Effects of a novel hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide, on pituitary hormone release in rats.

We have demonstrated that the novel hypothalamic peptide pituitary adenylate cyclase-activating polypeptide (PACAP-38; 0.1-100 nmol/l) caused an increase in the release of GH, ACTH, LH and alpha-subunit and accumulation of intracellular cyclic AMP from dispersed rat anterior pituitary cells in static culture for 24 h. There were no significant effects on TSH or prolactin release over the same time-period. PACAP-38 (10 nmol/l) increased the release of GH by 1.3-fold (P less than 0.05), ACTH by 1.9-fold (P less than 0.05), LH by 3.5-fold (P less than 0.001) and alpha-subunit by 2.0-fold (P less than 0.005) and the accumulation of intracellular cyclic AMP by greater than 2-fold (P less than 0.001) after 24 h. However, the time-course for the effect of PACAP-38 (1 mmol/l) on hormone release and intracellular cyclic AMP levels showed a temporal dissociation. The effect of PACAP-38 on GH and ACTH levels did not reach significance until 24 h whereas the effect of PACAP-38 on LH and alpha-subunit release reached significance after 4 h implying a different mechanism of action for their release. To investigate the PACAP-induced secretion of LH and alpha-subunit further, we examined the effects of PACAP after down-regulation of protein kinase C (PKC). PACAP-38 at a dose maximal for the stimulation of LH and alpha-subunit release (10 nmol/l) added together with the PKC activator, 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 0.1 mumol/l) had no greater effect on LH and alpha-subunit release than TPA alone over a 4 h incubation period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endocrine and immunohistochemical studies on thyrotropin (TSH)-secreting pituitary adenomas: responses of TSH, alpha-subunit, and growth hormone to hypothalamic releasing hormones and their distribution in adenoma cells.

Endocrine and immunohistochemical studies were performed in two cases of TSH-secreting pituitary adenomas. The patients had elevated serum TSH and alpha-subunit concentrations despite high serum thyroid hormone levels. In addition, one patient (no. 1) had elevated serum GH levels with clinical evidence of acromegaly. GH-releasing hormone infusion increased serum levels of TSH, alpha-subunit and GH in the two patients. TRH injection increased serum TSH levels in both patients and, concomitantly, serum alpha-subunit and GH levels in patient 1. Basal TSH levels and their responses to TRH changed reciprocally to changes in serum thyroid hormone levels, although TRH-induced GH release did not. The administration of GnRH also increased serum TSH, alpha-subunit, and GH levels in patient 1. In accordance with these in vivo results, pituitary adenoma cells in culture obtained from patient 1 responded to GH-releasing hormone, TRH, or GnRH to secrete TSH, alpha-subunit, and GH. Incubation of cells with dexamethasone resulted in inhibition of TSH and stimulation of GH secretion without a significant change in alpha-subunit secretion. On the basis of light microscopic and electron microscopic double gold immunohistochemistry, the tumor from patient 1 was a bimorphous adenoma composed of two separate cell types: cells with TSH beta-subunit (TSH beta) and alpha-subunit, and those with GH and alpha-subunit. The remainder consisted mainly of cells with TSH beta and alpha-subunit. The coproduction of the unusual combination of two hormones such as GH and alpha-subunit in a single-type of adenoma cell and the coexistence of thyrotrophs and somatotrophs in one pituitary adenoma along with the aberrant responses of TSH beta, alpha-subunit, and GH to multiple hypothalamic hormones suggest the dedifferentiation of pituitary cells to multipotential progenitor cells by neoplastic transformation.     

Development of hypothalamic control of growth hormone secretion in the rat

The development of hypothalamic control of GH in the late prenatal and early postnatal periods in the rat was studied by employing a static system for the incubation of pituitaries. The basal secretion of GH into the medium after a 3-h incubation period showed a gradual increase from day 18 prenatally to day 1 postnatally. This was followed by a gradual decline in GH release on postnatal days 5 and 8. There was a sustained rise in the total pituitary GH content from prenatal day 18 to postnatal day 8. The percentage of the total GH that was released into the medium was high from fetal pituitaries and lower from neonatal pituitaries. TRH (100 ng/ml) stimulated GH secretion starting on prenatal day 21. This TRH effect persisted through day 8 postnatally. Hypothalamic extracts from fetuses and neonates stimulated the secretion of GH when coincubated with pituitaries of the same age and with adult male rat pituitaries. Similarly, adult male rat hypothalamic extract stimulated the secretion of GH from pituitaries of 1-day-old neonates. Pronase treatment of neonatal hypothalamic extract completely abolished its stimulatory effect on GH release. Incubation of 1-day postnatal pituitaries with cerebral cortical extract obtained from neonates of the same age did not alter the secretion of GH; however, cerebral cortical extract from adult males did cause a significant stimulation of GH release from the neonatal pituitaries. Somatostatin (100 ng/ml) failed to inhibit GH release by pituitaries until day 5 postnatally, but a 10-fold increase in the concentration of somatostatin significantly inhibited GH secretion from pituitaries of rats as early as day 21 prenatally. Coincubation of hypothalamic extract with the high concentration of somatostatin significantly attenuated the effect of the extract in stimulating GH release from pituitaries of 1-day-old rats. The results suggest that the high circulating levels of GH during the late prenatal and early neonatal periods are maintained by a combination of factors including the release of a hypothalamic peptidergic GH-releasing factor, the relative insensitivity of the pituitary to somatostatin, and changes in the relative size of storage vs. releasable pools of GH during development.     

Effects of rat growth hormone (rGH)-releasing factor and somatostatin on the release and synthesis of rGH in dispersed pituitary cells

The effects of rat hypothalamic GH-releasing factor (GRF) and somatostatin (SRIF) on the release and biosynthesis of rat GH were studied by RIA and quantitative immunoprecipitation using monolayer cultures of rat anterior pituitary cells. In kinetic studies, GRF stimulation of GH release appeared at the first sampling time (20-min incubation) and the effect began to diminish after 2-h incubation with GRF. On the other hand, total (cell plus medium) content of GH significantly increased only after 24-h incubation. To examine the GH-synthesizing effect of GRF more directly, newly synthesized GH labeled by [35S]methionine during incubation with GRF was quantified by immunoprecipitation. The amount of immunoprecipitable GH increased significantly and specifically (compared with the total amount of labeled proteins) also only after 24-h incubation. When GH pools were labeled with [35S]methionine under different schedules, the basal release of newly synthesized GH, which was labeled for 1 h immediately before chase incubation was lower during the first 15 min than stored GH which had been labeled earlier. Basal newly synthesized GH secretion exceeded stored GH secretion after 30 min. GRF stimulated the release of GH from both pools but the stimulation of stored GH was greater. In this system, SRIF suppressed both the basal and stimulated release of GH but did not modify GH biosynthesis under either condition. Newly synthesized GH showed significant degradation during 24-h incubation; neither GRF nor SRIF affected the rate of GH degradation during the same incubation period. These results indicate that 1) GRF stimulates both release and synthesis of GH; 2) these two effects have different kinetics and different sensitivities to SRIF; and 3) GRF stimulates the release of GH from heterogeneous pools disproportionally.     

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)     

The effects of hypothalamic extracts and TRH on the growth and hormone production of GH3 cells in culture

Hypothalamic factors were tested for their effects on the production of hormones and the growth of GH3 cells, cloned rat pituitary cells producing prolactin (Prl) and growth hormone (GH). Hypothalamic extracts (HE) (0.05 mg/ml) and TRH (0.3 microM) stimulated the synthesis of Prl to levels of 306% and 360%, respectively, of the control culture in a medium containing 0.5% foetal bovine serum (FBS) during a 24 h incubation. They did not affect the rate of GH production. The thymidine uptake was suppressed to 57% and 46% of the control by the addition of HE and TRH, respectively. They also inhibited the growth of GH3 to 70% and 74% of the control culture during an 8-day incubation period. On the other hand, LRH affected neither the rate of hormone production nor the thymidine uptake. Somatostatin suppressed the synthesis of Prl and GH, but it did not affect the incorporation of thymidine into the cells. The gel filtration studies of HE revealed that the inhibitory effects of HE on the thymidine uptake were dependent on two substances, TRH and an unknown factor(s) of high molecular nature. The relationship between hormone synthesis and DNA synthesis will be discussed on the basis of the TRH-induced effects on Prl production and DNA synthesis in GH3 cells.     

Cachectin alters anterior pituitary hormone release by a direct action in vitro.

Cachectin (tumor necrosis factor) is a powerful macrophage hormone released during infection, which circulates in blood to produce diverse effects in the organism. We examined the effect of cachectin on release of anterior pituitary hormones from either hemipituitaries or dispersed pituitary cells incubated in vitro. The action of cachectin on dispersed cells was demonstrable only after 2 hr of incubation. With this incubation time, the protein produced a dose-related stimulation of release of adrenocorticotropin (ACTH), growth hormone (GH), and thyrotropin (TSH), but not of prolactin (Prl), from both hemipituitaries and dispersed cells. The doses required for stimulation were low in the case of hemipituitaries, usually of the order of 10(-12) M, whereas they were higher by one or two orders of magnitude with the dispersed pituitary cells. This may be related either to loss of receptors for the protein during the dispersion procedure or to the fact that in the hemipituitary system cell interactions are facilitated because the cells are close to each other. In the dispersed cell system cachectin evoked a dose-related decrease in cyclic AMP content. Incubation with somatostatin lowered the cyclic AMP content of the cells and depressed GH output without altering output of TSH or Prl. When somatostatin and cachectin were incubated together with the cells, the suppression of cyclic AMP production was abolished; TSH and Prl release were stimulated, but the action of cachectin to stimulate GH release was blocked. The stimulation of Prl release by cachectin in the presence of somatostatin may be related to the elevation of cyclic AMP, a known stimulator of Prl release. The cyclooxygenase inhibitor indomethacin nearly completely blocked the stimulatory effect of cachectin on release of GH and TSH from dispersed pituitary cells but had only a slight and nonsignificant attenuating effect on its ACTH-releasing action. These results suggest that at least part of the stimulatory action of the peptide on pituitary hormone release is brought about by prostaglandins. The failure of indomethacin to block the release of ACTH induced by cachectin suggests that other mechanisms may be involved in the release of ACTH induced by this peptide. Since the concentrations of cachectin required to stimulate pituitary hormone release are similar to those that are encountered in plasma during infection, it is likely that this direct pituitary action has pathophysiological significance.     

Anterior pituitary hormone control by interleukin 2.

Several monokines, proteins secreted by monocytes and macrophages, alter release of hormones from the anterior pituitary. We report here the ability of femtomolar concentrations of interleukin 2 (IL-2), a lymphokine released from T lymphocytes, to alter directly pituitary hormone release. The effects of concentrations of IL-2 ranging from 10(-17) to 10(-9) M on anterior pituitary hormone release were evaluated in vitro. Hemipituitaries were preincubated in 1 ml of Krebs-Ringer bicarbonate buffer (KRB) followed by incubation for 1 or 2 hr with KRB or KRB containing different concentrations of IL-2. This was followed by incubation for 30 min in 56 mM potassium medium to study the effect of pretreatment with IL-2 on subsequent depolarization-induced hormone release. Prolactin (PRL), luteinizing hormone (LH), follicle-stimulating hormone (FSH), corticotropin (ACTH), growth hormone (GH), and thyrotropic hormone (TSH) released into the incubation medium were measured by radioimmunoassay. IL-2 stimulated the basal release of PRL at 1 or 2 hr but suppressed the subsequent depolarization-induced PRL release, perhaps because the readily releasable pool of PRL was exhausted. The minimal effective dose (MED) was 10(-15) M. Conversely, IL-2 significantly suppressed the basal release of LH and FSH at 1 or 2 hr, with a MED of 10(-16) M, thus demonstrating a reciprocal action of the cytokine on lactotrophs and gonadotrophs. The subsequent depolarization-induced release of LH and FSH was suppressed, indicative of a persistent inhibitory action of IL-2. IL-2 stimulated ACTH and TSH release at 1 hr and the MEDs were 10(-12) and 10(-15) M, respectively. Conversely, IL-2 significantly lowered the basal release of GH at 1 hr, with a MED of 10(-15) M. The release of GH was not altered at 2 hr. The high potassium-induced release of ACTH, TSH, and GH was not affected. The results demonstrate that IL-2 at picomolar concentrations affects the release of anterior pituitary hormones. This cytokine may serve as an important messenger from lymphocytes exerting a direct paracrine action on the pituitary by its release from lymphocytes in the gland or concentrations in the blood that reach the gland may be sufficient to activate it.     

Thyrotropin-releasing hormone stimulates growth hormone release from the anterior pituitary of hypothyroid rats in vitro

We have examined the interaction of thyroid hormone and TRH on GH release from rat pituitary monolayer cultures and perifused rat pituitary fragments. TRH (10(-9) and 10(-8)M) consistently stimulated the release of TSH and PRL, but not GH, in pituitary cell cultures of euthyroid male rats. Basal and TRH-stimulated TSH secretion were significantly increased in cells from thyroidectomized rats cultured in medium supplemented with hypothyroid serum, and a dose-related stimulation of GH release by 10(-9)-10(-8) M TRH was observed. The minimum duration of hypothyroidism required to demonstrate the onset of this GH stimulatory effect of TRH was 4 weeks, a period significantly longer than that required to cause intracellular GH depletion, decreased basal secretion of GH, elevated serum TSH, or increased basal secretion of TSH by cultured cells. In vivo T4 replacement of hypothyroid rats (20 micrograms/kg, ip, daily for 4 days) restored serum TSH, intracellular GH, and basal secretion of GH and TSH to normal levels, but suppressed only slightly the stimulatory effect of TRH on GH release. The GH response to TRH was maintained for up to 10 days of T4 replacement. In vitro addition of T3 (10(-6) M) during the 4-day primary culture period significantly stimulated basal GH release, but did not affect the GH response to TRH. A GH stimulatory effect of TRH was also demonstrated in cultured adenohypophyseal cells from rats rendered hypothyroid by oral administration of methimazole for 6 weeks. TRH stimulated GH secretion in perifused [3H]leucine-prelabeled anterior pituitary fragments from euthyroid rats. A 15-min pulse of 10(-8) M TRH stimulated the release of both immunoprecipitable [3H]rat GH and [3H]rat PRL. The GH release response was markedly enhanced in pituitary fragments from hypothyroid rats, and this enhanced response was significantly suppressed by T4 replacement for 4 days. The PRL response to TRH was enhanced to a lesser extent by thyroidectomy and was not affected by T4 replacement. These data suggest the existence of TRH receptors on somatotrophs which are suppressed by normal amounts of thyroid hormones and may provide an explanation for the TRH-stimulated GH secretion observed clinically in primary hypothyroidism.     

Iso stimulation of GH and cAMP: comparison of beta-adrenergic- to GRF-stimulated GH release and cAMP accumulation in monolayer cultures of anterior pituitary cells in vitro

Growth hormone (GH) release and cAMP content were measured in monolayer cultures of anterior pituitary cells after beta-adrenergic and GH-releasing factor (GRF) receptor activation. Isoproterenol (Iso, ED50-20 nM) was less potent than GRF (ED50-20 pM) in stimulating GH release. Iso caused a rapid stimulation of GH release that was maximal after 15 min and declined thereafter, while GRF caused a more gradual increase in GH secretion that was maximal after 30 min and remained elevated after 3 h. Both Iso- and GRF-stimulated GH release were preceded by an increase in cAMP content in the pituitary cells. Further, the addition of 3-isobutyl-1-methylxanthine (IBMX) to the medium enhanced the GH-stimulatory and cAMP-accumulating effects of both secretagogues. Experiments performed with native catecholamines and synthetic catecholamine agonists and antagonists indicated that the GH-stimulatory effect of Iso was mediated by a mixed population of beta 1-adrenergic and beta 2-adrenergic receptors. Additionally, experiments performed with cultured GH3 tumor cells, found that incubation with GRF, Iso, vasoactive intestinal polypeptide, forskolin, or cholera toxin caused an increase in cAMP content in the cells. However, compared to the responses observed in primary pituitary cultures the GH secretory response to these agents was comparatively small. Together, these studies suggest that a mixed population of beta 1-adrenergic and beta 2-adrenergic receptors may act, at least in part, on somatotrophs in the anterior pituitary to stimulate GH release. Although both GRF and beta 2-adrenergic receptor agents affect GH release through a common second messenger system, their differing pharmacokinetic properties suggest distinct intracellular mechanisms.     

Development of radioimmunoassay for bullfrog thyroid-stimulating hormone (TSH): effects of hypothalamic releasing hormones on the release of TSH from the pituitary in vitro

A bullfrog (Rana catesbeiana) thyroid-stimulating hormone (TSH) beta-subunit (TSHbeta) antiserum was produced by employing a C-terminal peptide synthesized on the basis of the amino acid sequence deduced from bullfrog TSHbeta cDNA. Immunohistochemical studies revealed that the bullfrog adenohypophyseal cells that immunologically reacted with the anti-bullfrog TSHbeta corresponded to those positively stained with an antiserum against human (h) TSHbeta. The antiserum was used for the development of a specific and sensitive radioimmunoassay (RIA) for the measurement of bullfrog TSH. The sensitivity of the RIA was 0.75+/-0.07ng TSH/100microl assay buffer. The interassay and intraassay coefficients of variation were 7.6 and 5.3%, respectively. Several dilutions of pituitary homogenates of larval and adult bullfrogs, or medium in which bullfrog pituitary cells were cultured, yielded dose-response curves that were parallel to the standard curve. Bullfrog prolactin, growth hormone, luteinizing hormone, follicle-stimulating hormone, and alpha-subunit derived from glycoprotein hormones did not react in this assay. Immunoassayable TSH in the pituitary culture medium was confirmed to exist in the form of TSHbeta coupled with the alpha-subunit by an immunoprecipitation experiment using the TSHbeta antiserum and an alpha-subunit antiserum. TSH released from pituitary cells into the medium was also confirmed to possess a considerable activity in stimulating the release of thyroxine from the thyroid glands of larval bullfrogs in vitro.The effects of hypothalamic hormones such as mammalian gonadotropin-releasing hormone (mGnRH), ovine corticotropin-releasing hormone (oCRH), and thyrotropin-releasing hormone (TRH) on the release of TSH by dispersed anterior pituitary cells of the bullfrog larvae and adults were also studied. CRH markedly stimulated the release of TSH from both adult and larval pituitary cells. Both TRH and GnRH moderately stimulated the release of TSH from adult pituitary cells but not from the larval cells. This is the first report on the development of an RIA for amphibian TSH, which has provided the direct evidence that the release of TSH from the amphibian pituitary is enhanced by the hypothalamic releasing hormones such as CRH, TRH, and GnRH.     

Long-term in-vitro treatment of human growth hormone (GH)-secreting pituitary adenoma cells with octreotide causes accumulation of intracellular GH and GH mRNA levels

OBJECTIVE: We studied the effects of long-term in-vitro exposure of human GH secreting pituitary adenoma cells to octreotide on GH release, intracellular GH concentrations and GH messenger ribonucleic acid (mRNA) levels. DESIGN: Human GH-secreting pituitary adenoma cells were cultured for periods from 4 days up to 3 weeks without or with octreotide (10 nM) and/or bromocriptine (10 nM). The effects of these drugs were measured on GH release, intracellular GH concentrations and intracellular GH mRNA levels. PATIENTS: Thirteen patients with GH-secreting pituitary adenomas were studied. Twelve patients were untreated, one had been pretreated with octreotide (12 weeks, 3 x 100 micrograms daily). MEASUREMENTS: GH, PRL, alpha-subunit and IGF-I concentrations in plasma, media and cell extracts were determined by immunoradiometric or radioimmuno-assays. GH mRNA levels were determined by automatic quantification of grain numbers in individual adenoma cells. RESULTS: Incubation of the adenoma cells for 4 days with 10 nM octreotide induced a dose-dependent inhibition of GH release and a parallel increase (increase varying between 124 and 617% of control) in the intracellular GH levels was observed in six of seven adenomas. In addition, bromocriptine, when effective in inhibiting GH release by the adenomas, also induced an increase in intracellular GH levels. Even after 3 weeks of exposure to 10 nM octreotide in vitro there was a statistically significant increase in intracellular GH levels (between 191 and 923% of control). Withdrawal of octreotide after 6 days of incubation resulted in a lowering of intracellular GH levels to control values, showing that the octreotide-induced increase in intracellular GH is reversible. In a 96-hour incubation with 10 nM octreotide, GH mRNA levels were increased in two, and slightly decreased in one of the three adenomas tested. This effect was time dependent in that there was no significant effect of 10 nM octreotide on GH mRNA levels in a 24-hour incubation. CONCLUSIONS: (1) Long-term in-vitro exposure of GH-adenoma cells to octreotide causes an increase in intracellular GH levels in the majority of the adenomas, probably because of an increase in GH mRNA levels in the adenoma cells; and (2) this considerable increase in intracellular GH levels may be one of the explanations for the relatively poor effect of octreotide on tumour shrinkage in patients with GH-secreting pituitary adenomas.     

Specific growth hormone receptors on human peripheral mononuclear cells: reexpression, identification, and characterization

Although specific GH receptors have been demonstrated in various tissues of a number of species, the presence of GH receptors on human peripheral mononuclear cells (PMC) is controversial. Binding of human GH (hGH) to its receptor as the hypothesized initial step of hormone action was consequently studied using mononuclear cells from peripheral venous blood of normal subjects. Specific binding of [125I]hGH was rapid, reversible, and time and temperature dependent. Specific GH binding to PMC was maximal after 8-24 h of preincubation. Binding of hormone was maximal at 37 C after incubation of cells for 2 h. Dissociation of GH was maximal at 37 C after the addition of 6 M NaCl. A linear relationship between specific GH binding and cell number was found. Saturation of GH binding to 10(6) PMC was obtained with 25 ng iodinated hormones. Half-maximal inhibition of GH binding occurred at 12-25 ng unlabeled hGH/tube. Hypothalamic and pituitary hormones as well as insulin did not interfere with specific hGH binding to PMC. Scatchard analysis of [125I]hGH binding to PMC revealed a receptor with a mean affinity constant of 1.5 +/- 0.2 (+/- SD) X 10(9)/M-1 (n = 72) and a maximal binding capacity of 7.1 +/- 2.0 X 10(-11) M/10(6) cells. The concentrations of calcium, sodium, and magnesium ions in the incubation medium strongly influenced GH binding, whereas pH or potassium concentration did not. As interassay variation of the binding assay was low (14% for total binding; 6% for specific hGH binding), this direct approach to study tissue receptors for hGH in a human in vitro test was reproducible and should encourage the investigation of receptor regulation as well as the study of binding in human disease.     

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.     

Protein kinase C activators and calcium-mobilizing agents synergistically increase GH, LH, and TSH secretion from anterior pituitary cells

A series of studies was designed to determine the effects of protein kinase C activators on TSH, LH, and GH release from anterior pituitary cells. A 15-min incubation of cultured pituitary cells with synthetic diacylglycerol or phorbol myristate acetate, stimulators of protein kinase C, increased GH, LH, and TSH release. Similarly phospholipase C, which liberates endogenous diacylglycerol, stimulated GH, LH, and TSH secretion. The potentiation of the effects of protein kinase C activators is achieved by calcium mobilization in various cell types. The results of the present studies show that calcium ionophore A23187 or calcium channel activator maitotoxin potentiate diacylglycerol-, phorbol ester-, or phospholipase C-induced GH, LH, or TSH release. These findings suggest that activation of protein kinase C by diacylglycerol and mobilization of calcium may be synergistically involved in the regulation of GH, LH, and TSH release.     

Mechanisms of TSH release: studies with forskolin, phorbol ester and A23187

We have investigated the interactions of intracellular messenger systems which may regulate thyrotrophin (TSH) release in primary cultures of rat anterior pituitary cells. Calcium ionophore A23187 was used to raise intracellular free calcium, the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) to activate protein kinase C, and forskolin to activate adenylate cyclase. Each of these agents stimulated TSH secretion in a dose-dependent manner, but the effect of forskolin was delayed for at least 6 h. The combined effects of A23187 and TPA on TSH secretion were simply additive, but forskolin synergistically enhanced the effect of A23187 and 55 mM potassium, but not that of TPA. Co-incubation of cells with 1 microM A23187 caused parallel upward shift of the TPA dose-response curve, and the further addition of 10 microM forskolin led to further upward shift. These results suggest that calcium mobilization and adenylate cyclase activation may interact synergistically to modulate TSH secretion. Raised cellular cyclic AMP content may amplify further the TSH release stimulated by the combination of protein kinase C activation and raised intracellular free calcium.     

The inhibition of growth hormone release by gastrin-releasing peptide involves somatostatin release

Injection of gastrin-releasing peptide-27 (GRP) into the third ventricle (IVT) has been shown previously to lower plasma GH levels and block the GH release induced by GRF, suggesting that GRP might act via stimulation of the release of somatostatin (SRIF) into hypophysial portal vessels. Several experiments were performed to test this hypothesis. In the first experiment rat median eminence (ME) fragments were incubated in medium containing concentrations of GRP ranging from 1 pM to 1 microM, and SRIF levels were measured after the 30-min incubation period. GRP significantly stimulated SRIF release at doses of 0.1 nM to 1 microM. Microinjection of SRIF antiserum (3 microliters) IVT prevented GRP (2 micrograms, IVT) from inhibiting the GH surge induced by GRF (1 microgram/kg, iv). A slight but significant decrease in basal plasma GH levels was observed after GRP administration even in the presence of SRIF antiserum. Finally, to rule out a GRP-GRF interaction at the pituitary level, tubes containing dispersed rat pituitary cells (2.5 x 10(5) cells/tube) were incubated for 1.5 h in medium containing various concentrations of GRF (0.4-40 nM) alone or with 0.1 microM GRP. The addition of GRP to the medium had no significant effect on the dose-dependent stimulation of GH release by GRF. The results of these studies demonstrate that GRP can directly stimulate SRIF release in vitro. They further suggest that SRIF is a component of the mechanism whereby GRP inhibits GH release in vivo. Finally, the possibility that GRP acts at the pituitary level to inhibit GH release by blocking GRF receptors on somatotrophs has been ruled out.