Intrahypothalamic actions of somatostatin and growth hormone releasing factor on growth hormone secretion

Possible effects of GRF on somatostatin neurons and of somatostatin on GRF neurons were examined by measuring the effects of localised intracerebral injections of these peptides on growth hormone (GH) secretion. Serial GH concentrations were measured in plasma in unanaesthetized male rats chronically prepared with venous and intracerebral cannulae, before and after treatment with bilateral intracerebral injections of somatostatin or GRF in the preoptic anterior hypothalamic area (PO/AHA) and medial basal hypothalamus. Injections of 0.1 and 1 nmol of GRF in medial basal hypothalamus or 10 nmol somatostatin in the PO/AHA, respectively, had stimulatory or inhibitory effects on GH, which were assumed to be due to diffusion of the peptide from the injection site to the median eminence and pituitary gland. Injection of lower doses of somatostatin around GRF neurons in the medial basal hypothalamus were without significant effect on secretion of GH, but 0.1 nmol somatostatin in the PO/AHA resulted in an increase in GH concentrations from 128 +/- 61 to 524 +/- 103 ng/ml, p less than 0.02. Injections of GRF in lower doses amongst somatostatin neurons in the PO/AH or amongst GRF neurons in the medial basal hypothalamus were both without effect on GH secretion. We conclude that somatostatin may stimulate GH secretion by an effect on or close to somatostatin neurons in the PO/AHA. Somatostatin, though present in terminals on GRF neurons, is without effect at this site in our model. Furthermore, we have been unable to demonstrate any significant intrahypothalamic effect of GRF on GH regulation.     

Gonadotropin-induced expression of receptors for growth hormone releasing factor in cultured granulosa cells.

The hypothalamic neuropeptide, GRF, is formed in the ovary and acts via specific receptors in granulosa cells to enhance cAMP production and steroidogenic responses to the pituitary gonadotropin, FSH. Granulosa cells cultured without hormonal treatment displayed low levels of binding sites for GRF and the related neuropeptide, vasoactive intestinal peptide. However, treatment with increasing concentrations (50-500 ng/ml) of FSH caused dose-dependent increases in cAMP production and expression of binding sites measured with radioiodinated [His1, Nle27]human GRF(1-32)NH2, with no change in binding affinity. The maximum increase in GRF binding sites (2.2-fold) was elicited by 250 ng/ml FSH after 72 h incubation. GRF binding sites were also increased by agents that elevate intracellular cAMP, including choleragen, vasoactive intestinal peptide, dibutyryl cAMP, and forskolin. Low doses of forskolin that did not alone increase [125I] [His1, Nle27] human GRF(1-32)NH2 binding potentiated the action of FSH on GRF binding sites, but the effects of maximal stimulatory doses of both agents were not additive. These findings demonstrate that FSH promotes the expression of GRF receptors in maturing granulosa cells through cAMP-dependent mechanisms. Since GRF enhances the actions of FSH on cAMP production and granulosa cell differentiation, and GRF receptors are increased by the cAMP-mediated actions of FSH, locally produced GRF could exert a positive autoregulatory action to accelerate follicular maturation by amplifying the granulosa cell response to FSH.     

Down-regulation of growth hormone releasing factor receptors following continuous infusion of growth hormone releasing factor in vivo

The in vivo chronic infusion of growth hormone releasing factor (GRF) results in a loss of the pituitary growth hormone (GH) response to GRF as well as in a substantial depletion of pituitary GH content. To evaluate if the loss in response is due to the down-regulation of GRF receptors the specific GRF binding capacity of pituitary homogenates prepared from rats infused with saline or GRF (1 or 15 micrograms/h for 24 h) was determined. The pituitary binding capacity of animals infused with GRF was significantly reduced as compared to animals infused with saline.     

Transforming growth factor beta and follicle-stimulating hormone promote rat granulosa cell proliferation

Rat granulosa cells isolated from the ovaries of diethylstilbestrol-primed immature rats were treated with estrogen, FSH, and growth factors to determine those factors that were required to promote DNA synthesis. Estrogen and FSH, previously shown to stimulate the incorporation of [3H]thymidine into rat granulosa cell DNA in vivo, were ineffective in vitro. Epidermal growth factor, insulin-like growth factor 1 (IGF1), and fibroblast growth factor did not influence DNA synthesis whereas transforming growth factor beta (TGF beta) alone had a significant effect. Neither estradiol-17 beta (5 X 10(-8)-5 X 10(-6) M) nor IGF1 augmented the actions of TGF beta and FSH. FSH did not influence the actions of epidermal growth factor or IGF1 but dramatically augmented the effect of TGF beta on DNA synthesis. FSH and TGF beta also stimulated [3H]thymidine incorporation into the DNA of granulosa cells isolated from immature rats not treated with diethylstilbestrol. The increase in [3H]thymidine incorporation into DNA stimulated by TGF beta and FSH resulted subsequently in an increase in cell number. The response of the cells to TGF beta in the presence of a constant level of FSH (10 ng/ml) was dose dependent, 2.5 ng/ml being the minimal effective concentration. In the presence of antibody specific for TGF beta the bioactivity of the TGF beta was neutralized indicating that the growth promoting activity was due to TGF beta and not due to contaminants. In this paper, we have shown that the combined actions of FSH and TGF beta influence DNA synthesis and the proliferation of rat granulosa cells. Interactions between FSH and TGF beta may be important in regulating aspects of rat granulosa cell growth in addition to exerting pronounced effects on cytodifferentiation.     

Hormonal control of epidermal growth factor receptors by gonadotropins during granulosa cell differentiation

The hormonal induction of epidermal growth factor (EGF) receptor formation was analyzed during the maturation of granulosa cells obtained from diethylstilbestrol-implanted immature rats. In the absence of FSH, EGF receptors (as measured by the binding of [125I]iodo-EGF to the intact cells) rose by 50% at 6 h of culture, but then declined to about 25-40% of their initial levels at 24-96 h of culture. Scatchard analyses demonstrated the presence of high affinity EGF-binding sites in both freshly prepared cells and after FSH treatment. FSH stimulated a dose-dependent increase in the EGF receptor content of granulosa cells during a 96-h culture period. Concentrations of FSH as low as 2.5-5 ng/ml elevated EGF receptor levels 2- to 3-fold compared to those in untreated control cells, and 30 ng/ml FSH caused a maximal 15-fold rise. FSH increased EGF receptor levels approximately 2-fold in the first 6 h of culture and by up to 7-fold at 96 h compared to levels in freshly prepared cells. FSH treatment did not change the binding affinity (Kd = 5-6 X 10(-11) M) of the EGF receptor, but increased the total number of EGF-binding sites. The stimulatory effects of FSH on EGF receptor expression were mimicked by other cAMP-inducing ligands, including 8-bromo-cAMP, forskolin, and choleragen. Ligands known to inhibit granulosa cell function, including GnRH agonists and the phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate, reduced the stimulation of EGF receptors by FSH. However, only 12-O-tetradecanoyl-phorbol 13-acetate suppressed the induction of EGF receptors by 8-bromo-cAMP. In granulosa cells cultured for 48 h with FSH, subsequent treatment with hCG for 24 h reduced EGF receptor content by 25%. Autoradiographic studies with [125I]iodo-EGF in ovarian thin sections demonstrated that EGF-binding sites were uniformly dispersed throughout the ovaries of diethylstilbestrol-implanted rats. Treatment with PMSG markedly increased EGF receptors in the outer walls of the growing follicles, while hCG treatment after PMSG caused a general decline in ovarian labeling. These results indicate that FSH maintains and increases the number of EGF receptors during granulosa cell differentiation, while LH/hCG reduces EGF-binding sites. Such changes in EGF receptors in the presence of endogenous growth factors may influence the number and selection of follicles destined for ovulation.     

Platelet-derived growth factor enhances granulosa cell luteinizing hormone receptor induction by follicle-stimulating hormone and serum

Platelet-derived growth factor potentiated FSH-dependent LH receptor induction in serum-free and serum-containing cultures of rat granulosa cells. Levels of LH receptor binding comparable to pre-ovulatory levels could be achieved in vitro using a combination of FSH, platelet-derived growth factor, and serum. Receptor sites induced under these conditions were capable of mediating an hCG-stimulated increase in progestin secretion. Our results in vitro suggest a possible role for platelet-derived growth factor or related compounds in granulosa cell differentiation in vivo.     

Epidermal growth factor influences growth and differentiation of rat granulosa cells

The interactions of epidermal growth factor (EGF) with transforming growth factor beta (TGF beta), insulin-like growth factor-I (IGF-I), and FSH in the modulation of DNA synthesis and differentiated functions were examined in cultures of granulosa cells isolated from the ovaries of immature rats primed with diethylstilbestrol. EGF alone or in the presence of FSH had no effect on [3H]thymidine incorporation into the DNA of granulosa cells; however, EGF inhibited FSH plus TGF beta-induced DNA synthesis. In contrast, when FSH was omitted from the culture medium, EGF acted in concert with TGF beta, and TGF beta plus IGF-I, to promote DNA synthesis. EGF therefore has opposing actions on DNA synthesis; it inhibits or stimulates depending upon the presence or absence of FSH and consequently upon the endocrine environment in the follicle. As shown previously EGF alone had no effect on basal aromatase activity. EGF however inhibited FSH-induced and FSH plus IGF-I-induced aromatase activity. In this paper we show that EGF also inhibited the FSH-induced aromatase activity in the presence of TGF beta, which augmented FSH action on this system. The action of EGF on 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) was different from the effect of EGF on aromatase. In the absence of FSH, EGF induced 3 beta-HSD activity in the presence or absence of TGF beta. EGF augmented the action of FSH on 3 beta-HSD, and this interaction was further enhanced by TGF beta. These observations emphasize the multifunctional nature of EGF in influencing the growth and differentiation of immature rat granulosa cells. EGF can inhibit or stimulate growth and differentiated functions (aromatase and 3 beta-HSD), the response depending on the context of the signals that the cell receives from its endocrine and microenvironment.     

Effects of a growth hormone releasing factor in man

The effects of the 44-amino acid growth hormone releasing factor (GRF-44) were tested in normal adult men and women. At a dose of 1 microgram/kg, intravenous boluses of GRF-44 stimulated prompt elevations of plasma GH, which in 5 men reached maximum levels of 34 +/- 28 (S.D.) ng/ml, and in 3 women in the mid-follicular phase, 53 +/- 10 ng/ml. The action of GRF was highly selective; there were no changes in plasma PRL, LH, FSH, TSH, or cortisol at this dose level. Side effects, mostly flushing and a sense of warmth of the face and chest, were mild and occurred only in a minority of subjects.     

Growth hormone response to growth hormone releasing factor in Alzheimer's disease

Because of the well-established reduction in the concentration of somatostatin in several brain areas of patients with histologically verified Alzheimer's disease, we sought to determine if growth hormone (GH) secretion is altered in Alzheimer's disease. In order to study this, we assessed the GH response to growth hormone releasing factor (GRF) in 8 patients with Alzheimer's disease and 8 age-matched controls. Although there was no difference between the magnitude of the GRF-induced GH response (delta max GH response or area under the curve) between the Alzheimer's disease patients and the controls, the Alzheimer's patients exhibited a delayed GH response to GRF.     

Hypothalamic interconnections of somatostatin and growth hormone releasing factor neurons

Combined immunohistochemical labelling for neurons containing growth hormone (GH) releasing factor (GRF) or somatostatin and single labelling immunohistochemistry combined with Fluorogold retrograde transport labelling were used to examine whether somatostatin or GRF neurons might be reciprocally innervated. Occasional somatostatin-immunoreactive neurons in the periventricular preoptic area were found to be closely approached by GRF-immunoreactive fibres, providing possible evidence of scant innervation of somatostatin neurons by GRF cells. In contrast, many GRF-immunoreactive neurons in the arcuate nucleus appeared to have somatostatin-immunoreactive fibres closely applied to their perikarya suggesting that GRF neurons might be innervated by somatostatin cells. Combined retrograde tracing and fluorescence immunohistochemistry revealed few somatostatin-immunoreactive neurons doubly labelled following injections of Fluorogold in the basal hypothalamus. Occasional GRF-immunoreactive neurons in the basal hypothalamus were doubly labelled following PO/AHA injections of Fluorogold. Numerous somatostatin-immunoractive perikarya were observed in the periventricular arcuate region in colchicine-pretreated animals. We conclude that GH-regulating neurons do not have strong reciprocal innervation. The innervation of GRF neurons by somatostatin fibres may be derived from local somatostatin neurons.     

Effects of a purified growth hormone releasing factor on growth hormone secretion and pituitary cyclic nucleotide content

We examined the effects of partially purified ovine GRF on medium growth hormone and tissue cyclic AMP and cyclic GMP content of incubated pituitary explants. Although hypothalamic extracts that contained numerous releasing factors had increased both cyclic AMP and cyclic GMP, the purified GRF promoted the accumulation of cyclic GMP, but not cyclic AMP. Six μg of the GRF increased medium growth hormone 240% above flasks containing only control buffer. These data support the concept that cyclic GMP is an important intracellular mediator of growth hormone release.     

Mechanisms of action of gonadotropin releasing hormone on rat granulosa cells

To elucidate the mechanisms of stimulatory actions of GnRH on rat granulosa cells (GC), we have compared the actions of a GnRH agonist with those of a tumor-promoting phorbol ester, 12-0-tetradecanoylphorbol 13-acetate (TPA) and Ca+2 ionophore, A23187. GC were obtained from immature (28-29 days old) rats 48 h after injection of 20 IU PMSG. Following prelabeling with 3[H]arachidonic acid (AA), the cells were incubated with the test substances for 10 min and AA release determined. A GnRH agonist, [D-Ala6, des-Gly-NH2(10)] GnRH ethylamide (GnRHa; 10 ng/ml) increased AA release 175% compared to the control value. AA release in the presence of GnRHa was larger than that due to 1 microM A23187 or 40 nM TPA alone. A23187 or TPA increased GnRHa-stimulated AA release further. GC were incubated with the test substances for longer time periods, i.e., up to 5 h. GnRHa caused a 4-fold increase in prostaglandin (PG) synthase activity at 5 h. GnRHa increased PGE accumulation to the same extent as TPA, but only increased PG synthase activity about half as much. In combination with TPA, GnRHa had no influence on TPA-stimulated PG synthase activity, but increased PGE accumulation to levels comparable to those with A23187 plus TPA. GnRHa caused a 2.5 fold increase in progesterone (P) accumulation, which was the same as TPA. P accumulation in the presence of GnRHa was affected by neither A23187 nor TPA. These data indicate that the combination of TPA and A23187 can substitute for GnRH action on PGE and P accumulation in rat GC.     

Evidence for a growth hormone releasing factor mediating alpha-adrenergic influence on growth hormone secretion in the rat

The effects of adrenergic receptor agonists on GH secretion were studied in adult, male rats pretreated with reserpine and somatostatin antiserum. Frequent blood samples were obtained from intra-aortic cannulae. Plasma GH was determined by radioimmunoassay. Reserpine (10 mg/kg i.p.) caused a complete suppression of the normal, pulsatile secretion of GH in all animals. Administration of somatostatin antiserum resulted in rapid elevations of plasma GH in reserpine-pretreated rats with peak levels at 30 min. GH levels then fell but remained slightly elevated for the duration of the sampling period (8 h). Apomorphine (0.5 mg/kg i.p.) had no effect on plasma GH levels, whereas clonidine (0.5 mg/kg i.p.) induced release of GH in both antiserum treated and control rats. The results indicate that the alpha-adrenergic influence on the secretion of GH is mediated not by inhibition of somatostatin release but rather by effects on the release of a GHRF.     

Ectopic growth hormone releasing factor stimulates growth hormone release from human somatotroph adenomas in vitro

The effects of an ectopic growth hormone releasing factor (E-GHRF) from a carcinoid tumor were studied in three human GH-secreting pituitary adenomas in monolayer culture. GH release by the adenomas was stimulated by 55 +/- 10%, 57 +/- 8%, and 59 +/- 17% during a 4 h exposure to E-GHRF at concentrations of 3, 0.8, and 24 Units/ml, respectively. Two of the tumors also secreted prolactin (PRL) and in one, E-GHRF (0.8 Units/ml) stimulated PRL release by 48 +/- 8%. GH and PRL release were both suppressed by somatostatin (10(-8)M) in two of the adenomas. These results 1) demonstrate for the first time E-GHRF stimulation of GH release from human pituitary tissue in vitro, 2) support an etiologic role for E-GHRF in the development of acromegaly, and 3) indicate that GH-secreting adenomas retain the capability of responding to GHRF.     

The effect of repeated administration of hexarelin, a growth hormone releasing peptide, and growth hormone releasing hormone on growth hormone responsivity

OBJECTIVE Hexarelin is a synthetic six-amino-acid compound capable of releasing GH in animals and in man. Its mechanism of action is not understood and little is known about the GH response after repeated administration. The aim of this study was to determine the GH response to the administration of two intravenous boluses of hexarelin, growth hormone releasing hormone (GHRH) or hexarelin with GHRH. DESIGN Single boluses of hexarelin (1 μg/kg), GHRH-(1–29)-NH₂ (1 μg/kg) or hexarelin with GHRH-(1–29)-NH₂ were administered intravenously. Each study was performed on two further occasions, with a second bolus being administered 60 or 120 minutes after the first. A control study was performed giving saline intravenously. Studies were performed in a random order. SUBJECTS Six healthy adult males (25.4–34.1 years) were studied. MEASUREMENTS Serum GH was measured by radioimmunoassay. GH secretion rates were derived from the measured serum GH concentrations using the technique of deconvolution analysis. RESULTS The peak GH secretion rate following the first intravenous bolus of hexarelin was greater than that following the first bolus of GHRH-(1–29)-NH₂ (P < 0.001), and was greatest following the administration of hexarelin with GHRH-(1–29)-NH₂ (P < 0.001). The coadministration of the two secretagogues resulted in peak GH secretion rates significantly greater than the arithmetic sum of those following their isolated administration (P = 0.001), demonstrating synergism. Compared to saline, the administration of a second bolus of hexarelin, GHRH-(1–29)-NH₂ or both resulted in significant further GH secretion (P = 0.02, P = 0.002, P = 0.03, respectively). The administration of a second bolus of hexarelin or hexarelin with GHRH-(1–29)-NH₂ 120 minutes after the first bolus resulted in lower peak GH secretion rates (P = 0.03). The reductions in peak GH secretion rates following the 60-minute boluses were not statistically significant. The peak GH secretion rates following the first GHRH-(1–29)-NH₂ boluses were similar to those following the 60 and 120-minute GHRH-(1–29)-NH₂ boluses (P = NS). Irrespective of the interval between the boluses of hexarelin with GHRH-(1–29)-NH₂, the peak GH secretion rates following the second boluses were not significantly different from the arithmetic sum of those following the administration of the second boluses of hexarelin or GHRH-(1–29)-NH₂, indicating loss of synergism on repeated administration. CONCLUSIONS This study shows that hexarelin is a potent GH secretagogue active after two successive doses; the magnitude of the GH response to the second dose was influenced by the dosing interval. Hexarelin and GHRH-(1–29)-NH₂ are synergistic, a property which is lost after repeated administration. These findings may help our understanding of GHRPs and may have implications for the potential use of hexarelin and other GHRPs as therapeutic agents.