GHRH-stimulated GH Release Research Articles

Pituitary-Specific Expression and Pit-1 Regulation of the Rat Growth Hormone-Releasing Hormone Receptor Gene

The GHRH receptor is expressed in the somatotroph cell of the anterior pituitary, where it functions to mediate GHRH-stimulated GH release. To study pituitary and somatotroph cell-specific expression of this gene, a transgenic mouse model and complementary cell culture experiments were developed. The activity of the 1.6-kb proximal rat GHRH receptor promoter was examined in vivo by generating transgenic mice with the promoter directing expression of a luciferase reporter. The promoter directs tissue-specific expression; luciferase is highly expressed in the pituitary but absent from 14 other tissues. Immunocytochemistry experiments show that transgene expression is targeted to GH-expressing somatotroph cells. The transgene is 5-fold more highly expressed in males than females, and there is an increase in transgene expression leading up to the onset of puberty. The 1.6-kb promoter was further examined in cell culture experiments, which revealed that the promoter is selectively activated in pituitary cells and that promoter-reporter expression in nonpituitary cells can be enhanced by the pituitary-specific transcription factor Pit-1. EMSAs identified 10 short regions that specifically bind Pit-1 with highly variable relative affinities. The highest affinity site was previously identified and is required for Pit-1 activation of the promoter. Four additional sites contribute to Pit-1 regulation of the promoter and are important to achieving full activation of the gene. The results show that the 1.6-kb promoter is sufficient to direct tissue- and cell-specific expression in vivo and is regulated by Pit-1.

Identification of the Somatostatin Receptor Subtypes (sst) Mediating the Divergent, Stimulatory/Inhibitory Actions of Somatostatin on Growth Hormone Secretion

It is well established that somatostatin acts through G protein-coupled receptors, termed sst, to inhibit GH release. However in pigs somatostatin can stimulate or inhibit in vitro GH secretion in a dose- and somatotrope subpopulation-dependent manner. We report herein that somatostatin-stimulated GH release is blocked by pretreatment with GTPgamma-S, suggesting an involvement of G protein-coupled receptors. Consistent with this, an sst5 selective agonist stimulated spontaneous GH secretion at doses ranging 10(-13) to 10(-9) m, without influencing GHRH-induced GH release. Conversely, sst1-, sst2-, sst3-, and sst4-specific agonists inhibited GHRH-evoked GH release but not basal GH secretion. Examination of the effects of sst-specific agonists on two subpopulations of somatotrope cells separated by density gradient centrifugation [low- (LD) and high-density (HD) cells] showed that only a low dose of the sst5 agonist stimulated GH release in LD somatotropes, whereas both low and high doses of this agonist stimulated GH release in HD cells. In marked contrast, sst1 and sst2 agonists blocked GHRH-stimulated GH release in LD cells at all doses tested, whereas only a high dose of the sst2 agonist inhibited GHRH-induced GH release in HD somatotropes. Interestingly, sst expression pattern in these subpopulations correlates with the distinct actions of sst-selective agonists; specifically, sst5 is more abundant in HD somatotropes, whereas sst1 and sst2 mRNA predominate in LD cells. These results indicate that in the pig, sst1 and sst2 are the primary mediators of the inhibitory effects of somatostatin, whereas sst5 or an sst5-related mechanism mediates the stimulatory action of somatostatin on GH release.

Intermedin/Adrenomedullin-2 Inhibits Growth Hormone Release from Cultured, Primary Anterior Pituitary Cells

Intermedin (IMD), a novel member of the adrenomedullin (AM), calcitonin gene-related peptide (CGRP), amylin (AMY) peptide family, has been reported to act promiscuously at all the known receptors for these peptides. Like AM and CGRP, IMD acts in the circulation to decrease blood pressure and in the brain to inhibit food intake, effects that could be explained by activation of the known CGRP, AM, or AMY receptors. Because AM, CGRP, and AMY have been reported to affect hormone secretion from the anterior pituitary gland, we examined the effects of IMD on GH, ACTH, and prolactin secretion from dispersed anterior pituitary cells harvested from adult male rats. IMD, in log molar concentrations ranging from 1.0 pm to 100 nm, failed to significantly alter basal release of the three hormones. Similarly, IMD failed to significantly alter CRH-stimulated ACTH or TRH-stimulated prolactin secretion in vitro. However, IMD concentration-dependently inhibited GHRH-stimulated GH release from these cell cultures. The effects of IMD, although requiring higher concentrations, were as efficacious as those of somatostatin and, like somatostatin, may be mediated, at least in part, by decreasing cAMP accumulation. These actions of IMD were not shared by other members of the AM-CGRP-AMY family of peptides, suggesting the presence of a novel, unique IMD receptor in the anterior pituitary gland and a potential neuroendocrine action of IMD to interact with the hypothalamic mechanisms controlling growth and metabolism.

Homologous and heterologous in vitro regulation of pig pituitary somatostatin receptor subtypes, sst1, sst2 and sst5 mRNA.

Somatostatin (SRIF) is commonly regarded as an inhibitor of GH release in rodents and humans. However, in pigs, SRIF can stimulate the release of GH at low (picomolar) doses, while inhibiting GHRH-stimulated GH release at high (nanomolar) doses in primary pituitary cell cultures. One possible mechanism by which pig cells respond differently to the actions of SRIF is by differential expression and regulation of SRIF receptor subtypes. As no information is available on the homologous regulation of SRIF receptors in pigs, we examined the acute (4 h) in vitro effects of SRIF on mRNA levels of SRIF receptors sst1, sst2 and sst5 by multiplex RT-PCR. These particular sst subtypes were selected because all three have been implicated in the inhibitory effects of SRIF on GH release in both rodents and humans. At a high dose (10(-7) M), SRIF stimulated the expression of sst1, sst2 and sst5 in pig pituitary cell cultures. At a low dose (10(-13) M), SRIF markedly increased sst1, without affecting sst2 or sst5. Given that our laboratory has shown SRIF at high and low doses stimulates cAMP production in a subpopulation of pig somatotropes, we sought to determine if this signaling pathway may be responsible for the stimulatory effect of SRIF on its own receptor expression. The receptor-independent cAMP activator forskolin elevated sst1 and sst2 mRNA levels but did not affect sst5 expression, suggesting the stimulatory actions of high- and low-dose SRIF on sst1 and high-dose SRIF on sst2 mRNA levels can be mediated by activation of cAMP, whereas the stimulatory effect of high-dose SRIF on sst5 mRNA is elicited by a cAMP-independent pathway. Interestingly, both GHRH (10(-8) M) and ghrelin (10(-6) M), which release GH in pig pituitary cell cultures via cAMP-dependent mechanisms, decreased sst5 without altering sst1 or sst2 mRNA levels. Since the actions of GHRH and ghrelin on sst expression markedly contrasted with that observed for SRIF and forskolin these results clearly indicate GHRH and ghrelin are regulating sst5 mRNA levels by a cAMP-independent signaling pathway. Taken together, our results demonstrate that expression of pig SRIF receptors is under a complex, receptor subtype-selective regulation, wherein the concerted actions of key regulators of somatotrope function would play divergent and dose-dependent effects.

Effects of hypothalamic neuropeptides on extracellular signal-regulated kinase (ERK1 and ERK2) cascade in human tumoral pituitary cells.

The G protein-coupled receptor (GPCR) activation has been demonstrated to affect the ERK1/2 cascade in different cell lines. We investigated the effects of hypothalamic neuropeptides acting via GPCR on this pathway in GH-secreting (GH-oma) and nonsecreting (NFPA) pituitary adenomas. GHRH increased ERK1/2 activity (236 +/- 80%) in both gsp- and gsp+ GH-omas, this effect being almost completely abolished by protein kinase C (PKC) blockade. Both GnRH and pituitary adenylate-activating peptide caused a similar PKC-dependent activation of ERK1/2 in most NFPA. Increasing cAMP by forskolin caused a protein kinase A-dependent increase of ERK activity (287 +/- 37%) in GH-omas and had no effect in NFPA. ERK cascade blockade in GH-omas did not affect basal and GHRH-stimulated GH release, whereas it totally prevented the 3-fold increase in cyclin D1 protein expression induced by GHRH. In conclusion, this study demonstrated that in pituitary adenomas the activation of GPCR by neurohormones caused a PKC-dependent activation of ERK1/2 cascade that, at least in GH-omas, resulted to be involved in cyclin D1 induction by GHRH. Moreover, a stimulatory effect of the protein kinase A-dependent pathway on ERK1/2 cascade occurred selectively in GH-omas, probably contributing to the mitogenic potential of the cAMP pathway in this cell type.

Somatostatin infusion withdrawal: studies in the acute and recovery phase of anorexia nervosa, and in obesity.

Changes in GH/IGF-I axis activity occur in both anorexia nervosa (AN) and obesity (OB). A GH hypersecretory state with very low plasma IGF-I levels is present in AN, whereas in morbid OB, GH secretion is dull and plasma IGF-I levels are generally preserved. Endogenous GHRH activity in AN and OB has never been directly studied, although indirect evidence would indicate that GHRH function is altered in either condition, possibly enhanced and reduced respectively. Somatostatin (SS) infusion withdrawal (SSIW) is followed by a rebound rise of plasma GH in animals and humans, an event which, allegedly, is mediated by endogenous GHRH release. In the present study, 28 young women, eight with active AN (A-AN), six with AN in the recovery phase (R-AN), eight with morbid OB, and six healthy age-matched normal weight subjects (NW), were studied. All subjects underwent, on different occasions, the following two tests: (i) acute GHRH injection (1 microg/kg, i.v.); (ii) infusion of SS (9 microg/kg per h i.v. over 60 min), with blood samples drawn prior to and at different intervals after drug injections. Plasma GH levels were measured at each time interval in all sessions, and, in addition, baseline plasma estradiol, free triiodothyronine, TSH, IGF-I and insulin were measured at -30 min. Baseline plasma GH concentrations were significantly higher in A-AN than in NW (4.7+/-0.7 vs 2.1+/-0.6 microg/l, P<0.01). Baseline GH levels in R-AN were also higher than in NW, but the difference did not reach statistical significance (5.6+/-1.7 microg/l, not significant (NS)). Baseline plasma GH concentrations were significantly lower in OB than in NW (0.3+/-0.1 microg/l, P<0.01). GHRH-stimulated GH release was significantly higher in A-AN than in NW (mean change in area under the curve (DeltaAUC) 1904.9+/-626.1 vs 613.9+/-75.9 microg/l per min, P<0.01), whereas no statistically significant difference was present between R-AN and NW (mean DeltaAUC 638.2+/-293.0 microg/l per min, NS); in OB, GHRH failed to evoke a plasma GH rise (mean DeltaAUC 239.8+/-89.9 microg/l per min vs A-AN, R-AN, and NW, P<0.01). SS infusion markedly reduced plasma GH concentrations in both A-AN and R-AN and, to a lesser extent, in NW, but failed to do so in OB. In A-AN, SSIW was followed by a plasma GH rise markedly higher than that present in NW (mean DeltaAUC 193.0+/-42.3 vs 60.1+/-11.4 microg/l per min, P<0.01), whereas in R-AN the GH response after SSIW was nearly superimposable on that registered in NW (mean DeltaAUC 72.9+/-22.8 microg/l per min, NS). There were no changes in plasma GH levels after SSIW in OB (mean DeltaAUC 22.8+/-9.7 microg/l per min). In all groups, DeltaAUCs of the GH response to GHRH and after SSIW were highly positively correlated (r=0.7, P<0.01). These data support the view that a high endogenous GHRH tone, which subsides in the recovery phase of the disease, is present in AN, whereas GHRH hypofunction, possibly associated with pituitary impairment, might indicate OB.

Increased lactotrophs despite decreased somatotrophs in the dwarf (dw/dw) rat: a defect in the regulation of lactotroph/somatotroph cell fate?

The dwarf (dw/dw) rat differs from all other rodent models of GH deficiency in that its pituitary prolactin (PRL) content is normal or even increased. We have now studied this throughout postnatal development, using a combination of immunocytochemistry, RIA and fluorescence-activated cell sorting (FACS) and analysis. Compared with normal Albino Swiss (AS) rats, adult dw/dw rats showed a profound reduction in pituitary GH content accompanied by increased PRL content, significantly so in females (AS vs dw/dw; P<0.01). Somatotroph hypoplasia was evident in the adult dw/dw rats, with most GH(+ve) cells showing weak immunostaining, whereas many more strongly stained PRL cells were evident in pituitary sections from dw/dw rats. Facs analysis confirmed both somatotroph hypoplasia and relative lactotroph hyperplasia in dw/dw rats at all ages studied (9-144 days); the difference in somatotrophs increased with age whereas the difference in lactotrophs declined with age. At 9 days, the percentage of lactotrophs was 10-fold higher in dw/dw rats than in AS rats. Young dw/dw rats also had a higher proportion of mammosomatotrophs than AS rats, although this difference disappeared as the mammosomatotroph proportions increased with age in both strains. GHRH released GH from both dw/dw and as cells maintained in culture for 5 days. The sensitivity to GHRH and the amount of GH released was lower in the dw/dw cultures, mostly explained by their fewer GH cells and lower initial GH content. GHRH increased cAMP in as but not in dw/dw cultures, even when these were greatly enriched for dw/dw somatotrophs by FACS sorting prior to culture. These results suggest that GHRH-induced cAMP stimulation is required for trophic effects on GH synthesis and somatotroph proliferation, but is not required for GHRH-stimulated GH release. The inverse changes in somatotroph and lactotroph numbers suggest that the dw/dw mutation disturbs the mechanism that specifies and retains appropriate numbers of somatotrophs in their differentiated state, and results in a higher proportion of the remaining cells progressing to lactotrophs. The dw/dw phenotype is thus not confined to somatotrophs.

Accelerated Escape from GH Autonegative Feedback in Midpuberty in Males: Evidence for Time-Delimited GH-Induced Somatostatinergic Outflow in Adolescent Boys

A single injected pulse of GH inhibits the time-delayed secretion of GH in the adult by way of central mechanisms that drive somatostatin and repress GHRH outflow. The marked amplification of spontaneous GH pulse amplitude in puberty poses an autoregulatory paradox. We postulated that this disparity might reflect unique relief of GH-induced autonegative feedback during this window of development. The present study contrasts GH autonegative feedback in: 1) normal prepubertal boys (PP) (n = 6; Tanner genital stage I, chronologically aged 8 yr, 9 months to 10 yr, 1 month; median bone age 8.5 yr); 2) longitudinally identified midpubertal boys (MP) (n = 6; Tanner genital stages III/IV, aged 12 yr, 6 months to 15 yr, 6 months; median bone age 15 yr); and 3) healthy young men (YM) (n = 6, aged 18–24 yr; bone age >18 yr). Subjects each underwent four randomly ordered tandem peptide infusions on separate mornings while fasting: i.e. 1) saline/saline infused iv bolus at 0830 h and 1030 h; 2) saline/GHRH (0.3 μg/kg iv bolus) at the foregoing times; 3) recombinant human (rh) GH (3 μg/kg as a 6-min square-wave iv pulse)/saline; and 4) rhGH and GHRH. To monitor GH autofeedback effects, blood samples were obtained every 10 min for 5.5 h beginning at 0800 h (30 min before GH or saline infusion). Serum GH concentrations were quantitated by ultrasensitive chemiluminometry (threshold 0.005 μg/liter). On the day of successive saline/saline infusion, MP boys maintained higher serum concentrations of: 1) GH (μg/liter), 2.2 ± 0.25, compared with PP (0.61 ± 0.10) or YM (0.88 ± 0.36) (P = 0.011); 2) IGF-I (μg/liter), 493 ± 49 vs. PP (134 ± 16) and YM (242 ± 22) (P < 0.001); 3) T (ng/dl), 524 ± 58 vs. PP (<20) (P < 0.001); and 4) E2 (pg/ml),19 ± 3 vs. PP (< 10) (P = 0.030) (mean ± sem). Consecutive saline/GHRH infusion elicited comparable peak (absolute maximal) serum GH concentrations (micrograms per liter) in the three study groups, i.e. 18 ± 5.0 (PP), 9.6 ± 1.7 (MP), and 14 ± 5.3 (YM) (each P < 0.01 vs. saline; P = NS cohort effect). Injection of rhGH attenuated subsequent GHRH-stimulated peak serum GH concentrations (micrograms per liter) to 7.8 ± 1.9 (PP), 5.8 ± 1.2 (MP), and 4.8 ± 1.1 (YM) (each P < 0.01 vs. saline; P = NS pubertal effect). GH autofeedback reduced non-GHRH-stimulated (basal) serum GH concentrations by 0.74 ± 0.28 (PP), 5.7 ± 1.7 (MP) and 1.4 ± 0.27 (YM) fold, compared with saline (P = 0.016 for MP vs. PP or YM). In addition to greater fractional autoinhibition, MP boys exhibited markedly accentuated postnadir escape (4.6-fold steeper slope) of suppressed GH concentrations (P < 0.001 vs. PP or YM). Linear regression analysis of data from all 18 subjects revealed that the fasting IGF-I concentration negatively predicted fold-autoinhibition of GHRH-stimulated peak GH release (r = −0.847, P = 0.006) and positively forecast fold-autoinhibition of basal GH release (r = +0.869, P < 0.001). In contrast, the kinetics of rhGH did not differ among the three study cohorts. In summary, boys in midpuberty manifest equivalent responsiveness to exogenous GHRH-stimulated GH secretion; heightened susceptibility to rhGH-induced fractional inhibition of endogenous secretagogue-driven GH release, compared with the prepubertal or adult male; and accelerated recovery of GH output after acute autonegative feedback. This novel tripartite mechanism could engender recurrent high-amplitude GH secretory bursts that mark sex hormone-dependent activation of the human somatotropic axis.

Normal human pituitary gland and pituitary adenomas express cannabinoid receptor type 1 and synthesize endogenous cannabinoids: first evidence for a direct role of cannabinoids on hormone modulation at the human pituitary level.

Little is known about the expression and function of cannabinoid receptor type 1 (CB1) in the human pituitary gland. The aim of this study was to investigate CB1 expression in human normal and tumoral pituitaries by in situ hybridization and immunohistochemistry using an antibody against CB1. CB1 was found in corticotrophs, mammotrophs, somatotrophs, and folliculostellate cells in the anterior lobe of normal pituitary. After examination of 42 pituitary adenomas, CB1 was detected in acromegaly-associated pituitary adenomas, Cushing's adenomas, and prolactinomas, whereas faint or no expression was found in nonfunctioning pituitary adenomas. Experiments with cultured pituitary adenoma cells showed that the CB1 agonist WIN 55,212--2 inhibited GH secretion in most of acromegaly-associated pituitary adenomas tested and that the CB1 antagonist SR 141716A was generally able to reverse this effect. Moreover, WIN 55,212--2 was able to suppress GHRH-stimulated GH release, and this effect was not blocked by coincubation with SR 141716A, possibly indicating a non-CB1-mediated effect. In contrast, WIN 55,212--2 was ineffective on GH-releasing peptide-stimulated GH release. In four Cushing's adenomas tested, WIN 55,212--2 was not able to modify basal ACTH secretion. However, simultaneous application of CRF and WIN 55,212--2 resulted in a synergistic effect on ACTH secretion, and this effect could be abolished by SR 141716A, demonstrating a CB1-mediated effect. In the single case of prolactinomas tested, WIN 55,212--2 was able to inhibit basal secretion of PRL. Finally, the presence of endocannabinoids (anandamide and 2-arachidonoylglycerol) was investigated in normal and tumoral pituitaries. All tumoral samples had higher contents of anandamide and 2-arachidonoylglycerol compared with the normal hypophysis. Moreover, endocannabinoid content in the different pituitary adenomas correlated with the presence of CB1, being elevated in the tumoral samples positive for CB1 and lower in the samples in which no or low levels of CB1 were found. The results of this study point to a direct role of cannabinoids in the regulation of human pituitary hormone secretion.

HIV gp120 inhibits the somatotropic axis: a possible GH-releasing hormone receptor mechanism for the pathogenesis of AIDS wasting.

AIDS is often associated with growth retardation in children and wasting in adults. The dissociated envelope protein of the HIV (HIV-1), gp120, can be found in significant concentrations in the parenchyma and cerebrospinal fluid of brains in infected individuals, even in the earliest stages of HIV-1 disease. On the basis of this and the fact that we observed pentapeptide sequence homology between GH-releasing hormone (GHRH) and the V2 receptor-binding region of gp120, we initiated experiments to determine whether gp120 could affect GH secretion and growth in vivo and/or interact with anterior pituitary GHRH receptors in vitro. Although acute IV administration of gp120 in conscious rats had no effect on plasma GH levels, acute administration of gp120 (400 ng) into the brain significantly suppressed pulsatile GH release over a 6-h period compared with saline-injected controls. Furthermore, the putative gp120 antagonist, Peptide T (DAPTA), prevented the suppression of GH by gp120. In support of these in vivo findings, gp120 also significantly (P < 0.05) suppressed GHRH-stimulated GH release in static cultures of dispersed pituitary cells and from cells undergoing perifusion with the peptides. DAPTA prevented the GH suppression by gp120 in both of the pituitary cell paradigms. Furthermore, chronic administration of gp120 into the third ventricle significantly reduced body weight in juvenile rats, compared with saline-injected controls. Thus, gp120 appears to act both at the hypothalamus and pituitary to suppress GH release, and its action at these two locations is associated with a significant loss in body weight in chronically treated young animals. These findings may suggest a specific mechanism for the pathogenesis of wasting in HIV-1 patients that involves blockade of endogenous GHRH receptors by gp120.

Responsiveness of chicken embryonic somatotropes to somatostatin (SRIF) and IGF-I

We have previously demonstrated that chicken embryonic somatotropes are responsive to GHRH shortly after they differentiate. In contrast, relatively little is known about the regulation of GH secretion by somatostatin (SRIF) and IGF-I during chicken embryonic development. In the present study, anterior pituitary cells were isolated from day. 16, 18 and 20 embryos and subjected to reverse hemolytic plaque assays (RHPAs) for chicken GH to assess the effect of SRIF and IGF-I on basal GH release and SRIF on GHRH-stimulated GH secretion. We found that all three ages responded to SRIF, under both basal and stimulated conditions. SRIF inhibition of basal GH release was evident for day 18 and 20 cells by 9 h, while 18 h were required for day 16 cells. After 18 h, 10(-11) M SRIF depressed basal GH secretion by day 16 and 18 cells, while 10(-9) M SRIF was required to depress GH plaque percentages by day 20 cells. GHRH stimulated GH release from all ages tested. After 2 h, SRIF partially suppressed GHRH-stimulated GH release by day 20 cells. After 6 h, day 18 cells responded to SRIF, reducing the percentage of plaque-forming cells under GHRH stimulated conditions. After 18 h, the percentage of day 16 cells forming GH plaques was reduced for cells treated with GHRH and SRIF, compared with cells treated with GHRH alone. All ages examined also responded to IGF-I. After 36 h, GH release by day 16 and 18 cells was decreased when exposed to IGF-I. While IGF-I decreased the relative amount of GH secreted per somatotrope on day 20, a paradoxical increase in the percentage of cells secreting GH was noted. These results indicate that anterior pituitary somatotropes are responsive to SRIF and IGF-I during late embryonic development of chickens.

Involvement of nitric oxide in the interferon- γ-induced inhibition of growth hormone and prolactin secretion in anterior pituitary cell cultures

In previous work it was shown that the immune cytokine interferon- γ (IFN- γ) inhibits hormone secretion in anterior pituitary (AP) cell cultures, an action most likely mediated by folliculostellate (FS) cells. In the present study, we wanted to investigate whether nitric oxide (NO) is involved in this inhibitory action of IFN- γ. NO synthase (NOS) inhibitors with affinity for the inducible (iNOS) and the constitutive (cNOS) isoform such as N G-monomethyl- l-arginine ( l-NMMA) and S-methyl- l-thiocitrulline (SMLT) dose-dependently blocked the inhibitory action of IFN- γ on GHRH-stimulated GH secretion, and partially reversed the inhibitory effect on basal prolactin (PRL) release. In the absence of IFN- γ these inhibitors significantly augmented basal PRL release and slightly enhanced GHRH-stimulated GH release. l- N 6-(1-iminoethyl)lysine ( l-NIL), a NOS inhibitor with preferential affinity for iNOS, abrogated the IFN- γ effect on GHRH-stimulated GH secretion and partially reversed IFN- γ inhibition of PRL release. However, l-NIL did not exert a stimulatory effect on basal PRL and GHRH-stimulated GH release by its own. 2,4-diamino-6-hydroxypyrimidine (DAHP), a NOS inhibitor by interfering with tetrahydrobiopterin (BH4) cofactor availability, showed the same activity profile as l-NIL. NOS inhibitors blocked or reduced the production of NO as detected by measuring nitrite (NO − 2) levels in AP cell cultures and cGMP levels in the NO-reporter cell line RFL-6. The NOS inhibiting action of l-NMMA was confirmed by competition experiments with the natural NOS substrate l-arginine. Thus, in culture medium with lower amounts of l-arginine, l-NMMA blocked the IFN- γ-induced inhibition of GHRH-stimulated GH release at a lower dose. The inhibition of PRL and GH release by IFN- γ was markedly reduced in l-arginine-depleted medium. The NO donor sodium nitroprusside (SNP) mimicked the inhibitory action of IFN- γ on GHRH-stimulated GH and basal PRL release. Similarly to IFN- γ, SNP did not affect basal GH release. As previously reported, inhibition by IFN- γ occurred only in AP cell populations containing a minimal proportion of FS cells. As studied in different cell populations obtained by unit gravity sedimentation in a serum albumin gradient, l-NMMA reversed the IFN- γ effect in the same populations enriched in FS cells. Interestingly, in the absence of IFN- γ l-NMMA strongly stimulated basal PRL release in the population most enriched in FS cells. It is concluded that IFN- γ through activation of the iNOS pathway probably in FS cells enhances the production of NO and that this effect is responsible for the inhibitory action of IFN- γ on GHRH-stimulated GH release and partially for the IFN- γ-induced decrease in basal PRL release. On the other hand, NO, likely produced by cNOS, appears to exert a tonic inhibitory effect on GHRH-stimulated GH and basal PRL release. It seems therefore that low amounts of NO produced constitutively may take charge of subtle physiological adaptations, and higher levels of NO produced by iNOS under the influence of IFN- γ may attenuate PRL and GH release during emergency conditions of immune and inflammatory reactions.

Tyrosine kinase inhibitors enhance GHRH-stimulated cAMP accumulation and GH release in rat anterior pituitary cells.

In this study, the role of tyrosine phosphorylation in agonist-stimulated cAMP accumulation and GH release in rat anterior pituitary cells was investigated. It was found that genistein, a tyrosine kinase inhibitor, while having no effect on its own, potentiated GHRH-stimulated cAMP accumulation in a concentration-dependent manner. In comparison, daidzein, an inactive analogue of genistein, was ineffective and vanadate, a phosphotyrosine phosphatase inhibitor, reduced GHRH-stimulated cAMP accumulation. Additional structurally unrelated tyrosine kinase inhibitors, erbstatin and tyrphostins, also potentiated GHRH-stimulated cAMP accumulation. To determine the site of action of the tyrosine kinase inhibitors, pituitary adenylate cyclase-activating polypeptide (PACAP), cholera toxin and forskolin were used to increase cAMP accumulation. Genistein enhanced the PACAP-, cholera toxin- or forskolin-stimulated cAMP accumulation, suggesting that the site of action is at the post-receptor level. However, when the phosphodiesterase was inhibited by isobutylmethylxanthine, genistein did not potentiate and vanadate did not inhibit GHRH-stimulated cAMP accumulation, indicating that phosphodiesterase is a probable site of action for the inhibitor. Genistein and erbstatin also enhanced GHRH-stimulated GH release and the effect of vanadate was inhibitory. These results indicate that tyrosine kinase inhibitors enhance cAMP accumulation through their action on phosphodiesterase activity in rat anterior pituitary cells and the tyrosine kinase pathway appears to be involved in the control of GH release.

The effect of gonadotropin-releasing hormone agonists on adrenocorticotropin and cortisol secretion in adult premenopausal women.

GnRH agonists are known to suppress LH, FSH, and subsequent ovarian estradiol production by down-regulation of pituitary gonadotropin receptors. Previous investigations have demonstrated that GnRH agonists also suppress GHRH-stimulated GH release in normal men and women and PRL levels in subjects with hyperprolactinemia. Little is known about the effects of GnRH agonists on the hypothalamic-pituitary-adrenal axis. The purpose of the present investigation was to determine the secretion of ACTH and cortisol after an iv infusion of hCRH in control women (n = 11) and in women undergoing treatment with GnRH agonists (n = 10). The plasma and serum levels of ACTH and cortisol increased after infusion of CRH in all women. The basal and CRH-stimulated plasma levels of ACTH and cortisol at each time point were not statistically different between GnRH agonist-treated women and controls. Thus, the chronic use of GnRH agonists is known to suppress the hypothalamic-pituitary-ovarian axis and is associated with GH and PRL suppression as well, but does not apparently alter the hypothalamic-pituitary-adrenal axis.

On the role of the peptide galanin in regulation of growth hormone secretion

The effects of the peptide galanin on growth hormone secretion were studied in vitro using cultured rat and human anterior pituitary cells, and in vivo by iv administration of galanin in both rats and humans. Galanin in concentrations from 10 nmol/l to 1 mumol/l did not alter basal GH release, but slightly inhibited GHRH-stimulated GH release from cultured rat anterior pituitary cells. Galanin (1 mumol/l) did not significantly change basal or GHRH-stimulated GH secretion from cultured human anterior pituitary cells. In contrast, iv injection of 1 microgram (300 pmol) galanin to rats induced an increase in plasma GH that was reproducible at repetitive injections. The galanin-induced GH release in rats was of a lower magnitude than the increase in plasma GH after iv injections of GHRH, and was seen with a 5-15 min delay in comparison to iv administered GHRH. In man, iv infusions of galanin (40 pmol.kg-1.min-1.(40 min)) also caused a significant increase in plasma GH, but it occurred 25-30 min after the beginning of the infusion. These results suggest an indirect action of galanin on GH release in both rats and humans, i.e. galanin does not directly affect the somatotropes. In agreement with a central action, no binding sites for galanin could be demonstrated in the rat anterior pituitary by autoradiography. Since galanin did not affect somatostatin release from fragments of rat mediobasal hypothalamus, the stimulatory effects of galanin on GH release are most likely mediated via a stimulatory effect on GHRH neurons.

Effects of cytidine 5'-diphosphocholine administration on basal and growth hormone-releasing hormone-induced growth hormone secretion in elderly subjects

The basal and GH-releasing hormone-stimulated secretion of GH declines in the elderly. We tested the ability of cytidine 5'-diphosphocholine, a drug used in the treatment of stroke and Parkinson's disease, to alter GH secretion in 11 healthy elderly volunteers, aged 69-84. Each subject received an iv infusion of 2 g of cytidine 5'-diphosphocholine or normal saline. GHRH and TRH were also administered during cytidine 5'-diphosphocholine infusions. The infusion of cytidine 5'-diphosphocholine induced a 4-fold (p less than 0.05) increase in serum GH levels over basal values. A small increase in GH was seen after GHRH administration. However, the addition of GHRH to the cytidine 5'-diphosphocholine infusion resulted in a GH response which was significantly greater than that seen after GHRH alone; the integrated concentration of GH was more than 2-fold greater in the cytidine 5'-diphosphocholine treated group (706.85 +/- 185.1 vs 248.9 +/- 61.4 micrograms.l-1.(120 min)-1; p = 0.01). The PRL and TSH responses to TRH were not significantly affected by cytidine 5'-diphosphocholine infusion, indicating that dopaminergic mechanisms are not involved. These studies demonstrate that cytidine 5'-diphosphocholine can enhance basal and GHRH-stimulated GH release in the elderly, but the mechanism of action of the drug remains unclear.

THE EFFECT OF GALANIN ON BASELINE AND GHRH‐INDUCED GROWTH HORMONE SECRETION IN OBESE CHILDREN

We have evaluated the effect of the administration of galanin (Gal), a newly identified hypothalamic peptide, on baseline and GHRH-induced GH rise in five obese children and in seven controls. The GH response to GHRH (hpGRF(1-29), 1 microgram/kg i.v.), and to Gal (15 micrograms/kg/h for 1 h), evaluated both as the maximum GH peak and as integrated area under the curve (AUC), was significantly lower in the obese children than in the controls. Simultaneous administration of Gal plus GHRH significantly increased the GH response to GHRH in all the obese subjects, so that their mean peak GH levels and AUC after Gal plus GHRH were similar to those of the control children after GHRH. Also, in control children Gal caused a significant augmentation of the GH response to GHRH. Mean peak GH levels and mean AUC after Gal plus GHRH were significantly higher in the controls than in the obese children given the same treatment. Our data indicate that obese children have a blunted GH response to Gal, which, however, is able to enhance the GH response to GHRH. This observation strengthens the view that the mechanism of action of Gal involves modulation of endogenous somatostatin (SRIH) release. In addition, similarity between the effects of Gal and pyridostigmine on baseline and GHRH-stimulated GH release in obese children may indicate the existence of a cholinergic link in the action of Gal.

ROLE OF CHOLINERGIC MUSCARINIC PATHWAYS ON THE FREE FATTY ACID INHIBITION OF GH RESPONSES TO GHRH IN NORMAL MEN

In order to explore the mechanisms by which free fatty acids (FFA) inhibit GH secretion, we studied the effect of the acetylcholinesterase inhibitor pyridostigmine (120 mg p.o.) on the FFA blockade of GH responses to the administration of GHRH (100 micrograms i.v.) in seven normal subjects. GHRH-induced GH secretion was significantly reduced following elevation of circulating FFA levels by lipid-heparin infusion and significantly potentiated by previous pyridostigmine treatment. Peak GH levels following combined administration of pyridostigmine plus lipid-heparin plus GHRH were significantly higher (P less than 0.01) than after GHRH alone and significantly lower than after pyridostigmine plus GHRH (P less than 0.01). In conclusion, central cholinergic activation by pyridostigmine, with the presumed reduction in somatostatin discharge, reversed the blocking effect of FFA on GHRH-stimulated GH release. Conversely, FFA were able to reduce even a maximal GH stimulation by pyridostigmine plus GHRH.

Effect of oral contraceptives on adrenocorticotropin and growth hormone secretion following CRH and GHRH administration

Previous investigations have demonstrated that basal ACTH plasma levels are reduced and that GH levels are either increased or unchanged in women taking oral contraceptives. The purpose of the present investigation was to determine the secretion of ACTH and GH following an intravenous infusion of human corticotropin releasing hormone (CRH) and human growth hormone releasing hormone (GHRH) in control women (N = 8) and in women taking a triphasic oral contraceptive (N = 9). The studies were initiated between 7:00 and 9:00 a.m. and all women were fasting. An intravenous catheter attached to a 3-way stopcock was used for blood sampling and to inject a bolus of CRH and GHRH ( 1μg kg ). Plasma samples were frozen immediately and stored at −70 °C until assayed for content of ACTH and GH by radioimmunoassay. The plasma levels of ACTH and GH increased following infusion of CRH and GHRH in all women. The mean plasma levels of growth hormone were not statistically different in oral contraceptive users compared to normal women. In contrast, ACTH plasma levels in oral contraceptive users were reduced approximately 25% overall, and significantly lower (p<0.04) at 120 minutes following the CRH infusion compared to controls. In conclusion, the GHRH-stimulated GH release was similar in normal women and oral contraceptive users. CRH-stimulated ACTH release was modestly reduced in oral contraceptive users compared to normal women suggesting that estrogens and progestogens may have a suppressive effect on the release of ACTH by the pituitary.

Differential growth hormone responses to glucose and growth hormone releasing hormone in lactating dairy cows

Dairy cows in early lactation were reported to secrete growth hormone in response to declining glucose concentrations at day 5 but not day 30 post-partum, whereas GH responses to TRH were reported to be enhanced after day 30 post partum. The present study examined GH response following glucose infusion and the effect of GHRH as well as effects of SRIH on GHRH-stimulated GH release. Declining plasma glucose concentrations after glucose infusion stimulated GH release at day 5 post partum but not in nonpregnant, nonlactating cows or in cows at days 30 and 90 post partum. GHRH stimulated GH release on all days tested, but the response was highest at day 30 post partum when compared to other days. Somatostatin infusion inhibited GHRH effects on GH concentrations only at day 30 post partum and in nonpregnant, nonlactating cows. Thus, a differential response of the GH regulatory system could be demonstrated between days 5 and 30 post partum utilizing different stimuli. Evaluation of plasma glucose and free fatty acid concentrations on days 5, 10, 20, and 30 post partum revealed a progressive decrease in FFA but not glucose as lactation progressed. Decreased plasma FFA concentrations were paralleled by a decrease in basal GH, somatomedin-C and epinephrine. Thus, a decline in FFA may be responsible for the disparity between effects of GHRH and glucose on GH release between days 5 and 30 post partum.