Effect of insulin-like growth factors on human foetal, adult normal and tumour pituitary function in tissue culture

To determine the direct effects of insulin-like growth factors (IGFs) on hormone release by the human pituitary gland, human foetal, adult normal and tumour pituitary tissues were maintained in culture for 2 to 4 weeks and tested with acute (3 h) exposures to different preparations of IGF peptides. Adult normal pituitaries and adenomas were tested with a semipurified preparation of IGFs, free of immunoreactive insulin, containing IGF-I and IGF-II in a ratio of approximately 1:4. Human foetal pituitaries were tested with the semipurified IGFs as well as more purified preparations of IGF-I and IGF-II. Culture media were assayed for hGH, hPrl, hACTH and hLH using specific radioimmunoassays. Both foetal (n = 16 (No. of pituitaries), 33 (No. of observations] and normal adult (n = 3, 16) human pituitaries cultures responded to the semipurified IGFs (2-25 ngEq/ml for foetal and 2-4 ngEq/ml for adult pituitaries) with a significant decrease in hGH release compared to basal (P less than 0.01) whereas the GH-secreting pituitary tumours showed no effect when tested with from 2 to 25 ngEq/ml (n = 8, 129, NS). The effect of IGFs on human foetal somatotrope activity was dose-related for both the semipurified IGFs (2-25 ngEq/ml, n = 16, 33) and IGF-I or IGF-II (10-100 ng/ml; n = 3, 18).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of prolactin versus growth hormone on islet function and the importance of using homologous mammosomatotropic hormones

The purpose of this study was to determine the effects of homologous rat PRL (rPRL) and rat GH (rGH) on islet B-cell function in neonatal and adult rat islets in vitro. In neonatal rat islets, exposure to rPRL for more than 24 h was necessary for a stimulatory effect on insulin secretion. By day 4, insulin secretion was 3.8-fold greater in the islets cultured with rPRL. rGH had a modest effect on insulin secretion, and this effect was additive with that of rPRL. Both rPRL and rGH increased islet insulin content and [3H]thymidine incorporation. After removal of rPRL, more than 24 h were necessary to detect a reversal in the level of insulin secretion. In addition, even after 5 days without rPRL the previously treated islets still had elevated levels of insulin secretion. In a dose-response study of rPRL on insulin secretion, a detectable effect was observed at 62.5 ng/ml, with a half-maximal effect of approximately 100 ng/ml. Glucose oxidation by neonatal islets was enhanced by rPRL treatment, but not by rGH treatment. In adult rat islets, exposure to rPRL, but not to rGH, enhanced insulin secretion. In contrast, when using heterologous human GH, results similar to those obtained with rPRL, but not rGH, were observed. The results from these experiments indicate that rPRL and rGH have both individual and shared regulatory effects on rat islets. However, it is rPRL and not rGH that has the primary influence on insulin secretion. When interpreting studies examining the effect of GH and PRL on islet function, it is important to consider whether homologous hormones are used.     

Growth hormone and insulin-like growth factors I and II produce distinct alterations in glucose metabolism in 3T3-F442A adipocytes.

In 3T3-F442A adipocytes, human growth hormone (hGH) stimulates glucose oxidation in 4 hr. A maximal increase is evident at hGH concentrations of 50-100 ng/ml and rarely exceeds 50% above control. The stimulation is transient; after 48 hr of incubation with GH, glucose oxidation is significantly suppressed to 35% below control values. In view of the concept that insulin-like growth factors (IGF) may mediate the effects of GH, we compared the effects of hGH (500 ng/ml) and several preparations of IGF on glucose metabolism in 3T3 adipocytes. After 4 hr of incubation, IGF-I from human plasma stimulated glucose oxidation in a dose-related manner, producing a 10-fold increase at 50 ng/ml. Methionyl-IGF-I produced by recombinant DNA techniques was 85-88% as effective as IGF-I. IGF-II stimulated glucose oxidation 3-fold at 50 ng/ml after 4 hr of incubation. In contrast to the suppression observed with hGH after 48 hr, all three of the IGF preparations stimulated glucose oxidation after 48 hr of incubation and were as effective as they were after 4 hr. When each of the IGF preparations was tested (at 5 ng/ml) in combination with hGH, both the stimulatory and suppressive effects of GH were superimposed on the stimulation by the IGFs. Thus, the stimulatory properties of IGF differed from those of GH in that the maximum extent to which IGF increased glucose oxidation, compared with hGH, was as much as 20-fold greater. Furthermore, all of the IGF preparations stimulated glucose oxidation after 48 hr under conditions in which hGH suppressed glucose metabolism. Thus, it is unlikely that extracellular IGFs mediate the effects of hGH on glucose metabolism in 3T3-F442A adipocytes.     

A comparison between the effects of growth hormone on prolactin receptors and estrogen receptors in rat liver

Human GH (hGH) increased hepatic PRL and estrogen receptors in hypophysectomized (Hx) female rats after continuous infusion of the hormone (5 micrograms/h) using osmotic minipumps, but not after the infrequent administration of the same daily dose (120 micrograms) of the hormone by sc injections (60 micrograms/12 h). The effects of hGH on body weight (an increase) and tibial epiphyseal zones (a widening) were observed regardless of the mode of hGH administration. Thus, it would seem as if the mode of administration of hGH is of importance for the type of biological effect exerted by the hormone. Furthermore, rGH was effective in increasing both PRL and estrogen receptors in the liver of Hx ovariectomized (HxOx) rats, whereas rat PRL (rPRL) was much less efficient. Finally, rat GH (rGH) suppressed the concentration of a nonreceptor estrogen-binding protein in the liver of HxOx rats; the protein occurred at a low concentration in Ox rats and was markedly increased after hypophysectomy rPRL was inefficient in modulating the concentration of estrogen-binding protein. Thus, rGH, but not rPRL, seems to regulate both sexually differentiated (PRL receptors and estrogen-binding protein) and sexually nondifferentiated (estrogen receptor) functions in the rat liver. The mechanism behind this dual effect of rGH is obscure, but may possibly be related to the presence of isohormones of rGH with different effects on the liver.     

Differential implication of deoxyribonucleic acid methylation in rat prolactin and rat growth hormone gene expressions: a comparison between rat pituitary cell strains

In order to assess the potential role of DNA methylation in the expression of rat PRL (rPRL) as compared to rat GH (rGH) gene, the cleavage patterns generated by the isoschizomeric restriction enzymes HpaII and MspI were examined in DNA isolated from rat pituitary cell lines producing either high levels of rPRL (GH3B6) or of rGH (GC) and in a stable variant cell strain which produces minute amounts of both hormones (GH3CDL cells). The rPRL and the rGH genes were found hypomethylated in GH3B6 and GC cells, respectively, whereas in GH3CDL cells both genes were methylated, indicating a correlation between the extent of gene methylation and the level of expression. However the use of 5-azacytidine (5-azaC), which decreases DNA methylation, suggested a variable importance of gene methylation in the control of rPRL and rGH gene expression. 5-AzaC was unable to increase rPRL production to a detectable level in GC cells, whereas the cytidine analog markedly increased rPRL production and rGH production in GH3CDL cells. Further analysis using GH3CDL cells showed that the extent of the 5-azaC-induced rPRL and rGH gene demethylation was consistent with the 5-azaC-induced increase of gene expressions. However, in these cells, the stimulation of rPRL and rGH production unexpectedly increased as a function of time elapsed after drug withdrawal. The maximal stimulation, 30-fold and 7-fold, respectively, was observed 3 weeks after a 60-h exposure to 5-azaC. This pattern suggests that other events are required for the full expression of rPRL and rGH genes in addition to their own demethylation.     

Effects of insulin-like growth factors (IGF-I and -II) on growth hormone and prolactin release and gene expression in euryhaline tilapia,Oreochromis mossambicus

We investigated in vitro effects of insulin-like growth factors (IGF-I and -II) on growth hormone (GH) and prolactin (PRL) release and gene expression in euryhaline tilapia, Oreochromis mossambicus. Pituitaries were removed from freshwater-acclimated adult males and incubated for 2-24h in the presence of human IGF-I or -II at doses ranging from 1-1000 ng/ml (0.13-130 nM). IGF-I at concentrations higher than 10 ng/ml and IGF-II higher than 100 ng/ml significantly inhibited GH release after 8, 16, and 24h. No effect of IGFs was seen during the first 4h of incubation. IGFs at the same concentrations also significantly attenuated GH gene expression after 24h, although no effect was seen at 2h. By contrast, PRL(188) release was stimulated significantly and in a dose-related manner by IGF-I at concentrations higher than 10 ng/ml and by IGF-II at concentrations higher than 100 ng/ml within 2h. No stimulation was observed after 4h. Similarly, both IGFs at concentrations higher than 10 ng/ml increased PRL(177) release within 2h. However, no significant effect of IGF-I or -II was observed on mRNA levels of both PRLs after 2 and 24h at all concentrations examined. These results clearly indicate differential regulation of GH and PRL release and synthesis by IGFs in the tilapia pituitary, i.e., rapid-acting, stimulatory effects of IGFs on PRL release and slow-acting, inhibitory effects on GH release and synthesis.     

Regulation of growth hormone release: evidence against negative feedback in rat pituitary cells

To determine if the anterior pituitary gland is the site of negative feedback inhibition of GH release, we studied the effect of GH and multiplication-stimulating activity (MSA), a member of the somatomedin family, on isolated rat anterior pituitary cells in primary culture. The effect of GH was examined in two ways: 1) by adding to the cultures human GH (10 ng/ml to 20 microgram/ml) which was biologically active in the rat but not cross-reactive in the rat GH (rGH) RIA, and 2) by comparing rGH secretion in cultures of different cell densities. No suppression of either basal or prostaglandin E1-stimulated rGH release was found. An enhancement observed in serum-free conditions at high human GH concentrations was interpreted as a nonspecific response to protein, because bovine serum albumin produced the same effect. When added in the presence of serum, MSA (1--500 ng/ml) had no effect on rGH secretion. In the absence of serum, there were 71% and 30% increases in the basal and prostaglandin E1-stimulated rates of hormone release, respectively, possibly attributable to a trophic effect of MSA. Six other hormones having structural or functional similarity to either GH or somatomedin also failed to inhibit rGH secretion. Our results do not support the hypothesis that GH or somatomedin exerts a negative feedback effect on GH release directly on the anterior pituitary gland. Most likely, the hypothalamus or a higher brain center is the site for such regulation.     

Monensin influences basal and human growth hormone-releasing hormone 44-induced release of stored and new rat growth hormone and prolactin

When previous data suggested a growth hormone-releasing factor (GRF)-sensitive branch in intracellular hormone processing, the monensin-sensitive Golgi apparatus seemed a likely candidate. We examined monensin's effect on basal and GRF-stimulated release of newly synthesized and stored rat growth hormone (rGH) and rat prolactin (rPRL). 14C-Pre-labeled, perifused rat pituitary fragments were exposed to [3H]leucine in 0-10 microM monensin; a pulse of 3 nM GRF assessed subsequent secretory responsivity. Monensin dose-dependently reduced basal release of stored [14C]rGH and [14C]rPRL. GRF-stimulated release of stored [14C]hormone was doubled after 0.03 microM and 0.1 microM monensin; higher concentrations diminished stored hormone release. Low concentrations of monensin accelerated basal (0.03 microM and 0.1 microM) and GRF-stimulated (0.03 microM) [3H]rGH and [3H]rPRL release without altering recovery; higher monensin concentrations (greater than or equal to 1 microM) reduced basal, and abolished GRF-stimulated, new hormone release and reduced total [3H]rGH and [3H]rPRL recovery. These data are consistent with a GRF-sensitive and monensin-influenced branch in intracellular hormone processing that regulates the fraction of new hormone exiting the cell without prior immersion in storage compartments.     

Hormone receptors in livers of GH3 tumor-bearing rats: the predominant effect of growth hormone and testosterone.

The relationship between serum levels of rat GH (rGH), rat PRL (rPRL), corticosterone, estrogen, and testosterone and the liver-binding sites specific to [125I]iodo-human GH (hGH), -human PRL (hPRL), and -rPRL were investigated in normal rats, in rats bearing the GH- and PRL-secreting tumor (GH3), and in rats 14 days after tumor removal. The GH3 tumor elevated serum levels of rGH and rPRL and concomitantly increased the hepatic binding of radioiodinated hGH, hPRL, and rPRL; male rats had a greater increase than female rats. The increased binding was due to an increase in the specific membrane-binding sites, the receptors, whereas the affinity constant of binding (Ka) was not altered. In both male and female rats, the specific binding of receptors and the total binding capacities of livers correlated positively with serum levels of rGH (P less than 0.02) and inversely with serum levels of testosterone (P less than 0.05). There was insignificant or no correlation between rPRL or other steroids and the total liver-binding capacities. These results suggested that GH was the primary inducer and testosterone was the predominant regulator of the hepatic receptors in rats. The observed sex difference in the increase of receptors by GH3 tumor indicated that in normal and tumor-removed male rats, testosterone had suppressed the induction of hepatic receptors.     

Human growth hormone and somatomedin C suppress the spontaneous release of growth hormone in unanesthetized rats

To investigate the feedback control of GH secretion, we examined the effects of human GH (hGH) and somatomedin C (SmC) on spontaneous GH secretory surges in unanesthetized, freely moving rats. Under pentobarbital anesthesia a right atrial catheter and an intracerebroventricular cannula were placed 7-10 days before the experimentation. For iv studies, hGH (0.3 U/ml.h) was infused for 6 h after an iv loading dose (0.3 U) at the beginning of the experiments. For intraventricular injections, hGH (0.1 U/10 microliter) or SmC (500 ng/10 microliter) were injected into the lateral ventricle 2 h before the experiments. The equivalent dose of crystalline BSA diluted in the same vehicle solutions was administered to the same rat as a control on a separate day. Venous blood samples were collected every 20 min for 6 h. Intravenous and intraventricular administration of crystalline BSA did not affect the typical rat GH (rGH) surges which appeared about every 3 h and reached peak values of more than 300 ng/ml. The iv infusion of hGH significantly inhibited the amplitude of rGH surges compared to controls (planimetric areas under the secretory profile 752 +/- 172 vs. 1921 +/- 183, P less than 0.01, n = 6). rGH secretion was similarly inhibited by intraventricular hGH (701 +/- 127 vs. 2208 + 225, P less than 0.01, n = 6) and by intraventricular SmC (537 +/- 70 vs. 1503 +/- 114, P less than 0.01, n = 6). These findings suggest that both GH and SmC are active in the feedback regulation of rGH secretion.     

Basal and dibutyryl cyclic AMP-stimulated release of newly synthesized and stored growth hormone from perifused rat pituitaries.

The basal rate of pre-labeled stored 14C-labeled rat growth hormone ([14C]rGH) release from perifused rat pituitary explants is a constant fraction of pituitary GH content, suggesting random release from the storage pool. Basal release of newly synthesized [3H]rGH occurs in two phases: 1) immediate and associated with [3H]rGH synthesis, and 2) late (delayed by 60 min) and independent of concurrent [3H]rGH synthesis. Dibutyryl cyclic AMP (10(-2)M)-stimulated release of stored [14C]rGH is characterized by an initial acute rise followed by a second phase of continuous rapid release. Immediate and late release of new [3H]rGH is increased by dibutyryl cyclic AMP, and the late phase of [3H]rGH is less delayed. Simultaneous exposure of pituitary explants to [3H]alanine and [14C]leucine resulted in the release of immunoprecipitable rGH whose ratio of incorporated 3H and 14C varied with time. The observed changes suggest that after it is synthesized, a GH molecule may either be released directly or be processed into the somatotroph's storage compartment. In addition, stored GH is composed of two pools, one of which is immediately releasable. The differential incorporation of [3H]alanine and [14C]leucine into "big" and "small" rGH, together with the ability to differentially displace 3H-labeled "big" and 14C-labeled "small" rGH from the GH antibody suggest that "big" rGH is a heterogenous molecule including "small" rGH and another peptide rather than simply a dimer of "small" rGH.     

Release of stored, pre-labeled growth hormone and prolactin from perifused rat pituitary: effect of human pancreatic growth hormone-releasing factor-44

Human pancreatic growth hormone-releasing factor-44 (hpGRF-44) differentially stimulates release of stored and newly synthesized rGH without altering rGH synthesis over 3 h in static in vitro incubation; hpGRF-44 also stimulates release of stored, but not newly synthesized, rPRL. To study the time course of pre-labeled, stored hormone release without pharmacologically interrupting synthesis, the current experiments were performed in perifusion. Fifteen minute pulses of 0.1 to 10 nM hpGRF-44 stimulated stored [3H]rGH release (to 890% of base); 1.0 to 10 nM hpGRF-44 stimulated stored [3H]rPRL release (to 440% of base). Pulses of 0.1 to 1.0 mM (Bu) 2cAMP also stimulated release of [3H]rGH (to 570% of base) and [3H]rPRL (to 410% of base). However, peak [3H]rGH and [3H]rPRL responses to hpGRF-44 required 10 min, while peak responses to (Bu) 2cAMP required 25 min. Continuous hpGRF-44 stimulated an initial surge of stored [3H]rGH release which was not sustained; the diminishing release was not explained by hpGRF-44 degradation. Total radioimmunoassayable (RIA) hormone release roughly paralleled release of stored immunoprecipitable (IPn) hormone. Conclusions: in pituitary perifusion, hpGRF-44 stimulates release of both stored rGH and rPRL as shown in static incubation, but the response is biphasic: initial rapid release is followed by a progressively lesser response; and the response is both more acute and less well sustained than that resulting from exposure to (Bu) 2cAMP.     

Pituitary immediate release pools of growth hormone and prolactin are preferentially refilled by new rather than stored hormone

Pituitary stores of rat GH (rGH) and PRL (rPRL) are divisible into immediately releasable and more stable compartments representing either compartmentalized hormone within individual cells of a homogeneous population or responses of specialized cell subsets in a functionally heterogeneous population. In addition, newly synthesized rGH and rPRL can be processed either into intracellular storage or toward direct release. Fractional assignment of new hormone to these two paths can be influenced in the somatotroph by GHRH and may also represent either intracellular processes or functional heterogeneity of cells. We investigated the source, newly synthesized or stored, of hormone refilling the somatotroph and lactotroph immediately releasable pools (IRP) after their discharge by 21 mM potassium ion, 1 mM (Bu)2cAMP, 3 nM human GHRH-44, or 3 microM prostaglandin E1. Experiments were performed using perifused pituitary fragments exposed sequentially to [14C]- and [3H]leucine in association with stimulation by two 30-min pulses of the same secretagogue. Therefore, only [14C]hormone was available for release by the first stimulus, whereas both [14C]- and [3H]hormone were available for release by the second stimulus. Analysis was by specific immunoprecipitation. The first episode of stored [14C]rGH release exceeded the second episode of stored [14C]rGH release in response to each secretagogue. However, release of newly synthesized [3H]rGH in response to the second episode of stimulation exceeded the simultaneous release of stored [14C]rGH while matching or exceeding the [14C]rGH release that had occurred in the same experiment in response to the first episode of stimulation. Refilling both GH and PRL IRP stores drew primarily upon newly synthesized hormone, but with different secretagogue-specific patterns. These data confirm differential handling of new and stored rGH and rPRL within the pituitary. They are consistent with either (1) the enhanced shunting of newly synthesized hormone to IRPs within cells that are capable of compartmentalized intracellular hormone storage, or (2) the relatively complete discharge of a subset of somatotrophs and lactotrophs that are specialized to deliver pulsed hormone release, after which they are refilled by newly synthesized hormone.     

Growth hormone-releasing factor-44 specificity for components of somatotroph and lactotroph immediate release pool substructures

Rat somatotroph and lactotroph hormone storage is divisible into at least two functional compartments: an immediate release pool (IRP) and a pool that responds to prolonged stimulation. An IRP substructure has been defined by release in response to potassium ion (K+), prostaglandin E1 (PGE1), and Bu2cAMP. The somatotroph IRP is expandable; the lactotroph IRP is fixed in size. The present experiments examined which IRP components contribute to the rapid release of stored GH in response to GH-releasing factor-44 (GRF). Release of stored PRL was monitored for comparison. In vitro prelabeling defined stored rat (r) GH and rPRL. Release in response to 21 mM K+, 3 microM PGE1, 1 mM Bu2cAMP, and/or 3 nM GRF was monitored with a perifusion-immunoprecipitation system. After 120 min of basal perifusion, tissue was exposed to one of the four secretagogues for 90 min. During a second 90-min period a second secretagogue was added while exposure to the first secretagogue continued. We demonstrated that 21 mM K+ reduces peak rGH release in response to 3 nM GRF by 52%, whereas GRF does not reduce rGH release in response to K+; 3 microM PGE1 reduces rGH release in response to GRF by only 19% although GRF reduces rGH release in response to PGE1 by 88%; 1 mM Bu2cAMP reduces rGH release in response to GRF by 87%, and GRF eliminates rGH release in response to Bu2cAMP (1.2% of control value); combined K+ plus Bu2cAMP reduce rGH release in response to GRF to 2.5% of the control value, whereas after GRF pretreatment rGH release in response to combined K+ plus Bu2cAMP is 93% of the control value; and combined PGE1 and Bu2cAMP reduce the response to GRF to 17% of the control value. Effects on rPRL release are qualitatively similar. We conclude that immediate GRF-stimulated release of stored rGH originates in the somatotroph IRP components defined by responses to PGE1 and Bu2cAMP; it derives only slightly, if at all, from the IRP component defined by the response to K+. The smaller GRF-stimulated release of IRP rPRL is similarly derived.     

Combined effects of human growth hormone (GH)-releasing factor-44 (GRF) and somatostatin (SRIF) on post-SRIF rebound release of GH and prolactin: a model for GRF-SRIF modulation of secretion

Somatostatin (SRIF) and GRFs play key roles in regulating GH secretion. We previously presented a model of SRIF-cAMP interaction; SRIF blocks rat (r) GH release without preventing its accumulation in a potentially releasable pool. This phenomenon may represent a mechanism whereby tonic SRIF inhibition and its subsequent reduction or withdrawal can modulate the magnitude if not the initiation of rGH pulses. Herein we test that model using human GRF-44 (hGRF-44). Tritium-prelabeled rat anterior pituitary fragments were perifused until stored [3H]rGH and [3H]rPRL release rates were stable. SRIF (10 or 25 nM), with and without hGRF-44 (3 or 10 nM), was added in short (1-h hGRF-44) and long (3-h hGRF-44) protocols; SRIF was then withdrawn while hGRF-44 was continued. Release of stored prelabeled [3H]rGH and [3H]rPRL was assessed by immunoprecipitation. Effects on PRL release were followed for comparison. SRIF-induced inhibition of release was only partially reversed by hGRF-44. At these concentrations and so long as SRIF was present, hGRF-44 could not stimulate the rate of hormone release to values above pre-SRIF basal rates. On the other hand, the amplitude of post-SRIF rebound release was increased by prolonging exposure to SRIF alone, by including hGRF-44 with SRIF, by increasing the amount of hGRF-44 included with SRIF, by prolonging exposure to hGRF-44 plus SRIF, and by using a smaller concentration of SRIF during exposure to hGRF-44. Interaction of hGRF-44-SRIF effects generated peak rates of hormone release after SRIF withdrawal which exceeded the maximum rates achieved using hGRF-44 alone in this system. Lactotroph responses were much smaller, but qualitatively resembled somatotroph responses. We conclude that the interplay of simultaneous hGRF-44 and SRIF effects can regulate the amplitude of rGH pulses. Although GRF can initiate physiological GH release, and GRF antisera can block GH pulses, we suggest that the surge of release that follows reduction of SRIF-induced inhibitory tone in vitro represents a potential mechanism that could contribute to the initiation of some pulses of release. Finally, we also present a theoretical model of secretagogue interactions at the cellular level to explain our results. The model is compatible with either a homogeneous cell population in which each secretory cell has multiple capabilities or a heterogeneous cell population composed of cell subgroups with complementary secretory abilities.     

Induction of hepatic receptors for growth hormone (GH) and prolactin by GH infusion is sex independent

To determine whether induction of rat liver GH and PRL receptors by GH infusion is dependent upon the sex of the animal or whether or not the pituitary is intact, rat GH (rGH) or rat PRL (rPRL) was infused at approximately 200 micrograms/day for 7 days into male and female, intact and hypophysectomized rats, and the binding of radioiodinated bovine GH (bGH) and ovine PRL (oPRL) to liver microsomal membranes was measured. In females, bGH binding was reduced by hypophysectomy whether or not membranes were MgCl2 treated to remove endogenous ligand. However, in males, hypophysectomy caused an apparent 3-fold induction of bGH binding sites, which was absent in MgCl2-treated membranes, suggesting that the effect was due to receptor occupancy by endogenous rGH in the intact males. Hypophysectomy also lowered oPRL binding in females but had no effect in males. Infusion of rGH significantly induced binding sites for bGH and oPRL in all treatment groups, independently of sex or the presence of the pituitary, whereas rPRL infusion had no effect on either receptor type except for mild induction of bGH binding in hypophysectomized females. Serum somatomedin-C (SM-C), reduced 95% by hypophysectomy, was restored by rGH, but not rPRL, infusion. However, in intact animals of both sexes, rGH infusion significantly lowered SM-C levels by 30-40%; thus both bGH and oPRL binding in individual pituitary-intact rats were negatively correlated with serum SM-C. In contrast, in rGH-treated hypophysectomized rats, induced GH (but not PRL) binding sites showed significant positive correlation with SM-C levels. These results indicate that the induction of GH and PRL receptors by rGH occurs independently of SM-C generation, but suggest that newly induced GH receptors in GH-treated hypophysectomized rats may be involved in SM-C generation.     

In vitro regulation of growth hormone (GH) release from ovine pituitary cells during fetal and neonatal development: effects of GH-releasing factor, somatostatin, and insulin-like growth factor I

Using a monolayer approach, we have examined the acute (3 h) effects of GRF, somatostatin (SRIF), and insulin-like growth factor I (IGF-I) on GH release from pituitary cells of male and female 70-, 100-, and 130-day-old fetuses and newborn lambs and of prepubertal male lambs. GRF stimulated basal GH release in a dose-dependent (10(-12)-10(-8) M) manner at each stage in development. There was no linear relationship between maximal response and increasing age of the donor animals. The ED50 values for GRF were similar in all groups, except in the pituitaries from male and female 130-day-old fetuses, where the ED50 values were significantly higher. SRIF elicited a dose-related (10(-10)-10(-6) M) inhibition of basal GH secretion at each stage of fetal life and in the prepubertal period; although the response was lower in the youngest fetal pituitaries, there was no significant change in maximal response during the fetal or prepubertal period. No effect of SRIF on basal GH secretion was observed in newborn lambs. However, SRIF (10(-7) M) was able to block GRF (10(-8) M)-stimulated GH release in 100- and 130-day-old fetal and prepubertal as well as newborn lamb pituitary cells. Plasma IGF-I concentrations increased from 15.0 +/- 0.7 (mean +/- SE) and 13.8 +/- 0.9 ng/ml for male and female animals, respectively, at 70 days gestation to 55.8 +/- 3.2 and 51.8 +/- 11.1 ng/ml at the time of birth. The increase was much more pronounced in prepubertal lambs, especially in male animals, where IGF-I levels reached 300.8 +/- 37.7 ng/ml. IGF-I (100 ng/ml) had no effect on basal GH release in 70- and 100-day-old fetal, newborn, and prepubertal lamb pituitary cultures, but significantly inhibited basal GH secretion from 130-day-old fetal cells. This dose of IGF-I had no effect on GRF (10(-9) M)-stimulated GH release at 70 days gestation. It significantly inhibited this effect at 100 days and in prepubertal lamb cells. In 130-day-old fetal and newborn lamb pituitary cultures, IGF-I completely blocked the GH response to GRF.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction of ethanol with signal transduction mechanisms mediating growth hormone release by rat pituitary cells in vitro.

The effects of ethanol on signal transduction mechanisms of rat GH (rGH) release were investigated in primary culture of rat anterior pituitary cells. Ethanol (30, 100, and 300 mM) had no significant effect on basal rGH release or cell content after a 4-h incubation or on intracellular cAMP levels at 30 min. Ethanol did not alter rGRH (10(-11) M)-stimulated rGH release, but at concentrations of 100 and 300 mM it inhibited rGRH (10(-9) M)-stimulated rGH release by 12% (P less than 0.05) and 54% (P less than 0.01). In contrast, a dose-dependent stimulatory effect was observed on rGRH-induced cAMP accumulation. Ethanol enhanced the inhibitory effect of SRIH (10(-11) and 10(-9) M) on rGH release by up to 24% (P less than 0.01). Stimulation of rGH release by cAMP derivatives and forskolin was partially inhibited by ethanol, as was cAMP accumulation after forskolin treatment. Cholera toxin-stimulated rGH release was also inhibited by ethanol, whereas cAMP accumulation was increased. At the higher concentrations, ethanol enhanced rGH release after protein kinase-C activation by phorbol ester and after stimulation of calcium influx with Ca ionophore. No significant ethanol effect was noted on prostaglandin E2-stimulated rGH release, and ethanol did not alter rGH mRNA levels or proliferation of a pituitary somatomammotroph cell line. The results indicate that ethanol exerts multiple effects on systems mediating GH release from the pituitary in vitro. However, the inhibitory influence of ethanol on GH secretion is related primarily to the adenylate cyclase-cAMP pathway, which represents the major signal transducing system in the somatotroph.     

The hypothalamic mechanisms of the hypophysiotropic action of ghrelin

Ghrelin is an endogenous ligand for the growth hormone secretagogue (GHS) receptor, expressed in the hypothalamus and pituitary. Ghrelin, like synthetic GHSs, stimulates food intake and growth hormone (GH) release following systemic or intracerebroventricular administration. In addition to GH stimulation, ghrelin and synthetic GHSs are reported to stimulate the hypothalamo-pituitary-adrenal (HPA) axis in vivo. The aims of this study were to elucidate the hypothalamic mechanisms of the hypophysiotropic actions of ghrelin in vitro and to assess the relative contribution of hypothalamic and systemic actions of ghrelin on the HPA axis in vivo. Ghrelin (100 and 1,000 nM) stimulated significant release of GH-releasing hormone (GHRH) from hypothalamic explants (100 nM: 39.4 +/- 8.3 vs. basal 18.3 +/- 3.5 fmol/explant, n = 49, p < 0.05) but did not affect either basal or 28 mM KCl-stimulated somatostatin release. Ghrelin (10, 100 and 1,000 nM) stimulated the release of both corticotropin-releasing hormone (CRH) (100 nM: 6.0 +/- 0.8 vs. basal 4.2 +/- 0.5 pmol/explant, n = 49, p < 0.05) and arginine vasopressin (AVP) (100 nM: 49.2 +/- 5.9 vs. basal 35.0 +/- 3.3 fmol/explant, n = 48, p < 0.05), whilst ghrelin (100 and 1,000 nM) also stimulated the release of neuropeptide Y (NPY) (100 nM: 111.4 +/- 25.0 vs. basal 54.4 +/- 9.0 fmol/explant, n = 26, p < 0.05) from hypothalamic explants in vitro. The HPA axis was stimulated in vivo following acute intracerebroventricular administration of ghrelin 2 nmol [adrenocorticotropic hormone (ACTH) 38.2 +/- 3.9 vs. saline 18.2 +/- 2.0 pg/ml, p < 0.01; corticosterone 310.1 +/- 32.8 ng/ml vs. saline 167.4 +/- 40.7 ng/ml, p < 0.05], but not following intraperitoneal administration of ghrelin 30 nmol, suggesting a hypothalamic site of action. These data suggest that the mechanisms of GH and ACTH regulation by ghrelin may include hypothalamic release of GHRH, CRH, AVP and NPY.