Gonadotropin-releasing hormone overcomes follicle-stimulating hormone''s inhibition of insulin-like growth factor-5 synthesis and promotion of its degradation in rat granulosa cells

The effect of a gonadotropin-releasing hormone-agonist (GnRH-a) on the synthesis of insulin-like growth factor-binding protein-5 (IGFBP-5), a physiological marker for atresia, was investigated. Granulosa cells obtained from diethylstilbestrol (DES)-treated immature female rats were cultured in serum-free medium for 72 h with GnRH-a and the conditioned media were subjected to immunoblot analysis using rat IGFBP-5 specific antibody. GnRH-a caused a dose-dependent (ED₅₀ = 8.6 × 10⁻¹¹ M) accumulation of IGFBP-5, which migrated as 35 (non-glycosylated) and 36 kDa (glycosylated) bands under reducing conditions. A maximally effective dose of GnRH-a (10⁻⁹ M) caused a 4-fold increase in IGFBP-5 accumulation. In contrast, increasing doses of porcine follicle-stimulating hormone (pFSH) caused a biphasic effect on IGFBP-5 accumulation. A low dose of pFSH (0.25 ng/ml) increased and higher doses of pFSH (22.5 ng/ml) decreased the 35 and 36 kDa IGFBP-5 bands. In the presence of high doses of pFSH (20.75 ng/ml), a 22 kDa band corresponding to a cleaved IGFBP-5 fragment appeared in the media. When the granulosa cells were cultured with a saturating dose of pFSH, co-addition of GnRH-a dose dependently inhibited the FSH effects (ED₅₀ = (2.3–3.7) × 10⁻¹⁰ M). The GnRH-a effects were completely blocked by co-incubation with GnRH-antagonist. IGFBP-5 mRNA accumulation levels were increased by GnRH-a in a dose dependent manner. These results using cultured rat granulosa cells demonstrate that: (1) GnRH-a coordinately stimulates the expression of IGFBP-5 protein and IGFBP-5 mRNA; (2) GnRH-a abolishes the action of FSH to inhibit IGFBP-5 production and to induce IGFBP-5 protease activity; and (3) these GnRH-a effects are blocked by GnRH-antagonist. These results support the hypothesis that GnRH may be involved in the atretic process, by increasing IGFBP-5 production directly by itself and indirectly by altering the effects of FSH.     

Growth hormone-releasing hormone stimulates mitogen-activated protein kinase

GH-releasing hormone (GHRH) can induce proliferation of somatotroph cells. The pathway involving adenylyl cyclase/cAMP/protein kinase A pathway in its target cells seems to be important for this action, or at least it is deregulated in some somatotroph pituitary adenomas. We studied in this work whether GHRH can also stimulate mitogen-activated protein (MAP) kinase. GHRH can activate MAP kinase both in pituitary cells and in a cell line overexpressing the GHRH receptor. Although both protein kinase A and protein kinase C could activate MAP kinase in the CHO cell line studied, neither protein kinase A nor protein kinase C appears to be required for GHRH activation of MAP kinase in this system. However, sequestration of the betagamma-subunits of the G protein coupled to the receptor inhibits MAP kinase activation mediated by GHRH. This pathway also involves p21ras and a phosphatidylinositol 3-kinase, probably phosphatidylinositol 3-kinase-gamma. Despite the involvement of p21ras, the protein kinase Raf-1 is not hyperphosphorylated in response to GHRH, contrary to what usually occurs when the Ras-Raf-MAP kinase pathway is activated. In summary, this work describes for the first time the activation of MAP kinase by GHRH and outlines a path for this activation that is different from the cAMP-dependent mechanism that has been traditionally described as mediating the mitogenic actions of GHRH.     

A new approach to the treatment of acute myeloid leukaemia targeting the receptor for growth hormone‐releasing hormone

Growth hormone‐releasing hormone (GHRH) is secreted by the hypothalamus and acts on the pituitary gland to stimulate the release of growth hormone (GH). GHRH can also be produced by human cancers, in which it functions as an autocrine/paracrine growth factor. We have previously shown that synthetic antagonistic analogues of GHRH are able to successfully suppress the growth of 60 different human cancer cell lines representing over 20 cancers. Nevertheless, the expression of GHRH and its receptors in leukaemias has never been examined. Our study demonstrates the presence of GHRH receptor (GHRH‐R) on 3 of 4 human acute myeloid leukaemia (AML) cell lines—K‐562, THP‐1, and KG‐1a—and significant inhibition of proliferation of these three cell lines in vitro following incubation with the GHRH antagonist MIA‐602. We further show that this inhibition of proliferation is associated with the upregulation of pro‐apoptotic genes and inhibition of Akt signalling in leukaemic cells. Treatment with MIA‐602 of mice bearing xenografts of these human AML cell lines drastically reduced tumour growth. The expression of GHRH‐R was further confirmed in 9 of 9 samples from patients with AML. These findings offer a new therapeutic approach to this malignancy and suggest a possible role of GHRH‐R signalling in the pathology of AML.     

Lactogenic hormone responsive element reporter gene activation assay for human growth hormone.

We investigated the bioactivity of GH and compared with their immunoactivity in GH bioassay system using lactogenic hormone responsive element (LHRE) reporter gene in Chinese hamster ovary cells transiently co-transfected with human GH receptor cDNA and LHRE/TK-luciferase reporter gene (LHRE/Luc). The recombinant and serum GH but not prolactin almost equally were able to induce LHRE/Luc in a significant and dose-dependent manner, which were equally suppressed by anti-GH. Recombinant GH binding protein (GHBP) at 100 ng/ml but not at 20 ng/ml slightly attenuated GH-induced LHRE/Luc. The serum GH bioactivity (ng/ml) in patients with acromegaly were equal near to their immunoactivity, whereas the bioactivity of the serum GH in a short child with mutant GH (R77C) revealed lower than their immunoactivity. The bioactivity of the recombinant mutant GH was as half as that of wild type GH, thus confirming an antagonistic property of mutant GH. LHRE reporter gene activation assay is useful to measure the GH bioactivity in addition to the conventional bioassay using cell proliferation.     

Hormonal control of glucose production and pyruvate kinase activity in isolated rat liver cells: influence of hypothyroidism

Hormonal control of glucose production and of -pyruvate kinase activity has been measured in isolated liver cells from fed control and thyroidectomized rats. In hypothyroid rats, sensitivity to isoproterenol as measured by these parameters was increased: the apparent K_0.5 for isoproterenol-induced stimulation of glucose production decreased from 8.0 ± 3 × 10⁻⁶ M in control rats to 2.0 ± 0.2 × 10⁻⁸ M in hypothyroid rats (P < 0.001) and the apparent K_0.5 for inhibition of -pyruvate kinase was 5 ± 2 X 10⁻⁷ M vs. 7 ± 2 × 10⁻⁹ M (P < 0.001) in control and thyroidectomized rats, respectively. Utilisation of specific adrenergic antagonists confirmed increased β-adrenergic responsiveness in hypothyroid rats. This phenomenon was not reversed by 3 days of T₃ treatment (10 μg/100 g body weight). Sensitivity to the -agonist was unchanged by thyroid status. Stimulation of glucose production and inhibition of -pyruvate kinase activity by glucagon and their reversal by insulin were not affected by hypothyroidism. The dose-response curve to vasopressin and its maximal effect measured on stimulation of glucose production were unchanged in thyroidectomized rats. Thus, hypothyroidism produces a specific enhancement of liver β-adrenergic responsiveness without affecting sensitivity to glucagon, insulin and vasopressin.     

Injury produced by free fatty acids to lysosomes and mitochondria in cultured heart muscle and endothelial cells

Four-day primary coverglass cultures of rat heart muscle (M) and endothelioid (E) cells were treated for 30 min with stearic, oleic or linoleic acids in a FFA/albumin ratio of 6:1 at concentrations from 5 × 10⁻⁶ M to 5 × 10⁻⁴ M. Labilization of lysosomes and mitochondria was measured by acid phosphatase and succinic dehydrogenase staining activity respectively.

Stearate or linoleate, but not oleate, labilized M cell lysosomes at 5 × 10⁻⁶ M. Lysosomes of E cells were not significantly affected by any of the FFA at 5 × 10⁻⁴ M. The order of activity of these FFA for M cell lysosomes was LINOLEATE = stearate > oleate.

Both E and M cell mitochondria were significantly labilized by oleate or linoleate, 5 × 10⁻⁶ M, and by stearate, 5 × 10⁻⁵ M. The order of activity was linoleate > oleate > stearate.

Treatment of cultures for 24 hr with 50 μg/ml of hydrocortisone before the FFA at 5 × 10⁻⁵ M provided significant protection only against stearate-induced lysosomal labilization.     

Growth hormone-releasing hormone as an agonist of the ghrelin receptor GHS-R1a

Ghrelin synergizes with growth hormone-releasing hormone (GHRH) to potentiate growth hormone (GH) response through a mechanism not yet fully characterized. This study was conducted to analyze the role of GHRH as a potential ligand of the ghrelin receptor, GHS-R1a. The results show that hGHRH(1–29)NH₂ (GHRH) induces a dose-dependent calcium mobilization in HEK 293 cells stably transfected with GHS-R1a an effect not observed in wild-type HEK 293 cells. This calcium rise is also observed using the GHRH receptor agonists JI-34 and JI-36. Radioligand binding and cross-linking studies revealed that calcium response to GHRH is mediated by the ghrelin receptor GHS-R1a. GHRH activates the signaling route of inositol phosphate and potentiates the maximal response to ghrelin measured in inositol phosphate turnover. The presence of GHRH increases the binding capacity of ¹²⁵I-ghrelin in a dose dependent-fashion showing a positive binding cooperativity. In addition, confocal microscopy in CHO cells transfected with GHS-R1a tagged with enhanced green fluorescent protein shows that GHRH activates the GHS-R1a endocytosis. Furthermore, the selective GHRH-R antagonists, JV-1–42 and JMR-132, act also as antagonists of the ghrelin receptor GHS-R1a. Our findings suggest that GHRH interacts with ghrelin receptor GHS-R1a, and, in consequence, modifies the ghrelin-associated intracellular signaling pathway. This interaction may represent a form of regulation, which could play a putative role in the physiology of GH regulation and appetite control.     

Role of selected endogenous peptides in growth hormone-releasing hexapeptide activity: analysis of growth hormone-releasing hormone, thyroid hormone-releasing hormone, and gonadotropin-releasing hormone.

The purpose of this study was to evaluate the contribution of endogenous GH-releasing hormone (GHRH) to exogenous GH-releasing hexapeptide (GHRP-6) activity, and to determine whether TRH or GnRH are endogenous analogs of GHRP-6. The activity of GHRP-6, a synthetic GH secretagogue, was significantly attenuated in rats administered GHRH antiserum or alpha-methyl-rho-tyrosine to reduce endogenous GHRH concentrations, and also in rats administered 5-50 micrograms/kg of [N-Ac-Tyr1,D-Arg2]-GRF 1-29 amide to block pituitary GHRH receptors. However, GHRP-6 activity was potentiated in rats administered 150 micrograms/kg [N-Ac-Tyr1,D-Arg2]-GRF 1-29 amide, presumably due to partial agonist activity of the GHRH receptor antagonist at the higher dose. These data show that endogenous GHRH contributes to full expression of exogenous GHRP-6 activity in vivo. Like TRH, a subthreshold dose of GHRP-6 was significantly more effective in hypothyroid rats than in euthyroid rats. However, suprathreshold doses of GHRP-6 were less effective in hypothyroid rats. Unlike TRH, GHRP-6 had no effect on GH and prolactin release from GH3 cells, and TRH and GnRH were poor competitors for 3H-GHRP-6 binding sites on pituitary membranes. A GnRH receptor antagonist did not block GHRP-6 activity in vivo, and GnRH administered alone or in combination with GHRP-6, did not stimulate GH release. The results of this study suggest that synergy between GHRH and GHRP-6 seen in pharmacological studies is physiologically relevant, and that TRH and GnRH are not endogenous analogs of GHRP-6.     

Regulation of GHRH receptor gene expression in the neonatal and adult rat pituitary.

The growth hormone releasing hormone (GHRH) receptor gene is essential for normal growth, and its expression is developmentally regulated. The factors that control GHRH receptor expression in the neonatal pituitary are not well understood. This study focuses on the regulation of GHRH receptor gene expression by thyroid hormone, glucocorticoids, insulin-like growth factor-I (IGF-I) and IGF-II in rat pituitary cell cultures. In newborn pituitaries, both T3 and hydrocortisone (24 h) caused a dose-dependent increase in GHRH receptor mRNA abundance, reaching levels 4.8-fold (P<0.001) and 6.5-fold (P<0.001) over corresponding controls. T3 and hydrocortisone also stimulated GHRH receptor expression in adult (70 day) pituitary cell cultures, consistent with our earlier findings. IGF-I treatment suppressed the inductive effects of T3 (P<0.02) and hydrocortisone (P<0.03) on GHRH receptor expression in adult pituitaries but not in neonatal pituitaries. Unlike IGF-I, IGF-II treatment had no effect on T3-induced or hydrocortisone-induced GHRH receptor expression in either neonates or adults. Taken together, these results indicate that (1) thyroid hormone and hydrocortisone act directly at the neonatal pituitary as potent stimulators of GHRH receptor gene expression, (2) IGF-I, but not IGF-II, acts at the pituitary to suppress GHRH receptor mRNA expression and (3) the effects of IGF-I on GHRH receptor gene expression are developmentally determined.     

Modulation of cultured mouse leydig cells adenylate cyclase by forskolin and hCG

The diterpene, forskolin, stimulated cAMP accumulation about 15-fold over basal levels in purified mouse Leydig cells; however, it remained far less potent than hCG. Simultaneous addition of forskolin and hCG resulted in a striking synergistic stimulation of cAMP production. In contrast, forskolin-enhanced testosterone accumulation was never synergistic with that produced by maximal concentrations of hCG. hCG (3 × 10₋₉ M) lowered about 6-fold the ED₅₀ for forskolin-elicited cAMP accumulation and increased the maximal response to forskolin about 16-fold. Conversely, forskolin (10⁻⁶ M) reduced the ED₅₀ for hCG 2-fold but had a much smaller effect (2–3 fold) on maximal response. Moreover, pretreatment with hCG induced only a homologous desensitization of adenylate cyclase, whereas the enzyme became partially resistant to both hCG and forskolin in cells pretreated with forskolin. The homologous hCG-induced desensitization and the partial heterologous one induced by forskolin suggest that more than the catalytic unit of the cyclase is required for the diterpene activation.     

Transgenic mice expressing the human growth hormone gene provide a model system to study human growth hormone synthesis and secretion in non-tumor-derived pituitary cells: differential effects of dexamethasone and thyroid hormone

Growth hormone (GH) is regulated by pituitary and hypothalamic factors as well as peripheral endocrine factors including glucocorticoids and thyroid hormone. Studies on human GH are limited largely to the assessment of plasma levels in endocrine disorders. Thus, insight into the regulation of synthesis versus secretion has come mainly from studies done on non-human GH and/or pituitary tumor cells. However, primate and non-primate GH gene loci have differences in their structure and, by extension, regulation. We generated transgenic (171hGH/CS-TG) mice containing the intact hGH1 gene and locus control region, including sequences required for integration-independent and preferential pituitary expression. Here, we show hGH co-localizes with mouse (m) GH in somatotrophs in situ and in primary pituitary cells. Dexamethasone treatment increased hGH and mGH, as well as GH releasing hormone (GHRH) receptor RNA levels, and hGH release was stimulated by GHRH treatment. By contrast, triiodothyronine decreased or had no effect on hGH and mGH production, respectively, and the negative effect on hGH was also seen in the presence of dexamethasone. Thus, 171hGH/CS-TG mouse pituitary cultures represent a model system to investigate hormonal control of hGH synthesis and secretion.     

Failure to respond to growth hormone releasing hormone (GHRH) in acromegaly due to a GHRH secreting pancreatic tumor: dynamics of multiple endocrine testing

Growth hormone releasing hormone (GHRH) has recently been isolated and sequenced from pancreatic tumors secreting GHRH. Patients with untreated acromegaly due to a pituitary tumor respond to exogenous administration of GHRH with a further rise of their elevated basal growth hormone (GH) levels. For the first time, we report the effects of exogenously administered synthetic GHRH in a patient with acromegaly due to a GHRH secreting pancreatic tumor. The diagnosis was established by high peripheral IR-GHRH levels (1100 pg/ml) and an arterio- venous tumor gradient of IR-GHRH. In this patient GH failed to respond to 1 microgram/kg of exogenous GHRH with the pancreatic tumor in situ; however, further increase of serum GH levels occurred after TRH administration, hypoglycemia and oral glucose administration. After removal of the tumor, serum GH levels decreased and a normal response to GHRH and TRH were demonstrated. The extract of the tumor contained 1.7 micrograms IR-GHRH per g wet tissue. Thus, lack of response to exogenous GHRH in untreated acromegaly may indicate the presence of an ectopic GHRH producing tumor.     

Antagonism of endogenous growth hormone-releasing hormone (GHRH) leads to reduced proliferation and apoptosis in MDA231 breast cancer cells

GHRH, in addition to stimulating the release of growth hormone (GH) from the pituitary, is a trophic factor for pituitary somatotrophs. Growth hormone-releasing hormone is also expressed in the gonads, gastrointestinal tract, pancreas, thymus, and lymphocytes, as well as in tumors of the pancreas, lung, central nervous system, and breast. Since GHRH has mitogenic effects, we examined the hypothesis that GHRH is an autocrine/paracrine growth factor in neoplastic breast tissue. The effect of disrupting endogenous GHRH on cell growth and apoptosis of MDA231 cells was examined through the use of a competitive GHRH antagonist, [N-acetyl-Tyr1, D-Arg2] fragment 1–29Amide (GHRHa). Cell proliferation was determined by direct cell counting and tritiated thymidine incorporation. Apoptosis was analyzed by examination of DNA laddering and nuclear condensation. GHRHa resulted in a dose-dependent, transient, and reversible decrease in cell number, proliferation rate, and tritiated thymidine uptake. Conversely, GHRHa led to a marked and dose-dependent increase in both DNA laddering and nuclear condensation. These results indicate that disruption of endogenous GHRH action in MDA231 cells results in both decreased cellular proliferation and increased apoptosis. Taken together, the findings suggest that endogenous GHRH acts as an autocrine/paracrine factor in the regulation of growth of at least some breast cancer cell types.     

Growth hormone-releasing hormone and pituitary adenylate cyclase-activating polypeptide in the reproductive system

Growth hormone-releasing hormone (GHRH) and pituitary adenylate cyclase-activating polypeptide (PACAP) are both members of the glucagon superfamily that, with gonadotropins, act at central and peripheral levels as paracrine and autocrine coregulators of reproductive function. GHRH and PACAP are ancient peptides. Their original forms (both 27 amino acids long) were encoded by a single ancestral gene, several duplications of which led to the genes that encode the neuropeptides of the glucagon superfamily. In the male and female reproductive tracts, GHRH and PACAP interact with a subset of G protein-coupled receptors that are structurally similar to the PACAP receptor and variants of the vasoactive intestinal peptide receptor, and share several biological actions. These are related mainly to the modulation of cAMP-dependent and other signal transduction pathways in several cells of the pituitary–gonadal axis. The recent discovery that antagonists of GHRH and PACAP suppress the growth of human cancer cell lines that are derived from reproductive tissues indicates the potential importance of these peptides as local regulators of cell division, cell cycle arrest, differentiation and cell death.     

Regulation of somatotroph differentiation and growth hormone (GH) secretion by corticosterone and GH-releasing hormone during embryonic development

The role of extracellular factors in the regulation of anterior pituitary cell differentiation and GH secretion during embryonic development was investigated. Previously, we reported that somatotrophs become a significant population by embryonic day (e-) 16 of the chick and that corticosterone is the active compound responsible for the observed GH cell-differentiating activity of e-16 serum. More recently, the influence of hormone interactions on somatotroph differentiation and GH secretion during mid- to late embryogenesis was evaluated. Anterior pituitary cells from e-12, -14, and -17 chicks were cultured for 2, 3, and 6 days with corticosterone (10(-9) M) and GH-releasing hormone (GHRH; 10(-10)-10(-7) M) alone and in combination. Medium samples were analyzed for GH concentrations, and recovered cells were subjected to GH reverse hemolytic plaque assay for determination of somatotroph percentages and the relative amount of GH secretion from individual somatotrophs. GHRH significantly (P < 0.05) increased GH secretion from e-17, but not e-12 and e-14, pituitary cells during 2 and 3 days of culture. Corticosterone alone failed to increase GH secretion from e-12, -14, and -17 pituitary cells; however, corticosterone in combination with GHRH increased GH secretion from cells of all three ages. Culture with GHRH decreased percentages of e-17 GH-secreting cells in a concentration-dependent manner (from basal levels of 12.3 +/- 2.4% to 3.2 +/- 0.7% by 2 days), but did not affect percentages of e-12 and e-14 somatotrophs. Conversely, corticosterone increased percentages of e-12 and e-14 GH-secreting cells (by as much as 14- and 3-fold above basal levels, respectively), but did not alter the proportions of e-17 GH cells. Corticosterone in combination with GHRH was more effective than either hormone alone for increasing percentages of e-12 GH-secreting cells (from 9.6 +/- 0.8% with corticosterone to 15.9 +/- 1.5% with corticosterone plus GHRH), but this synergistic effect was not apparent until after 3 days of culture. Exposure to corticosterone in culture for 2, 3, and 6 days increased subsequent GH release from e-12 and e-14 pituitary cells during reverse hemolytic plaque assay. Combined treatment with corticosterone and GHRH further increased subsequent GH release from e-12 and e-14 cells. We conclude that glucocorticoids induce GH cell differentiation and that corticosterone and GHRH can interact at specific stages of embryonic development to regulate somatotroph differentiation and GH secretion.     

Growth hormone regulation in primary fetal and neonatal rat pituitary cell cultures: the role of thyroid hormone

GH is first detectable in the fetal rat pituitary between gestational days 18 and 19. The reasons for the GH surge soon after birth and subsequent postnatal decline to adult levels remain unclear. We therefore determined whether GH gene regulation in the developing pituitary could be distinguished from adult rat somatotroph function. In primary cultures of fetal and neonatal rat pituitary cells, GH secretion was detected by the 20th gestational day. These cells were stimulated by GH-releasing hormone (GHRH), but not by T3 or the morphogen retinoic acid. The stimulatory effect of T3 (0.25 mM) on GH secretion was detected only on the 2nd neonatal day and was similar to that seen in mature rat pituitary cell cultures. GHRH (10 nM) treatment for 24 h caused a 5-fold induction of GH secretion in pituitary cells derived from 2-, 5-, and 12-day-old neonatal rats. The presence or absence of T3 in the culture medium did not alter the response to GHRH. In contrast, only 2-fold induction of GH was observed in adult male pituitary cells during the same time course. Insulin-like growth factor-I (IGF-I; 6.5 nM), the peripheral target hormone for GH, resulted in a modest (20%) attenuation of GH secretion from pituitary cells derived from 20-day-old fetuses. IGF-I, however, produced a 70% reduction in GH levels in adult male pituitary cells grown under similar conditions. The effects of IGF-I on adult pituitary cells grown in T3-depleted medium were blunted. Addition of T3 partially restored the responsiveness of these cells to IGF-I. The results suggest that the high circulating GH levels in the fetal and neonatal rat may be secondary to relative insensitivity of the immature somatotroph to the inhibitory actions of IGF-I in addition to enhanced responsiveness to GHRH compared with the adult rat pituitary. Relative thyroid hormone deficiency in the immature rat may be contributory to this early transient state of pituitary IGF-I resistance.     

Effect of continuous somatostatin and growth hormone-releasing hormone (GHRH) infusions on the subsequent growth hormone (GH) response to GHRH. Evidence for somatotroph desensitization independent of GH pool depletion

Continuous infusions of growth hormone-releasing hormone (GHRH) attenuate the subsequent growth hormone (GH) response to GHRH. To test whether this phenomenon can occur in the absence of GH pool depletion, we examined the effects of continuous infusions of 10 nM GHRH and of 10 nM somatostatin (SRIH), separately or in combination, on dispersed, perifused rat anterior pituitary cells. Columns of these cells were given either GHRH alone for 5 h, GHRH and SRIH together for 3 h followed by GHRH alone, or SRIH alone for 3 h followed by GHRH or medium. SRIH blunted both basal GH release and the GH response to GHRH, without affecting the subsequent GH responses to GHRH. The GHRH infusions attenuated the subsequent GH response to GHRH, even when GH release was initially prevented by the concurrent infusion of SRIH. Furthermore, the degree of attenuation was similar in the presence or absence of SRIH, suggesting that pool depletion plays little role in the desensitization process under these experimental conditions. The results are consistent with the hypothesis that a short-term infusion of GHRH leads to attenuation of the GH response in rat anterior pituitary cells primarily through receptor effects rather than through GH pool depletion.