Ingestive responses to administration of stress hormones in baboons.

Experimental stress and the administration of the stress hormone ACTH have been reported to stimulate sodium appetite in many nonprimate species. Experiments were conducted to determine whether prolonged intracerebroventricular infusions of the neuropeptides corticotropin-releasing factor (CRF) and urocortin (Ucn), or systemic administration of ACTH, affected ingestive behaviors in a nonhuman primate, the baboon. Intracerebroventricular infusions of CRF or Ucn significantly decreased daily food intake. The decrease with Ucn continued into the postinfusion period. These infusions did not alter daily water intake. Daily voluntary intake of 300 mM NaCl solution was not increased, and there was evidence of reductions on days 2-4 of the infusions. Intramuscular injections of porcine ACTH or synthetic ACTH (Synacthen) for 5 days did not affect daily NaCl intake, although the doses were sufficient to increase cortisol secretion and arterial blood pressure. Sodium depletion by 3 days of furosemide injections did induce a characteristic sodium appetite in the same baboons. These results demonstrate the anorexigenic action of CRF and Ucn in this primate. Also, CRF, Ucn, and ACTH did not stimulate sodium appetite at the doses used.

Mice that lack corticotropin-releasing factor (CRF) receptors type 1 show a blunted ACTH response to acute alcohol despite up-regulated constitutive hypothalamic CRF gene expression.

BACKGROUND: The purpose of this work was to determine the influence of acute alcohol treatment, injected intraperitoneally, on the hypothalamic-pituitary-adrenal axis of mice that lack type 1 receptor for corticotropin-releasing factor (CRFR1). METHODS: CRFR1-deficient (CRFR1-/-), heterozygous (CRFR1+/-), and wild-type (CRFR1+/+) mice were generated and maintained under standard conditions. Homozygous, heterozygous, and wild-type offspring were identified by polymerase chain reaction analysis of tail DNA. Experiments were performed on 9- to 16-week-old male and female mice. All blood samples were obtained by rapid decapitation of conscious mice conducted between 10 AM-12 PM. Blood sample collection was completed within 20 to 30 sec of disturbing the animals, and all samples were terminal. Preliminary experiments were conducted to determine the time-course of the ACTH and hypothalamic responses to alcohol in all three groups of mice, and a single time point (30 min and 2 hr, respectively), corresponding to peak responses, was chosen to measure the corresponding parameters in all subsequent studies. RESULTS: In vehicle-injected animals, basal ACTH and corticosterone levels were statistically comparable in heterozygotes and mice with a null allele for the CRFR1 gene, although values of this latter hormone were slightly lower in the mutants. Alcohol (4.0 g/kg) elicited the expected significant (p < 0.01) increase in plasma ACTH and corticosterone levels in heterozygous mice. These responses were virtually abolished or markedly decreased, respectively, in CRFR1-deficient animals. As previously reported, constitutive CRF mRNA levels were elevated in the paraventricular nucleus (PVN) of the hypothalamus in mice that lacked CRFR1, compared to wild-type control mice. Interestingly, this was not the case for transcripts of the immediate early gene NGFI-B. When measured 2 hr after alcohol, PVN NGFI-B gene expression was significantly (p < 0.01) increased in both control and mutant mice, as were CRF mRNA levels in mutant mice, but the hypothalamic responses of the mutants were larger (p < 0.01) than those of the control mice. This difference may be due, at least in part, to the lack of steroid feedback in the mutants. CONCLUSION: These results indicate that although the intraperitoneal injection of alcohol remains capable of eliciting PVN CRF neuronal activation in mice that lack CRFR1, the ACTH and corticosterone responses are significantly blunted, a phenomenon believed to be due to the lack of CRFR1 in the pituitary of these animals.

Fos induction in selective hypothalamic neuroendocrine and medullary nuclei by intravenous injection of urocortin and corticotropin-releasing factor in rats.

CRF and urocortin, administrated systemically, exert peripheral biological actions which may be mediated by brain pathways. We identified brain neuronal activation induced by intravenous (i.v.) injection of CRF and urocortin in conscious rats by monitoring Fos expression 60 min later. Both peptides (850 pmol/kg, i.v.) increased the number of Fos immunoreactive cells in the paraventricular nucleus of the hypothalamus, supraoptic nucleus, central amygdala, nucleus tractus solitarius and area postrema compared with vehicle injection. Urocortin induced a 4-fold increase in the number of Fos-positive cells in the supraoptic nucleus and a 3.4-fold increase in the lateral magnocellular part of the paraventricular nucleus compared with CRF. Urocortin also elicited Fos expression in the accessory hypothalamic neurosecretory nuclei, ependyma lining the ventricles and choroid plexus which was not observed after CRF. The intensity and pattern of the Fos response were dose-related (85, 255 and 850 pmol/kg, i.v.) and urocortin was more potent than CRF. Neither CRF nor urocortin induced Fos expression in the lateral septal nucleus, Edinger-Westphal nucleus, dorsal raphe nucleus, locus coeruleus, or hypoglossal nucleus. These results show that urocortin, and less potently CRF, injected into the circulation at picomolar doses activate selective brain nuclei involved in the modulation of autonomic/endocrine function; in addition, urocortin induces a distinct activation of hypothalamic neuroendocrine neurons.

Carbon monoxide modulates secretion of corticotropin-releasing factor from rat hypothalamic cell cultures.

The present study examined the actions of the putative gaseous neurotransmitter carbon monoxide (CO) on secretion of corticotropin-releasing factor (CRF) from cultured primary rat hypothalamic cells. 6 h treatment of cells with 100% gaseous CO, or with the heme analog, hematin (100 microM) which produces CO as a by-product of its metabolism, increased basal secretion of CRF to 207 +/- 8% and 200 +/- 65% of control respectively. Zinc protoporphyrin IX (ZnPPIX) (0.3-100 microM), a selective inhibitor of CO formation, decreased CRF secretion in a dose-dependent manner. The changes in CRF secretion observed with hematin were attenuated during concurrent treatment with ZnPPIX. These studies suggest that basal secretion of CRF in the rat hypothalamus may be regulated by CO.

Protooncogene junB as a target for activin actions.

Activin, a member of the transforming growth factor-beta family of peptides, is implicated in the regulation of cell growth and differentiation in a variety of biological systems. We have sought to identify immediate early genes whose altered expression may provide a common nuclear event involved in activin-regulated phenotypic changes in many cell types. In both human K562 myelogenous leukemia and rat PC12 pheochromocytoma cells, activin treatment caused transient transcription-dependent and protein synthesis-independent increases of junB messenger RNA (mRNA) within 1 h, whereas neither c-jun nor c-fos mRNA were inducible. In K562 cells, this selective junB mRNA induction was synergistically augmented by treatment with 12-O-tetradecanoyl phorbol-13-acetate but not affected by forskolin. Furthermore, in PC12 cells, the up-regulation of junB mRNA by activin was observable even after high-dose treatment with 12-O-tetradecanoyl phorbol-13-acetate for 48 h, indicating that junB mRNA expression by activin is independent of both A- and C-kinases. Our report suggests that induction of this ubiquitous gene product may be a critical event shared by a set of activin-responsive tissues.

Secretion of melanin-concentrating hormone and neuropeptide-EI from cultured rat hypothalamic cells.

Rat melanin-concentrating hormone (MCH) is a homolog of the peptide first isolated from salmon pituitary glands which produces melanosome aggregation within melanophores of teleost fish as well as interacting with the hypothalamic-pituitary axis. We have previously characterized the rat and human MCH counterparts as identical 19-amino acid peptides showing approximately 70% peptide sequence identity to salmon MCH. Immunoreactivity for MCH has been found in high concentrations within cell bodies of the dorsolateral hypothalamus, with projections to the hippocampus, brainstem, posterior pituitary, and cerebral cortex. We have adapted a cultured cell model for studying MCH secretory responses of hypothalamic cells obtained from 7-day-old rats. MCH and the MCH precursor-derived peptide neuropeptide glutamic acid isoleucine (NEI) were secreted from these cells after 2 days of culture and for up to 22-24 days of culture. The secreted peptides were confirmed by HPLC analysis and RIA to be identical to MCH isolated from rat hypothalamic tissue and to the sequences of MCH and NEI predicted from the MCH precursor. MCH and NEI secretion was stimulated 3- to 5-fold by 8-bromo-cAMP and 8-bromo-cGMP. Dexamethasone produced a dose-dependent increase in cell content of both MCH and NEI and an increase in MCH secretion. The present study is the first to demonstrate the existence of the predicted peptide NEI in a biological system and indicates that cultured neonatal rat hypothalamic cells are a useful model for the study of MCH/NEI release in vitro.

Isolation and characterization of hypothalamic growth-hormone releasing factor from common carp, Cyprinus carpio.

A growth hormone-releasing factor (GRF)-like peptide was isolated from the hypothalamus of common carp, Cyprinus carpio, by acid extraction, gel filtration chromatography, immunoaffinity chromatography using antiserum directed against rat GRF, and multiple steps of HPLC using octadecyl columns. Based on Edman degradation and peptide mapping, this teleost GRF was established to be a 45-residue peptide with the following primary structure: His-Ala-Asp-Gly-Met-Phe-Asn-Lys-Ala-Tyr-Arg-Lys-Ala-Leu-Gly-Gln-Leu-Ser- Ala-Arg - Lys-Tyr-Leu-His-Thr-Leu-Met-Ala-Lys-Arg-Val-Gly-Gly-Gly-Ser-Met-Ile-Glu- Asp-Asp-Asn-Glu-Pro-Leu-Ser. Carp GRF is closely related structurally to peptides of the glucagon-secretin superfamily, and more particularly to mammalian vasoactive intestinal peptide (VIP) precursors and the N-terminal portion of mammalian GRFs. A synthetic replicate of this peptide is highly potent [50% effective dose (ED50) approximately 0.08 nM] in stimulating GH release from cultured goldfish pituitary glands and in elevating serum GH levels 30 min after injection (0.1 micrograms/g) in goldfish.

Rat melanin-concentrating hormone messenger ribonucleic acid expression: marked changes during development and after stress and glucocorticoid stimuli.

Melanin-concentrating hormone (MCH) is a cyclic neuropeptide first isolated from fish and rats. MCH may be involved in the control of the hypothalamic-pituitary-adrenocortical axis and, more generally, of specific goal-oriented behaviors and homeostatic functions in mammals. In this paper we examine 1) the cellular distribution of MCH gene transcripts in the rat central nervous system, 2) the changes in neuronal expression of MCH mRNA during rat development, and 3) the effects of stress and hormonal stimuli on rat MCH (rMCH) gene activity. Northern blot analysis and in situ hybridization histochemistry show that mature rMCH mRNA (1.0 kilobase) is very abundant in the zona incerta and the dorsolateral hypothalamus. While this is in agreement with previous peptide mapping by immunohistochemical techniques, a surprising new result is that a few clusters of rMCH mRNA-containing cells are found outside the hypothalamus, in the olfactory tubercle and the pontine tegmentum. Developmentally, rMCH mRNA is detected on embryonic day 18; its level increases gradually during early postnatal life and rises abruptly at weaning to reach a constant value in adult rats. In addition, striking variations in rMCH mRNA length occur during postnatal development and are found to be variations in the polyadenylate tail. Interestingly, this structural modification appears to be independent of the increase in rMCH mRNA levels. The regulation of rMCH mRNA expression by glucocorticoids and chronic stress is examined by Northern blot analysis. Chronic intermittent footshock stress causes a 58% or 29% decrease in rMCH mRNA content in the whole hypothalamus after a 1- or 3-day regimen, respectively. In contrast, the rMCH mRNA level returns to normal after a 7-day regimen. Two weeks after adrenalectomy (ADX) the whole hypothalamus rMCH mRNA content decreases 2.5-fold, but rises close to the control value 3 weeks after ADX. Dexamethasone administration 2 weeks after ADX not only reverses the fall in rMCH mRNA, it even provokes a slight increase (123% of control). No change in rMCH mRNA length is observed after chronic stress or ADX and dexamethasone injection. These results provide evidence for a negative regulation of rMCH gene expression by stress and suggest a major role for glucocorticoids in a positive feedback control of rMCH gene activity.

The melanin-concentrating hormone system of the rat brain: an immuno- and hybridization histochemical characterization.

In addition to a nonadecapeptide homologous to the teleost melanin-concentrating hormone (MCH), the amino acid sequence predicted from a rat prepro-MCH (ppMCH) cDNA suggested that at least one (neuropeptide EI, or NEI), and possibly a second (NGE), additional neuropeptide may be encoded by this precursor. Cross-reactivity with epitopes of NEI or NGE can account for reported localization of alpha-MSH, rat CRF, and human GRF in rat dorsolateral hypothalamic neurons. We have used antisera raised against rat MCH and NEI in immunohistochemical studies at the light and electron microscopic levels, along with hybridization histochemical localization of ppMCH mRNA, to define the organization of this system. As expected, ppMCH mRNA is prominently expressed in cells in the lateral hypothalamic area and zona incerta. The MCH and NEI peptides were extensively colocalized in neurons in both of these areas. In addition, smaller cell groups in the olfactory tubercle and pontine tegmentum were also positively hybridized for ppMCH mRNA and immunostained for MCH and NEI. Fibers stained for MCH and NEI were similarly, and very broadly, distributed throughout the central nervous system in patterns that generally conformed with known projection fields of the lateral hypothalamic area and zona incerta. A differential distribution was seen in at least one region, the interanterodorsal nucleus of the thalamus, which contained a prominent terminal field stained for MCH but not NEI. At the electron microscopic level, MCH-stained perikarya displayed a prominent staining associated with the Golgi apparatus; this was not encountered in NEI-stained cells. Both peptides were distributed similarly in terminals in the lateral hypothalamic area and median eminence, with staining associated principally with dense-cored vesicles. The results suggest that ppMCH-derived peptides may serve as neurotransmitters or modulators of prominence in a surprisingly expansive projection field of incerto-hypothalamic neurons. The terminal distributions of this system seem most compatible with functional roles in generalized arousal and sensorimotor integration, processes previously implicated as being subject to modulation by the lateral hypothalamic area.

Central activin administration modulates corticotropin-releasing hormone and adrenocorticotropin secretion.

A broad and diffuse neuronal network conveys information reflecting the state of the internal and external environment to the neurosecretory hypothalamus. Recently, we identified an inhibin-beta A- (I beta A) immunoreactive terminal field within the CRF-rich portion of the dorsomedial paraventricular nucleus which originates from a cell group in the commissural portion of the nucleus of the solitary tract (NTS). The NTS receives baroreceptor input, somatosensory input via the spinosolitary tract, and sensory information from the oral, thoracic, and abdominal cavities and, thus, is positioned to serve as a primary relay for visceral sensory inputs to neurons critical to the function of the hypothalamic-pituitary-adrenal (HPA) axis. Although these NTS cells contain multiple putative transmitters, we present evidence that activin, an inhibin-beta A dimer, plays a modulatory role in HPA axis function via facilitation of CRF release. First, intraventricular injection of activin-A (0-3 nmol), but not the related inhibin heterodimer, evoked dose-related 1.7- to 2.8-fold elevations of circulating ACTH levels in male rats. Second, analysis of hypophysial-portal plasma after bilateral paraventricular nucleus microinfusion of activin-A revealed a dose-related facilitation of CRF secretion up to 4-fold above preinjection levels which was unaccompanied by changes in arginine vasopressin levels. Finally, activin-A also enhanced CRF secretion from neonatal hypothalamic cells in primary culture with an EC50 dose of approximately 0.25 nM. Overall, these observations provide evidence of both an anatomical and a pharmacological substrate for activin-mediated central modulation of HPA axis function.

Differential regulation of corticotropin-releasing factor mRNA in rat brain regions by glucocorticoids and stress.

The regulation of corticotropin-releasing factor (CRF) mRNA expression in the rat brain by glucocorticoids and stress was examined by Northern blot analysis and in situ hybridization histochemistry. Rats either were exposed to a single electrical footshock session and killed 2, 4, 12, or 24 hr later (acute stress), or were subjected to the same regimen twice daily for 3 or 7 d and killed on the day following the last session (chronic stress). Rats placed in the experimental chamber but not administered shock comprised a "sham-handling" group. Chronic (7 d) intermittent footshock stress resulted in an 84 +/- 26% (P less than 0.05) increase in CRF mRNA levels in the whole hypothalamus as detected by Northern blot analysis and a 97 +/- 29% (P less than 0.05) increase in the paraventricular nucleus (PVN) as detected using in situ hybridization. No significant change in CRF mRNA levels was observed in the hypothalamus at any time up to 24 hr after a single exposure to footshock stress. A different pattern of results was obtained in other CRF-expressing cell groups. In Barrington's nucleus (a pontine micturition center), both acute and chronic stress produced significant increases in CRF mRNA, while in the olfactory bulb, both paradigms resulted in decreased levels. By Northern blot analysis, CRF mRNA in the olfactory bulb declined steadily, beginning at 4 hr after acute stress, and reached significance at 24 hr (69.2 +/- 1.9% of control, P less than 0.05). Levels from chronically (7 d) stressed animals declined to 54.1 +/- 5.1% of control value (P less than 0.05). Analysis of hybridization histochemical material revealed that both the number of positively hybridized cells and the number of silver grains per cell in the mitral and external plexiform layers of the bulb decreased following acute and chronic stress. However, CRF mRNA levels in the olfactory bulb were decreased to a comparable extent in the sham-handling group, suggesting that exposure to a novel environment can effect a decrease in CRF mRNA levels in the olfactory bulb. To provide comparisons with the effects of manipulation of glucocorticoid status, comparable analyses were carried out in separate groups of animals following adrenalectomy (ADX) with and without corticosteroid replacement. After ADX, CRF mRNA levels in the whole hypothalamus increased 60 +/- 5% (P less than 0.05) and were normalized following dexamethasone replacement. In contrast to the hypothalamus, no effects of steroid manipulation on CRF mRNA levels in the olfactory bulb, midbrain, cerebral cortex, or brain stem were detected.(ABSTRACT TRUNCATED AT 400 WORDS)

Structure of the human melanin concentrating hormone mRNA.

The melanin-concentrating hormone (MCH) is a cyclic neuropeptide which induces skin paling and may be involved in the control of the pituitary adrenal axis in teleost fishes. We have recently cloned and characterized the salmon and rat MCH mRNAs and we report in the present paper the cloning and sequencing of the human counterpart. The deduced human MCH (hMCH) precursor is 165 amino acids long and as for rat and salmon, encodes the MCH peptide at the C-terminus. The human and rat MCH precursors are very similar to one another but differ extensively from the salmon counterpart. Strong sequence conservation was found in the regions of mammalian prohormones encoding the novel putative neuropeptides named NGE and NEI which we had originally identified in the rat MCH precursor. Furthermore, sequence identities, with perhaps functional implications, were found among the MCH, human ANF, and aplysia peptide A hormone precursors.

Characterization of the glucose-dependent release of growth hormone-releasing factor and somatostatin from superfused rat hypothalami.

The glucose-dependent secretion of the neuropeptides, growth hormone-releasing factor (GRF) and somatostatin (SRIF), by hypothalamic fragments was studied in vitro using a superfusion system. After equilibration of mediobasal hypothalami in HEPES-buffered Krebs-Ringer solution containing 5.5 mM glucose, glucose levels in the superfusion medium were altered. Lowering the glucose concentration in the medium from 5.5 to 2.7 or 1.1 mM provoked a rapid increase in GRF and SRIF release in a concentration and Ca2+-dependent manner. At 1.1 mM glucose, neuropeptide secretion was elevated 3- to 4-fold. The increase of GRF and SRIF release induced by low glucose was transient since stimulated neuropeptide secretion declined to basal levels in the continued presence of low glucose. Furthermore, after reequilibration in 5.5 mM glucose, no second stimulation of neuropeptide release could be induced by reduced glucose. Intracellular glucopenia induced by addition of 2-deoxy-D-glucose (16.5 mM) to the superfusion medium containing 5.5 mM glucose, also evoked increases in GRF and SRIF release. The sensitivity of GRF and SRIF neurons to glucose was absent in the postnatal period until day 9 after birth and then gradually increased. The parallel increases of GRF and SRIF release in response to low glucose observed in the present in vitro study, together with the suppression of plasma GH levels occurring in hypoglycemia in the rat, suggest that, in this condition, the inhibition of GH release induced by elevated SRIF levels predominates whereas the increase of GRF release might serve to attenuate this effect of SRIF.

In the mouse, the activation of the hypothalamic-pituitary-adrenal axis by a lipopolysaccharide (endotoxin) is mediated through interleukin-1.

The mechanisms through which endotoxin stimulates the hypothalamic-pituitary-adrenal axis are not well understood. In the studies reported here we tested the hypothesis that endotoxin increases plasma ACTH levels by releasing interleukin-1 (Il-1). Two experimental tools reported to interfere with the biological activity of IL-1 were used: antibodies directed against IL-1 receptors and alpha MSH. In a first series of experiments, adult male mice were injected with a lipopolysaccharide (LPS; 25 micrograms), antibodies against IL-1 receptor, alpha MSH (1-30 micrograms), or LPS and either IL-1 antibodies or alpha MSH. All treatments were administered ip. The endotoxin LPS caused a marked increase in plasma ACTH levels, measured 6 h later. Both alpha MSH and the Il-1 receptor antibodies, while having no effect by themselves, significantly (P less than or equal to 0.01) blocked LPS-induced ACTH release. In a second series of experiments, mice were injected ip with 500 ng recombinant human (rh) Il-1 alpha or 100 ng rhIl-1 beta in the presence or absence of alpha MSH (1-30 micrograms, ip). While not altering ACTH secretion induced by rhIl-1 alpha, 10-30 micrograms alpha MSH significantly (P less than or equal to 0.01) interfered with the effect of rhIl-1 beta. These results suggest 1) that endotoxin activates the hypothalamic-pituitary-adrenal axis through a mechanism involving the activation of interleukin-1 receptors; and 2) that the effect of rhIl-1 beta, but not -alpha, on ACTH secretion by the mouse can be partially blocked by alpha MSH.

Characterization of melanin-concentrating hormone from rat hypothalamus.

A melanin-concentrating hormone (MCH)-like peptide was isolated from rat hypothalamus by acid extraction, gel filtration chromatography, immunoaffinity chromatography using antiserum directed against salmon MCH, and two steps of HPLC using octadecyl columns. Several zones of immunoreactivity were isolated, and Edman degradation in a gas phase sequencer indicated that the amino acid sequence of all zones was identical. Rat hypothalamic MCH is a nonadecapeptide which differs from salmon MCH by an N-terminal extension of two amino acids and four additional substitutions. Rat MCH has the following primary structure: Asp-Phe-Asp-Met-Leu-Arg-Cys-Met-Leu-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Gln- Val.

Secretion of corticotropin-releasing factor from cultured rat hypothalamic cells: effects of catecholamines.

An understanding of the regulation of CRF secretion in rats is currently incomplete, in part due to the lack of sensitive in vitro models available for studying this neuropeptide. In particular, the effects of catecholamines on CRF secretion, and the receptor subtypes mediating these actions have long been the subject of much debate. A cultured cell model has been adapted for studying secretory responses of hypothalamic cells of 1-week-old rats. Between 7-16 days in monolayer culture the cells secreted detectable levels of immunoreactive CRF, and this release was paralleled by the appearance of punctate bead-like regions of immunoreactivity along fine cellular processes. CRF secretion was increased up to 4-fold by norepinephrine (EC50, approximately 0.5 microM). The increase in CRF secretion produced by norepinephrine was blocked by the beta-receptor antagonist propranolol, but not by the alpha-antagonist prazosin. Moreover, the beta-receptor agonist isoproterenol significantly elevated CRF secretion, whereas the alpha-agonist phenylephrine was without effect, except at high concentrations. Addition of phenylephrine, however, potentiated the effect of isoproterenol, but this response was still significantly less than that produced by norepinephrine. Forskolin (EC50, approximately 0.7 microM) and the active phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (EC50, approximately 40 nM) also increased CRF secretion by 3- to 4-fold. Inactive phorbol derivatives had no effect on CRF release from these cultures. The results indicate that cultured neonatal rat hypothalamic cells are a powerful model for the study of CRF release in vitro, and that norepinephrine acts directly at the isolated cell level to stimulate secretion of this peptide, primarily by activating beta-adrenoceptors. The results also suggest that at least two functional second messenger systems (adenylate cyclase and protein kinase-C) are involved in CRF secretion and are already functional in the neonatal hypothalamus.

Oxytocin mediates the hypothalamic action of vasoactive intestinal peptide to stimulate prolactin secretion.

The ability of centrally administered vasoactive intestinal peptide (VIP) to stimulate PRL secretion when injected intracerebroventricularly could be due to leakage to the pituitary, where it is known to exert direct PRL-releasing activity, or to a hypothalamic action on its own release or that of another possible PRL-releasing factor. When 3 micrograms VIP were injected into the third ventricle of conscious ovariectomized rats, a significant (P less than 0.005) and transient elevation of plasma oxytocin (OT) levels was observed. When OVX rats were injected iv with 1 ml anti-OT serum 30 min before the central administration of 3 micrograms VIP, the PRL surge seen after VIP injection in normal rabbit serum-treated controls was completely absent. The PRL surge seen after central VIP administration was not significantly altered by iv saline infusion (1 ml over 30 min) or by infusion of a VIP antagonist [D-4-Cl-Phe6,Leu17]VIP at a dose of 0.5 microgram/kg.min in 1 ml saline for 30 min before the VIP injection. This was not due to the inability of the VIP antagonist to block the PRL-releasing factor activity of VIP, since it significantly antagonized that action both in vitro and in vivo in the suckling stimulation paradigm. However, the PRL surge was completely absent in ovariectomized rats pretreated by iv infusion of an OT antagonist, [deamino Cys1,D-Trp2,Val4,Orn8]OT, at a similar dose. This recruitment of OT by VIP indicates that it may act at more than one locus within the hypothalamo-pituitary axis to insure the coordinated control of PRL secretion.

Growth hormone-releasing factor secretion from fetal hypothalamic cell cultures is modulated by forskolin, phorbol esters, and muscimol.

The regulation of GRF secretion was studied using a fetal rat hypothalamic cell culture system. The cells were subjected to short term release experiments on days 10-18 after plating, and GRF secretion was assessed by RIA. The identity of GRF immunoreactivity in the incubation medium was confirmed by reverse phase liquid chromatographic analysis. Depolarization of the cells with 56 mM K+ evoked a 4-fold increase in basal GRF release. When cultures were pretreated for 6 days with the adenylate cyclase activator forskolin, basal GRF release was augmented in subsequent release experiments to levels 2-fold greater than those in the control cultures. In nonpretreated cultures, forskolin (1-100 microM) and the protein kinase C activator phorbol 12-myristate 13-acetate (10 nM-1 microM), stimulated basal GRF release in a dose-dependent fashion. The Ca2+ channel blocker verapamil (100 microM) significantly inhibited the GRF response to both forskolin and phorbol 12-myristate 13-acetate. The gamma-aminobutyric acid (GABA) agonist muscimol (0.1-10 microM) inhibited forskolin-stimulated, but not K+ stimulated, GRF release in a dose-dependent manner. This inhibition was reversed by the GABA antagonists bicuculline and picrotoxinin. Muscimol (10 microM) slightly suppressed basal GRF release. The present findings suggest that GRF secretion can be evoked by agents known to increase intracellular cAMP levels or activate protein kinase-C. They also support a role for GABA in the inhibitory control of GRF secretion.

Regulation of corticotropin-releasing factor secretion in vitro by glucose.

The neurosecretory responses of the isolated rat hypothalamus were assessed in vitro. Rat hypothalamic blocks were incubated for 30 min in a N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered salt solution with 5.5 mM glucose (base-line collection period). The blocks were transferred to fresh buffer with a new concentration of glucose with or without various additions (test period); corticotropin-releasing factor (CRF) and other hormones in the media were determined by radioimmunoassay. CRF secretion was maximally increased to approximately 200% of base line at glucose concentrations less than 4 mM and decreased to 65% of base line at higher glucose concentrations. The increase in CRF secretion at low glucose (0.55 or 1.38 mM) was Ca2+ dependent and completely reversible. Hexamethonium, cyproheptadine, and atropine partially blocked the CRF response to 0.55 mM glucose. Glucose concentrations from 0 to 11 mM had no effect on the CRF response to 47.5 mM KCl. The inhibitory effects of high glucose were completely reversed by the addition of 2-deoxy-D-glucose (3-49 mM). Glucose levels did not alter secretion of either gonadotropin-releasing hormone or arginine vasopressin from hypothalamic blocks. The results suggest that the isolated rat hypothalamus is extremely sensitive to the level of glucose and that CRF is rapidly and reversibly secreted in response to slight reductions in glucose concentrations. These concentrations are consistent with those observed during moderate to severe hypoglycemia in vivo. The rise in glucocorticoids observed in vivo during hypoglycemia may result at least in part from the ability of the hypothalamus to directly sense glucose levels and promote secretion of CRF.

Inhibin production by primary Sertoli cell-enriched cultures: regulation by follicle-stimulating hormone, androgens, and epidermal growth factor.

Inhibin levels in the serum-free media of primary Sertoli cell-enriched (SCE) cultures were studied as a function of time and hormonal treatment. SCE cultures were established from 20-day-old rats and maintained in SF media supplemented with insulin, transferrin, epidermal growth factor (EGF), and bacitracin. Radioimmunoassayable inhibin was measured using an antibody directed against a synthetic porcine inhibin-alpha (pI alpha) and measured using this same synthetic peptide as well as a highly purified ovine inhibin standard. Results of these determinations are expressed in terms of a synthetic peptide as femtomoles of pI alpha-(1-26)-G-Y-per ml/10(6) cells. Inhibin levels (+/- SEM) that accumulated at this rate in media from control cultures were 184.9 +/- 6.1 and 167.4 +/- 5.2 on days 0-4 and 4-8, respectively. When FSH (oFSH 17; 1-1000 ng/ml) was added, a dose-dependent increase in inhibin levels was significant at all time points beyond the initial 24 h. The simultaneous addition of 2 x 10(-7) M testosterone (T) with low doses of FSH addition of 2 x 10(-7) m testosterone (T) with low doses of FSH suppressed the inhibin response to FSH, but when T was combined with 300 ng/ml FSH, there was no effect of T on inhibin levels compared to FSH alone, regardless of time in culture. In spite of the modest effect of T, androstenedione (A; 2 x 10(-13) to x 10(-5) M) produced a dose-dependent suppression of inhibin levels. This inhibition was also observed at all doses of FSH. The action of A was not due to its conversion to estrogens, as 17 beta-estradiol had no effect on inhibin production by SCE cultures in either the presence or absence of FSH. The effect of EGF, a component of the basal serum-free medium, was next examined; it produced a 1.5-fold higher level of inhibin (188.7 +/- 9.5) compared to cultures without EGF (135.6 +/- 5.0). When SCE cultures were plated with FSH plus EGF, the stimulation of inhibin levels was additive. We conclude that in Sertoli cell cultures established from immature rats (1) the accumulation of inhibin in medium declines from 90 fmol/10(6) cells.day (initial 24 h) to 40-50 fmol/10(6) cells.day (over the first 48 h) and continues to accumulate in the medium for 8 days of culture; (2) FSH regulates the production of inhibin by Sertoli cells; the best dose response is observed over a 3-day period.(ABSTRACT TRUNCATED AT 400 WORDS)