Growth hormone-releasing factor regulates growth hormone mRNA in primary cultures of rat pituitary cells.

A peptide with high intrinsic activity for specifically stimulating the secretion of immunoreactive growth hormone (GH; somatotropin) has been characterized and reproduced by total synthesis. This peptide, human pancreatic growth hormone-releasing factor, 44-amino-acid form (hpGRF1-44-NH2), was isolated from a tumor localized in the pancreas of a patient with acromegaly. We report here the effect of this growth hormone-releasing factor (GRF) on GH release and the GH mRNA levels in monolayer cultures of rat pituitary. The cytoplasmic dot hybridization technique was used to examine the effect of GRF on GH mRNA levels. Incubation of rat pituitary cultures with GRF for 72 hr resulted in a 2- to 2.5-fold increase in GH mRNA levels, and the maximal levels of stimulation were achieved at GRF concentrations as low as 1 fM. GRF did not stimulate prolactin release, nor did it affect specific prolactin mRNA levels in the pituitary cultures. We conclude that GRF is a potent and specific GH secretagogue that also affects specifically GH mRNA levels in normal pituitary cells.     

Growth hormone-releasing factor desensitization in rat anterior pituitary cells in vitro

Preincubation of rat pituitary cells in primary culture with rat GH-releasing factor (rGRF) resulted in substantial desensitization to subsequent GRF stimulation. rGRF-directed GH release and intracellular cAMP accumulation decreased in the desensitized cells. Whereas prior treatment of rat pituitary cells caused partial depletion of intracellular GH levels, diminished cellular reserves could not entirely account for the decreased GH release. Cells that had been preexposed to 10 nM rGRF for 4 h demonstrated at 30-50% depletion of intracellular GH; subsequent stimulation of those cells with 10 nM rGRF elicited GH release which was only 5% of that seen in cells that were not desensitized [control, 112 +/- 3.2 ng/well (+/- SEM); GRF-stimulated, 435 +/- 32 ng/well; GRF-pretreated, control, 63 +/- 3 ng/well, GRF-pretreated, GRF-stimulated, 73 +/- 3.4 ng/well]. Despite the marked depletion of cellular GH stores and the greatly diminished rGRF-stimulated GH release in cells that had been preexposed to rGRF, both forskolin and (Bu)2cAMP were able to induce a 2-fold stimulation of GH release. Incubation of the rGRF-pretreated cells with fresh medium which lacked rGRF resulted in gradual recovery of the ability of rGRF to stimulate GH release without complete reconstitution of the intracellular GH stores. These results indicate that exposure of rat pituitary cells to rGRF results in 1) partial depletion of intracellular GH stores; 2) a diminished ability of a subsequent rGRF challenge to elicit GH secretion and intracellular cAMP accumulation, and 3) a sustained ability of forskolin and (Bu)2cAMP to stimulate GH release, indicating that rGRF desensitization occurs in vitro.     

Growth hormone-releasing hormone and gonadotropin-releasing hormone stimulate nitric oxide production in 17beta-estradiol-primed rat anterior pituitary cells

It was reported that neuronal nitric oxide synthase (nNOS) was expressed only in gonadotrophs and folliculo-stellate cells in the anterior lobe of the pituitary gland. However, recent studies have demonstrated the occurrence of nNOS in the somatotrophs and lactotrophs. In the present study, we investigated effects of growth hormone-releasing hormone (GHRH), gonadotropin-releasing hormone (GnRH), and 17β-estradiol on nitric oxide (NO) release in cultured rat anterior pituitary cells in vitro. The NO ₂ ⁻ level in the incubation medium of the rat anterior pituitary cells was dependent on the cell density. Pretreatment with 10 μM 17β-estradiol resulted in an increase in medium NO ₂ ⁻ level. GHRH and GnRH failed to change medium NO ₂ ⁻ levels, but they elicited increases in medium NO ₂ ⁻ levels in estrogen-treated cells. The GHRH-induced increase in NO ₂ ⁻ level was inhibited by Nχ-nitro-l-arginine methyl ester, a NOS inhibitor. These findings suggest that GnRH and GHRH could activate nNOS in the gonadotrophs and the somatotrophs, respectively.     

Growth hormone-releasing peptide-2 (GHRP-2) does not act via the human growth hormone-releasing factor receptor in GC cells

Effect of growth hormone-releasing peptide-2 (GHRP-2) on ovine somatotrophs is abolished by a growth hormone-releasing factor (GRF) receptor antagonist, which raises the possibility that GHRP-2 may act on GRF receptors. In the present study, we used rat pituitary GC cells with or without stable transfection of cDNA coding for the human GRF receptor (GC/R⁺ or GC/R⁻) to determine whether or not GHRP-2 acts via the GRF receptor. Northern blot analysis indicated that GRF receptor mRNA was undetectable in GC/R⁻ cells, whereas a high level of expression occurred in GC/R⁺ cells that were transfected by GRF receptor cDNA. In GC/R⁻ cells, incubation with up to 10⁻⁷ M of either hGRF or GHRP-2 did not alter the intracellular cAMP, [Ca²⁺]i, or GH secretion. In GC/R⁺ cells, hGRF (10⁻¹¹–10⁻⁷ M) increased cAMP levels in a concentration-dependent manner up to 20-fold. This increase in cAMP levels was blocked by a GRF receptor antagonist, [Ac-Tyr¹, d-Arg²]-GRF 1–29, but not by a Ca²⁺ channel blocker, NiCl₂ (0.5 mM). GH secretion and [Ca²⁺]i were, however, not increased by hGRF. Incubation of the transfected cells with 10⁻¹¹–10⁻⁸ M GHRP-2 did not modify intracellular cAMP levels. This result suggests that GHRP-2 does not act through the GRF receptor.     

Growth hormone-releasing factor production by fetal rat cerebrocortical and hypothalamic cells in primary culture

Recent data from our laboratory and others have shown radioimmunoassayable GRF (IR-GRF) in the rat brain cortex. In the present study the ontogenesis of immunoreactive rat(r) GRF (IR-GRF) in long term dissociated fetal rat cerebrocortical and hypothalamic cell cultures and the regulation of its secretion by potassium depolarization and calcium channel-blocking agents were investigated. The chromatographic profiles of IR-rGRF from cell extracts were determined and compared with those from in vivo cerebrocortical and hypothalamic tissues. Mechanically dispersed cultured telencephalic and diencephalic cells from 17- to 21-day-old fetal rats showed a progressive increase in IR-rGRF, reaching maximum values (media plus cells) between 775-1020 (pg/mg protein) in hypothalamic cells on days 10-20 and between 450 and 950 pg/mg protein in cortical cells on days 25-30. IR-rGRF from acidic extracts of cells and adult cortical and hypothalamic tissues adsorbed onto octadecylsilyl-silica columns corresponds primarily to rGRF-(1-43)OH on HPLC. In gel filtration chromatography, almost all IR-rGRF from cultured cerebrocortical cells and fetal and adult cortical tissues coeluted with rGRF-(1-43)OH. IR-rGRF from cultured hypothalamic cells showed an additional component with a higher mol wt eluting in the void volume. To determine the influence of membrane depolarization of rGRF release, potassium (K+) concentrations in the medium were increased to 30 and 56 mM, inducing a marked increase in medium rGRF concentrations. Verapamil, a Ca2+ channel blocker (20 microns) reverses the effect of 56 mM potassium depolarization, suggesting that it is at least partially dependent upon Ca2+ transport. These data indicate that fetal rat cerebrocortical and hypothalamic cells in culture produce and release rGRF in response to depolarizing agents. The presence of rGRF in cortical tissue suggests that there are other physiological roles besides those implicated in the stimulation of GH secretion.     

Growth hormone-releasing hormone stimulates cAMP release in superfused rat pituitary cells.

The release of growth hormone (GH) and cAMP was studied in superfused rat pituitary cells by infusing growth hormone-releasing hormone (GHRH) at different doses or a combination of GHRH and somatostatin 14 (SS-14). Three-minute pulses of GHRH caused a dose-dependent GH and cAMP release (effective concentration of 50% of the maximal biological effect is 0.21 nM and 52.5 nM, respectively). The lowest effective doses of GHRH in the superfusion system were 0.03 nM for GH release and 0.3 nM for cAMP discharge when 3-min pulses were applied. The amount of cAMP liberated from the cells was not proportional to GH release: cAMP responses to low doses of GHRH were disproportionally small, and the gradual increase in the release of cAMP after high doses of GHRH was not followed by a parallel rise in GH release. The desensitization induced by repeated pulses or prolonged infusion of GHRH resulted in a greater reduction in GH release than in cAMP liberation. A simultaneous infusion of SS-14 completely blocked GH release stimulated by GHRH but did not inhibit the immediate release of cAMP caused by GHRH. An abrupt decrease in GHRH-stimulated GH release induced by SS-14 was followed by only a minimal reduction in cAMP liberation 9 min later. Our findings indicate that a discharge of cAMP is stimulated after a GHRH pulse, but this effect alone cannot maintain the release of GH. Other steps of the signal transduction mechanisms that are independent of the cAMP route may participate in the process of GH release. The nature of the mechanisms involved in the mediation of GH release may vary with the doses of GHRH used.     

Dopamine does not attenuate phosphoinositide hydrolysis in rat anterior pituitary cells

The hydrolysis of membrane phosphatidylinositol to yield [3H]labelled inositol phosphates by anterior pituitary cells was stimulated significantly by angiotensin II, TRH and neurotensin over a broad range of concentrations. These secretagogues also stimulated release of prolactin. Although the coincident incubation of dopamine with these agents resulted in a marked diminution of prolactin release, no concomitant reduction in inositol phosphate production was observed. In addition, bromocriptine, a potent agonist of dopamine, also proved ineffective in blunting stimulated phosphatidylinositol catabolism. Although it slightly inhibited basal rates of inositol tris-, bis- and monophosphate production, these results show that the secretagogue-mediated enhancement of phosphatidylinositol catabolism may be correlated with an increased release of prolactin and that the inhibition of hormone release produced by dopamine is not achieved by reducing basal or secretagogue-mediated inositol phosphate production.     

Platelet-derived growth factor does not stimulate prostacyclin synthesis by cultured endothelial cells

We examined the effect of highly purified platelet-derived growth factor (PDGF) on prostacyclin (PGI2) release by cultured human umbilical vein and bovine aortic endothelial cells. PDGF tested at concentrations equal to or exceeding those observed in serum did not increase endothelial cell PGI2 synthesis as measured by radioimmunoassay of its metabolite, 6-keto-PGF1 alpha. In contrast, cells incubated with 20% human whole blood serum (WBS) demonstrated significantly increased PGI2 production (fivefold stimulation). Addition of anti-PDGF antibody to the 20% WBS did not attenuate the increased synthesis of PGI2. Incubation with 20% plasma-derived serum (PDS) that was deficient in PDGF produced stimulation of PGI2 release similar to 20% WBS. These results demonstrate that PDGF does not cause increased PGI2 synthesis in cultured human endothelial cells of human or bovine origin, and further suggest that the stimulation observed with serum is not due to a platelet-release product.     

Growth hormone-releasing factor binding sites in rat anterior pituitary membrane homogenates: modulation by glucocorticoids

Specific high affinity binding sites for growth hormone releasing factor (GRF) were described in rat anterior pituitary homogenates with use of the analog [His1-mono-125I-Tyr10,Nle27]-hGRF(1-32)-NH2 as radioligand. Computerized analysis of competition experiments indicated one class of specific high affinity binding sites with a dissociation constant of 0.19 nM. The relative binding affinities of rGRF, hGRF(1-40) and various analogs correlated well with their in vitro biological potencies. Further, the number binding sites was drastically decreased after adrenalectomy; chronic dexamethasone treatment of these animals restored GRF binding capacity to control without changing binding affinity. These results may in part explain the enhanced responsiveness of the somatotroph after dexamethasone treatment.     

Electrophysiological responses of rat pituitary cells in somatotroph-enriched primary culture to human growth-hormone releasing factor

The in vitro effects of human growth hormone releasing factor (hGRF, 1-44) were studied in somatotroph-enriched cultures (75-85%) obtained from adult male rat pituitaries. Cells perifused with hGRF showed an up to 800% increase in growth hormone (GH) release over basal values in a dose-dependent manner. Calcium current blockers (5 mM of Co2+, Ni2+ or Cd2+) completely inhibited this stimulating effect but sodium-free (choline) medium did not. Using a single-intracellular-electrode recording technique, it was found that hGRF induced a dose-dependent depolarizing response concomitant with a decrease in membrane resistance in 38% of the cells recorded (98 of 258 cells). The reversal potential of this response was close to -40 mV. This depolarizing response was recorded in both excitable and unexcitable cells with no marked difference. Co2+ and Ni2+ (5 mM) completely and reversibly inhibited the membrane response to hGRF. Application of hGRF and somatostatin (SRIF), a hormone that inhibits GH release, to the same cell cultures showed the existence of two subpopulations: one was responsive to both hGRF and SRIF (53%, n = 62), another was only responsive to SRIF (47%, n = 62). Human GRF did not affect prolactin release and did not modify the electrical properties of cells responding to dopamine and therefore considered as lactotrophs. These results suggest that (1) hGRF leads to an increase in growth hormone release and a modification of membrane electrical properties by means of an extracellular Ca2+-dependent pathway, and (2) according to their responses to SRIF and hGRF, there are at least two subpopulations of somatotrophs.     

Influence of sex steroid hormones on rat growth hormone-releasing factor and somatostatin in dispersed pituitary cells

The modulatory effects of glucocorticoid and sex steroid hormones on the effects of rat GH-releasing factor (GRF) and somatostatin (SRIF) on GH release and biosynthesis were studied in monolayer cultures of rat anterior pituitary cells with RIA and quantitative immunoprecipitation methods. Dexamethasone (10(-7) M), a potent synthetic glucocorticoid, increased both the sensitivity and maximum response of GH release stimulated by GRF. Progesterone (10(-7) M) also enhanced GH release stimulated by GRF. The stimulatory effects of dexamethasone and progesterone were dose dependent and required a latent period of at least 24 h to be evident. Testosterone, dihydrotestosterone, and 17 beta-estradiol showed no apparent influence on GRF-induced GH release under the same conditions. None of the hormones studied showed significant influences on basal or SRIF-suppressed GH release. Progesterone added to the maximally effective concentrations of dexamethasone had no additional effects on GRF-induced GH release. The effect of progesterone was attenuated by both 5 alpha-dihydronorethindrone, a progesterone antagonist and 17 alpha-methyltestosterone, a glucocorticoid antagonist. In terms of GH synthesis, stimulatory effects of GRF on GH synthesis were apparent only when pituitary cells were pretreated with dexamethasone. These results indicate that: pretreatment with glucocorticoid or progesterone enhances the effects of GRF on GH release and/or synthesis; these two steroids share at least one common step to enhance GRF effects; and steroid hormones have little influence on basal or SRIF-suppressed GH release.     

Angiotensin peptides stimulate phosphoinositide breakdown and prolactin release in anterior pituitary cells in culture

We investigated the effects of angiotensin peptides on the breakdown of specific membrane phospholipids, the inositol lipids, in anterior pituitary cells in culture, measuring the water-soluble products (inositol phosphates) produced during the cleavage of phosphoinositides by phospholipase C. Both angiotensin II and angiotensin I in the presence of 10 mM LiCl potently increased, in a concentration-dependent manner, total [3H]inositol phosphate and PRL release in cultured rat anterior pituitary cells. The release of LH, TSH, or GH was not significantly enhanced by the peptides. The effect on inositol phosphate accumulation was significant at 0.01 nM, and maximal stimulation (approximately 5-fold increase) occurred at 10 nM, with an ED50 of about 0.3 nM. The stimulatory effects of both angiotensin II and angiotensin I were antagonized by the receptor antagonists saralasin and Sar1,Ile8-angiotensin II. Moreover, 1 microM captopril, an inhibitor of angiotensin-converting enzyme, antagonized the effects of 0.1 and 1 nM angiotensin I, suggesting that the effect of angiotensin I on phosphoinositide breakdown and PRL release is dependent on prior conversion of angiotensin I to angiotensin II. The effect of angiotensin II was very rapid. Fractionation of the water-soluble inositol phosphates showed that angiotensin II significantly increased inositol bisphosphate and inositol triphosphate at 10 sec, whereas inositol monophosphate was increased only after 40 sec. These data indicate that in the pituitary, and presumably in the lactotroph, the binding of angiotensin II to specific membrane receptors provokes increased polyphosphoinositide hydrolysis, leading to increased production of intracellular messengers, i.e. inositol triphosphate and 1,2-diacylglycerol, responsible for the stimulation of PRL release.     

Synergistic interaction in bovine pituitary cultures between growth hormone-releasing factor and other hypophysiotrophic factors

Synthetic human pancreatic GH-releasing factor (1-44)NH2 (GRF) and acetylcholine (ACh) were shown to evoke a dose-related release of GH from cultured bovine pituitary cells with half-maximal effective doses of 0.3 and 500 nmol/l respectively. Concentrations of ACh (10 mumol/l) and GRF (25 nmol/l) which were shown to give near maximal responses when presented alone, produced highly synergistic responses when tested in combination. This synergism was related to the ACh concentration employed, and both the ACh-induced release and ACh-induced synergism were abolished by the muscarinic antagonist, atropine. A synergistic interaction was also demonstrated between GRF and concentrations of thyrotrophin-releasing hormone (TRH) and bombesin which, in the absence of GRF, failed to elicit significant GH release. Acetylcholine stimulated a similar dose-dependent release of prolactin, but GRF was ineffectual in either directly stimulating prolactin release or affecting the response to ACh or TRH. No synergistic interaction could be detected between combinations of ACh and TRH or between ACh and bombesin. The data suggest that, in the somatotroph, GRF acts through a different second messenger pathway to ACh, TRH and bombesin and that these two pathways can be activated to produce a potentiated response. Growth hormone-releasing factor is, therefore, not only a specific GH secretagogue, but may act in concert with other hypophysiotrophic factors to regulate GH secretion from the bovine anterior pituitary.     

Growth hormone induction of insulin-like growth factor I messenger RNA in primary cultures of rat liver cells

Primary liver cells from adult rats were used to study the hormonal regulation of mRNA coding for insulin-like growth factor I (IGF-I). IGF-I mRNA could be detected at a low concentration in liver cells prepared from hypophysectomized rats and cultivated for 3 days. When these cells were exposed to GH on the second day, a dose-dependent increase in IGF-I mRNA was observed. The continuous presence of serum was not a prerequisite for this response, since GH also induced IGF-I mRNA in a defined hormone-supplemented medium. It is concluded that GH can induced IGF-I mRNA in the liver by a direct mechanism.     

Nucleotide regulation of growth hormone-releasing factor binding to rat pituitary receptors

The specific binding of a GRF radioligand, [His1,125I-Tyr10,Nle27]hGRF-1-32NH2, to rat pituitary homogenates is reduced by the addition of GTP and its nonhydrolyzable analogs 5'-guanylylimidodiphosphate (GppNHp) and guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S). GDP and cAMP had no effect while the nonhydrolyzable ATP analogs 5'-adenylylimidodiphosphate and adenosine 5'-O-(3-thiotriphosphate) did elicit a significant reduction in GRF binding. The effect of GppNHp was half-maximal at 0.2 microM, and the maximum inhibition achieved was 85%. The effect of 0.1 microM GppNHp on GRF competitive displacement experiments indicated a significant reduction in affinity for the ligand (Kd = 0.51 +/- 0.11 nM in the absence of GppNHp and 2.1 +/- 1.1 nM in its presence) without an effect on receptor number. The GRF radioligand dissociates slowly from its receptor (t1/2 = 250 +/- 50 min), but the addition of 0.1 microM GppNHp converts approximately half of the receptors present to a more rapidly dissociating form (t1/2 = 9 +/- 10 min). These results are consistent with existing models for receptor-G-protein interactions, and thus, we conclude that transduction of the GRF response across the cell membrane involves a guanine nucleotide-binding protein, presumably Gs.     

Growth hormone-releasing factor stimulates calcium entry in the GH3 pituitary cell line

The GH3 pituitary cell line has been extensively used to study various aspects of the stimulus secretion coupling process. It is known that GH3 cells release PRL and GH in the basal state and in response to various secretagogues. However, this cell line was considered unsuitable as a model for studying the effects of GHRF since the neuropeptide did not affect GH secretion or gene expression. This suggested that the GH3 cells may lack GHRF receptors. The present study investigates the effect of GHRF on free intracellular Ca2+ concentrations in GH3 cells. Cytosolic free calcium concentrations ([Ca2+]i) were monitored in individual cells by microspectrofluorimetry using the fluorescent dye indo 1. When the cells were challenged with a brief application of GHRF (100 nM; 15 sec), 36 out of 59 of these cells responded within a few seconds by a marked increase in [Ca2+]i. GHRF enhanced the frequency of [Ca2+]i oscillations in spontaneously active cells or triggered [Ca2+]i oscillations in inactive cells. The response to GHRF was totally blocked by external Ca2+ free solutions and Ca2+ channel blockers. Combined electrophysiological and fluorescent experiments were carried out in 16 cells. Eleven responded to GHRF. In all cases, the Ca2+ transients triggered by GHRF were associated with action potentials. The Ca2+ responses observed in our experiments clearly show that GH3 cells possess membrane receptors to GHRF. Thus, it is likely that the lack of secretory response observed in GH3 cells does not result from the absence of binding sites to the peptide. It is more likely to be related to alterations of transduction mechanisms resulting in uncoupling between stimulation and secretion.