Interaction of ethanol with signal transduction mechanisms mediating growth hormone release by rat pituitary cells in vitro.

The effects of ethanol on signal transduction mechanisms of rat GH (rGH) release were investigated in primary culture of rat anterior pituitary cells. Ethanol (30, 100, and 300 mM) had no significant effect on basal rGH release or cell content after a 4-h incubation or on intracellular cAMP levels at 30 min. Ethanol did not alter rGRH (10(-11) M)-stimulated rGH release, but at concentrations of 100 and 300 mM it inhibited rGRH (10(-9) M)-stimulated rGH release by 12% (P less than 0.05) and 54% (P less than 0.01). In contrast, a dose-dependent stimulatory effect was observed on rGRH-induced cAMP accumulation. Ethanol enhanced the inhibitory effect of SRIH (10(-11) and 10(-9) M) on rGH release by up to 24% (P less than 0.01). Stimulation of rGH release by cAMP derivatives and forskolin was partially inhibited by ethanol, as was cAMP accumulation after forskolin treatment. Cholera toxin-stimulated rGH release was also inhibited by ethanol, whereas cAMP accumulation was increased. At the higher concentrations, ethanol enhanced rGH release after protein kinase-C activation by phorbol ester and after stimulation of calcium influx with Ca ionophore. No significant ethanol effect was noted on prostaglandin E2-stimulated rGH release, and ethanol did not alter rGH mRNA levels or proliferation of a pituitary somatomammotroph cell line. The results indicate that ethanol exerts multiple effects on systems mediating GH release from the pituitary in vitro. However, the inhibitory influence of ethanol on GH secretion is related primarily to the adenylate cyclase-cAMP pathway, which represents the major signal transducing system in the somatotroph.     

Roles and mechanisms of action of pituitary adenylate cyclase-activating polypeptide (PACAP) in growth hormone and prolactin secretion.

It appears that PACAP has a direct action on somatotrophs to release GH. The intracellular signal transduction mechanisms for PACAP might be similar to but partly distinct from those for GRH. PACAP might play a role in GH secretion induced by serotoninergic mechanisms but not in ultradian rhythm of GH secretion in the rat. PACAP can stimulate PRL release from the pituitary in rats possibly through indirect paracrine effect. In addition, PACAP might participate in regulation of PRL secretion via hypothalamic VIP.     

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.     

Effects of a novel hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide, on pituitary hormone release in rats.

We have demonstrated that the novel hypothalamic peptide pituitary adenylate cyclase-activating polypeptide (PACAP-38; 0.1-100 nmol/l) caused an increase in the release of GH, ACTH, LH and alpha-subunit and accumulation of intracellular cyclic AMP from dispersed rat anterior pituitary cells in static culture for 24 h. There were no significant effects on TSH or prolactin release over the same time-period. PACAP-38 (10 nmol/l) increased the release of GH by 1.3-fold (P less than 0.05), ACTH by 1.9-fold (P less than 0.05), LH by 3.5-fold (P less than 0.001) and alpha-subunit by 2.0-fold (P less than 0.005) and the accumulation of intracellular cyclic AMP by greater than 2-fold (P less than 0.001) after 24 h. However, the time-course for the effect of PACAP-38 (1 mmol/l) on hormone release and intracellular cyclic AMP levels showed a temporal dissociation. The effect of PACAP-38 on GH and ACTH levels did not reach significance until 24 h whereas the effect of PACAP-38 on LH and alpha-subunit release reached significance after 4 h implying a different mechanism of action for their release. To investigate the PACAP-induced secretion of LH and alpha-subunit further, we examined the effects of PACAP after down-regulation of protein kinase C (PKC). PACAP-38 at a dose maximal for the stimulation of LH and alpha-subunit release (10 nmol/l) added together with the PKC activator, 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 0.1 mumol/l) had no greater effect on LH and alpha-subunit release than TPA alone over a 4 h incubation period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pituitary adenylate cyclase-activating polypeptide, interleukin-6 and glucocorticoids regulate the release of vascular endothelial growth factor in pituitary folliculostellate cells

There is increasing evidence that hormones play an important role in the control of endothelial cell function and growth by regulating the production of vascular endothelial growth factor (VEGF). VEGF regulates vascular permeability and represents the most powerful growth factor for endothelial cells. In the normal anterior pituitary, VEGF has been detected only in folliculostellate (FS) cells. In the present study, the regulation of the release of VEGF from FS-like mouse TtT/GF cells, and from FS cells of rat pituitary monolayer cell cultures was investigated using a specific VEGF ELISA. Basal release of VEGF was demonstrated in cultures of both TtT/GF cells and rat pituitary cells. Interestingly, the VEGF secretion was stimulated by both forms of pituitary adenylate cyclase-activating polypeptide (PACAP-38 and PACAP-27), indicating that this hypothalamic peptide regulates endothelial cell function and growth within the pituitary. VEGF secretion was also stimulated by interleukin-6 (IL-6) whereas basal, IL-6- and PACAP-stimulated secretion was inhibited by the synthetic glucocorticoid dexamethasone. The inhibitory action of dexamethasone was reversed by the glucocorticoid receptor antagonist RU486, suggesting that in FS cells functional glucocorticoid receptors mediate the inhibitory action of glucocorticoids on the VEGF secretion. The endocrine and auto-/paracrine control of VEGF production in pituitary FS cells by PACAP, IL-6 and glucocorticoids may play an important role both in angiogenesis and vascular permeability regulation within the pituitary under physiological and pathophysiological conditions.     

Interactions of ovarian steroids with pituitary adenylate cyclase-activating polypeptide and GnRH in anterior pituitary cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) releases LH and FSH from anterior pituitary cells. Although this effect is relatively weak, it has a strong sensitizing action on GnRH-induced gonadotropin secretion. Here we investigated the possibility that ovarian steroids, which are well-known modulators of LH secretion, interact with PACAP and GnRH in pituitary gonadotrophs. Rat pituitary cells were treated for 48 h with vehicle, 1 nmol/l estradiol, 1 nmol/l estradiol + 100 nmol/l progesterone or 48 h with 1 nmol/l estradiol and 4 h with 100 nmol/l progesterone. The cells were stimulated for 3 h with 1 nmol/l GnRH or 100 nmol/l PACAP. Estradiol treatment alone enhanced basal as well as GnRH- or PACAP-stimulated LH secretion. LH release was facilitated by additional short-term progesterone treatment. Long-term treatment with estradiol and progesterone led to reduced LH responses to GnRH and PACAP. Neither treatment paradigms affected cAMP production. However, estradiol treatment led to enhanced cAMP accumulation in quiescent or GnRH-stimulated cells. PACAP-induced increases of cAMP production were inhibited by estradiol treatment. After 7-h preincubation with 10 nmol/l PACAP, cells responded with enhanced LH secretion to GnRH stimulation. When steroid pretreatment was performed the responsiveness of gonadotrophs to low concentrations of GnRH was still increased. In contrast, at high concentrations of GnRH the sensitizing action of PACAP on agonist-induced LH secretion was lost in steroid-treated cells. There were no significant differences between the steroid treatment paradigms. It is concluded that estradiol but not progesterone acts as a modulator of adenylyl cyclase in gonadotrophs. The stimulatory effect of estradiol is thought to be involved in its sensitizing action on agonist-induced LH secretion. The inhibitory effect of estradiol on PACAP-stimulated adenylyl cyclase activities seems to be responsible for the loss of its action to sensitize LH secretory responses to GnRH.     

Neuropeptides of the pituitary adenylate cyclase-activating polypeptide/vasoactive intestinal polypeptide/growth hormone-releasing hormone/secretin family in testis

Mammalian testicular development and the maintenance of spermatogenesis are hormone-dependent processes that are controlled by the pituitary gonadotropins and testosterone. Recent studies have demonstrated the presence of many neuropeptides and their receptors in the testis, suggesting that these peptides operate as local regulators of testicular germ cell development and function. Among these testicular neuropeptides, the peptides that belong to the pituitary adenylate cyclase-activating polypeptide (PACAP) family, particularly growth hormone-releasing hormone and secretin, appear to show some unique common features in terms of intratesticular localization and the time of expression during the spermatogenic cycle. However, their precise physiologic roles and mechanisms of action remain unknown. This review analyzes the available information on the functional interactions among the testicular cells that appear to be mediated by locally produced neuropeptides, with a special emphasis on the peptides of the PACAP family.     

Pituitary adenylate cyclase-activating polypeptide stimulates calcium mobilization in amphibian pituitary cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38-amino acid peptide of the glucagon-secretin-vasoactive intestinal polypeptide superfamily. Although PACAP is a potent stimulator of adenylate cyclase activity in the adenohypophysis, the precise target cells for PACAP in the anterior pituitary remain unknown. The aim of the present study was to investigate whether PACAP could stimulate calcium mobilization in individual cells of the pituitary and to determine the type of cells that responded to PACAP. Enzymatically dispersed frog distal pituitary cells were plated on photoetched coverslips and cultured for 3-7 days. The cells were loaded with the fluorescent calcium indicator indo-1, and changes in intracellular calcium concentrations ([Ca2+]i) were monitored using dual wavelength microfluorimetry. The individual cells were localized with the aid of the alpha/numeric grid of the coverslips and identified retrospectively by immunofluorescence. Approximately 45% of GH and PRL cells and 25% of ACTH and TSH cells responded to PACAP (10(-5) M) ejection by an elevation of [Ca2+]i. Only 16% of gonadotropes were stimulated by PACAP. The time course of [Ca2+]i variations showed three different patterns: transient spikes, sustained stimulations, and oscillatory responses. In addition, heterogenous responses were observed within each cell type. These data provide evidence for the involvement of calcium mobilization in the mechanism of action of PACAP on pituitary cells. The results also indicate that in frogs, PACAP may stimulate the secretory activity of GH and PRL cells and, to a lesser extent, ACTH, TSH, and gonadotrope cells.     

Feeding and metabolism in mice lacking pituitary adenylate cyclase-activating polypeptide

Disruption of the pituitary adenylate cyclase-activating polypeptide (PACAP) gene in mice has demonstrated a role for this highly conserved neuropeptide in the regulation of metabolism and temperature control. Localization of PACAP neurons within hypothalamic nuclei that regulate appetite suggest PACAP may affect feeding and thus energy balance. We used PACAP-null mice to address this question, examining both food intake and energy expenditure. PACAP-null mice were leaner than wild-type littermates due to decreased adiposity and displayed increased insulin sensitivity. The lean phenotype in the PACAP-null mice was completely eliminated if animals were fed a high-fat diet or housed near thermoneutrality (28 C). Further metabolic analyses of PACAP-null mice housed at 21 C indicated that the reduced body weight could not be explained by decreased food intake, increased metabolic rate, or increased locomotor activity. The thyroid hormone axis of PACAP-null mice was affected, because mRNA levels of hypothalamic TRH and brown adipose tissue type 2 deiodinase were reduced in PACAP-null mice housed at room temperature, and brain deiodinase activity was lower in PACAP-null mice after an acute cold challenge compared with wild-type controls. These results demonstrate that PACAP is not required for the regulation of food intake yet is necessary to maintain normal energy homeostasis, likely playing a role in central cold-sensing mechanisms.     

Prolonged stimulation with thyrotropin-releasing hormone and pituitary adenylate cyclase-activating polypeptide desensitize their receptor functions in prolactin-producing GH3 cells

We used somatolactotroph GH3 cells to examine changes in response to stimulation with thyrotropin-releasing hormone (TRH) and pituitary adenylate cyclase-activating polypeptide (PACAP) after sustained treatment with these peptides. TRH and PACAP increased prolactin promoter activity in mock- and PACAP type 1 receptor (PAC1R)-transfected cells. When the cells were pretreated with TRH for 48 h, the response of the prolactin promoter to both TRH and PACAP was diminished. Similarly, in PAC1R-transfected GH3 cells pretreated with PACAP, the effects of TRH and PACAP on the prolactin promoter were eliminated. The stimulation of prolactin mRNA expression by TRH and PACAP was eliminated by prolonged pretreatment with these peptides in PAC1R-transfected cells. Both the serum response element (SRE) promoters and cAMP response element (CRE) promoters were activated by TRH and PACAP in either mock- or PAC1R-transfected cells. Pretreatment for 48 h with TRH also eliminated the effects of TRH and PACAP on the SRE and CRE promoters, and pretreatment of PAC1R-transfected cells with PACAP for 48 h reduced the responses of the SRE and CRE promoters to TRH and PACAP. These observations demonstrated that sustained stimulation with TRH and PACAP desensitizes their own and each other’s receptors.     

Molecular evolution of the growth hormone-releasing hormone/pituitary adenylate cyclase-activating polypeptide gene family. Functional implication in the regulation of growth hormone secretion

Growth hormone-releasing hormone (GHRH) and pituitary adenylate cyclase-activating polypeptide (PACAP) belong to the same superfamily of regulatory neuropeptides and have both been characterized on the basis of their hypophysiotropic activities. This review describes the molecular evolution of the GHRH/PACAP gene family from urochordates to mammals and presents the hypothesis that the respective roles of GHRH and PACAP in the control of GH secretion are totally inverted in phylogenetically distant groups of vertebrates. In mammals, GHRH and PACAP originate from distinct precursors whereas, in all submammalian taxa investigated so far, including birds, amphibians and fish, a single precursor encompasses a GHRH-like peptide and PACAP. In mammals, GHRH-containing neurons are confined to the infundibular and dorsomedial nuclei of the hypothalamus while PACAP-producing neurons are widely distributed in hypothalamic and extrahypothalamic areas. In fish, both GHRH- and PACAP-immunoreactive neurons are restricted to the diencephalon and directly innervate the adenohypophysis. In mammals and birds, GHRH plays a predominant role in the control of GH secretion. In amphibians, both GHRH and PACAP are potent stimulators of GH release. In fish, PACAP strongly activates GH release whereas GHRH has little or no effect on GH secretion. The GHRH/PACAP family of peptides thus provides a unique model in which to investigate the structural and functional facets of evolution.     

Ghrelin-induced growth hormone release from goldfish pituitary cells involves voltage-sensitive calcium channels

Ghrelin (GRL) is a stimulator of growth hormone (GH) release in many organisms, including goldfish. As a first study to examine the signalling mechanisms mediating GRL action on GH release in goldfish, we tested the hypothesis that GLR induces GH release from goldfish pituitary cells by enhancing Ca²⁺ entry through L-type voltage-sensitive Ca²⁺ channels (LVSCCs) using perifusion GH release and fura-2/AM Ca²⁺-imaging experiments. Goldfish (g)GRL₁₉ at 1 nM elicited reversible and repeatable GH responses from dispersed goldfish mixed pituitary cultures. However, the lack of a dose-response relationship in sequential treatments with decreasing concentrations of gGRL₁₉ (ranging from 10 to 0.01 nM) implicated rapid desensitization of the GH response. Sequential applications of gGRL₁₉ (1 nM) and salmon GnRH (100 nM), a known Ca²⁺-dependent stimulator of GH release, increased intracellular free Ca²⁺ levels ([Ca²⁺]_i) from the same identified somatotropes, suggesting co-expression of GRL and GnRH receptors on single cells. In contrast, 1 nM gGRL₁₉ failed to elicit GH release and elevation in [Ca²⁺]_i when the cells are incubated with nominally Ca²⁺-free media. When GH release and [Ca²⁺]_i increases were already stimulated by the LVSCC agonist Bay K8644 (10 μM), addition of 1 nM gGRL₁₉ did not further elevate these responses. Finally, the LVSCC inhibitors nifedipine (1 μM) and verapamil (1 μM) abolished 1 nM gGRL₁₉-induced GH release responses while nifedipine eliminated gGRL₁₉-induced [Ca²⁺]_i increase. Taken together, the results of this study provide evidence that entry of extracellular Ca²⁺ through LVSCCs is a key component of the GRL signalling pathway leading to GH release in the goldfish pituitary.     

Anti-inflammatory role in septic shock of pituitary adenylate cyclase-activating polypeptide receptor

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are two mediators synthesized by immune cells, specially under inflammatory and antigen stimulation conditions. Reports have shown that neuropeptides attenuate the deleterious consequences of septic shock both by down-regulating the production of proinflammatory mediators and by stimulating the production of anti-inflammatory cytokines by activated macrophages. In this study, we used a knockout for the PACAP receptor (PAC1(-/-)) to demonstrate an important protective role for PAC1 receptor in endotoxic shock. Moreover, our results indicate that PAC1 receptor acts in vivo as an anti-inflammatory receptor, at least in part, by attenuating lipopolysaccharide (LPS)-induced production of proinflammatory IL-6, which appears to be the main cytokine regulating the expression of the majority of the acute phase protein genes, which are an important deleterious component of septic shock. Besides, our findings point to endogenously produced VIP and PACAP as participants of the natural anti-inflammatory machinery. Because VIP and PACAP are two attractive candidates for the development of therapies against acute and chronic inflammatory diseases, septic shock, and autoimmune diseases, this paper represents a contribution to the understanding of the mechanism of action of these anti-inflammatory agents.     

垂体腺苷酸环化酶激活多肽对胰腺癌细胞的生长调控

目的 观察垂体腺苷酸环化酶激活多肽（pituitary adenylate cyclase activating polypeptide,PACAP)对人胰腺癌细胞株的生长调控作用;并确定神经鞘磷脂是否作为第二信使参与受体后信息传导。方法 人胰腺癌细胞株JF305,HS766T,ASPC－1进行细胞培养,传代。分别加入不同浓度的PACAP1－38（10^-6-10^-12mol/L)于三种癌细胞中。应用MTT法观察细胞增程度。薄层层析法测定细胞神经鞘磷脂。放射免疫法测定细胞内cAMP含量。Fura-2/AM测定细胞内游离Ca^2 浓度。结果 PACAP1－38促进JF305,HS766T,ASPC－1细胞的生长;PACAP1－38增加细胞内神经鞘磷脂、cAMP、Ca^2 的生长;生长抑素可明显抑制PACAP1－38诱导的JF305细胞的生长等作用。结论 PACAP1－38促进人胰腺癌细胞株的增殖。PACAP受体后信息传递途径;（1）腺苷 酸环化酶途径;（2）钙－钙调素途径。神经鞘磷脂作为第二信使也参与此过程。     

Gonadal steroid modulation of signal transduction and luteinizing hormone release in cultured chicken pituitary cells

The mechanism whereby gonadal steroids modulate GnRH-stimulated LH secretion by primary cultures of chicken pituitary cells was investigated. Estradiol (10(-8) M), testosterone (10(-7) M), and progesterone (10(-7) M) inhibited LH release stimulated by GnRH (10(-7) M) by 56%, 61%, and 53%, respectively, and the inhibitory effects required prolonged preincubation (24-48 h) with the steroids. The steroids inhibited the spike (0-3 min) and plateau (9-30 min) phases of LH release to a similar degree. The ED50 values of estradiol, testosterone, and progesterone for inhibition of GnRH-stimulated LH release were 7 x 10(-11), 2 x 10(-9), and 1 x 10(-9) M, respectively. Estradiol, testosterone, and progesterone inhibited the maximal LH response to GnRH, but the ED50 of GnRH (4 x 10(-9) M) was not altered by steroid pretreatment. Steroid pretreatment did not cause a change in cellular LH content, suggesting that the steroids do not inhibit LH synthesis. Combinations of two or three of the steroids were not additive, suggesting that all three steroids affect GnRH-stimulated LH release via the same mechanism. In experiments investigating their mechanism of action, the steroids inhibited LH release stimulated by GnRH and Ca2+ ionophore A23187, but generally had no effect on the responses to phorbol ester (12-O-tetradecanoylphorbol-13-acetate), forskolin, K+, Bay K8644, or veratridine. Estradiol inhibited GnRH-stimulated 45Ca2+ efflux, but its inhibitory effect on GnRH-induced inositol phosphate production was not significant. Estradiol had no effect on binding of 125I-[His5,D-Tyr6]GnRH to a pituitary cell preparation. These findings suggest that the site of steroid modulation of GnRH action is distal to binding of GnRH to its receptor, and that the inhibitory effects are exerted at two intracellular sites: 1) the coupling events linking receptor activation to mobilization of Ca2+, and 2) a site distal to Ca2+ mobilization.     

Stage-dependent regulation of ovarian pituitary adenylate cyclase-activating polypeptide mRNA levels by GnRH in cultured rat granulosa cells

The present study was designed to test whether GnRH regulates pituitary adenylate cyclase-activating polypeptide mRNA levels in a stage-dependent manner during follicle development in the rat ovary. The granulosa cells of preovulatory and immature follicles obtained from PMSG- and estrogen-treated rats, respectively, were cultured in serum-free conditions in the presence of various hormones. GnRH receptor mRNA expression was detected in both preovulatory and immature granulosa cells and was down-regulated by gonadotropins. Treatment of preovulatory granulosa cells with GnRH agonist stimulated pituitary adenylate cyclase-activating polypeptide mRNA levels in a dose-dependent manner. In situ hybridization analysis of cultured preovulatory follicles revealed that GnRH-induced pituitary adenylate cyclase- activating polypeptide signals were detected in granulosa cells, but not thecal cells. In immature granulosa cells, cotreatment with GnRH agonist suppressed FSH-stimulated pituitary adenylate cyclase-activating polypeptide mRNA levels in a dose-dependent manner, whereas treatment with GnRH alone had no effect. Furthermore, treatment with GnRH antagonist inhibited LH-induced pituitary adenylate cyclase-activating polypeptide gene expression in preovulatory granulosa cells, whereas it stimulated FSH-induced pituitary adenylate cyclase-activating polypeptide gene expression in immature granulosa cells. Interestingly, GnRH-stimulated pituitary adenylate cyclase-activating polypeptide mRNA levels in preovulatory granulosa cells was inhibited by arachidonyltri fluoromethyl ketone, an inhibitor of phospholipase A(2), but not by an inhibitor of protein kinase A or C. Lastly, treatment of preovulatory follicles with pituitary adenylate cyclase-activating polypeptide antagonist suppressed GnRH-stimulated progesterone production during 6--9 h of culture. Taken together, these results demonstrate the stage-dependent regulation of pituitary adenylate cyclase-activating polypeptide mRNA levels by GnRH, the stimulatory and inhibitory effect in granulosa cells of preovulatory and immature follicles, respectively.     

Pituitary adenylate cyclase-activating polypeptide regulates [Ca]i and electrical activity in pituitary cells through cell type-specific mechanisms

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a recently identified hypothalamic factor that acts on a variety of anterior pituitary cell types. It is clear, however, that its actions are not mediated by the same intracellular signaling mechanisms in each cell type. The signaling pathways by which PACAP regulates changes in [Ca²⁺], and electrical activity in rat somatotrophs and gonadotrophs is described in the present article. Finally, the possibility that the differences in PACAP-regulated signaling in anterior pituitary cells is due to the differential expression and coupling of PACAP receptor subtypes is discussed.     

Regulation of growth hormone release: evidence against negative feedback in rat pituitary cells

To determine if the anterior pituitary gland is the site of negative feedback inhibition of GH release, we studied the effect of GH and multiplication-stimulating activity (MSA), a member of the somatomedin family, on isolated rat anterior pituitary cells in primary culture. The effect of GH was examined in two ways: 1) by adding to the cultures human GH (10 ng/ml to 20 microgram/ml) which was biologically active in the rat but not cross-reactive in the rat GH (rGH) RIA, and 2) by comparing rGH secretion in cultures of different cell densities. No suppression of either basal or prostaglandin E1-stimulated rGH release was found. An enhancement observed in serum-free conditions at high human GH concentrations was interpreted as a nonspecific response to protein, because bovine serum albumin produced the same effect. When added in the presence of serum, MSA (1--500 ng/ml) had no effect on rGH secretion. In the absence of serum, there were 71% and 30% increases in the basal and prostaglandin E1-stimulated rates of hormone release, respectively, possibly attributable to a trophic effect of MSA. Six other hormones having structural or functional similarity to either GH or somatomedin also failed to inhibit rGH secretion. Our results do not support the hypothesis that GH or somatomedin exerts a negative feedback effect on GH release directly on the anterior pituitary gland. Most likely, the hypothalamus or a higher brain center is the site for such regulation.