Differential inhibition of dopamine and bromocriptine on induced prolactin release: multiple sites for the inhibition of dopamine.

Effects of dopamine and bromocriptine on TRH- or dibutyryladenosine 3',5'-cyclic monophosphate (dbcAMP)-induced prolactin release from primary cultured rat pituitary cells were studied using a perifusion system. TRH (100 nmol/l) stimulated prolactin release from basal concentrations of 33.8 +/- 0.5 to 151.2 +/- 28.0 ng/ml (net increase) or 447% increase. Dopamine inhibited the basal release of prolactin throughout the experiment, but TRH (100 nmol/l) was still able to stimulate prolactin release under the influence of dopamine. The increment in prolactin release was inversely proportional to the dopamine concentration. When TRH (100 nmol/l) was introduced during a perifusion period with bromocriptine 1 nmol/l, the prolactin concentration was increased to 110.9% of basal levels. The stimulatory effect of TRH under the influence of bromocriptine (1 nmol/l) was significantly lower than that without bromocriptine (control), although the higher concentrations of bromocriptine (10 and 100 nmol/l) did not further reduce the peak concentration of TRH-induced prolactin release. During a perifusion period with a low concentration of dopamine (1 nmol/l plus 0.1 mmol/l ascorbic acid), introduction of dbcAMP (3 mmol/l) stimulated prolactin release to 48% of basal concentration. A higher concentration of dopamine further reduced the stimulatory effect of prolactin release. Bromocriptine impeded the stimulatory effect of dbcAMP (3 mmol/l) on prolactin release in a similar manner as dopamine. Since a higher concentration of bromocriptine (10 and 100 nmol/l) did not further inhibit the TRH-induced prolactin release whereas a higher concentration of dopamine did, it is concluded that dopamine acts through additional mechanism(s) other than the D2 receptor transduction system.     

Actions of dopamine on prolactin secretion and cyclic AMP metabolism in ovine pituitary cells

Prolactin secretion from cultured sheep pituitary cells was inhibited by low concentrations of dopamine (0.1 nM-0.1 microM) with a half-maximal effect at 3 nM. At a maximally effective dose (0.1 microM) dopamine significantly inhibited prolactin secretion within 5 min. with an 80% inhibition of basal secretion over 2 h. Basal prolactin secretion was stimulated by the addition of methylisobutylxanthine (MIX) (0.3-1.0 mM) and 8-bromo-cyclic AMP (2 mM), but cholera toxin (3 micrograms/ml) and prostaglandin E2 (0.1-1.0 microM), which also raised cellular cyclic AMP levels, had no effect on prolactin release. The inhibition of prolactin release by dopamine (0.1 microM) was not affected by any of these compounds. Dopamine inhibited MIX-induced cyclic AMP accumulation over a similar concentration range to the inhibition of secretion, but had no effect on the changes in cyclic AMP concentration produced by cholera toxin and prostaglandin E2. Overall the results with sheep pituitary cells suggest that lowered cyclic AMP levels do not mediate the inhibitory effects of dopamine on basal prolactin secretion, but that changes in cellular cyclic AMP levels may alter the secretion of this hormone, and dopamine may affect pituitary cell cyclic AMP concentrations in some circumstances.     

Effect of reserpine on the inhibition of prolactin released from different pituitary constructs in vitro by dopamine, bromocriptine and apomorphine.

Hourly release of Prolactin by pituitary constructs 1 whole pituitary (PI), adenohypophysis (P-N) and pituitary-hypothalamus co-incubate (PHC) were compared. Adenohypophysis secreted significantly more prolactin than PI and PHC, while PHC secreted significantly less than PI. Co-incubation of (P-N) with posterior pituitary reduced the elevated secretion of prolactin. Addition of dopamine (10(-7) M), bromocriptine (10(-7) M) and apomorphine (5 x 10(-8) M) to these constructs did not affect the release of prolactin from PI but inhibited the same from (P-N) and PHC. Treatment with reserpine increased serum prolactin levels but intrapituitary prolactin contents were decreased. Hourly release of prolactin from pituitary constructs derived from reserpine-treated rats was significantly reduced as compared to ascorbic acid--treated controls. Inclusion of dopamine (10(-7) M), bromocriptine (10(-7) M) and apomorphine (5 x 10(-8) M) in these constructs inhibited prolactin secretion further. In vitro addition of perphenazine stimulated the release of prolactin by PHC but was without any effect on PI and (P-N). The data are interpreted to suggest that dopamine in posterior pituitary may be an important determinant of hypothalamic modulation of prolactin secretion.     

Involvement of calcium ions in dopamine inhibition of prolactin secretion from sheep pituitary cells

The involvement of calcium in the regulation of prolactin secretion and a possible inhibitory mechanism of action for dopamine have been investigated. Basal prolactin secretion from cultured ovine pituitary cells was dependent on the concentration of calcium ions (Ca²⁺) in the medium and was inhibited by the presence of verapamil (10 μM). The divalent cation ionophore A23187 (1 μM) caused a rapid stimulation of prolactin release from the cells. The effect was essentially complete within 10 min and subsequently secretion of prolactin occurred at close to the basal rate. A23187 had no effect on cell cyclic AMP levels. Dopamine (0.1 μM) but not verapamil (10 μM) inhibited the A23187 (10 μM) induced release of prolactin. Inhibition of basal and A23187 (1 μM) stimulated prolactin secretion occurred over a similar range of dopamine concentrations. The dopamine receptor antagonist haloperidol (1 μM) reversed the inhibitory effect of dopamine (0.1 μM) on A23187-stimulated prolactin release. These results provide evidence to support the concept that control of Ca²⁺ handling by lactotrophs may be of fundamental importance in the regulation of prolactin secretion.     

Demonstration of specific dopamine receptors on human pituitary adenomas

Dopamine receptors on human pituitary adenoma membranes were characterized using [3H] spiperone as the radioligand. The specific [3H]spiperone binding sites on prolactin (PRL)-secreting adenoma membranes were recognized as a dopamine receptor, based upon the data showing high affinity binding, saturability, specificity, temperature dependence, and reversibility. All of 14 PRL-secreting adenomas had high affinity dopamine receptors, with a dissociation constant (Kd) of 0.85 +/- 0.11 nmol/l (mean +/- SEM) and a maximal binding capacity (Bmax) of 428 +/- 48.6 fmol/mg protein. Among 14 growth hormone (GH)-secreting adenomas examined, 8 (57%) had dopamine receptors with a Kd of 1.90 +/- 0.47 nmol/l and a Bmax of 131 +/- 36.9 fmol/mg protein. Furthermore, 15 of 24 (58%) nonsecreting pituitary adenomas also had dopamine receptors with a Kd of 1.86 +/- 0.37 nmol/l and a Bmax of 162 +/- 26.0 fmol/mg protein. These results indicate that some GH-secreting adenomas as well as some nonsecreting pituitary adenomas contain dopamine receptors. But their affinity and number of binding sites are significantly lower (P less than 0.05) and fewer (P less than 0.001) respectively, than those in PRL-secreting adenomas.     

The functional state of the dopamine receptor in the anterior pituitary is in the high affinity form

In order to determine whether the high affinity state or the low affinity state of the dopamine receptor mediated the inhibition of release of PRL by dopamine agonists, a large number of dopaminergic agonists (n = 31) and antagonists (n = 24) were tested for their potencies to inhibit the binding of [3H] spiperone to porcine anterior pituitary tissue, and for their potencies to affect the release of PRL from rat anterior pituitary cells in culture. All agonists (except bromocriptine, ergocryptine, and dihydroergocryptine) inhibited [3H]spiperone binding in two phases: one phase occurred at nanomolar or subnanomolar concentrations (representing the high affinity state of the dopamine receptor) and the other phase occurred at much higher concentrations of agonist (the low affinity state of the dopamine receptor). The dissociation constants (K) for each drug at each state were derived by computer, with the program LIGAND. It was observed that the agonist K values for the high affinity state were virtually identical with those agonist concentrations inhibiting PRL release; the K values for the low affinity state were about 2 orders higher. These data suggest that the high affinity state of the D2 dopamine receptor is the functional state which mediates the inhibition of PRL release.     

Differences in the interaction between dopamine and estradiol on prolactin release by cultured normal and tumorous human pituitary cells

We studied the interaction between dopamine and estradiol on PRL release by cultured normal and tumorous PRL-secreting cells prepared from human pituitaries. If pituitary glands were obtained within 3 h after sudden death of previously normal individuals, the viability of isolated pituitary cells prepared by dispersion with dispase was more than 75%. After 4 days of culture, dopamine (500 nM) inhibited PRL release by cells prepared from four normal pituitaries by 24 +/- 3% (+/- SEM). Pretreatment of the cells with 100 nM estradiol did not alter dopamine-mediated inhibition of PRL release. Estradiol alone increased basal PRL release and cell PRL content. Cultured PRL-secreting pituitary tumor cells, obtained by transsphenoidal operation from four patients, were similarly sensitive to dopamine. Estradiol stimulated tumor cell PRL release and content, but significantly diminished the inhibitory effect of dopamine. The estrogen receptor blocker tamoxifen did not alter PRL release, but it did reverse the estradiol-induced insensitivity of the prolactinoma cells to the dopamine agonist bromocriptine. In conclusion, these in vitro results indicate that estrogens do not antagonize the effect of dopamine on normal human PRL-secreting pituitary cells. In human pituitary tumor cells, however, estradiol decreased the sensitivity of PRL release to dopamine (agonists), and the estrogen action can be acutely reversed by tamoxifen.     

Actions of pertussis toxin on the inhibitory effects of dopamine and somatostatin on prolactin and growth hormone release from ovine anterior pituitary cells

Forskolin and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate stimulate prolactin and GH release from ovine anterior pituitary cells cultured in vitro. Dopamine and somatostatin inhibit release of prolactin and GH respectively, after stimulation by these agents, but without effects on intracellular cyclic AMP concentrations. In each case the inhibitory effects were reversed by pretreatment of cells with pertussis toxin, in a dose-related fashion (1-100 ng/ml), again without affecting cyclic AMP levels. The results suggest that the inhibitory effects of dopamine and somatostatin in this system are mediated by one or more pertussis toxin-sensitive G proteins, and that these act by a mechanism which does not involve inhibition of adenylate cyclase.     

Serotonin and dopamine receptors in the rat pituitary gland: autoradiographic identification, characterization, and localization

S2 serotonin and D2 dopamine receptors were identified, characterized, and localized in rat pituitary gland by quantitative light microscopic autoradiography. [3H]Spiperone was used to localize S2 serotonin and D2 dopamine receptors. A high concentration of D2 dopamine receptors [1 microM 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (ADTN)- or sulpiride-displaceable [3H]spiperone binding] was found in the rat intermediate lobe with much lower concentrations present in the anterior and posterior lobes. Significant densities of cinanserin-displaceable [3H]spiperone binding sites (i.e. S2 serotonin receptors) were present in all three lobes of the pituitary gland. [125I]Lysergic acid ([125I]LSD) was used to characterize further and selectively visualize S2 serotonin receptors in the rat pituitary. Data analysis by densitometry showed that [125I]LSD binding the rat intermediate pituitary was saturable and of high affinity with an apparent dissociation constant (Kd) of 1.2 nM. Data from competition studies using a variety of compounds showed a S2 serotonin receptor profile at this [125I]LSD binding site in rat pituitary. The highest concentration of [125I]LSD binding sites was found in the intermediate lobe with progressively lower concentrations present in the posterior and anterior lobes, respectively. There is a uniform pattern of distribution of S2 serotonin and D2 dopamine receptors within each lobe of the rat pituitary gland. The results of the present study provide the first identification of S2 serotonin receptors in the pituitary and confirm the heterogeneous distribution of D2 dopamine receptors within the rat pituitary. These data provide further evidence for the importance of dopamine in regulating pituitary function and suggest a physiological role for serotonin in regulating pituitary hormone secretion.     

Suppression of prolactin release and mRNA accumulation by two novel dopamine agonist agents

Two novel dopamine agonist drugs, CV 205-502 and CQP 201-403, have been investigated to compare their effects on prolactin secretion and prolactin mRNA accumulation in cultured rat pituitary cells. Both drugs gave dose-dependent suppression of prolactin release over a 24 h incubation period: when each drug was used at 100 nmol/l CV 205-502 and CQP 201-403 induced suppression to 8.9 +/- 1.7 and 10.2 +/- 1.8% of control release, respectively, compared to 26.7 +/- 4.8% of control with 100 nmol/l bromocriptine. There was no consistent effect on growth hormone release. Cytoplasmic accumulation of prolactin mRNA was also inhibited by both drugs at this concentration, to 50.2 +/- 5.5% of control values by CV 205-502 and to 67.4 +/- 8% of control by CQP 201-403, and to a similar extent by 100 nmol/l bromocriptine (50.6 +/- 9.1% of control). None of the drugs had any significant effect on GH mRNA levels. These data suggest that the agents exert their effect at a pretranslational stage of prolactin synthesis, as well as at the level of hormone release.     

NON-FUNCTIONING PITUITARY ADENOMAS DO NOT REGRESS DURING BROMOCRIPTINE THERAPY BUT POSSESS MEMBRANE-BOUND DOPAMINE RECEPTORS WHICH BIND BROMOCRIPTINE

Dopaminergic binding to membranes from 20 well-characterized non-functioning pituitary tumours was investigated using the radioligand [3H]spiperone; nine had failed to regress during pre-operative bromocriptine therapy. Five macroprolactinomas and six normal pituitaries were similarly investigated. High affinity dopaminergic binding sites were defined in all tissues studied. Mean dissociation constants were similar for the three groups (0.92, 0.55 and 0.51 nmol/l, respectively) but mean site numbers were greater in the prolactinomas (698) than in non-functioning tumours (131) or normal pituitaries (136 fmol/mg protein). In a pool of non-functioning tumour membranes ligand competition experiments confirmed that binding was dopaminergic. Stereospecificity was demonstrated using the (+) and (-)isomers of butaclamol. Bromocriptine was present in three non-functioning tumours that had been exposed to the drug within 24 h of surgery and it largely prevented [3H]spiperone binding; membrane washing restored [3H]spiperone binding to control values. We conclude that non-functioning tumours possess high affinity membrane-bound dopaminergic binding sites similar to those in normal pituitary and macroprolactinomas, but apparently fewer in number than in the latter. Though bromocriptine binds to dopamine receptors on non-functioning tumours in vivo, this does not result in tumour regression.     

Regulation of intermediate pituitary corticotropin-releasing hormone receptors by dopamine.

It has been shown that chronic cold exposure results in selective CRH receptor up-regulation in the intermediate pituitary. Since the intermediate pituitary is under dopaminergic control, the participation of a dopaminergic mechanism in the effect of cold stress was studied in rats treated with dopaminergic agonists and antagonists. CRH receptors were measured by the binding of radioiodinated Tyr-ovine (o) CRH to neurointermediate pituitary membranes of slide-mounted sections. Cold exposure for 60 h caused the expected increase in CRH binding in neurointermediate lobe membranes. Administration of the dopaminergic agonist bromocriptine did not prevent the effect of cold stress, but increased CRH binding in control rats. The dopaminergic antagonist metoclopramide decreased intermediate pituitary CRH binding in control and cold-exposed rats. Bromocriptine administration for 1-8 days caused a progressive increase in the binding of [125I]Tyr-oCRH in neurointermediate pituitary membranes, despite atrophy of the intermediate zone. Scatchard analysis of the binding data indicated that the changes were due to variations in receptor concentration, without changes in affinity. No changes in anterior pituitary CRH receptors were observed with agonist or antagonist treatment. Autoradiographic analysis of CRH binding after 3 days of treatment with bromocriptine or haloperidol confirmed the results observed in membranes and demonstrated that changes in binding were confined to the intermediate lobe. The functional consequences of the changes in CRH binding were studied by analysis of adenylate cyclase activity in cells and homogenates of intermediate pituitaries of rats treated with bromocriptine. In 18-h cultured intermediate pituitary cells from rats treated with bromocriptine for 3 days, CRH-stimulated cAMP production, measured in the presence of phosphodiesterase inhibitors, was increased to levels only slightly higher than those in cells from control rats. Likewise, CRH-stimulated adenylate cyclase, measured by conversion of [32P]ATP to [32P] cAMP, was not significantly different in homogenates from microdissected intermediate lobes from control and bromocriptine-treated rats. The lack of parallel changes in adenylate cyclase responsiveness suggests only partial receptor coupling, probably reflecting an inhibitory effect of dopamine on components of the adenylate cyclase. This study demonstrates that in contrast to the recognized inhibitory effect on cell division and POMC mRNA expression, dopamine causes up-regulation of CRH receptors in the intermediate pituitary. The qualitatively similar and nonadditive effects of cold stress and dopaminergic agonists suggest that a dopaminergic mechanism may be involved in intermediate pituitary CRH receptor regulation during chronic cold stress.     

Dopamine agonists both stimulate and inhibit prolactin release in GH4ZR7 cells.

Prolactin secretion from the anterior pituitary gland is regulated by multiple factors including prolactin-release inhibiting factors (PIFs) and prolactin releasing factors. PIFs, however, usually dominate to exert a tonic inhibition in the biological system, and the physiological PIF is believed to be dopamine. However, there is accumulating evidence that dopamine can not only inhibit but also stimulate prolactin release. Many investigators believe that this is achieved by activating inhibitory and stimulatory subtypes of dopamine receptors. We tried to demonstrate that one subtype of dopamine receptors is capable of both inhibiting or stimulating prolactin release using GH(4)ZR(7) cells. GH(4)ZR(7) cells express only a short form of dopamine D(2) receptors (D(2s)). Low concentrations of three well-established D(2) receptor agonists (dopamine, apomorphine and bromocriptine) stimulated prolactin release from GH(4)ZR(7) cells while high concentrations inhibited the release. Haloperidol, a D(2) receptor antagonist, blocked the inhibitory action, but was unable to block the dopamine-induced stimulatory action. Pretreatment of cells with phenoxybenzamine, a receptor alkylating agent, abolished both the dopamine-induced stimulatory and inhibitory actions. Our results support the thesis that the stimulation of prolactin release induced by dopamine is mediated through dopamine D(2s) receptors since the GH(4)ZR(7) cells have only D(2s) receptors among dopamine receptors. We have concluded that the D(2s) receptor is capable of both stimulating and inhibiting prolactin release, probably via the activation of a G(s) protein by low concentrations and a G(i) protein by high concentrations of dopaminergic agents.     

Treatment of pituitary tumors: dopamine agonists

The neurotransmitter/neuromodulator dopamine plays an important role in both the central nervous system and the periphery. In the hypothalamopituitary system its function is a dominant and tonic inhibitory regulation of pituitary hormone secretion including prolactin- and proopiomelanocortin-derived hormones. It is well known that dopamine agonists, such as bromocriptine, pergolide, quinagolide, cabergoline, and lisuride, can inhibit PRL secretion by binding to the D(2) dopamine receptors located on normal as well as tumorous pituitary cells. Moreover, they can effectively decrease excessive PRL secretion as well as the size of the tumor in patients having prolactinoma. Furthermore, dopamine agonists can also be used in other pituitary tumors. The major requirement for its use is that the tumor cells should express D(2) receptors. Therefore, in addition to prolactinomas, targets of dopamine agonist therapy are somatotroph tumors, nonfunctioning pituitary tumors, corticotroph pituitary tumors, Nelson's syndrome, gonadotropinomas, and thyrotropin-secreting pituitary tumors. It is also an option for the treatment of pituitary disease during pregnancy. Differences between the effectiveness and the resistance of different dopaminergic agents as well as the future perspectives of them in the therapy of pituitary tumors are discussed.     

Studies of TRH-induced prolactin secretion and its inhibition by dopamine, using ovine pituitary cells

Prolactin secretion from ovine pituitary cell cultures was stimulated by thyrotropin-releasing hormone (TRH) (10(-10)-10(-7) M) with a half-maximal effect at approximately 2.5 X 10(-9) M. A maximally effective concentration of TRH produced a peak secretory response, 5-10-fold stimulation over basal release, within 15 min. Dopamine (10(-10)-10(-7) M) but not somatostatin caused a dose-related inhibition of TRH (10(-8) M) stimulated prolactin release. Both dopamine (10(-7) M) and somatostatin (10(-7) M) inhibited basal secretion from the cells. TRH did not significantly increase pituitary cell cyclic AMP levels under any of the conditions tested. Stimulation of prolactin secretion by TRH was not prevented when Ca2+ was omitted from the incubation medium. Dopamine inhibited secretion induced by TRH under low Ca2+ conditions. Our results are consistent with a hypothesis that TRH may stimulate prolactin secretion via release of intracellular Ca2+ rather than increased cellular Ca2+ uptake, and imply that dopamine inhibition involves a lowering of intracellular Ca2+ levels.     

Evidence for alteration in the processing of dopamine in the anterior pituitary gland of aged rats: receptors and intracellular compartmentalization of dopamine

Receptors for dopamine and the subcellular localization of dopamine in the anterior pituitary gland were studied in young cycling female rats and in aged, constant estrous female rats. Dopamine receptors were quantified in membrane preparations of anterior pituitary tissue using [3H] spiperone as the ligand. On the basis of saturation isotherms, it was calculated that the equilibrium dissociation constant (Kd) and binding capacity for [3H]spiperone binding to pituitary membranes from young rats were 34.2 pM and 82 fmol/mg protein, respectively. The relative binding capacity of membranes from aged rats was 35% greater than that of membranes from young rats. There was no difference in the Kd values in aged and young rats. When the relative binding of [3H]spiperone by anterior pituitary membranes from individual animals was quantified by incubation with a saturating concentration of the ligand, it was found that [3H]spiperone binding in aged rats was significantly greater than that in young rats. When the subcellular localization of dopamine in anterior pituitary tissue was examined by means of density gradient centrifugation, it was found that the subcellular distribution of dopamine in tissue of aged rats was quantitatively different from that in young rats. In young rats, a small amount of dopamine was associated with light particles, whereas a large amount of dopamine was associated with heavy particles, which cosedimented with PRL-containing granules. In aged rats, the amount of dopamine associated with light particles was 5 times that found in young rats, whereas the amount of dopamine associated with heavy particles was the same as that in young rats. We speculate that altered intracellular compartmentalization of dopamine, leading to a marked accumulation of dopamine in the light particles, is related to increased secretion of PRL in aged rats.     

Dopamine and somatostatin inhibit forskolin-stimulated prolactin and growth hormone secretion but not stimulated cyclic AMP levels in sheep anterior pituitary cell cultures

Forskolin, an activator of adenylate cyclase, has been used to investigate the effects of raising pituitary cell cyclic AMP concentrations on prolactin and growth hormone secretion and to examine the role of cyclic AMP in the inhibitory actions of dopamine and somatostatin. Incubation of cultured ovine pituitary cells with forskolin (0.1-10 microM; 30 min) produced a modest dose-related increase in prolactin release (120-140% of basal) but a much greater stimulation of growth hormone secretion (170-420% of basal). Cellular cyclic AMP concentrations were only increased in the presence of 1 and 10 microM forskolin (2-5.5 times basal). A study of the time course for forskolin (10 microM) action showed that stimulation of prolactin (1.5-fold) and growth hormone (4.7-fold) secretion occurred over 15 min; subsequently (15-60 min) the rate of prolactin secretion from forskolin-treated cells was equivalent to that measured in controls, while growth hormone release remained elevated. Cellular cyclic AMP concentrations were also rapidly stimulated by forskolin (10 microM); they reached a maximum (12 times control) within 15 min, and then declined (15-60 min) but remained elevated relative to those in untreated cells (4.9 times control at 60 min). Dopamine (0.1 microM) inhibited basal secretion of both prolactin and growth hormone. In the presence of forskolin (0.1-10 microM), dopamine (0.1 microM) inhibited prolactin secretion to below the basal level and considerably attenuated the stimulation of growth hormone secretion. Similarly, somatostatin suppressed both basal and forskolin-induced prolactin and growth hormone secretion. However, neither dopamine nor somatostatin significantly decreased the stimulatory effect of forskolin on cellular cyclic AMP accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dibutyryl cyclic AMP, adenosine and guanosine blockade of the dopamine, ergocryptine and apomorphine inhibition of prolactin release in vitro.

Rat anterior hemipituitaries were incubated in Krebs-Ringer bicarbonate containing [3H]leucine. Newly synthesized [3H]prolactin and [3H]GH in the pituitary and incubation medium were assayed, as was the radioimmunoassayable prolactin released into the medium during a 5-h incubation. Dopamine (7.5 X 10(-8)M), ergocryptine (4 X 10(-10) M) and apomorphine (6 X 10(-8)M) all significantly inhibited both radioimmunoassayable prolactin release and newly-labeled [3H]prolactin release without affecting [3H]GH release. Conversely, dibutyryl cyclic AMP (2.5 mM) stimulated radioimmunoassayable prolactin release as well as [3H]prolactin and [3H]GH release. The addition of 2.5 mM dibutyryl cyclic AMP to media containing dopamine, ergocryptine or apomorphine completely restored both radioimmunoassayable prolactin release and [3H]prolactin release to at least control levels. Dopamine, ergocryptine and apomorphine all inhibited incorporation of [3H]leucine into prolactin but not into GH, whereas 2.5 mM dibltyryl cyclic AMP with any one of the inhibitors restored total incorporation into [3H]prolactin to levels insignificantly lower than the nucleotide-stimulated incorporation. Adenosine and guanosine at 2.5 mM also stimulated incorporation into [3H]prolactin and blocked the inhibitory effects of apomorphine upon [3H]prolactin synthesis and release. These nucleosides also stimulated [3H]GH release; and guanosine, but not adenosine, stimulated incorporation into [3H]GH. The ability of dibutryl cyclic AMP to block the effects of dopamine, ergocryptine and apomorphine upon prolactin release is consistent with these three inhibitors acting by a common mechanism. Cyclic AMP could be hypothesized as a second messenger for prolactin release, but the ability of adenosine and guanosine to mimic almost perfectly the effects of this cyclic nucleotide does not allow any conclusive interpretation.     

Effects of dopamine and somatostatin on phorbol ester-stimulated prolactin and growth hormone secretion

Incubation of cultured ovine pituitary cells with the tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) (0.1-100 nM), caused a dose-related stimulation of both growth hormone (ED50 approximately 4 nM) and prolactin (ED50 approximately 14 nM) secretion. Stimulation by TPA (100 nM) produced a substantial 10-fold increase in growth hormone with a smaller, 2-fold rise in prolactin secretion over 30 min; significant effects on the release of both hormones occurred within 2 min. Treatment with TPA also produced a small, time- and concentration-dependent rise in cellular cyclic AMP content which reached, at maximum, a level 20-30% over basal values. Non-tumor-promoting phorbol esters did not stimulate the secretion of either growth hormone or prolactin. In the presence of TPA (10 nM), dopamine (1-1000 nM) suppressed prolactin secretion to a level close to that observed for maximal inhibition of unstimulated cells. At high concentrations (0.1-1.0 microM) dopamine also partially attenuated (by 43%) the TPA-induced stimulation of growth hormone secretion. Somatostatin (0.01-1.0 microM) completely inhibited the substantial (approximately 9-fold) TPA-induced stimulation of growth hormone secretion (inhibitory ED50 approximately 47 nM), and also suppressed TPA-stimulated prolactin secretion to the control level. Our results suggest that activation of protein kinase-C may be involved in the stimulatory regulation of both growth hormone and prolactin secretion in sheep pituitary cells. Failure of TPA to attenuate the inhibitory activity of dopamine and somatostatin suggests that inhibitory regulation occurs at, or beyond, the point in the secretory process regulated by protein kinase-C.     

D2 Dopamine receptor subtype mediates the inhibitory effect of dopamine on TRH-induced prolactin release from the bullfrog pituitary

Dopamine receptors in mammals are known to consist of two D1-like receptors (D1 and D5) and three D2-like receptors (D2, D3 and D4). The aim of this study was to determine the dopamine receptor subtype that mediates the inhibitory action of dopamine on the release of prolactin (PRL) from the amphibian pituitary. Distal lobes of the bullfrog (Rana catesbeiana) were perifused and the amount of PRL released in the effluent medium was measured by means of a homologous enzyme-immunoassay. TRH stimulated the release of PRL from perifused pituitaries. Dopamine suppressed TRH-induced elevation of PRL release. Quinpirole (a D2 receptor agonist) also suppressed the stimulatory effect of TRH on the release of PRL, whereas SKF-38393 (a D1 receptor agonist) exhibited no such an effect. The inhibitory action of dopamine on TRH-induced PRL release from the pituitary was nullified by the addition of L-741,626 (a selective D2 receptor antagonist) to the medium, but not by the addition of SCH-23390 (a selective D1 receptor antagonist). These data indicate that the inhibitory effect of dopamine on TRH-evoked PRL release from the bullfrog pituitary gland is mediated through D2 dopamine receptors.