measurements of electrical activity and intracellular calcium in perforated cells revealed that apomorphine promptly abolished spontaneous firing of action potentials and accompanied Ca2+ transients (Fig. 3BGo). TRH also inhibited spontaneous electrical activity but transiently, followed 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 hyperpolarization coincided with the spike response in [Ca2+]i, whereas the sustained spiking coincided with the plateau rise in [Ca2+]i (Fig. 3AGo). The inhibitory effect of apomorphine (Fig. 3CGo) and bromocriptine (Fig. 3DGo) on spontaneous Ca2+ transients was also observed in intact cells loaded with fura-2. In these experiments, TRH was applied at the end of 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).


Figure 3
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  		FIG. 3. Dopamine agonists inhibit spontaneous electrical activity and calcium transients in single cells. A and B, Simultaneous measurements of electrical activity and calcium signals in perforated cells with activated TRH (A) and dopamine (B) receptors. Horizontal black bars indicate duration of TRH and apomorphine applications. C and D, Inhibitory effect of apomorphine (C) and bromocriptine (D) on spontaneous calcium transients in intact cells. For this and the following figures, TRH was applied at the end of recording to identify lactotrophs. Agonists were present in incubation medium from the moment of their application (indicated by arrows) to the end of recording.

 
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. 4Go, A and C). Measurements of PRL content in the same samples revealed that the increase in cAMP production was accompanied by stimulation of PRL release (Fig. 4Go, B and D). Forskolin treatment also elevated [Ca2+]i in single lactotrophs (Fig. 4EGo). Application of bromocriptine and apomorphine attenuated AC activity in the presence of forskolin, but the residual cAMP production was severalfold higher than in untreated cells (Fig. 4Go, A and C; basal = 82 ± 7, forskolin = 1377 ± 203, and forskolin plus apomorphine = 416 ± 33 pmol/min; (n = 4). At such elevated cAMP levels, both agonists inhibited PRL release (Fig. 4Go, B and D) in a manner highly comparable to that observed in controls (Fig. 2Go, A and C). Apomorphine also abolished stimulatory effect of forskolin on Ca2+ transients in a majority of TRH-responsive cells (Fig. 4EGo). In the absence of apomorphine, there was a sustain elevation in cAMP production, VGCI, and PRL release in forskolin-treated cells (24). These results indicate that inhibition of VGCI alone is sufficient to block rapid PRL release.

Dopamine inhibits Ca2+ transients and PRL release in cells with inhibited inward rectifier K+ (Kir) channels
The role of dopamine in activation of Kir channels in pituitary lactotrophs is well established (19, 20). Extracellular Cs+ in 2–5 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. 5AGo) and apomorphine-induced (Fig. 5CGo) inhibition of PRL release. In the presence of Cs+, agonist-induced inhibition of AC activity was also preserved (Fig. 5Go, 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. 5EGo). The same effects were also observed in the presence of 2 mM Cs+ (data not shown). Dopamine-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 µM dopamine, perifused pituitary cells released 8.2 ± 1.3 ng/min in the absence of Ba2+ 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 1Go). The inhibitory effect of apomorphine and bromocriptine on spontaneous Ca2+ transients was lost in PTX-treated cells (Fig. 6Go, A and B). However, the same PTX treatment could exert only a partial loss of inhibitory effects of bromocriptine and apomorphine on PRL release (Fig. 6Go, C and D, and Table 2Go). To examine the possibility that the PTX dose used in these experiments was not sufficient to fully block Gi/o signaling pathway, we performed dose-dependent studies. As shown in Fig. 6EGo, no further reduction in effectiveness of dopamine was observed in cells in static cultures treated with PTX in 0.5 and 1 µg/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.


Figure 6
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  		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 apomorphine (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 2Go. E, Dose-dependent effects of PTX on basal and dopamine (DA)-induced inhibition of PRL release in static cultures of pituitary cells. In all experiments, cells were treated with PTX overnight. In A–D, cells were treated with 250 ng/ml PTX.

 
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. 7AGo, depolarization-induced Ca2+ influx was reduced by 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. 7BGo), confirming the hypothesis that dopamine can also inhibit PRL release in a Ca2+-independent manner.


Figure 7
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  		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 number 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.

 
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, β-arrestin-dependent pathways, the latter being discovered recently by Caron’s group (30). The emerging components of this pathway include Akt (a member of serine/threonine kinase family), protein phosphatase-2A, and glycogen synthase kinase (GSK) (39). Via its pleckstrin homology domain, Akt is regulated 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. 8CGo). Lithium antagonizes dopamine-dependent 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. 8Go, A and C) and with (Fig. 8BGo) 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. 9Go, left panel). On the other hand, Li+ stimulated Akt phosphorylation (Fig. 9Go, center panel), which is consistent with observations in other cell types (41). Akt is also present in pituitary cells, but we could not observe the presence of phosphorylating forms of this protein, suggesting that in our experimental conditions, dopamine cannot signal through the Akt/β-arrestin signaling complex by dephosphorylating Akt on Thr308.


Figure 8
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  		FIG. 8. Dopamine-induced inhibition of basal PRL release in cells with inhibited PI3-kinase and Akt/β-arrestin signaling pathways. A 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 cells without (A) and with (B) blocked Gi/o signaling pathway (250 ng/ml PTX, overnight). Traces shown are representative from three similar experiments. C, The lack of effects of 1 µM wortmannin (WT), an inhibitor of PI3-kinase, and 3 mM Li+, on dopamine (DA)-induced inhibition of basal PRL release. Traces shown are pooled data from five independent experiments. D, Effects of acute treatment with 100 nM PMA on 1 µM dopamine (DA)-induced inhibition of PRL release in cells with and without blocked Gi/o signaling pathway. PMA was applied 30 min before and during application of dopamine. Traces shown are representative from three similar experiments.

 

Figure 9
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  		FIG. 9. Wortmannin and lithium sensitivity of Akt phosphorylation. Western blot showing the relative levels of total and phospho-Akt (Ser-473 and Thr-308) in extracts from HEK293 and pituitary cells. A phospho-independent antibody was used as a loading control. HEK293 cells were treated with wortmannin (WT) for 10 min only (left panel) or first with 1 µM wortmannin for 10 min and then with 3 mM Li+ for an additional 10 min (center panel). Pituitary cells were treated with 1 µM wortmannin for 10 min, followed by treatment with 1 µM dopamine (DA) or 100 ng/ml epidermal growth factor (EGF) for 5 min.

 
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{alpha} subunits (42, 43). As shown in Fig. 8DGo, the inhibitory effect of dopamine on PRL secretion was dramatically reduced in PMA-treated cells with operative Gi/o signaling pathway. Dopamine-induced inhibition of secretion was further reduced in PTX- plus PMA-treated cells (Fig. 8DGo). These results are consistent with the possibility that Gz protein-dependent signaling pathway accounts for the PTX-insensitive component in dopamine-induced rapid inhibition of PRL release.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The fusion of secretory vesicles with the plasma membrane is termed regulated exocytosis and leads to release of hormones from endocrine cells and neurotransmitters from neurons (12). Exocytosis is mediated by complex protein machinery, which is conserved in organisms ranging from yeast to mammals and includes docking, ATP-dependent priming and fusion of vesicle membranes through interactions of proteins and their modulations by intracellular messengers (44, 45, 46). Calcium is the primary intracellular signaling molecule controlling hormone release. An increase in [Ca2+]i is required for two steps in regulated exocytosis: the priming of secretory vesicles, which occurs on the time-scale of tens of seconds, and triggering the fusion, which occurs within 1–2 sec (12). Both the depolarization-driven Ca2+ entry (47) and Ca2+ mobilization from intracellular stores (48) can trigger the fusion of primed secretory vesicles. Other signaling pathways, including cAMP/protein kinase A (23, 49), diacylglycerol/protein kinase C (50, 51, 52), and phosphatidylinositol bisphosphate/PI3-kinase (46, 53), also contribute to the control of exocytosis.

Lactotrophs exhibit spontaneous firing of action potentials, 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, spontaneous, or basal and in vivo can be achieved by removing the hypothalamo-pituitary connection or by transplantation of pituitary glands under the kidney capsule (2). Slow spontaneous secretion from single lactotrophs could be monitored with confocal microscopy using FM4-64-loaded cells (54, 55). In vivo and in vitro, basal PRL release is modulated by numerous G protein-coupled receptors; it is inhibited by dopamine, somatostatin, and endothelin receptors and stimulated by TRH, pituitary adenylate cyclase-activating peptide, vasopressin-oxytocin, galanin, and angiotensin II receptors (1). The parallelism we observed in the actions of dopamine on cAMP/Ca2+ signaling and secretion is in full agreement with the literature, describing inhibition of AC (10, 22) and Cav channels (15, 56) and activation of Kir channels, which in turn inhibits VGCI (20). These findings suggest that basal PRL release could reflect changes in intracellular concentrations of cAMP and Ca2+.

Here we confirmed the PTX-sensitive coupling of dopamine receptors to ACs as well as the PTX-sensitive inhibition of VGCI, suggesting the major role of Gi/o signaling pathway in control of these two intracellular messengers. Our results indicate that dopamine agonists can inhibit PRL secretion not only when the coupling of these receptors to AC is attenuated but also when the activity of this enzyme is elevated. We further show that Kir channels contribute to the control of basal [Ca2+]i and PRL release, a finding consistent with observations in GH3 cells (38), but that dopamine-induced inhibition of spontaneous VGCI was preserved in cells bathed in the presence of blockers of these channels. Thus, dopamine-induced inhibition of the Cav channel gating alone is sufficient to block spontaneous Ca2+ influx. Finally, we show that dopamine receptors can inhibit PRL release independently of the status of VGCI, at least in part. This was experimentally achieved by PTX treatment, which effectively blocked dopamine-induced inhibition of spontaneous and depolarization-induced VGCI but was unable to abolish dopamine-induced inhibition of PRL release at elevated [Ca2+]i levels.

At the present time, we do not know by which mechanism dopamine inhibits PRL release in cells with blocked PTX-sensitive signaling pathway. In addition to Gi/o signaling pathway, dopamine receptors also signal through a recently discovered signaling complex composed of Akt, protein phosphatase-2A, and β-arrestin (30). Formation of this complex leads to inactivation of Akt, mediated by protein phosphatase-2A-induced dephosphorylation of Thr-308, and activation of GSK-3 (39). PI3-kinase affects Akt via its pleckstrin homology domain (40). In our hands, however, wortmannin did not influence dopamine-induced inhibition of PRL release. In the mouse striatum, dopamine-stimulated dephosphorylation of Akt leads to a reduction in kinase activity and activation of GSK-3, and Li+ antagonizes dopamine-dependent effects mediated by Akt/GSK-3 signaling cascade (41). In perifused pituitary cells with and without blocked Gi/o signaling pathway, we observed no effects of extracellular Li+ on dopamine- and bromocriptine-induced inhibition of PRL release. Furthermore, we were unable to observe the presence of phosphorylated forms of Akt in resting cells. These observations argue against the role of β-arrestin-Akt signaling pathway in rapid inhibition of PRL release by dopamine, but more work is required to clarify the potential role of this G protein-independent signaling pathway on D2 dopamine receptor-mediated regulation of PRL synthesis and sustained release. In that respect, it is of relevance to mention that Akt regulates PRL promoter activity (32) and that the novel signaling pathway of dopamine receptors is operative during prolonged stimulation of the D2 class of receptors (41).

Anterior pituitary cells also express Gz{alpha} (29), and the coupling of dopamine receptors (including the D2 subtype) with these proteins is well established (26, 27, 28). Consistent with this finding, we observed the presence of PTX-sensitive and -insensitive components in dopamine-induced inhibition of basal AC activity. Furthermore, the heterotrimeric Gz proteins provide a pathway through which endothelin receptors can stop PRL release for a prolonged period of time. Two lines of evidence supported this conclusion. First, down-regulating the Gz{alpha} expression led to abolition of the inhibitory phase in PRL release. Second, in experiments with phorbol esters, which silence the Gz signaling pathway through protein kinase C-dependent phosphorylation of the Gz{alpha} subunits (42, 43), the inhibitory action of endothelin-1 was attenuated (29). In parallel to that, the PTX-insensitive dopaminergic inhibition of PRL release was also remarkably attenuated in cells treated with phorbol esters. These results are consistent with a hypothesis that dopamine inhibits PRL release through the Gz signaling pathway, but additional work is required to demonstrate it.

The PTX-insensitive inhibition of PRL release by dopamine could reflect desensitization of Ca2+-secretion coupling, as indicated by the ability of dopamine to inhibit PRL release in PTX-treated cells, which showed no inhibition of spontaneous electrical activity. In the case of endothelin receptors, both arms of Gz proteins, {alpha} and β{gamma}, were suggested to contribute to inhibition of PRL release downstream of VGCI (57). The contribution of Gz{alpha}-mediated attenuation of AC activity in pituitary cells appeared not to be sufficient in dopamine-induced rapid inhibition of PRL release, because such inhibition was preserved in forskolin-treated cells. Thus, liberation of Gzβ{gamma} may represent the primary factor in dopamine-induced inhibition of Ca2+-secretion coupling. Such a conclusion is in accordance with the finding in other cell types about the role of Gβ{gamma} subunits in control of secretion independently of their actions on AC, phospholipase C-β2, and several tyrosine kinases (58). Recent experiments by Martin’s group in permeabilized PC12 cells also demonstrated that G protein β{gamma} directly regulates soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein fusion machinery to modulate secretory granule exocytosis. This action occurs rapidly and affects ATP-primed secretory vesicles (59).

The presence of the PTX-insensitive step in dopamine action on PRL release does not argue against the relevance of AC, Kir channels, and Cav channels in this process in vivo but demonstrates that pharmacological exclusion of a single pathway does not provide a valid experimental model for a system simultaneously controlled by multiple pathways. In vitro, dopamine-mediated control of VGCI is more than sufficient to block PRL release, as is shown in experiments with removal of extracellular Ca2+ (13, 14). The triple control of VGCI by inhibiting AC and Cav and activating Kir channels, combined with the mechanism for desensitization of Ca2+-secretion coupling, not only secures inhibition of PRL release but also provides the possibility for maintaining such inhibition when cells are exposed to multiple agonists. For example, the Gz signaling pathway probably contributes to the control of PRL release by dopamine when the electrical activity in lactotrophs is stimulated by other factors.

In conclusion, here we show that dopamine receptors activate multiple signaling pathways in lactotrophs and that such redundancy reinforces the blockade of basal PRL release (Fig. 10Go). Our results indicate that dopamine rapidly reduced basal cAMP production, a second messenger involved 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 action 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 channels, 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 mechanism by which dopamine inhibits PRL secretion downstream of VGCI.


Figure 10
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  		FIG. 10. Activation of multiple signaling pathways by dopamine-2 receptors in lactotrophs. Arrows indicate stimulatory actions, and circles indicate inhibitory actions.

 


   Acknowledgments
 
We are thankful to Dr. Fredrick Van Goor for electrophysiological measurements and Dr. Tamas Balla for help with Akt experiments.


   Footnotes
 
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 nothing to declare.

First Published Online December 20, 2007

1 A.E.G.-I. and T.M. contributed equally to this work. Back

Abbreviations: AC, Adenylyl cyclase; [Ca2+]i, intracellular calcium concentration; Cav, voltage-gated Ca2+; GSK, glycogen synthase kinase; Kir, inward rectifier K+; PI3, phosphoinositide 3; PMA, phorbol 12-myristate 13-acetate; PRL, prolactin; PTX, pertussis toxin; VGCI, voltage-gated Ca2+ influx; Vm, membrane potential.

Received July 18, 2007.

Accepted for publication December 10, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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