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.     

Effect of GnRH-associated peptide on prolactin secretion from human lactotrope adenoma cells in culture

The effect of GnRH-associated peptide on PRL secretion by human pituitary lactotropes in culture was studied. Pituitary adenomas obtained at selective transsphenoidal adenomectomy from a patient with prolactinoma, and two patients with mixed GH- and PRL-secreting pituitary adenomas were cultured in monolayer. When cells were incubated with dopamine (10 nmol/l), a significant inhibition in PRL secretion was observed in all the experiments, which was blocked by co-incubation with haloperidol. In mixed GH- and PRL-secreting adenoma cells, dopamine likewise decreased GH secretion. Incubation of cells with synthetic GnRH-associated peptide at concentrations up to 100 nmol/l, on the other hand, failed to affect both PRL and GH secretion. These results suggest that synthetic GnRH-associated peptide has no inhibitory effect on PRL secretion in human pituitary lactotropes.     

Characterization of dopamine and estrogen interaction on primate prolactin secretion with pituitary cells cultured on extracellular matrix and with pituitary stalk-transected monkeys

To determine the direct effects of estrogen and dopamine on primate PRL secretion, cultures of dispersed monkey pituitary cells were maintained in serum-free medium on an extracellular matrix secreted by bovine corneal endothelial cells, and female monkeys with pituitary stalk-transections were prepared. Dispersed pituitary cells from male and female monkeys were cultured in a 1:1 mixture of Dulbecco's Modified Eagle's Medium H-16 and Ham's F-12 medium containing insulin, transferrin, selenium, and cadmium with or without 10.0 nM estradiol. When possible, a sister culture was established in 10% charcoal-treated fetal calf serum with or without estrogen. On days 4, 8, and 12 after plating, the incubation medium was replaced with identical medium containing increasing concentrations of dopamine with ascorbate for 6 h. Medium PRL concentrations were determined by RIA. Half-maximal inhibition (IC50) of PRL secretion occurred at about 10.0 nM dopamine with all treatments. Dopamine suppressed PRL secretion to a greater extent on day 4 when estrogen was present in the serum-free medium. There was no difference in the percent inhibition of PRL on days 8 and 12, as the serum-free cultures without estrogen appeared to increase in sensitivity. In cultures maintained in 10% charcoal-treated serum, estrogen had little effect on the dose-response inhibition of PRL on days 4 and 8, but by day 12, there was significantly less inhibition at the higher doses of dopamine. Dopamine infusion in pituitary stalk-transected (SS) monkeys caused a dose-related inhibition of PRL secretion. Serum PRL levels decreased 80% with 100 nM dopamine, and the IC50 occurred with 10 nM dopamine. In SS monkeys receiving 2 months of estrogen treatment, dopamine infusion inhibited PRL secretion to a greater extent at the lowest dose of dopamine, although the IC50 did not change. These experiments suggest that estrogen does not decrease the effectiveness of dopamine as an inhibitor of PRL secretion in SS monkeys or from primate pituitary cells cultured for 12 days in serum-free medium. Extended maintenance of cells in medium containing serum may alter this response to dopamine.     

The inhibition of phosphatidylinositol turnover: a possible postreceptor mechanism for the prolactin secretion-inhibiting effect of dopamine

We studied the association between the inhibition of phosphatidylinositol (PI) turnover and the inhibition of PRL secretion in the presence of dopamine. The incorporation of radiolabeled phosphate into anterior pituitary gland PI as well as serum PRL levels were significantly (P less than 0.01) greater in female than in male rats. No significant sex-related difference was found in the incorporation by pituitary tissue of 32P into phosphatidylcholine (PC) or phosphatidylethanolamine (PE). Dopamine decreased the incorporation of 32P into PI, but not into PC or PE, by female rat pituitary glands; this effect was reversed by two dopamine receptor-blocking agents, haloperidol and pimozide. After dopamine was removed from the incubation medium, basal 32P incorporation into PI was restored within 10 min. The administration of bromocriptine (500 micrograms/kg, ip, 4 h earlier) significantly reduced pituitary PI turnover. Conversely, in vivo injection of alpha-methyl-p-tyrosine (alpha MpT; 200 mg, ip, 2.5 h before death), an inhibitor of catecholamine biosynthesis, dramatically increased serum PRL levels. In vitro incorporation of 32P into PI, but not into PC or PE, increased (+130%) when these glands were incubated for 30 min with radiolabeled phosphate. The in vitro addition of 0.5 microM dopamine to glands from alpha MpT-treated rats counteracted the stimulation of 32P incorporation into PI produced by alph MpT treatment. In rats bearing the transplantable PRL-secreting tumor MtTW15, the hyperprolactinemia produced by the tumor stimulates hypothalamic turnover of dopamine, with a consequent inhibition of pituitary gland PRL secretion. 32P incorporation into PI, but not into PC or PE, was significantly (P less than 0.01) inhibited (-41%) in pituitary glands from these rats. The injection of alpha MpT (200 mg/kg, ip) or haloperidol (2 mg/kg, ip) 12 and 3 h before death into MtTW15 tumor-bearing rats abolished the inhibition of 32P incorporation into pituitary PI. Dopamine also decreased PI turnover in the 7315a PRL-secreting pituitary tumor. Our data indicate that the PI cycle may be an intracellular mechanism controlling PRL release in the rat and that the changes in its cleavage and turnover may be an early postreceptor event responsible for the inhibition of PRL secretion produced by factors such as dopamine.     

Pharmacologic evidence that a D2 receptor subtype mediates dopaminergic stimulation of prolactin secretion from the anterior pituitary gland.

The ability of low concentrations of dopamine (DA) to stimulate the secretion of prolactin (PRL) was examined in perifused or monolayer cultures of anterior pituitary cells. In cultures perifused with media containing 100 nM DA, changing the DA concentration to either 1 or 100 pM caused a significant dose-dependent stimulatory PRL secretory response within 6 min when compared to the PRL secretory response to removal of DA altogether. Picomolar concentrations of DA caused a biphasic PRL secretory response. This response is characterized by an immediate increase in the rate of PRL secretion similar to that seen when the cells were treated with 100 nM thyrotropin-releasing hormone followed by a decrease in the rate of PRL secretion to levels comparable to cells receiving media alone. In a monolayer culture system DA, at concentrations between 10 nM and 1.0 pM, caused significant stimulation of PRL secretion relative to media alone. Maximal stimulation occurred at nanomolar concentrations of DA (approximately 60% greater than control). Although the D2 agonists, bromocriptine and 2-(N-phenethyl-N-propyl)-amino-5-hydroxytetralin hydrochloride (PPHT) caused significant (p less than 0.05) inhibition of PRL secretion at nanomolar concentrations and above, neither had stimulatory activity. The D1 agonists, SKF 38393 and SKF 82958, had no effect on PRL secretion when tested at 0.1 pM to 1 microM. These data suggest that DA not only inhibits PRL secretion in vitro, but also stimulates PRL secretion at relatively low concentrations. Stimulation is mediated by a DA receptor which is neither recognized by D2 nor D1 agonists, suggesting a possible third DA receptor subtype.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relaxin stimulates prolactin secretion from anterior pituitary cells

The anterior pituitary has recently been implicated as a relaxin target issue because of the cAMP elevation after relaxin treatment. We attempted to correlate this finding with an endocrine response to relaxin in rats. Anterior pituitary cells were enzymatically dispersed and subjected to the reverse hemolytic plaque assay. PRL secretion was significantly stimulated 1.31-fold by human relaxin at the lowest concentration studied (30 pM) and maximally stimulated 1.65-fold at 0.3 nM relaxin. Antibodies directed against relaxin inhibited this effect, as did the PRL inhibitory hormone, dopamine. In contrast to the response of PRL cells, there was no effect or a slight inhibition of LH release after incubation with relaxin. In conclusion, we propose that one of the pituitary cell types responsive to relaxin in culture is the PRL-secreting mammotroph.     

Antipsychotic drugs inhibit prolactin release from rat anterior pituitary cells in culture by a mechanism not involving the dopamine receptor.

Bromocriptine, a dopamine agonist, inhibited secretion of PRL and did not affect GH release from rat anterior pituitary cells in culture. The reversal of this inhibition of PRL release by butaclamol, a dopamine antagonist, was stereospecific; 10 nM d-butaclamol completely reversed the inhibition caused by 10 nM bromocriptine, while l-butaclamol had no effect at concentrations up to 10 microM. However, both enantiomers at 10 microM inhibited PRL release to 30% and GH release to 91% of control values. Two other dopamine antagonists also inhibited hormone release. Haloperidol (10 microM) inhibited PRL release to 23% of control values and did not affect GH release; 3.3 microM pimozide inhibited PRL and GH release to 18% and 38% of control values, respectively. These data indicate that, the inhibition of PRL by antipsychotic drugs is not mediated through the dopamine receptor.     

Dopamine inhibits prolactin secretion stimulated by the calcium channel agonist Bay-K-8644 through a pertussis toxin-sensitive G protein in anterior pituitary cells

In primary culture of anterior pituitary cells, BAY-K-8644, a calcium channel agonist, stimulated PRL secretion by 83% with EC50 of 18 nM. This effect was blocked by nifedipine, a calcium channel antagonist. The stimulations of PRL secretion induced by potassium (50 mM) and BAY-K-8644 were additive. Dopamine inhibited basal as well as BAY-K-8644-stimulated PRL secretion by 64% and 75%, respectively, and with respective EC50 values of 4.5 and 0.6 nM. In the presence of 50 mM K+, dopamine only partially blocks the dose-dependent stimulation of PRL secretion induced by the calcium channel agonist. The inhibitory dopamine effect was blocked by (+)butaclamol, a specific dopamine receptor antagonist. The dopamine response was also blocked by 1-sulpiride, a specific dopamine D2 receptor antagonist, and mimicked by RU 24926, a specific dopamine D2 receptor agonist, suggesting that the dopamine effect on BAY-K-8644-stimulated PRL secretion was mediated through a D2 dopamine receptor. Although unknown, the mechanism by which dopamine inhibited the BAY-K-8644-stimulated PRL secretion involves a GTP binding protein sensitive to Bordetella pertussis toxin. In fact, the dopamine inhibition of PRL secretion induced by the calcium channel agonist was blocked by the pretreatment of cells with the toxin. These results suggest that dopamine D2 receptors in lactotroph cells modulate calcium influx through a GTP binding protein.     

Effects of 17 beta-estradiol on angiotensin II receptors and prolactin release in cultured pituitary cells

Angiotensin II (AII) binds to specific receptors in the lactotroph and stimulates PRL secretion from isolated rat pituitary cells. Since estrogens exert major regulatory actions on PRL secretion, the effects of estradiol (E2) on pituitary AII receptors and PRL responses were studied in vivo and in cultured rat anterior pituitary cells. In female rats, treatment with E2-containing Silastic capsules for 4 days caused a significant increase in PRA from 1.3 to 3 ng/ml X min and a 38% decrease in the binding of [125I]AII to anterior pituitary membrane-rich fractions (P less than 0.01). In vitro studies showed that treatment of cultured anterior pituitary cells with 1 nM E2 for 4 days caused a 57 +/- 6% decrease in AII receptor concentration with no change in binding affinity. Reduction of AII receptors by E2 in 4-day cultures was dose dependent and was demonstrable with E2 concentrations that occur in plasma during the estrous cycle (0.01-1 nM). The decrease in AII receptors in cells incubated with 1 nM E2 was near maximum after 24 h of culture, and results were similar when receptor concentrations were calculated per unit protein or per cell. Despite the substantial decrease in AII receptors, E2 treatment did not specifically decrease the responsiveness of the pituitary cells to AII stimulation. Thus, PRL responses to AII (10 nM) or TRH (100 nM) were unchanged after 1 day of E2 treatment and were increased after 4 days of treatment. These findings demonstrate that E2 has a direct inhibitory action on expression of pituitary AII receptors that is not accompanied by a decrease in AII-stimulated PRL secretion. In the rat pituitary, estrogen modulation of postreceptor events is the predominant determinant of lactotroph responsiveness during stimulation of PRL release by AII.     

Studies on the effects of catecholestrogens on prolactin secretion by cultured normal rat anterior pituitary cells

The catecholestrogens 2-hydroxyesterone (2-OHE1) and 2-hydroxyestradiol (2-OHE2) are shown to directly inhibit PRL release by cultured normal rat anterior pituitary cells if they have been cultured in charcoal-treated estrogen-stripped fetal calf serum. Both catecholestrogens affect PRL release in a dose-dependent bimodal way. 2-OHE1 (1 microM) maximally inhibits PRL release by 38%, but 10 microM of the substance has no effect. In contrast, 0.1 microM 2-OHE2 maximally inhibits PRL release by 37%, while the effect of 1 microM of the compound was significantly less (-26%). The effect of the catecholestrogens on PRL release by cultured pituitary cells was significant after 2 h and was still effective after 48 h. The mechanism of action of 2-OHE1 and 2-OHE2 probably does not involve dopamine receptors, as 50 nM haloperidol did not alter the PRL release inhibitory effect of these substances. Moreover, 2-OHE1 and 2-OHE2 did not affect dopamine-mediated inhibition of PRL release. The total amount of PRL (medium plus cells) present in cultures of normal pituitary cells exposed to 2-OHE1 or 2-OHE2 was identical to that in control cells. TRH (100 nM)-stimulated PRL release from cells exposed to catecholestrogens was significantly higher than that from control cells. Addition of the calcium entry-blocking agent verapamil prevented the inhibitory effect of dopamine on PRL release, but did not prevent the effects of 2-OHE1 and 2-OHE2. Preincubation of cultured normal rat pituitary cells for 30 min with 50 nM estradiol completely prevented the inhibitory effects of 2-OHE1 and 2-OHE2 on PRL release. In conclusion, the acute inhibitory effects of the catecholestrogens 2-OHE1 and 2-OHE2 on PRL release by cultured normal rat pituitary cells do not involve dopamine receptors or calcium transport over the cell membrane. These effects are only demonstrable after culture of the cells in charcoal-treated fetal calf serum and can be completely prevented by short term preincubation with estradiol. Our observations support the suggestion that the action of estradiol on lactotropic cells may be modulated by its metabolites, which have opposite effects on PRL release.     

Identification by the sequential cell immunoblot assay of a subpopulation of rat dopamine-unresponsive lactotrophs

The responsiveness to dopamine of PRL secretion from individual lactotrophs of female rats was investigated by the use of a newly developed sequential cell immunoblot assay. In this assay, PRL secretion from the same single lactotrophs that had been cultured on plastic coverslips was quantified before and after dopamine treatment by a combination of direct absorption of PRL secreted on protein-blotting transfer membranes, immunostaining, and microscopic image analysis. The assay was sensitive enough to detect 0.03 fg PRL/pixel and was specific for PRL. The range of PRL secretion from single pituitary cells in culture was 0.03-1.92 pg/cell.h. PRL secretion was increased with time of incubation and reached a maximum by 80-160 min. There was a significant correlation in PRL secretion from the same lactotrophs between the first and second 60-min incubations. When medium used in the second incubation contained no dopamine, amounts of PRL secreted during the second incubation period were 21-204% of those secreted during the first incubation period. Inclusion of 10(-8)-10(-6) M dopamine in the second incubation medium increased in a dose-dependent manner the proportion of lactotrophs whose PRL secretion was suppressed significantly compared with that during the first incubation period. However, PRL secretion from approximately 6% of the total lactotrophs was not suppressed even by dopamine at concentrations over 10(-6) M. The present study demonstrates that the sequential cell immunoblot assay is a useful means to quantify repeatedly hormone secretion from individual endocrine cells in culture. Furthermore, these results suggest that there is a subpopulation of rat PRL-secreting lactotrophs that are unresponsive to dopamine.     

Growth hormone secretion by cultured rat anterior pituitary cells. Effects of culture conditions and dexamethasone

Optimal conditions were sought for the study of GH secretion by cultured normal pituitary cells. Dispersed rat pituitary cells were cultured for 1 week in four different media supplemented with 10% fetal calf serum. Minimal essential medium resulted in high GH content and secretion during a 4-h incubation period, whereas GH secretion was lower (P less than 0.05) for cells cultured in medium 199, Ham's F-10, and RPMI-1640. GH secretion/24 h declined gradually with time. After 2 weeks in culture hormone secretion amounted to 30% of secretion on day 1, but after 3 weeks GH secretion was still measurable. GH recovery during the 3-weeks culture period was more than 600% of the amount initially plated. GH secretion was positively correlated with the bicarbonate concentration between 0.85 and 2.2 g/liter NaHCO3. When pituitary cells were cultured in concentrations varying from 0.5 X 10(5) to 10 X 10(5) cells per dish, GH secretion and content per cell were constant, suggesting that no direct autofeedback occurred in cultures with high cell densities and thus high medium GH. Dexamethasone stimulated GH secretion and content in a dose-dependent way (0.1 nM-10 microM). The stimulatory effect of 100 nM dexamethasone occurred within 24-48 h. After 7 days of treatment with 100 nM dexamethasone, GH secretion had increased to 190% and GH content to 230% of control. In contrast to the effects on GH, dexamethasone suppressed PRL secretion in a dose-dependent way, but this effect was seen only after 7 days of treatment and not after 4 days of treatment. Cycloheximide and actinomycin D prevented the stimulatory effect of dexamethasone on GH secretion. However, 24 h after cessation of cycloheximide treatment GH secretion was stimulated by dexamethasone. Four days of treatment with 100 nM dexamethasone did not affect the GH response to somatostatin, prostaglandin E1, and theophylline, nor the PRL response to dopamine, TRH, and theophylline. Thus, culture conditions may affect GH production, and dexamethasone can be used to culture somatotrophs for longer periods with steady GH production and normal responsiveness.     

Bombesin stimulates prolactin and growth hormone release by pituitary cells in culture

Bombesin (BBS) has been previously shown to stimulate the secretion of PRL and GH in steroid-primed rats. To determine whether these effects were mediated by the central nervous system or were due to direct action on the pituitary gland, we studied the interaction of BBS with GH4C1 cells, a clonal strain of rat pituitary cells which synthesizes and secretes PRL and GH. The addition of 100 nM BBS to GH4C1 cells for 60 min increased PRL release to 140 +/- 3% of the control value (mean +/- SE) and GH release to 133 +/- 5% of the control value. Stimulation of hormone secretion was observed within 15 min of treatment with 100 nM BBS and continued for at least 2 h. Half-maximal stimulation of PRL release occurred with 0.5 nM BBS, and a maximal effect was observed with 10 nM peptide. The BBS analogs ranatensin, litorin, and [Tyr4]BBS, each at a concentration of 100 nM, caused the same stimulation of PRL release as maximal concentrations of BBS itself. BBS stimulated hormone release selectively in two of five different clonal pituitary cell strains examined. Pretreatment of GH4C1 cells with 1 nM estradiol and/or 100 nM insulin resulted in more powerful stimulation of PRL release by both TRH and BBS. When epidermal growth factor and vasoactive intestinal peptide were added simultaneously with BBS, PRL release was greater than in the presence of either peptide alone. In contrast, the stimulatory effects of TRH and BBS were not additive. Somatostatin inhibited both basal and stimulated PRL release. Thus, low concentrations of BBS can directly stimulate PRL and GH release by a clonal pituitary cell strain in culture. These results suggest that BBS may stimulate PRL and GH secretion in vivo by direct action on the pituitary gland.     

Regulation of prolactin secretion in patients with Cushing's disease. A comparative study on the effects of dexamethasone, lysine vasopressin and ACTH on prolactin secretion by the rat pituitary gland in vitro

In 15 untreated patients with Cushing's disease the regulation of prolactin (PRL) was evaluated. Plasma PRL was 11.5 +/- 4.8 vs. 5.3 +/- 3.6 ng/ml (patients with Cushing's disease vs. control; mean +/- S.D.; p less than 0.001). The maximal increment of plasma PRL in response to TRH was 32.3 +/- 17.3 vs. 27.9 +/- 17.2 ng/ml (NS); the maximal increment of plasma PRL in response to an insulin-induced hypoglycemia was 3.8 +/- 4.6 vs. 22.7 +/- 12.4 ng/ml (p less than 0.001). Additionally the effect of dexamethasone, lysine vasopressin and ACTH on the secretion of PRL by rat pituitary glands in vitro was studied. Dexamethasone (1.25--10 microM) inhibited the secretion of PRL. However, in the presence of dexamethasone modulation of PRL release by TRH and dopamine remained unaltered. Lysine vasopressin (5 nM - 5 microM) and ACTH (0.5--12.5 microM) did not have a direct effect on PRL release by normal rat pituitary glands in vitro and these substances also did not interfere with dopamine-mediated inhibition of PRL release. Conclusions: In Cushing's disease the PRL responses to TRH (normal) and to insulin-induced hypoglycemia (blunted) are differentially affected. Therefore, hypercortisolism probably selectively interferes with the regulation of PRL secretion at a suprahypophyseal level. It is concluded that TRH and dopamine regulate PRL release at sites which are not under corticosteroid regulation, while corticosteroids modulate PRL secretion in response to stress.     

Transient dopaminergic inhibition of prolactin release from hybrid cells derived by fusion of normal rat pituitary and GH4C1 tumor cells

The clonal rat pituitary tumor cell line GH4C1 secretes PRL but does not respond to dopamine, a physiological inhibitor of PRL. In an attempt to generate a dopamine-responsive cell line, GH4C1 cells, which lack the enzyme hypoxanthine-guanine phosphoribosyltransferase, were fused with cells from the pituitary glands of lactating rats to generate cell hybrids. The GH4C1 cells were fused with dispersed normal pituitary cells by either chemical fusion in 40% polyethylene glycol or electrofusion. The fused cells were grown in medium with hypoxanthine, aminopterin, and thymidine (HAT) for 4 weeks to select for hybrid cells. Control fusions between GH4C1 cells only or normal cells only did not produce viable colonies. Of 36 HAT-selected colonies, 3 responded to 10 nM bromocryptine (BCR) with inhibition of TRH-stimulated PRL release. These hybrid colonies had an inhibitory response similar to that of normal pituitary cells in culture. Both TRH- and vasoactive intestinal peptide-stimulated PRL release were inhibited to basal levels by 10 nM BCR, with an IC50 for BCR of approximately 0.25 nM. Basal hormone release was not inhibited by BCR. The BCR-sensitive hybrid cells grew more slowly than the parental GH4C1 line both in culture and when passaged in female Wistar-Furth rats. The response of the hybrid cells to the dopamine agonist and the characteristic of slow growth were lost after 9 months of continuous culture and after freezing cells. The parental GH4C1 cells were grown in female Wistar-Furth rats, the resulting tumors were dissociated, and the cells were grown in culture. This resulted in a brief establishment of the dopamine response. Stimulated PRL and GH release from freshly dispersed GH4C1 tumor cells was inhibited by BCR at concentrations from 0.1-10 nM, and spiroperidol reversed the inhibition. The inhibitory response to the dopaminergic agonist was lost quickly as the cells were carried in culture. These results demonstrate that GH4C1 cells may have the genetic information necessary for dopaminergic inhibition of PRL synthesis, but that the dopamine response is not observed under standard tissue culture conditions.     

Stimulation of prolactin secretion after short term or pulsatile exposure to dopamine in superfused anterior pituitary cell aggregates

The dynamics of dopamine (DA) action on PRL release was studied in superfused rat anterior pituitary cell aggregates, cultured for for 5 days either in conventional or in serum-free defined medium. In aggregates cultured in conventional medium 0.1-1 nM DA applied for 20 min provoked a rapid and concentration-dependent inhibition of PRL release, lasting only a few minutes, after which there was a gradual rise in secretion up to near baseline levels. A sustained inhibition was obtained from DA concentrations more than or equal to 10 nM. When DA, used at the latter concentration, was withdrawn from the superfusion medium, a marked rebound secretion of PRL occurred, exceeding basal release for as long as 40-50 min. Rebound secretion was not followed by a compensatory fall in secretion rate. After a 10-min pulse of 10 or 30 nM DA, the amount of PRL released above baseline was considerably higher than the amount of PRL not released during the time DA was present. The latter stimulation of PRL release was not seen after a 40- or 90-min exposure time to DA. However, when DA was given for 40 min in 10 pulses of 4 min (4 min DA on 4 min DA off), a clear-cut stimulation of PRL release followed the termination of the pulses. When the serum used in the culture medium was extracted with dextran-coated charcoal, post-DA rebound secretion of PRL was markedly diminished. The latter secretion pattern partially reappeared when the extracted serum was supplemented with 10 nM dexamethasone. DA had similar effects on PRL release in aggregates cultured in serum-free defined medium. Dexamethasone did not affect DA-inhibition but strongly stimulated post-DA rebound, and this effect was potentiated by T3 present in the defined medium. There was three to four times more PRL secreted in excess of basal release than was inhibited during exposure to DA. The present data suggest a dual action of DA on PRL release: inhibition during tonic exposure to the catecholamine and inhibition-mediated stimulation after pulsatile exposure.     

Studies on the conditions determining the inhibitory effect of somatostatin on adrenocorticotropin, prolactin and thyrotropin release by cultured rat pituitary cells

Somatostatin (SRIH) is a physiological inhibitor of growth hormone (GH) secretion, but its role in the regulation of adrenocorticotropic hormone (ACTH), prolactin (PRL) and thyroid-stimulating hormone (TSH) release is unclear. SRIH (1 pM to 1 microM) did not affect basal and corticotropin-releasing hormone (CRH)-stimulated ACTH release by normal rat pituitary cells cultured in medium with 10% fetal calf serum (FCS). In cells deprived of serum for 48 h, or preincubated with the glucocorticoid-receptor-blocking agent, RU 38486, CRH-stimulated ACTH release was significantly suppressed by 1 pM to 0.10 nM SRIH. Preincubation with 5 nM dexamethasone completely abolished this inhibitory effect of SRIH on ACTH release. PRL release by pituitary cells cultured in phenol red-free culture medium with 10% estrogen-stripped FCS showed a very low sensitivity to SRIH. Increasing concentrations of 10 and 50 pM and 1 nM estradiol made PRL release by these cells significantly less sensitive to 50 nM dopamine, whereas the sensitivity to SRIH increased to a similar extent. In all instances dopamine and SRIH together exerted additive inhibitory effects, the extent of which remained similar under all conditions. After a 2-hour incubation, thyrotropin-releasing hormone-stimulated TSH secretion was significantly suppressed by 100 nM and 1 microM SRIH only in cells cultured in medium with 10% hypothyroid serum, and not in cells cultured in medium with 10% FCS. Such a difference in the sensitivity of thyrotrophs to SRIH disappeared during longer incubation. Conclusions: (1) ACTH release by normal corticotrophs is only sensitive to SRIH in the absence of the physiological peripheral feedback regulation by glucocorticoids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acute effect of basic fibroblast growth factor on secretion of prolactin as assessed by the reverse hemolytic plaque assay

The effect of basic fibroblast growth factor (bFGF) on acute secretion of PRL by pituitary lactotrophs was examined under basal conditions and after treatment with TRH or dopamine. We used the reverse hemolytic plaque assay (RHPA) to determine the amount of PRL secreted per lactotroph and the percentage of pituitary cells secreting PRL. Young (2- to 3-month-old) female Sprague-Dawley rats were ovariectomized and 1 week later implanted with a Silastic capsule containing 180 micrograms/ml estradiol in sesame oil. Three days later, rats were killed, anterior pituitaries were removed, and cells were enzymatically dispersed and prepared for use in the RHPA. In Exp I, time and dose responses to bFGF were determined using the RHPA. Basic FGF reduced (P less than 0.0001) the mean basal secretion of prolactin per lactotroph. The effect was similar at 30, 60, 120, and 240 min of incubation. The reduction in PRL was greatest at 3.36 x 10(-6) M, while lesser reductions were observed at 1.68 x 10(-6) and 5.60 x 10(-7) M. A dose of 3.36 x 10(-6) M (60 ng/ml) and an incubation time of 60 min were subsequently used in Exp II. In Exp II, we examined the effects of bFGF on TRH stimulation and dopamine inhibition of PRL secretion. PRL secretion was maximally stimulated (P less than 0.01) by 10(-7) M TRH. Basic FGF blocked the TRH-induced increase in PRL secretion. PRL secretion was maximally reduced (P less than 0.001) by 10(-5) M dopamine. Coincubation of bFGF with dopamine reduced (P less than 0.01) the mean plaque area to the same extent as dopamine alone. In each experimental situation changes in mean plaque area reflected a shift in the frequency distribution of the plaque area. Neither bFGF, TRH, dopamine, nor the combined treatments influenced the percentage of pituitary cells secreting PRL compared to basal conditions. We have demonstrated that 1) bFGF reduces the basal secretion of PRL in an acute manner; 2) bFGF blocks the TRH-induced increase in PRL; and 3) bFGF does not potentiate the inhibitory effect of dopamine on PRL secretion and, therefore, may act in part through the same inhibitory pathway as dopamine. We conclude from these data that bFGF, or related factors, could play a role in the regulation of PRL secretion.     

Cortistatin inhibits growth hormone release from human fetal and adenoma pituitary cells and prolactin secretion from cultured prolactinomas

CONTEXT: Cortistatin (CST) is a neuropeptide that shares high homology with somatostatin and binds with high affinity to all somatostatin receptor (SSTR) subtypes. Many of its endocrine and biological activities overlap with those of somatostatin. OBJECTIVE/DESIGN: The objective of the study was to assess the direct in vitro effects of CST on human pituitary hormone secretion. SETTING: This study was performed in the endocrine laboratory of a tertiary academic medical center. MATERIALS: Primary cell cultures of human fetal (21-25 wk gestation) pituitary tissues and cultured hormone-secreting adenoma cells were used in this study. INTERVENTIONS: Cell cultures were incubated with CST-14 or CST-17, somatostatin, GHRH, SSTR analogs, and ghrelin analogs, and hormone secretion was analyzed. OUTCOME MEASURES: GH and prolactin (PRL) medium concentrations were tested by hormone assay, and SSTR mRNA was tested by RT-PCR. RESULTS: CST-14 (10 nm) inhibited GH secretion by up to 65% in all fetal pituitary specimens after 4-h incubation (P < 0.05). CST-14 or CST-17 (10 nm) inhibited basal GH secretion in six of the 13 GH-cell adenomas and two of the three GH-PRL mixed adenomas. CST-17 (100 nm) suppressed the GH response to GHRH and ghrelin analog (10 nm each) by 30-50% in adenomas (P < 0.05). Three PRL-adenomas treated with CST-17 (10 nm) showed a 20-40% inhibition of PRL release (P < 0.05), whereas in three others no suppression or mild response was achieved at this concentration. A comparable inhibition of PRL secretion was obtained with SSTR5-selective analog but significantly less with SSTR2-preferential compounds. RT-PCR revealed the expression of both SSTR2 and SSTR5 in all GH-cell and mixed adenomas studied and all PRL-secreting adenomas studied, except for two of the CST-resistant prolactinomas, in which SSTR5 was absent. CONCLUSIONS: This is the first report of in vitro CST suppression of human GH and PRL in cultured pituitary tissues. The regulation of PRL release from cultured adenomas appears to be primarily mediated by SSTR5.     

The stimulatory and inhibitory effects of dopamine on prolactin secretion involve different G-proteins.

The reverse hemolytic plaque assay (RHPA) was used in this study to further characterize the mechanism whereby low concentrations of dopamine (DA) stimulate PRL secretion in vitro. Female Sprague-Dawley rats were used as a source of anterior pituitary cells for the RHPA. Pituitary cells were infused into Cunningham chambers along with a suspension of protein-A-coated ovine red blood cells. Excess cells were rinsed from the chambers leaving a monolayer of cells attached to the glass. The cells were then incubated with solutions containing PRL antiserum (1:40) and various concentrations of DA. After 4 h, a solution containing guinea pig complement (1:60) was infused into the chambers. Thirty minutes later, the cells were fixed and plaques (zones of hemolysis) surrounding PRL-producing cells (lactotrophs) were measured and used as an index of the amount of PRL secreted. Control cells that received no DA had a mean plaque area of 8,000 microns 2 and two distinct subpopulations of plaque sizes. This biphasic population of cells consisted of a small and a large plaque producing population. The mean plaque area surrounding lactotrophs was significantly (P less than 0.05) decreased if 1 microM or 10 microM DA was present (4,500 microns 2 and 3,500 microns 2, respectively). These cells which received inhibitory concentrations of DA demonstrated a monophasic distribution of plaque-forming cells. On the other hand, mean plaque area was significantly (P less than 0.05) increased if 0.1 nM or 1 nM DA was presented to the cells (15,000 microns 2 and 14,500 microns 2, respectively). These cells receiving stimulatory doses of DA exhibited a multiphasic distribution of plaque-forming cells. The possibility that the two physiological opposing actions of DA on PRL secretion might be mediated by different GTP binding proteins was also examined using cholera toxin (CTX) and pertussis toxin (PTX). Anterior pituitary cells were pretreated with either CTX (50 micrograms/ml) or PTX (5 micrograms/ml) for 1 h before initiation of the RHPA. In the RHPA, cells received no DA, a stimulatory dose of DA (0.1 nM), or a inhibitory dose of DA (10 microM). The effects of toxin pretreatment on mean plaque area of DA-treated cells was determined. PTX pretreatment significantly attenuated the inhibitory effects of DA while having no effect on the stimulatory effects of DA on PRL secretion. CTX significantly (P less than 0.05) potentiated the stimulatory effects of DA on PRL secretion and had no effect on inhibition.(ABSTRACT TRUNCATED AT 400 WORDS)