Dexamethasone is a potent stimulator of growth hormone-releasing factor-induced cyclic AMP accumulation in the adenohypophysis

The stimulatory effect of maximal concentrations of synthetic human pancreatic growth hormone (GH)-releasing factor (GRF)(1-40)NH on cyclic AMP accumulation in rat anterior pituitary cells in culture is 4.5-fold increased following a 48-h preincubation with the potent glucocorticoid dexamethasone while the sensitivity of GRF action is increased by approximately 4-fold. Dexamethasone pretreatment, on the other hand, has no effect on basal cyclic AMP levels but approximately doubles both basal and GRF-induced GH release. The present data suggest that the potent stimulatory effect of glucocorticoids on GH secretion is exerted on the adenylate cyclase system at a step preceding cyclic AMP formation.     

Somatostatin prevents the desensitizing action of growth hormone-releasing factor on growth hormone release

We have investigated the effect of prior exposure to somatostatin (SRIF) alone or in combination with growth hormone-releasing factor (GRF) on the subsequent cyclic AMP and GH responses to GRF in rat anterior pituitary cells in primary culture. The maximal 4.5-fold stimulation of GH release induced by a 3-hr incubation with GRF is reduced by 60% following a prior 3-hr exposure to 30 nM GRF. A 3-hr preincubation with GRF in the presence of 30 nM SRIF doubles spontaneous GH release while the maximal amount of GH released during a subsequent 3-hr exposure to GRF is similar to that measured in cells pretreated with control medium, thus completely preventing the loss of GH responsiveness induced by prior exposure to GRF. The prevention by SRIF of the desensitizing action of GRF on GH release is not observed on the cyclic AMP response which remains almost completely inhibited in GRF-pretreated cells. Similar protective effects are obtained when SRIF is incubated with prostaglandin E2 (PGE2), thus completely preventing the desensitizing action of PGE2 on GH release. Prior treatment with pertussis toxin completely prevents the protective action of SRIF on GH responsiveness. Pretreatment with GRF + SRIF increases by 85 and 60% the maximal amount of GH release induced by cholera toxin and 8-bromoadenosine 3',5'-monophosphate, respectively. The post-SRIF rebound effect on GH release occurs mainly during the first 30 min following withdrawal of the tetradecapeptide. The present data demonstrate that simultaneous preincubation with SRIF and GRF prevents the marked inhibition of GH release during subsequent exposure to GRF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypothalamic growth hormone-releasing factor (GRF) participates in the negative feedback regulation of growth hormone secretion

Effects of growth hormone (GH) excess on immunoreactive hypothalamic GH-releasing factor (GRF) and somatostatin (SRIF) were studied in rats. Hypothalamic GRF content significantly reduced after 7-day daily treatment with 160 micrograms of rat GH or after inoculation of GH-secreting rat pituitary tumors, MtT-F4 for 9 or 13 days and GH3 for 3 months. Basal and 59 mM K+-evoked release of GRF from incubated hypothalami diminished, more than the content, by 43-51% in MtT-F4 tumor- or by 67-83% in GH3 tumor-bearing rats. In contrast, there was a small but significant increase in content or release of SRIF in rats harboring the GH3 or MtT-F4 tumor, respectively. These results indicate the existence of a negative feedback loop via hypothalamic GRF as well as SRIF in control of GH secretion.     

Stimulation of growth hormone release in dwarf and normal chickens by thyrotrophin releasing hormone (TRH) or human pancreatic growth hormone releasing factor (hpGRF)

The effect of thyrotrophin releasing hormone (TRH) or human pancreatic growth hormone releasing factor (hpGRF) on growth hormone (GH) release was studied in both dwarf and normal Rhode Island Red chickens with a similar genotype except for a sex-linked dw gene. Both TRH (10 micrograms/kg) and hpGRF (20 micrograms/kg) injections stimulated plasma GH release within 15 min in young and adult chickens. The increase in GH release was higher in young cockerels than that in adult chickens. The age-related decline in the response to TRH stimulation was observed in both strains, while hpGRF was a still potent GH-releaser in adult chickens. The maximal and long acting response was observed in young dwarf chickens, suggesting differences in GH pools releasable by TRH and GRF in the anterior pituitary gland. The pituitary gland was stimulated directly by perifusion with hpGRF (1 microgram/ml and 10 micrograms/ml) or TRH (1 microgram/ml). Repeated perifusion of GRF at 40 min intervals blunted further increase in GH release, but successive perifusion with TRH stimulated GH release. The results suggest the possibility that desensitization to the effects of hpGRF occurs in vitro and that the extent of response depends on the number of receptors for hpGRF or TRH and/or the amount of GH stored in the pituitary gland.     

Growth hormone-releasing factor releases ACTH from an AtT-20 mouse pituitary tumor cell line but not from normal pituitary cells

Corticotropin-releasing factor (CRF) and both human pancreatic growth hormone-releasing factor (hp-GRF) and rat hypothalamic GRF (rh-GRF) stimulated ACTH release from neoplastic AtT-20 mouse pituitary tumor cells in a dose-dependent fashion, with CRF inducing a 10-fold increase and GRF a maximal increment of approximately one-half that of CRF. Neither rh-GRF nor hp-GRF induced ACTH release in normal anterior pituitary cells. Pretreatment with either dexamethasone or somatostatin prior to the addition of rh-GRF inhibited the increase in ACTH release. Both ovine CRF and rh-GRF stimulated adenosine 3,5-monophosphate production in AtT-20 cells. The weak but clearly discernible effect of GRF on ACTH release from AtT-20 cells may be due to an abnormality in the AtT-20 cell receptor.     

Dynamic of the GRF-induced GH response in genetically obese Zucker rats: influence of central and peripheral factors

To determine the time onset of the growth hormone (GH) alteration in the genetically obese rat, we studied the in vivo and in vitro rat growth hormone releasing factor (rGRF(1-29)NH2)-induced GH secretion in 6- and 8-week-old lean and obese male Zucker rats. Under sodium pentobarbital anesthesia, rGRF(1-29)NH2 (GRF) was injected intravenously at two doses: 0.8 and 4.0 micrograms/kg b.w. Basal serum GH concentrations were similar in lean and obese age-matched animals. The GH response to both GRF doses tested was unchanged in 6-week-old obese rats as compared to their lean litter mates. In contrast, a significant decrease of the GH secretion in response to 4.0 micrograms/kg b.w. GRF was observed in the 8-week-old obese rats. The effect of GRF (1.56, 6.25 and 12.5 pM) was further studied in vitro, in a perifusion system of freshly dispersed anterior pituitary cells of lean and obese Zucker rats. Basal GH release was similar in the 6-week-old animal group. In contrast, it was significantly decreased in 8-week-old obese rats as compared to their lean litter mates. Stimulated GH response to 1.56 and 6.25 pM GRF was significantly greater in the 6-week-old obese group than in the age-matched control group. In contrast, the GH response to all GRF concentrations tested was significantly decreased in the 8-week-old obese rats as compared to their respective lean siblings. In 8-week-old obese rats, a decrease of GH pituitary content and an increase of hypothalamic somatostatin (SRIF) concentration were observed. Insulin and free fatty acid serum were significantly increased in 8-week-old obese rats. In contrast, lower insulin-like growth factor I serum levels were observed in the obese animals as compared to their lean litter mates. Finally, to further clarify the role of the periphery in the inhibition of GH secretion observed in the 8-week-old fatty rats, we exposed cultured pituitary cells of 8-week-old lean animals to 17% serum of their obese litter mates. A significant decrease of GRF-stimulated GH secretion of lean rat pituitary cells exposed to the obese serum was noted (P less than 0.05). This study demonstrates that, in the obese Zucker rat, an alteration of the GH response to GRF is evident by the 8th week of life. This defective GH secretion could be related to peripheral and central abnormalities.     

Partial purification and characterization of a novel growth hormone-releasing factor (GRF) from teleost brain related to the rat hypothalamic peptide

A growth hormone-releasing factor (GRF)-like molecule has been partially purified and characterized from acid extracts of codfish (Gadhus morhua) brain using immunoaffinity and gel chromatography, followed by HPLC. This material has a mol.wt. which is similar to known mammalian forms of GRF but is immunologically and/or chromatographically distinct from previously described GRF peptides. However, it is related to rat(r) GRF(1-43) since it causes marked displacement in the rGRF RIA. Codfish GRF is a highly specific and potent hypophysiotropic factor as shown by its ability to stimulate the release of GH, but no other hormone, from rat anterior pituitary cells in vitro. These findings suggest that, phylogenetically, GRF is an ancient molecule with its biologic activity and certain immunoreactive domain(s) conserved, at least, from teleost to mammal.     

In vitro release of growth hormone-releasing factor (GRF) from the hypothalamus: somatostatin inhibits GRF release.

The manner of release of growth hormone-releasing factor (GRF) from the rat hypothalamus was studied in a perifusion system using a highly sensitive radioimmunoassay for rat GRF. The recovery of GRF in this system was 50-60%. The release of GRF from the rat hypothalamic blocks was almost stable for 20-240 min after the start of the perifusion and was stimulated by depolarization induced by high K+ concentration. The release of GRF was inhibited by somatostatin at concentrations of 10(-11) to 10(-8) M with maximum inhibition to 52.5% of the basal release at a concentration of 10(-9) M. These results suggest that this system is useful in studying the regulatory mechanism of GRF release and that, in addition to its action on the pituitary, somatostatin appears to act at the level of the hypothalamus in inhibiting GRF release in the regulation of GH secretion.     

In vivo and in vitro effects of cholecystokinin octapeptide on the release of growth hormone in rats

The effect of cholecystokinin octapeptide (CCK-8) on the release of growth hormone (GH) in rats was studied in vivo and in vitro. Intravenous injection of 5 micrograms/100 g BW of CCK-8 resulted in significant increase in the plasma GH level after 10 and 20 min. CCK-8 at concentrations of 10(-11)M to 10(-7)M also caused dose-dependent stimulation of GH release from dispersed cells of rat anterior pituitary. On the other hand, somatostatin (SRIF) inhibited GH release from dispersed cells of rat anterior pituitary in a dose-related manner at concentrations of 10(-7)M to 10(-9)M. Release of GH from the cells was increased by addition of K+ at high concentration (50 mM) in a Ca++-dependent manner. Addition of 10(-3)M verapamil to the incubation medium inhibited CCK-8-induced GH release from the cells. Addition of SRIF (10(-7)M) to the incubation medium inhibited GH release from the cells induced by CCK-8 or high K+ (50 mM). These results indicate that CCK-8 acts directly on the anterior pituitary cells to stimulate GH release and that calcium ion is involved in the mechanism of this effect.     

Growth hormone releasing effect of hpGRF-40 in rats at different time intervals following ablation of the mediobasal hypothalamus

We have investigated the effect of hypothalamo-pituitary disconnection in the rat on the growth hormone (GH) responsiveness to human pancreatic GH-releasing factor (hpGRF). Adult female rats, sham-operated (sham-op) or bearing a complete mechanical ablation of the mediobasal hypothalamus (MBH-A) were challenged, while under urethane anesthesia, with hpGRF-40 (20,100,500 ng/rat i.v.) at different time intervals after surgery. In sham-op rats only 500 ng/rat of hpGRF-40 stimulated GH release, while in 1-and 7-day MBH-A rats the stimulation also occurred with the lower hpGRF doses and the rise in plasma GH was greater than in sham-op controls. Twenty-one and 42 days after the placing of the lesions the GH response to hpGRF-40 was still present at the 500 ng/rat dose, though it was smaller than in sham-op controls. Evaluation of pituitary GH content demonstrated a progressive and rapid decline starting the first day after the placing of the lesions. These data indicate that GH responsiveness to hpGRF is: 1) enhanced in the anterior pituitary shortly after hypothalamo-pituitary disconnection and, 2) despite a striking reduction of the pituitary GH stores, it is maintained after these lesions.The physiologic growth hormone (GH) releaser in the rat is GH-releasing factor and, recently, a group of peptides has been characterized from human pancreatic tumors (hpGRFs) (1,2) which are potent and specific GH-releasers in both animals (3) and man (4). The availability of these peptides, which show a high degree of homology with the physiologic rat hypothalamic GRF (5), offers the unique opportunity to assess somatotrope responsiveness to GRF molecules in rats with hypothalamo-pituitary disconnection.In this study we have first evaluated the GH pituitary responsiveness to increasing doses of hpGRF-40 in rats following mechanical ablation of the mediobasal hypothalamus (6). These rats, by definition, lack the effect of both central nervous system (CNS) inhibitory (e.g. somatostatin) and stimulatory (e.g. GRF) influences to GH release. With the aim to ascertain how the lack of these two opposing inputs reflects on the secretory capacity of the somatotropes, we also investigated the GH response to hpGRF-40 at different time intervals after the lesioning. In a study in rats with electrolytic lesions of the ventromedial-arcuate region of the hypothalamus Tannenbaum et al (7) had shown persistence of the GH response to huge doses of a hpGRF analog.     

The effects of testosterone and estrogen on the pituitary growth hormone response to growth hormone-releasing factor

The effects of testosterone and estrogen on the pituitary growth hormone response to hypothalamic growth hormone-releasing factor (GRF) were evaluated in vivo using male and female rats and in vitro using a pituitary cell monolayer culture system. In vivo the increase in plasma growth hormone (GH) concentration in response to a 500 ng/kg dose of GRF was similar in gonadectomized male and female rats. Pretreatment of intact and gonadectomized male rats with testosterone caused significant enhancement of the pituitary GH response to GRF, whereas pretreatment of gonadectomized female rats with 17 beta-estradiol did not alter the response. The GH response to GRF was not different between prepubertal (i.e., 30-day-old) male and female rats. However, following puberty (i.e., by 60 days of age), the response in male rats was significantly greater than that observed in female rats. The in vitro preincubation of anterior pituitary cells with either testosterone or 17 beta-estradiol did not cause any shift in the dose-response curve between GRF and GH. These results demonstrated that androgens play an active role in modulating the pituitary response to GRF in vivo.     

Influence of endogenous growth hormone-releasing factor (GRF) on the secretion of GH during the perinatal period in the rat

Passive immunization of pregnant rats with a specific antiserum to rat GRF (GRF-AS) is followed by a decrease in fetal serum GH on the 19th day of gestation. A significant reduction in serum GH is still observed in older fetuses and newborn rats. Pituitary GH content increases in 19- and 20-day-old fetuses after GRF-AS administration to their mothers. These results suggest that endogenous fetal hypothalamic GRF (or placenta GRF) play a physiological role in the secretion of pituitary GH as early as the 19th day of fetal life and may be responsible for the peak of GH release that occurs in fetuses at the end of gestation.     

A cellular basis for functionally releasable pools in somatotropes

In an attempt to define the cellular basis for the phenomenon of releasable pools, we compared the effects of two growth hormone (GH)-releasing peptides which differentially influence the dynamics of GH release. Monodispersed anterior pituitary cells from neonatal male rats were subjected to reverse hemolytic plaque assays for GH in the presence or absence of GH-releasing peptide (GHRP-6, an enkephalin-like hexapeptide) and rat GH-releasing factor (GRF). GRF increased the rate of plaque formation (an index of the rate of hormone release) from almost all somatotropes, whereas GHRP-6 influenced only half of these cells. Analysis of plaque sizes (which provides a relative index of the cumulative amount of hormone released per cell) revealed that GRF produced a bimodal frequency distribution of plaque sizes, demonstrating that some somatotropes released more hormone than others after treatment with a maximal dose of this secretagogue. This pattern contrasted with those of untreated and GHRP-6 treated somatotropes which each produced unimodal frequency distributions that were skewed to the left (toward smaller plaques) and were virtually superimposable at the end of a 4 h incubation. However, GHRP-6 greatly accelerated the rate at which the final size distribution pattern was attained. Taken together, these results suggest that GHRP-6 causes the immediate release of a limited pool of GH which is present only in a discrete subpopulation of somatotropes that respond to GRF. This pool may be identical to that which is released over a more prolonged period under basal conditions. Moreover, GRF appears to access a more substantial pool of hormone which is not released by GHRP-6. This pool is present in a small minority of somatotropes but probably accounts for a larger portion of the GH released by pituitaries stimulated with GRF.     

Rat hypothalamic GRF elicits its biologic action in GH3 cells by interaction with VIP- preferring receptor site(s)

GH3 cells can be used effectively to study the in vitro mechanism of action of GRF. In these cells, there is a time and concentration-dependent release of cAMP into the medium. Rat hypothalamic GRF, (rGRF) is 7 to 10 fold more active than human hypothalamic GRF (hGRF). VIP, a peptide which is structurally homologous to GRF, stimulates cAMP efflux in GH3 cells, with a higher affinity than hGRF or rGRF. We propose that in contradistinction to the normal rat pituitary, the stimulation of cAMP release by GRF in GH3 cells occurs via activation of VIP-preferring receptors and that GRF (rGRF in particular) behaves as a partial VIP agonist.     

Growth hormone releasing factor (GRF) increases free arachidonate levels in the pituitary: a role for lipoxygenase products

GRF, a specific stimulator of GH release, increased in a concentration- and time-dependent manner pituitary [3H]-arachidonate levels in vitro. This effect was antagonized by 100 nM somatostatin. Exogenous arachidonate also stimulated GH release in vitro. Quinacrine, a phospholipase A2 inhibitor, reduced both basal and GRF-stimulated free arachidonate levels as well as GH release. The cyclooxygenase inhibitor indomethacin was ineffective, while BW755c, which also inhibits the lipoxygenase pathway, produced a further increase in the levels of the fatty acid stimulated by GRF and potently reduced GH release. These results provide additional evidence for the involvement of arachidonate metabolism in the hormone-releasing effect of GRF at the somatotroph.     

Ontogeny of the response to growth hormone-releasing factor

Primary cell cultures were prepared from fetal, neonatal and adult rat pituitaries and evaluated for their ability to secrete growth hormone (GH) in response to growth hormone-releasing factor (GRF). Pituitary cells prepared from fetuses at days 19 and 21 of gestation, neonatal animals at the day of birth (day 0) or the following day (day 1) and peripubertal male rats showed full dose response curves to GRF with maximal GH release when stimulated with 1 X 10(-10) M rat GRF. At this concentration of GRF, the amount of GH released was not different from that elicited by activation of adenylate cyclase with 1 X 10(-5) M forskolin. In contradistinction, a preparation of cells from fetuses at day 18 of gestation did not show the same release of GH when challenged with 1 X 10(-10) M GRF and forskolin (0.057 +/- 0.001, compared to 0.076 +/- 0.003 micrograms/10(5) cells per 4.5 h), although the cells clearly responded to both secretagogues (basal levels of GH, 0.029 +/- 0.002 micrograms/10(5) cells per 4.5 h). While cells prepared from fetuses at day 21 of gestation or from animals after birth released 5-10% of their total cellular GH content, those prepared from 18- and 19-day fetuses released as much as 40% of their total GH suggesting there is a maturation of intracellular GH processing that occurs late in gestation. The results show that, in late pregnancy, the rat fetal pituitary is highly responsive to growth hormone-releasing factor and suggest that this peptide participates in regulating GH levels during the perinatal period.     

Galanin stimulates rat pituitary growth hormone secretion in vitro

The effect of galanin on growth hormone (GH) secretion was investigated in monolayer cultures of rat anterior pituitary cells. Galanin caused a gradual increase in GH concentrations into the culture medium that was maximal at 90 minutes and sustained after 180 minutes. The ED50 for galanin-stimulated GH secretion was approximately 200 nM compared to an ED50 for rat GH-releasing factor (rGRF)-stimulated GH secretion of 10pM. Galanin and rGRF were additive in increasing GH release into the incubation medium. These data indicate that porcine-derived galanin has a direct effect on pituitary GH secretion in vitro.     

Growth hormone-releasing factor (GRF) stimulates PGE2 production in rat anterior pituitary. Evidence for a PGE2 involvement in GRF-induced GH release

Rat anterior pituitaries were incubated over a 3-h period. Both PGE2 and GH were increased by GRF in a concentration-related manner (ED50: 3.5 nM and 6.5 nM, respectively). A significant correlation (r = 0.88, n = 127) was observed between GH and PGE2 release over the range of GRF concentrations tested. Among the five prostanoids analyzed, only PGE2 was selectively increased. Somatostatin lowered GH release, without any effect on PGE2 production. Indomethacin (Id) and Aspirin reduced significantly PGE2 synthesis and GRF-induced GH release. The inhibitory effect of Id was counteracted by addition of PGE2 to the medium. GRF and PGE2, at maximal concentrations, had a partial additive effect on GH release. The increase in PGE2 production and the reduced GH release in the presence of cyclooxygenase inhibitors suggest that PGE2 is involved in GRF-induced GH release.     

Protein kinase C enhances growth hormone releasing factor (1-40)-stimulated cyclic AMP levels in anterior pituitary. Actions of somatostatin and pertussis toxin

The brain peptide human growth hormone releasing factor (1-40) (GRF), which stimulates adenylate cyclase activity in the anterior pituitary, is the predominant hormone signal for pituitary growth hormone (GH) release. Activators of protein kinase C such as teleocidin and 4 beta-phorbol 12-myristate 13-acetate (PMA) double the cyclic AMP accumulation induced by GRF, with no apparent effect on GRF potency; an inactive 4-alpha-PMA has no such action in cultured anterior pituitary cells. This PMA potentiation can be measured as early as 60 s, is maximal by 15 min, and wanes such that by 3-4 h there is no such amplifying effect of PMA. PMA, phorbol 12,13-dibutyrate, and teleocidin ED50 values for potentiating GRF activity are similar to those obtained for direct protein kinase C activation. The major inhibitory peptide somatostatin reduced both GRF- and GRF + PMA-stimulated cyclic AMP accumulation. Pertussis toxin totally blocked this somatostatin action without affecting the degree of maximal GRF potentiation achieved with PMA. Thus, the pertussis toxin target(s) are required for somatostatin inhibition of the cyclic AMP generating system, but may not be involved in the PMA potentiation of GRF-stimulated cyclic AMP accumulation.     

Somatostatin as a physiological regulator of pulsatile growth hormone secretion

Somatostatin plays an important role in the regulation of the episodic and ultradian rhythm of growth hormone (GH) secretion. Passive immunization of rats with specific antibodies to the 14 and 28 amino acid sequences caused a significant GH elevation. The fact that somatostatin antiserum was unable to block episodic GH surges indicates that this hormone's release must be regulated by a dual mechanism. Indeed, GH-releasing factor (GRF) seems to be instrumental in the maintenance of pulsatile GH secretion. Moreover, exogenous GRF induced a further GH increase predominantly during the period of active secretion. Neutralization of endogenous somatostatin eliminated this time-dependent effect, indicating that this peptide blocks periodical spontaneous GH release. Food deprivation and changes in glucose homeostasis virtually obliterate the ultradian GH rhythm. In this context, peripheral somatostatin seems to play an important role. Also the central GRF/somatostatin interplay is responsible for a short-loop feedback control on pituitary somatotrops.