Stimulation of chicken growth hormone release by phorbol esters.

Synergism between thyrotropin-releasing hormone (TRH) and human pancreatic growth hormone-releasing factor (hpGRF) has been shown in a primary (48 hr) culture of chicken adenohypophyseal cells established in this laboratory. The purpose of the present study was to determine if phorbol esters acting alone or in concert with TRH or hpGRF affect chicken GH release. Collagenase-dissociated chicken adenohypophyseal cells were treated (2 hr) with combinations of TRH, hpGRF, phorbol esters (activators of protein kinase C; PKC), and pharmacologic agents that increase cAMP. Phorbol myristate acetate (PMA) or phorbol dibutyrate (PDBu) alone stimulated GH release in a dose-dependent manner; either phorbol ester (10(-6) M) increased GH release from 100 to 390% over the value obtained in the absence of test agents (control). Similarly, hpGRF (10(-9) M), 8 Br-cAMP (10(-3) M), forskolin (10(-6) M), or isobutylmethylxanthine (IBMX, 10(-3) M) alone elevated GH release by at least 60% over the control value. The combined effects of phorbol esters (either PMA or PDBu) and hpGRF, 8 Br-cAMP, or forskolin on GH release were additive. Only one combination, phorbol esters with IBMX, exerted synergistic effects on GH release. No synergy was shown between TRH (1.3 x 10(-9) M) and either phorbol ester. These findings are the first to implicate PKC in chicken GH release in vitro. In addition, these studies, together with previous results, suggest that TRH and hpGRF synergy occurs via a pathway that arises prior to activation of PKC.     

Possible participation of calcium in growth hormone release and in thyrotropin-releasing hormone and human pancreatic growth hormone-releasing factor synergy in a primary culture of chicken pituitary cells

We previously reported that thyrotropin-releasing hormone (TRH) and human pancreatic growth hormone-releasing factor (hpGRF) exert synergistic (greater than additive) effects on growth hormone (GH) release from chicken pituitary cells in primary culture. In the present studies the possible participation of calcium in GH release and in TRH and hpGRF synergy was investigated. Following dispersion with collagenase, cells were cultured for 48 hr prior to exposure (2 hr) to test agents. Cultured cells were exposed to a range of calcium concentrations (0, 0.02, 0.2, and 2.0 mM) in the presence and absence of secretagogues. These results demonstrated that basal GH release was not altered by the concentration of calcium in the medium: however, secretagogue-induced GH release required calcium. Thus, TRH, hpGRF, 8 Br-cAMP, or forskolin stimulated GH release in the absence of calcium. Furthermore, synergistic GH release evoked by TRH and hpGRF, 8 Br-cAMP, or forskolin was observed only at the highest calcium concentration (2.0 mM). In other studies, ionomycin (10(-5) M), a calcium ionophore, stimulated GH release to a value about 125% over the basal (absence of test agent) value. Ionomycin-induced GH release was not affected by TRH (5.0 ng/ml); the combined effects of ionomycin (10(-7)-10(-5) M) and hpGRF (5.0 ng/ml) on GH release were less than additive. However, ionomycin (10(-5) M) further increased GH release over that resulting from the synergistic action of TRH and hpGRF (5.0 ng/ml each). Verapamil (a calcium channel blocker) did not affect GH release induced by either TRH or hpGRF (5.0 ng/ml each). However, this agent did inhibit synergistic GH release evoked by TRH and hpGRF, 8 Br-cAMP, forskolin, or isobutylmethylxanthine. These results suggest that calcium participates in secretagogue-induced GH release from chicken somatotrophs in vitro.     

Growth hormone secretion from chicken adenohypophyseal cells in primary culture: effects of human pancreatic growth hormone-releasing factor, thyrotropin-releasing hormone, and somatostatin on growth hormone release

A primary culture of chicken adenohypophyseal cells has been developed to study the regulation of growth hormone (GH) secretion. Following collagenase dispersion, cells were exposed for 2 hr to vehicle (control) or test agents. Human pancreatic (tumor) growth hormone-releasing factor (hpGRF) and rat hypothalamic growth hormone-releasing factor stimulated GH release to similar levels. GH release was increased by the presence of dibutyryl cyclic AMP. Thyrotropin-releasing hormone (TRH) alone did not influence GH release; however, TRH plus hpGRF together exerted a synergistic (greater than additive) effect, increasing GH release by 100 to 300% over the sum of the values for each secretagogue acting alone. These relationships between TRH and hpGRF were further examined in cultured cells exposed to secretagogues for two consecutive 2-hr incubations. TRH pretreatment enhanced subsequent hpGRF-stimulated GH release by about 80% over that obtained if no secretagogue was present during the first incubation. In other experiments, somatostatin (SRIF) alone did not alter GH secretion. However, SRIF reduced hpGRF-stimulated GH release to levels found in controls. Furthermore, GH release stimulated by the presence of both TRH and hpGRF was lowered to control values by SRIF. The results of these studies demonstrate that a primary culture of chicken adenohypophyseal cells is a useful model for the study of GH secretion. Indeed, these results suggest that TRH and hpGRF regulate GH secretion by mechanisms which are not identical.     

In vivo and in vitro stimulation of growth hormone release in chickens by synthetic human pancreatic growth hormone releasing factor (hpGRFs)

The effect of the synthetic human pancreatic GH releasing factors (hpGRFs, hpGRF44 and hpGRF40) on GH release was studied both in vivo and in vitro. Four-week-old cockerels were injected iv with hpGRFs at 0.1 microgram, 1.0 microgram, and 10.0 micrograms/bird. The 0.1 microgram dose of hpGRFs had no effect on plasma GH levels, but the 1.0 microgram and 10.0 micrograms doses of hpGRFs caused a dose-dependent elevation of plasma GH. Plasma GH levels returned to basal 60 min after injection. HpGRFs, thyrotropin releasing hormone (TRH) and somatostatin (SRIF) were examined in a chicken in vitro pituitary cell culture system. In vitro hpGRFs and TRH caused a significant dose-dependent release of chicken GH, and the ability to stimulate GH was additive when hpGRF44 and TRH were added together in the culture medium. SRIF showed no consistent effect on the release of chicken GH, but it inhibited the stimulatory effect of hpGRF44 on chicken GH release. We conclude that hpGRF is a potent releaser of chicken GH in vivo and in vitro.     

Relationship between cyclic AMP-stimulated and native gonadotropin-releasing hormone-stimulated gonadotropin release in the goldfish.

The relationship between drugs elevating intracellular cAMP levels and gonadotropin (GTH)-releasing hormone (GnRH) in the stimulation of GTH secretion in the goldfish was investigated using dispersed goldfish pituitary cells in primary culture. In static incubation experiments, activation of adenylyl cyclase by forskolin and the inhibition of cAMP phosphodiesterase by 3 isobutyl-1-methylxanthine (IBMX) increased cAMP release and stimulated GTH secretion. The addition of membrane permeant cAMP analogs, 8-bromoadenosine 3':5'-cyclic monophosphate (8Br-cAMP), and dibutyryl cAMP also increased GTH release, suggesting that elevation of cAMP levels can induce GTH secretion. In the goldfish, dopamine is a physiological inhibitor of GTH release. Application of the dopamine agonist apomorphine decreased the GTH responses to forskolin, 8Br-cAMP, and salmon GTH-releasing hormone (sGnRH). The ability of agents that elevate cAMP levels to mimic GnRH action on GTH release suggests that cAMP may mediate GnRH-stimulated GTH secretion in the goldfish; however, this possibility was not substantiated by results from further experiments. In 2-hr static incubation studies, the GTH responses to sGnRH and chicken GnRH-II (cGnRH-II) were enhanced by coincubations with forskolin, IBMX, and 8Br-cAMP. The magnitudes of these enhancements were at least additive, if not synergistic. The levels of cAMP released into the media were unaffected by treatment with sGnRH and cGnRH-II, either in the absence or in the presence of IBMX. Replacement of normal testing media with Ca(2+)-deficient media (without Ca2+ salts and in the presence of 0.1 mM EGTA) decreased sGnRH and cGnRH-II stimulation of GTH release but did not affect forskolin and 8Br-cAMP actions. These results indicate that sGnRH and cGnRH-II stimulation of short term (less than or equal to 2-h) GTH release in the goldfish is not mediated by cAMP. The kinetics of the interactions between sGnRH, forskolin, and IBMX were also investigated in cell column perifusion studies. Applications of 5-min pulses of forskolin and IBMX stimulated rapid increases in GTH release; the latencies of these responses were similar to that observed with sGnRH. The simultaneous applications of sGnRH with either forskolin or IBMX resulted in GTH responses that were of greater magnitude and longer duration than those in response to sGnRH alone. These results together indicate that elevation of cAMP levels can potentiate the GTH response to the native GnRHs by increasing the magnitude of the acute GTH release and by prolonging the duration of GnRH action; however, cAMP does not appear to be involved directly in mediating GnRH stimulation of GTH release.(ABSTRACT TRUNCATED AT 400 WORDS)     

Is calcium or cyclic AMP involved in the inhibitory effect on pituitary hormone secretion of the tripeptide aldehyde proteinase inhibitors?

The mechanism by which tripeptide aldehyde proteinase inhibitors decrease prolactin (PRL) and growth hormone (GH) secretion was studied. Agents known to modify the intracellular levels of cyclic adenosine monophosphate (cAMP) or cytosolic free calcium were used in monolayer cultures of the rat anterior pituitary gland. The phosphodiesterase inhibitor isobutyl-methylxanthine (IBMX), 8-bromo-cAMP and forskolin all stimulated PRL release. Boc-D-Phe-Pro-arginal (Boc-DPPA) at 1 mmol/l concentration was a potent inhibitor of basal PRL release and significantly decreased the effect of 8-Br-cAMP, forskolin or IBMX (0.5 mmol/l). Forskolin (1 mumol/l) stimulated ACTH, PRL and GH release and all these effects were decreased by 100 mumol/l of Boc-D-Phe-Phe-lysinal (Boc-DPPL). Neither tripeptide aldehyde affected the forskolin-induced rise in intracellular cAMP. Growth hormone releasing factor (hpGRF, 1 nmol/l) stimulated both GH release and intracellular cAMP generation; Boc-DPPL (100 mumol/l) significantly decreased stimulated GH release without affecting cAMP accumulation. Increasing medium K+ to 10 times normal level stimulated PRL release presumably by enhancing Ca2+ entry into the cells and 1 mmol/l Boc-DPPA decreased high potassium-stimulated PRL release. The ionophore A-23187 stimulated PRL release at 10 mumol/l but not at 1 mumol/l. At 1 mumol/l A-23187 prevented the Boc-DPPA-induced inhibition of PRL release. These findings suggest that the tripeptide aldehyde proteinase inhibitors inhibit PRL and GH release at a site beyond cAMP formation.     

Possible involvement of adenylyl cyclase-cAMP-protein kinase a pathway in somatostatin inhibition of growth hormone release from chicken pituitary cells.

Somatostatin (SRIF) reduces growth hormone releasing hormone (GRF)-stimulated growth hormone (GH) release from avian and mammalian adenohypophyseal cells. The present studies examined the intracellular mechanisms mediating SRIF inhibition of GRF-stimulated GH release from chicken pituitary cells. Increases (P less than 0.05) in GH release were observed in the presence of (1) GRF; (2) the adenylyl cyclase stimulator, forskolin; (3) a cAMP analog, 8-bromo-cAMP; (4) the phosphodiesterase inhibitor 3-isobutyl-l-methyl-xanthine (IBMX) combined with GRF; (5) a tumor-promoting phorbol ester and protein kinase C activator, phorbol 12-myristate, 13-acetate (PMA); (6) a diacylglycerol analog, 1,2-dioctanoyl-glycerol (DiC8); and (7) a calcium ionophore, A23187, alone and in combination with PMA. Somatostatin (10 ng/ml) reduced the release of GH stimulated by GRF, forskolin, and 8-bromo cAMP and the GRF-provoked release of GH in the presence of IBMX (P less than 0.05). Somatostatin, however, did not influence GH release in the presence of the protein kinase C activators, PMA or DiC8, or the calcium ionophore A23187. These data suggest that SRIF inhibits GRF-provoked GH release by reducing the ability of the cAMP-protein kinase A but not of the calcium or protein kinase C intracellular message pathways to stimulate GH release.     

Human pancreatic tumor growth hormone (GH) - releasing factor and cyclic adenosine 3',5'- monophosphate evoke GH release from anterior pituitary cells: the effects of pertussis toxin, cholera toxin, forskolin, and cycloheximide

Both synthetic human pancreatic tumor GH-releasing factor (hpGRF) and prostaglandin E2 (PGE2) rapidly stimulate cellular cAMP accumulation in and GH release from primary cultures of rat anterior pituitary cells. SRIF inhibits both of these actins. A 1-h treatment with the protein synthesis inhibitor cycloheximide potentiates hpGRF-induced cAMP accumulation for hours and GH release for the first hour. This indicates that a rapidly turning over protein tonically mutes the degree of hpGRF-stimulated cAMP accumulation. Pretreatment of the cells with pertussis toxin amplifies hpGRF- and PGE2-stimulated cAMP levels and GH release; pertussis toxin also attenuates the ability of SRIF to affect these variables. This suggests that an inhibitory coupling protein contributes to these events. Finally, cholera toxin and forskolin are also potent stimulators of cAMP accumulation and GH release. We conclude that hpGRF-evoked GH release and the inhibitory action of SRIF are closely correlated with the cAMP-generating system.     

Hypothalamic hormones that release growth hormone stimulate hepatic 5'-monodeiodination activity in the chick embryo

Plasma GH, tri-iodothyronine (T3), thyroxine (T4) and liver 5'-monodeiodination (5'-D) activity were measured in 18-day-old chick embryos injected with thyrotrophin-releasing hormone (TRH) and human pancreatic growth hormone releasing factor (hpGRF). Injections of 0.1 and 1 microgram TRH and 1.5 micrograms hpGRF increased the concentration of plasma GH while injection of 15 micrograms hpGRF had no effect. Concentrations of plasma T3 were raised after injection of TRH or hpGRF. Injections of TRH but not of hpGRF raised the concentration of plasma T4. The increases in concentration of plasma T3 after injection of TRH or hpGRF were parallelled by increases in liver 5'-D activity. An injection of 0.25 micrograms T4 significantly raised the concentration of T4 in plasma but had no effect on plasma T3 or liver 5'-D activity. It is concluded that the release of chicken GH by TRH or hpGRF is responsible for the observed increase in plasma concentration of T3 and liver 5'-D activity.     

Pituitary growth hormone secretion in the turbot, a phylogenetically recent teleost, is regulated by a species-specific pattern of neuropeptides.

In mammals, growth hormone (GH) is under a dual hypothalamic control exerted by growth hormone-releasing hormone (GHRH) and somatostatin (SRIH). We investigated GH release in a pleuronectiform teleost, the turbot (Psetta maxima), using a serum-free primary culture of dispersed pituitary cells. Cells released GH for up to 12 days in culture, indicating that turbot somatotropes do not require releasing hormone for their regulation. SRIH dose-dependently inhibited GH release up to a maximal inhibitory effect of 95%. None of the potential stimulators tested induced any change in basal GH release. Also, neither forskolin, an activator of adenylate cyclase, nor phorbol ester (TPA), an activator of protein kinase C, were able to modify GH release, suggesting that spontaneous basal release already represents the maximal secretory capacity of turbot somatotropes. In contrast, forskolin and TPA were able to increase GH release in the presence of SRIH. In this condition (coincubation with SRIH), pituitary adenylate cyclase-activating polypeptide (PACAP) stimulated GH release, whereas none of the other neuropeptides tested (GHRHs; sea bream or salmon or chicken II GnRHs; TRH; CRH) had any significant effect. These data indicate that inhibitory control by SRIH may be the basic control of GH production in teleosts and lower vertebrates, while PACAP may represent the ancestral growth hormone-releasing factor in teleosts, a role taken over in higher vertebrates by GHRH.     

Cyclic AMP inhibits the synthesis and release of prolactin from human decidual cells

Exposure of human decidual cells for 0.5 h to dibutyryl cAMP, isobutyl-methylxanthine (IBMX), cholera toxin or forskolin caused a dose-dependent inhibition of prolactin release with maximal inhibition by each agent of 50-60%. Dibutyryl cAMP (5 mM), IBMX (0.5 mM), and cholera toxin (10 micrograms/ml) also inhibited prolactin synthesis to the same extent as prolactin release. Dibutyryl cAMP, IBMX, and cholera toxin, however, had no effect on the release of 35S-methionyl-prolactin from decidual cells preincubated for 24 h in medium with 35S-methionine. These agents, however, had no effects on the synthesis or release of TCA-precipitable 35S-decidual proteins and did not cause the degradation of intracellular or released prolactin. The demonstration that agents which increase intracellular cAMP levels inhibit the synthesis and release of decidual prolactin strongly implicates cAMP as a second messenger in the regulation of the synthesis and release of the hormone. The inhibitory effect of cAMP on prolactin release appears to be on the release from a rapidly releasable, newly synthesized intracellular prolactin pool.     

Calcium control of growth hormone release from chicken pituitary glands in vitro

The influence of calcium on the basal and stimulated release of growth hormone (GH) from chicken pituitary glands has been determined in vitro. Basal GH release occurred in Ca2+ deficient media, although it was increased in proportion to the medium Ca2+ concentration. Growth hormone release was stimulated by 10(-7)-10(-9) M thyrotrophin-releasing hormone (TRH), maximal stimulation being observed in the presence of 10(-8) M TRH and 1.5 mM Ca2+. Decreases in the Ca2+ concentration (to 0.75, 0.375, or 0 mM) suppressed the GH response to 10(-8) M TRH, as did increases (to 3.0 and 6.0 mM) in the Ca2+ concentration. These results suggest that GH release in chickens is regulated by Ca2+-dependent mechanisms.     

Penfluridol decreases secretagogue-induced TSH, GH, and LH secretion in vitro: a possible role for calcium-calmodulin

This study was designed to investigate the effects of penfluridol, a potent neuroleptic calmodulin inhibitor, on basal and secretagogue-stimulated secretion of thyroid-stimulating hormone (TSH), growth hormone (GH), and luteinizing hormone (LH). The drug had no effect on basal TSH or LH release, but decreased GH release in a dose-related manner. TSH, LH, and GH secretion stimulated by calcium ionophore A23187 or 50 mM K+ was decreased by penfluridol as was TSH and GH release stimulated by dibutyryl-cAMP (dbcAMP). Penfluridol reversibly abolished the stimulatory effect of thyrotropin-releasing hormone (TRH) on TSH release in perifused dispersed pituitary cells. The compound inhibited hormonal release without affecting hormonal synthesis and cellular morphology (trypan blue exclusion test). Penfluridol appears to inhibit hormonal secretion by interfering with the calcium-calmodulin system in the anterior pituitary; therefore calmodulin may be an important link in the stimulus-secretion coupling of adenohypophyseal hormones.     

Triiodothyronine (T3) inhibition of growth hormone secretion by chicken pituitary cells in vitro.

These studies examined the cellular basis for the inhibitory effects of triiodothyronine (T3) on growth hormone-releasing factor (GRF)-evoked growth hormone (GH) release from chicken anterior pituitary cells in vitro. A primary monolayer culture of anterior pituitaries from 4- to 8-week-old White Leghorn cockerels was performed as previously described by this laboratory. Following a 72-hr preincubation period, cells were washed and incubated (2 hr) with either secretagogues or media alone (control). T3 (20 ng/ml) or vehicle was added to cells during both the preincubation (48-72 hr) and incubation (2 hr period. Triiodothyronine reduced (P less than 0.05) GH release (ng/ml) in response to (1) GRF; (2) the adenylyl cyclase stimulator, forskolin; (3) the cAMP analog and protein kinase A activator, 8-bromo cAMP; and (4) the phorbol ester and protein kinase C activator, phorbol 12-myristate 13-acetate. Triiodothyronine reduced (P less than 0.05) the intracellular content of GH and total GH (released GH and intracellular GH) irrespectively of whether secretagogues were also present. When GH release was expressed as a percentage of total GH [released GH/(intracellular GH + released GH)], percentage GH released in response to GRF, or the protein kinase A, protein kinase C, or calcium pathway activators was not as great in T3-treated versus non-T3-treated cells. These data indicate that T3 inhibits GRF-evoked GH release by reducing the availability of intracellular stores of GH and by also inhibiting second messenger-stimulated GH release pathways.     

Comparative stimulation of growth hormone secretion in anaesthetized chickens by human pancreatic growth hormone-releasing factor (hpGRF) and thyrotrophin-releasing hormone (TRH)

Plasma concentrations of growth hormone (GH) were elevated in anaesthetized male domestic fowl following the intravenous administration of either synthetic human pancreatic GH-releasing factor 1-44 (NH2) (hpGRF) or synthetic thyrotrophin-releasing hormone (TRH). In 6-week-old chicks the plasma GH level was elevated between 5 and 10 min after the injection of hpGRF at doses between 1 and 80 micrograms/kg. The magnitude of the response increased with doses of hpGRF between 1 and 10 micrograms/kg but declined with higher doses. The GH concentration rapidly declined between 10 and 20 min and between 20 and 40 min after injection. The administration of TRH had similar effects on GH secretion, although the responses were greater than with comparable doses of hpGRF, and the most effective dose (1-1.4 micrograms/kg) was less than with hpGRF. In anaesthetized adult cockerels GH secretion was also increased by the administration of hpGRF (1-20 micrograms/kg) or TRH (0.1-80 micrograms/kg) and in both cases the dose-response relationship was biphasic. The maximal response to TRH in adult birds was again greater than that produced by hpGRF although the response was less than that elicited in immature birds and required a higher dose (20 micrograms/kg) of TRH. The optimal dose of hpGRF and the magnitude of the GH response induced in adult birds was comparable with that in immature chicks. These results demonstrate provocative effects of TRH and hpGRF on GH secretion in the domestic fowl. The sensitivity of the GH response to TRH suggests that it may have a physiological role in the hypothalamic control of GH secretion.     

Growth hormone secretion in anaesthetized fowl. 2. Influence of heterologous stimuli in birds refractory to human pancreatic growth hormone-releasing (hpGRF) or thyrotrophin releasing hormone (TRH)

In anaesthetized young (6 weeks old) and adult (22-30 weeks old) domestic fowl, the administration of thyrotrophin releasing hormone (TRH; 1.0 micrograms/kg in young birds; 10.0 micrograms/kg in adults) or human pancreatic growth hormone-releasing factor (hpGRF(1-44)NH2; 10.0 micrograms/kg in both cases) markedly increased the growth hormone (GH) concentration in plasma samples collected 10 min later. In birds injected with TRH, this stimulation of GH secretion attenuated the GH response to a second TRH challenge (given 15 or 60 min after the first in adult or young birds, respectively); similarly, hpGRF pretreatment blunted the GH response to a further hpGRF injection. However, the administration of hpGRF to both immature and adult birds made refractory to TRH challenge was followed by increased GH secretion and vice versa. Moreover, the GH secretory response to hpGRF in birds pretreated with TRH was greater (1.99-fold in young birds, 1.52-fold in adults) than the increase in plasma GH concentration following hpGRF administration in untreated birds. Similarly, prior exposure to hpGRF also increased the GH response to TRH stimulation (by 2.24-fold in the young, 3.56-fold in the adults). These results demonstrate that TRH not only overcomes GH refractoriness to hpGRF and vice versa, but the GH response to heterologous provocative stimuli is potentiated in birds refractory to TRH or hp GRF challenge.     

Stimulation of growth hormone secretion in dwarf chickens by thyrotrophin-releasing hormone (TRH) or human pancreatic growth-hormone-releasing factor (hpGRF)

The basal plasma growth hormone (GH) level in adult sex-linked dwarf hens was elevated in comparison with autosomal dwarf hens and with control (Cornell K strain) laying hens. The iv administration of thyrotrophin-releasing hormone (TRH) (10 μg/kg) had no effect on GH secretion in control hens but slightly (1.2-fold) and transiently (for 10 min) increased the GH level in the autosomal dwarfs and greatly (8.7-fold) increased the GH level in the sex-linked dwarfs, in which it remained elevated for at least 30 min after injection. The iv administration of human pancreatic GH-releasing factor (hpGRF) (10 μg/kg) stimulated GH release in each strain. The response in the sex-linked dwarfs was greater than that in the autosomal dwarfs and the control hens but less than that elicited by TRH. These results suggest that the increased basal GH level in the sex-linked dwarfs results from an increased responsiveness to provocative stimulation.     

A modulatory role for cyclic AMP in the control of thyrotrophin release: studies with forskolin and dibutyryl cyclic AMP

We have studied the effects of cyclic AMP (cAMP) on TSH secretion by cultured rat pituitary cells, using forskolin and dibutyryl cAMP (dbcAMP) to raise the cellular cAMP content by different mechanisms. Forskolin (10 mumol/l), a stimulator of adenylate cyclase, raised the cAMP content within 10 min, but had a more delayed effect on TSH release, with no significant stimulation for at least 6 h, but a clear dose-dependent effect at 24 h. Incubation with dbcAMP likewise increased TSH release after 6-24 h. By contrast, high cellular cAMP levels induced by either forskolin or dbcAMP augmented the TSH response to TRH at an early stage, before any detectable change in unstimulated TSH release. Pretreatment of cells with forskolin led to a parallel upward shift in the subsequent TRH dose-response curve, without a significant change in median effective dose or any change in cellular TSH content. These findings suggest that cAMP acts to increase the availability of TSH for acute release by TRH by modulation of an intracellular releasable hormone pool, and indicate synergistic interactions between the adenylate cyclase system and the phospholipid-calcium stimulus-release coupling mechanism of TRH.     

GRF and human somatotropic adenoma. In vivo and in vitro correlations between GH release and morphological and immunocytochemical aspects

Nine somatotropic adenomas identified by histological and immunocytochemical methods were studied in vitro during 40 days. The spontaneous release of GH decreased at various rates according to adenomas. A decrease in the size, number of secretory granules and in immunoreactivity with anti-hGH serum were also noted. When cortisol was added (350 nM), the GH secretion sustained for a longer time or even increased at its initial level. The effect of GRF (hpGRF1-44NH2, 10(-8)M) on GH release was tested by 3 hours incubation at 6-9 days of culture. The increase in GH varied from 106 to 420% compared to the basal release. It was similar to in vivo release in 3 out of 6 cases. The highest responses were found in densely granulated and strongly immunoreactive adenomas. The effect of GRF on the GH synthesis was studied by continuous incubation of GRF (10(-8)M) from the 10th the 40th day of culture. The quantity of GH in the culture medium was never higher than in culture mediums without GRF. No change in GH concentrations in mediums with or without cortisol was found when TRH (2.5, 10(-6)M) or TRH + GRF was added. No change in morphological and immunocytochemical cell characteristics were noted either with or without GRF. Thus, the variability of GH response to GRF in somatotropic adenomas in culture seems to be related to their morphofunctional heterogeneity. Under our experimental conditions, GRF stimulates the release of stored GH in tumoral cells but does not seem to stimulate its synthesis.     

Regulation of thyrotropin (TSH) release and production in monolayer cultures of transplantable TSH-producing mouse tumors.

Studies of TSH release and production were performed in short term monolayer cultures of transplantable, thyroid hormone responsive, thyrotropin (TSH) producing mouse pituitary tumors. These tumors contained large amounts of TSH, small amounts of growth hormone (GH) and no detectable luteinizing hormone (LH), indicating that the predominant hormone product of tumor cells was TSH. The TSH content per tumor cell was similar to that of the normal pituitary where thyrotrophs represent a small fraction of the total cells, suggesting that the TSH content per tumor cell was less than that of the normal thyrotroph. There was a time dependent release and production of TSH by tumor cells in monolayer culture. Thyrotropin releasing hormone (TRH) increased the release into the media and the production of TSH in a dose dependent manner. Maximum effects were noted at 0.2 ng/ml. Thyroid hormones and somatostatin inhibited both basal and TRH induced effects on both TSH release and production. TSH release as induced by TRH was calcium dependent. TSH release was stimulated by ouabain (10(-3)M) and potassium (57 mM), agents known to promote cellular calcium uptake in a calcium dependent manner. These studies indicate that tumor derived cells function in monolayer culture in a similar fashion to normal thyrotrophs. Studies were conducted to test the hypothesis that TRH action is mediated by adenosine 3',5' monophosphate (cAMP). Dibutyryl cAMP (6 mM) and theophylline (10 mM) increased TSH release suggesting that cAMP is involved in TSH release. However, TRH had no detectable effect on tumor cell adenylate cyclase activity or levels of cAMP. In contrast, PGE1 (1-10 mug/ml) stimulated adenylate cyclase activity and elevated cellular levels of cAMP without increasing TSH release. Thus, we are unable to confirm the postulate that cAMP is the intracellular mediator of TRH action.