Keith P. Ray

Effects of growth hormone-releasing factor, somatostatin and dopamine on growth hormone and prolactin secretion from cultured ovine pituitary cells.

A synthetic form of human pancreatic growth hormone releasing factor (GRF‐44‐NH2) was shown to be a potent stimulator of growth hormone (GH) secretion and cellular cyclic AMP levels in cultured sheep pituitary cells. A small dose‐dependent stimulation of prolactin secretion was also observed. Somatostatin (0.5 μM) completely blocked the maximal GRF (1 nM)‐stimulated secretion without a significant effect on cyclic AMP levels. Dopamine (0.1 μM) inhibited the GRF‐elevated GH secretion by 50% and lowered cyclic AMP levels by 30%. Dopamine (0.1 μM) inhibition of basal prolactin secretion was not affected by GRF (1 nM). The data support the hypothesis that cyclic AMP is involved in the action of GRF but suggest that somatostatin can inhibit GRF‐induced secretion of GH independently of cyclic AMP.

Monoclonal antibodies to bovine growth hormone potentiate effects of the hormone on somatomedin C levels and growth of hypophysectomized rats

Bovine growth hormone (50 micrograms/day) elevated somatomedin C levels in plasma of hypophysectomized rats: the effect was seen 12 h after the first injection and was sustained throughout a 7-day treatment period. When monoclonal antibodies to bovine growth hormone were administered with the hormone the stimulation of somatomedin C levels was markedly enhanced, as were effects on body weight. Each one of a panel of 7 monoclonal antibodies potentiated the actions of the hormone on growth and somatomedin C levels, though the extent and pattern of potentiation varied considerably from one antibody to another. Effects on growth and somatomedin C levels correlated fairly well, though there were some discrepancies.

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.

Effects of growth hormone-releasing factor and somatostatin on growth hormone secretion and cellular cyclic AMP levels. Cultured ovine and rat anterior pituitary cells show markedly different responses.

Human pancreatic growth hormone‐releasing factor (GRF‐44‐NH2) stimulated growth hormone (GH) secretion and intracellular cyclic AMP levels in cultured pituitary cells from both sheep and rat. Somatostatin (SRIF), over a wide range of doses and time, showed no significant effect on the elevated cyclic AMP levels in sheep cells, but did block the GH release in a dose‐dependent manner. In rat cells, however, SRIF inhibited GRF‐stimulated cyclic AMP levels by 75% maximum (still 8‐fold greater than the basal levels) and GH release to almost half the basal value. We conclude that somatostatin inhibits GRF‐elevated cyclic AMP levels in rat pituitary cells but not in sheep cells.

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)

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 microM). The divalent cation ionophore A23187 (1 microM) 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 microM) but not verapamil (10 microM) inhibited the A23187 (10 microM) induced release of prolactin. Inhibition of basal and A23187 (1 microM) stimulated prolactin secretion occurred over a similar range of dopamine concentrations. The dopamine receptor antagonist haloperidol (1 microM) reversed the inhibitory effect of dopamine (0.1 microM) 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.

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.

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.

Regulation of growth hormone secretion and cyclic AMP metabolism in ovine pituitary cells: interactions involved in activation induced by growth hormone-releasing hormone and phorbol esters

Growth hormone-releasing hormone (GHRH) and the phorbol ester tetradecanoylphorbol acetate (TPA) each stimulated a rapid and extensive (up to 15-fold) increase in the secretion of growth hormone from cultured ovine anterior pituitary cells. Effects of the releasing hormone on growth hormone secretion were associated with a concurrent, large increase in cellular cyclic AMP accumulation. TPA induced a much smaller (26-78%), though still significant, increase in cellular cyclic AMP levels. Forskolin and isobutylmethylxanthine (IBMX) also stimulated growth hormone secretion and cyclic AMP accumulation. When combined with a maximally effective concentration of GHRH these compounds did not further elevate growth hormone secretion even though they induced further increases in cyclic AMP concentration; this is consistent with activation occurring via a common cyclic AMP-dependent pathway. In contrast TPA when combined with maximally effective concentrations of either GHRH, forskolin or IBMX caused additional release of growth hormone, suggesting that the TPA-induced secretion involved a cyclic AMP-independent process. However, TPA also markedly potentiated the cellular cyclic AMP accumulation due to each of these agents. That TPA induced stimulation of basal and GHRH-stimulated cyclic AMP levels measured in the presence of IBMX suggests an action affecting cyclic AMP synthesis. Carbachol had no effect on basal or GHRH-stimulated growth hormone secretion or cyclic AMP levels. The two actions of TPA, one on secretion and one on cyclic AMP metabolism, may result from activation of some common event possibly involving protein kinase C. Our results suggest that GHRH and TPA activate independent pathways regulating growth hormone secretion.

Mechanisms involved in the effects of TRH on GHRH-stimulated growth hormone release from ovine and bovine pituitary cells

Incubation of cultured ovine pituitary cells with growth hormone-releasing hormone (GHRH) (10(-12)-10(-7) M) stimulated growth hormone secretion up to 3-fold. At a maximal stimulatory concentration of GHRH (10(-10) M), thyrotropin-releasing hormone (TRH) (10(-7) M) caused an inhibition of growth hormone release to approx. 50% of the response obtained with GHRH alone (during a 15 min incubation period). TRH also caused a small inhibition of the GHRH-stimulated cellular cyclic AMP level but this effect was only significant at a relatively high concentration of GHRH (10(-9) M). Incubation of cultured bovine pituitary cells with GHRH (10(-11)-10(-8) M) plus TRH (10(-7) M) caused a significant stimulation of growth hormone release by up to 40%, compared with the response obtained with GHRH alone (at all concentrations of GHRH). TRH (10(-7) M) had no effect on GHRH (10(-8) M)-stimulated cellular cyclic AMP levels in a partially purified bovine pituitary cell preparation. The effects of varying extracellular [Ca2+] (0.1-10 mM) on intracellular [Ca2+] and on the responsiveness to releasing hormones were also determined using ovine pituitary cells. GHRH (10(-10) M)-stimulated growth hormone release was inhibited when cells were incubated at both high (10 mM) and low (0.1 mM) [Ca2+] (compared with 1 mM or 3 mM Ca2+) with or without TRH (10(-7) M). At 1 mM Ca2+, TRH produced a synergistic effect with GHRH to stimulate growth hormone release. However, at 3 mM Ca2+ TRH inhibited GHRH-stimulated growth hormone release.(ABSTRACT TRUNCATED AT 250 WORDS)