Regulation of Pituitary Corticotropin Releasing Hormone (CRH) Receptor mRNA and CRH Binding During Adrenalectomy: Role of Glucocorticoids and Hypothalamic Factors

The role of glucocorticoids and hypothalamic factors on CRH receptor expression in the pituitary were studied by analysis of the effects of adrenalectomy and suppression of CRH and VP secretion by hypothalamic lesions in the rat. Consistent with previous in situ hybridization studies, Northern blots showed that pituitary CRH receptor mRNA decreased only transiently after adrenalectomy, falling to 51% of the control levels after 18 h, and returning to control values after 6 days (112%). The early decrease was prevented by dexamethasone injection, 100 &mgr;g, s.c. The role of increased levels of CRH and VP in the pituitary portal circulation on the transient decrease in CRH receptor mRNA levels after adrenalectomy were studied by in situ hybridization in rats subjected to PVN lesions or median eminence deafferentation by hypothalamic anterolateral cuts (ALC). PVN lesion (12 days) or ALC (8 days) resulted in undetectable irCRH and VP in the external zone of the median eminence and had no effect on basal levels of pituitary POMC mRNA, CRH binding and CRH receptor mRNA. In sham lesioned rats, adrenalectomy for 18 h or 4 days caused the expected increases in pituitary POMC hnRNA and mRNA, and decreases in CRH binding. CRH-R mRNA levels decreased by about 50% after 18 h adrenalectomy but returned to basal by 4 days. PVN lesion or ALC fully prevented the fall in CRH binding after 18 h or 4 days adrenalectomy and the increase in POMC mRNA after 4 days adrenalectomy, whereas only attenuated the decrease in CRH receptor mRNA and increase in POMC mRNA levels after 18 h adrenalectomy. Administration of a CRH antagonist did not affect CRH receptor mRNA and POMC hnRNA and mRNA indicating that residual CRH in the median eminence after hypothalamic surgery is not responsible for the effect of adrenalectomy. These studies confirm previous in situ hybridization studies showing that adrenalectomy causes transient decreases in pituitary CRH receptor mRNA levels. The data indicate that while increases in hypothalamic CRH secretion following glucocorticoid withdrawal mediate pituitary CRH receptor binding loss and the increase in POMC expression after long-term adrenalectomy, CRH only partially accounts for the early changes in CRH receptor mRNA and POMC mRNA.     

Neural Regulation of Corticotropin Releasing Hormone (CRH) and CRH Receptor mRNA in the Hypothalamic Paraventricular Nucleus in the Rat

The role of afferent innervation to the hypothalamic paraventricular nucleus (PVN) on CRH mRNA and CRH receptor mRNA levels was studied in control and stressed rats. Groups of rats were subjected to unilateral transection of the stria terminalis (ST), the medial forebrain bundle at the rostral hypothalamic level (RMFB), or the lower brainstem through the medulla oblongata between the obex and the locus coeruleus (CBs). Twelve days after surgery, each group of rats was further divided into controls (basal conditions) and stressed (1 h immobilization), before collecting brains for mRNA analysis by in situ hybridization histochemistry. While ST and RMFB cuts had no effect on basal CRH mRNA levels in the PVN, CBs cut decreased CRH mRNA in the PVN ipsilaterally to the knife cut but it was without effect on the contralateral side (– 40% and –37%vs contralateral and sham-operated, respectively, P&0.01). Acute stress (rats were killed 3 h after immobilization) increased CRH mRNA levels by about 30% bilaterally, an effect which was unchanged by any of the three hemisections. Under basal conditions, CRH receptor mRNA levels in the PVN were indistinguishable from the surrounding areas in sham-operated controls, ST and RMFB operated rats. However, brainstem hemisection resulted in clear expression of CRH receptor mRNA in areas consistent with the dorsal, medial-ventral and lateral parvicellular subdivisions of the PVN, ipsilateral to the transection. CRH neurons in these subdivisions project to the lower brainstem and the spinal cord. Expression of CRH receptor mRNA in the medial-dorsal and anterior parvicellular divisions (CRH neurons with median eminence projections) was not affected by CBs cut. In these subdivisions, immobilization stress markedly increased CRH receptor mRNA levels but it did not influence CBs cut-induced CRH receptor expression. ST and RMFB hemisections were without effect on PVN CRH receptor mRNA levels under basal or stress conditions. Oxytocin (OT) and vasopressin (VP) mRNA levels in the magnocellular subdivision of the PVN were unchanged after immobilization, or following ST, RMFB or CBs cuts, whereas OT mRNA in the medial-ventral and caudal parvicellular subdivisions was decreased by 52% after CBs cut. The data demonstrate that: 1) basal CRH mRNA levels in the PVN are under tonic stimulatory influence of the lower brainstem (and/or spinal cord) afferents; 2) CRH receptor mRNA expression in PVN subdivisions (pituitary vs lower brainstem/spinal cord projecting neurons) is under different control mechanisms, and 3) immobilization-induced changes in CRH mRNA and CRH receptor mRNA levels are mediated either by neural inputs from brain areas other than those investigated here, or by humoral factors.     

Stress-Specific Regulation of Corticotropin Releasing Hormone Receptor Expression in the Paraventricular and Supraoptic Nuclei of the Hypothalamus in the Rat

Corticotropin releasing hormone (CRH), a major regulator of pituitary ACTH secretion, also acts as a neurotransmitter in the brain. To determine whether CRH is involved in the regulation of hypothalamic function during stress, CRH receptor binding and CRH receptor mRNA levels were studied in the hypothalamus of rats subjected to different stress paradigms: immobilization, a physical-psychological model; water deprivation and 2% saline intake, osmotic models; and i.p. hypertonic saline injection, a combined physical-psychological and osmotic model. In agreement with the distribution of CRH receptor binding in the brain, in situ hybridization studies using ³⁵S-labeled cRNA probes revealed low levels of CRH receptor mRNA in the anterior hypothalamic area, which were unaffected after acute or chronic exposure to any of the stress paradigms used. Under basal conditions, there was no CRH binding or CRH receptor mRNA in the supraoptic (SON) or paraventricular (PVN) nuclei. However, 2 h after the initiation of acute immobilization, CRH receptor mRNA hybridization became evident in the parvicellular division of the PVN, with levels substantially increasing from 2 to 4 h, decreasing at 8 h and disappearing by 24 h. Identical hybridization patterns of CRH receptor mRNA were found in the parvicellular PVN after repeated immobilization; levels were similar to those after 2 h single stress following immobilization at 8-hourly intervals for 24 h (3 times), and very low, but clearly detectable 24 h after 8 or 14 days daily immobilization for 2 h. On the other hand, water deprivation for 24 or 60 h and intake of 2% NaCI for 12 days induced expression of CRH receptor mRNA in the SON and magnocellular PVN, but not in the parvicellular pars of the PVN. Both parvicellular and magnocellular hypothalamic areas showed CRH receptor mRNA following i.p. hypertonic saline injection, single (4 h after) or repeated at 8-hourly intervals for 24 h (3 injections), or one injection daily for 8 or 14 days. Consistent with the expression of CRH receptor mRNA, autoradiographic studies showed binding of ¹²⁵I-Tyr-oCRH in the parvicellular division of the PVN after immobilization; in the magnocellular division of the PVN after osmotic stimulation, and in the PVN and SON after i.p. hypertonic saline injection. The data show that stress-specific activation of the parvicellular and magnocellular systems is associated with CRH receptor expression, and suggest a role for CRH in the autoregulation of hypothalamic function.     

Dexamethasone Increases Growth Hormone (GH)-Releasing Hormone (GRH) Receptor mRNA Levels in Cultured Rat Anterior Pituitary Cells

To examine the effects of glucocorticoid (GC) on growth hormone (GH)-releasing hormone (GRH) receptor gene expression, a highly-sensitive and quantitative reverse-transcribed polymerase chain reaction (RT-PCR) method was used in this study. Rat anterior pituitary cells were isolated and cultured for 4 days. The cultured cells were treated with dexamethasone for 2, 6, and 24  h. GRH receptor mRNA levels were determined by competitive RT-PCR using a recombinant RNA as the competitor. Dexamethasone significantly increased GRH receptor mRNA levels at 5  nM after 6- and 24  h-incubations, and the maximal effect was found at 25  nM. The GC receptor-specific antagonist, RU 38486 completely eliminated the dexamethasone-induced enhancement of GRH receptor mRNA levels. Dexamethasone did not alter the mRNA levels of β -actin and prolactin at 5  nM for 24  h, whereas GH mRNA levels were significantly increased by the same treatment. The GH response to GRH was significantly enhanced by the 24-h incubation with 5  nM dexamethasone. These findings suggest that GC stimulates GRH receptor gene expression through the ligand-activated GC receptors in the rat somatotrophs. The direct effects of GC on the GRH receptor gene could explain the enhancement of GRH-induced GH secretion.     

PACAP Stimulation of Gonadotropin-II Secretion in Goldfish Pituitary Cells: Mechanisms of Action and Interaction with Gonadotropin Releasing Hormone Signalling

Pituitary adenylate cyclase-activating polypeptide (PACAP) has recently been shown to be a hypophysiotropic factor in the goldfish. In this study, we examined the mechanisms of PACAP action on goldfish maturational gonadotropin (GTH-II) release using primary cultures of pituitary cells. The GTH-II response to mammalian PACAP1-38 (mPACAP) was inhibited by a PACAP receptor antagonist suggesting a receptor-mediated action. Addition of either an adenylate cyclase inhibitor or a protein kinase A (PKA) inhibitor reduced the mPACAP-induced GTH-II release. In addition, when GTH-II release was already stimulated by either forskolin or 8-bromo-cAMP (8Br-cAMP), mPACAP did not further increase GTH-II secretion. These results strongly implicated the involvement of an adenylate cyclase/cAMP/PKA pathway in PACAP-stimulated GTH-II release. Although mPACAP induced a rise in intracellular Ca2+ level in identified gonadotropes, results with voltage-sensitive Ca2+ channel inhibitors indicated that the GTH-II responses to mPACAP, forskolin and 8Br-cAMP did not depend upon Ca2+ entry through these channels. Two protein kinase C (PKC) inhibitors did not affect mPACAP-elicited GTH-II release, and mPACAP further increased GTH-II secretion in the presence of PKC activators. These results indicate that PKC-dependent elements are not essential for the stimulatory action of mPACAP in gonadotropes. Interestingly, while GTH-II responses to a stimulatory concentration of mPACAP were additive to responses elicited by maximal effective concentrations of two endogenous gonadotropin releasing hormones (GnRHs), a subthreshold concentration of mPACAP potentiated GnRH and PKC activator stimulation of GTH-II secretion. Similarly, submaximal concentrations of forskolin potentiated the GTH-II response to the PKC activator, tetradecanoyl phorbol acetate. These data suggest that PACAP and its cAMP-dependent signalling mechanisms provide an alternate stimulatory input to goldfish gonadotropes and may influence the effectiveness of the major neuroendocrine control exerted by the PKC-dependent GnRH signalling pathway.     

Effects in vitro of New Growth Hormone Releasing Peptide (GHRP-1) on Growth Hormone Secretion from Ovine Pituitary Cells in Primary Culture

Continuous perifusion of pituitary cells was used to study the effects of a newly synthesized GHRP (GHRP-1 or KP 101) on growth hormone (GH) secretion from ovine pituitary cells and these have been compared to effects of growth hormone-releasing factor (GRF) and the original growth hormone-releasing peptide (GHRP-6). GH was continuously released at a constant rate during perifusion and secretion was increased by KP 101, GHRP-6 and GRF in a dose-dependent manner. The half-maximal effective dose of KP 101 and GHRP-6 was 10^?7 M, an order of magnitude higher than that for GRF. The maximal effects of KP 101 and GHRP-6 were similar but significantly less than the maximal effect of GRF. Blockade of calcium channels with Cd²⁺ (2 mM) totally and reversibly abolished the releasing effects of all three peptides. Like GHRP-6, the GH release induced by KP 101 was not affected by a GRF antagonist ([Ac-Tyr¹, D-Arg²]-GRF 1–29, 1 μM) which significantly reduced the effect of GRF on GH release. For each peptide, the response to a second application (1 h after the first application) was lower than the first response. When GRF (or KP 101, GHRP-6) was applied first and then KP 101 or GHRP-6 (or GRF) given 1 h later, the second response was not attenuated. Only a small additive effect on the release of GH by GRF was obtained by the co-administration of either KP 101 or GHRP-6. This result was achieved with maximal doses of the peptides, but not with half-maximal doses. These data indicate that KP 101 has similar potency and GH releasing properties to GHRP-6 but both are less potent than GRF. There is no synergistic effect of the peptides with GRF and only a small additive effect of KP 101 or GHRP-6 on GRF-stimulated GH release from ovine pituitary cells in vitro. KP 101 stimulates GH release by a mechanism that involves a common step employed also by GRF and GHRP-6, an increase in calcium influx. In addition, our data strongly suggest that KP 101 like GHRP-6 do not act through the GRF receptor and that there is no cross-desensitization the GRF elicited response.     

Corticotropin Releasing Factor (CRF) increases post-prandial duodenal motor activity in humans

The effects of peripherally administered Corticotropin Releasing Factor (CRF) on post-prandial gastrointestinal motility were studied in normal subjects. Pressure activity was monitored for 90 min pre-and 120 min post-prandially in the antrum and duodenum in 8 healthy male volunteers (mean age 45.5 years). Subjects received, on separate days, ovine CRF (0.6 nmol/kg) or vehicle, infused intravenously over 5 min, 15 min after the beginning of the meal. In all subjects, CRF infusion transiently increased the frequency of contractile events to the frequency of the duodenal slow wave (11.7 ± 0.3 cpm). The postprandial duodenal mobility index (MI) after CRF infusion was significantly greater (7.72 ± 0.29) when compared to vehicle infusion (4.34 ± 0.14) (mean ± SEM; P < 0.001). However, the fraction of propagated contractile events was not altered significantly after CRF when compared to vehicle. In contrast, the antral post-prandial MI was not affected by the CRF application. Serum cortisol levels increased significantly at 60 and 90 min post-CRF injection. These data indicate that CRF transiently switches the post-prandial duodenal motor activity to a band of non-propagated high frequency contractions, but does not affect antral contractions.     

Gonadotropin-Releasing Hormone and Thyrotropin-Releasing Hormone Regulation of G Protein Function in the Rat Anterior Pituitary Lobe

In order to evaluate the role of guanine nucleotide-dependent transducer proteins (G proteins) in hormone-mediated signal transduction in the anterior pituitary lobe, we examined the effect of gonadotropin-releasing hormone (GnRH) and thyrotropin-releasing hormone (TRH) on two parameters of G protein function, namely [³⁵ S]GTP_γS binding and low K_mGTPase activity. Plasma membranes were prepared from anterior pituitary lobes of adult male rats using conventional procedures. GTP binding was determined by incubating 2 to 5 μg membrane protein with approximately 100,000 cpm [³⁵ S]GTP_γS in a buffer containing 20 mM Tris- HCl, 1 mM EDTA, 1 mM dithiothreitol, and 100 mM NaCI at a pH of 7.4 for 10 or 15 min at 37 °C GnRH agonist and TRH stimulated high affinity [³⁵ S]GTP_γS binding in a concentration-dependent manner. GTP binding was maximally stimulated by GnRH agonist (1 μM) and TRH (0.1 μM) by up to 27% and 34%, respectively. A time-course study revealed that 1 μM GnRH agonist stimulated GTP binding by 30% at 15 min; 0.1 μM TRH stimulated GTP binding by 23% at 1 min, 18% at 5 min and 25% at 10 min. A stable GTP analog, 5′-guanylylimidodiphosphate, inhibited GnRH- as well as TRH-stimulated GTP binding. GnRH antagonist did not affect GTP binding. However, in the presence of the antagonist, stimulation of GTP binding by the GnRH agonist was completely blocked. The low K_mGTPase activity (EC 3.6.1.-), another parameter of G protein function, was assayed in 2 to 5 μg membrane protein using [γ-³² P]GTP at 37 °C in an ATP-regenerating buffer containing 1 μM unlabeled GTP. GnRH agonist (0.1 μM) and TRH (1 μM) maximally stimulated this GTPase activity by up to 50% and 40%, respectively. GnRH agonist (1 μM) stimulated the GTPase activity by 30% at 10 min and 48% at 30 min. TRH (1 μM) stimulated the GTPase activity at all time points monitored; stimulation was 46% at 5 min, 49% at 20 min, and 41% at 30 min. Interestingly, the GnRH antagonist stimulated GTPase activity by about 20%, but inhibited GnRH agonist-stimulated GTPase activity in a concentration-dependent manner. These results indicate that the binding of GnRH and TRH to their receptors results in interaction of the receptor with a G protein and activation of the G protein cycle.     

Corticotrophin-Releasing Hormone Stimulates Neurohypophyslal Hormone Release through an Interaction with the Intermediate Lobe of the Pituitary

Corticotrophin-releasing hormone is found co-localized with oxytocin in the magnocellular-neurohypophysical system but its function in this context is unknown. We tested its effects on neurohypophysical hormone secretion in vitro , in the presence and absence of the intermediate lobe of the pituitary. Corticotrophin-releasing hormone caused significant, calcium-dependent secretion of oxytocin and vasopressin from neural lobes in contact with intermediate lobes, i.e. neurointermediate lobes. This effect was inhibited by the dopamine agonist, bromocriptine. Corticotrophin-releasing hormone had no effect on isolated neural lobes in the absence of the intermediate lobe, but α- and γ-melanocyte-stimulating hormone produced an increase in secretion that was comparable in pattern and magnitude to the effect of corticotrophin-releasing hormone on neurointermediate lobes. These findings suggest that corticotrophin-releasing hormone released with oxytocin may act in a paracrine fashion to stimulate release of intermediate peptides which, in turn, can directly evoke release of oxytocin and vasopressin from neural lobe terminals.     

Neuronal Gonadotrophin‐Releasing Hormone (GnRH) and Astrocytic Gonadotrophin Inhibitory Hormone (GnIH) Immunoreactivity in the Adult Rat Hippocampus

Gonadotrophin‐releasing hormone (GnRH) and gonadotrophin inhibitory hormone (GnIH) are neuropeptides secreted by the hypothalamus that regulate reproduction. GnRH receptors are not only present in the anterior pituitary, but also are abundantly expressed in the hippocampus of rats, suggesting that GnRH regulates hippocampal function. GnIH inhibits pituitary gonadotrophin secretion and is also expressed in the hippocampus of a songbird; its role outside of the reproductive axis is not well established. In the present study, we employed immunohistochemistry to examine three forms of GnRH [mammalian GnRH‐I (mGnRH‐I), chicken GnRH‐II (cGnRH‐II) and lamprey GnRH‐III (lGnRH‐III)] and GnIH in the adult rat hippocampus. No mGnRH‐I and cGnRH‐II+ cell bodies were present in the hippocampus. Sparse mGnRH‐I and cGnRH‐II+ fibres were present within the CA1 and CA3 fields of the hippocampus, along the hippocampal fissure, and within the hilus of the dentate gyrus. No lGnRH‐III was present in the rodent hippocampus. GnIH‐immunoreactivity was present in the hippocampus in cell bodies that resembled astrocytes. Males had more GnIH+ cells in the hilus of the dentate gyrus than females. To confirm the GnIH+ cell body phenotype, we performed double‐label immunofluorescence against GnIH, glial fibrillary acidic protein (GFAP) and NeuN. Immunofluorescence revealed that all GnIH+ cell bodies in the hippocampus also contained GFAP, a marker of astrocytes. Taken together, these data suggest that GnRH does not reach GnRH receptors in the rat hippocampus primarily via synaptic release. By contrast, GnIH might be synthesised locally in the rat hippocampus by astrocytes. These data shed light on the sites of action and possible functions of GnRH and GnIH outside of the hypothalamic‐pituitary‐gonadal axis.     

The Interactive Language of the Hypothalamus for the Gonadotropin Releasing Hormone (GNRH) System

The enormous diversity in neurochemical signals employed within the network of afferents to GnRH neurons is well-documented. An examination of novel and accumulating knowledge on the operation of these messengers indicates the presence of an interactive language governing GnRH secretion. The basic operational structures identified to date to affirm this interactive form of communication summarized in this review are the following: (i) the demonstration of interconnections within various components of the afferent network; (ii) coexistence and possible co-release of excitatory and inhibitory neurotransmitters/ neuromodulators; (iii) co-action of various messengers at synaptic targets, and (4) modulation by gonadal steroids of the synthesis and release of signals and their receptors, and induction of synaptic plasticity for the timely relay of signals for GnRH secretion. Unraveling the molecular sequelae that promote this interactive communication to elicit periodic GnRH secretion is now a new challenge.     

Dynamic Changes in Gonadotropin Releasing Hormone Receptor mRNA Content in the Mediobasal Hypothalamus during the Rat Estrous Cycle

The purpose of the present study was to determine if GnRH receptor mRNA levels in the rat brain undergo changes during the estrous cycle. We focused on the arcuate and ventromedial nuclei of the hypothalamus and on the hippocampus which are sites in the rat central nervous system that have been shown to contain measurable amounts of GnRH receptor mRNA. Groups of regularly cycling female rats were decapitated at 08.00 and 17.00  h of each day of the estrous cycle, trunk blood was collected for radioimmunoassay analysis of circulating LH levels, and the brains were processed for ' in situ ' hybridization. A cDNA probe encoding the rat pituitary GnRH receptor was transcribed ' in vitro ' in the presence of ³³P-alpha UTP and used under saturating conditions to label GnRH receptor mRNA. The results show that in the arcuate and ventromedial nuclei GnRH receptor mRNA levels are relatively high during diestrus 1, they decline slightly during diestrus 2 before they rise to the highest levels at 08.00  h of proestrus. By 17.00  h of proestrus, GnRH receptor mRNA levels had declined to the lowest levels of the estrous cycle where they remain through the morning of estrus. The GnRH receptor mRNA levels rise again sharply during the afternoon of estrus. The changes in the hippocampus follow a similar pattern in that a decline in GnRH receptor mRNA levels to its lowest levels occurs between 08.00 and 17.00  h of proestrus. However, the changes in the hippocampus did not reach statistical significance.     

Growth Hormone (GH)-Releasing Peptide and GH Releasing Hormone Stimulate GH Release from Subpopulations of Somatotrophs in Rats

The synthetic hexapeptide GH-releasing peptide (His-D-Trp-Ala-Trp-D-Phe-Lys-NH2; GHRP-6) and GH releasing hormone (GHRH) are both potent stimulators of GH release in rats. Using reverse hemolytic plaque assay (RHPA), we have compared the effects of human GHRH and GHRP-6 on GH release from the dispersed individual cells of rat anterior pituitary. In a single RHPA, we quantified the percentage of plaque forming cells (% PFC) and their mean plaque area (MPA) after 30 min-incubation, and calculated a total secretion index (TSI) by multiplying % PFC and MPA. 10 nM GHRH and 100 nM GHRP-6 each caused a significant increase in % PFC (%) (GHRH 39.15, GHRP-6 29.4, vs vehicle 24.3, P<0.01), MPA (×10^?2 μm²) (GHRH 124.04, GHRP-6 94.80, vs vehicle 44.57, P<0.01) and TSI (×10^?2) (GHRH 54.46, GHRP-6 32.87, vs vehicle 10.84, P<0.01). Simultaneous addition of both secretagogues caused a further increase in GH release (%PFC 46.4, MPA 142.55, TSI 69.82, P<0.01 vs vehicle), although the effect was additive but not synergistic. Somatostatin analog, SMS201–995 (SMS) partially suppressed all parameters in GH secretion after stimulation by GHRH and/or GHRP-6. A double RHPA was then performed to test whether all somatotrophs respond equally to GHRH and GHRP-6 or some cells formed plaques only by either GHRH or GHRP-6. There were somatotrophs responsive to only GHRH (23.3%vs control 6.2%, P<0.01), those responsive to only GHRP-6 (11.9%vs control 6.1%, P<0.01), and those responsive to both GHRH and GHRP-6 (7.8%vs control 0.2%, P<0.01). These results confirmed the previous findings that GHRP-6 and GHRH directly but independently stimulate GH release from the pituitary cells, and further suggest the presence of at least three functionally distinct somatotroph subpopulations concerning the responsiveness to GHRP-6 and GHRH in rats.     

Regulation of Pituitary Vasopressin V1b Receptor mRNA during Stress in the Rat

Previous studies have shown a parallel relationship between pituitary vasopressin (VP) receptor content and responsiveness of the corticotroph during chronic stress. The regulation of pituitary VP receptors was further studied by analysis of V1b VP receptor mRNA levels in pituitaries of rats subjected to chronic immobilization, i.p. hypertonic saline injection (physical stress paradigms associated with increased pituitary responsiveness), and water deprivation, or to 2% saline in the drinking water (osmotic stress paradigms associated with decreased pituitary responsiveness). Northern blot hybridization with a 363 bp ³²P-labelled fragment of the rV1b receptor cDNA coding sequence revealed two bands of about 3.7 and 3.2 Kb, whereas a probe directed to the 5′ untranslated region recognized only the 3.7 Kb band. Repeated i.p. hypertonic saline injection, 3 times in 24 h at 8 h intervals, or daily for 8 days, increased the intensity of the 3.7 Kb band by 155 ± 17.5% (P<0.01) and 118 ± 14.6% (P<0.01), respectively, while the 3.2Kb band increased by 122 ± 39.3% (P<0.01) only after 3 times injection. Smaller increases of 39 ± 11 and 33 ± 9% (P<0.05) in the 3.7 Kb band were found after repeated immobilization 3 times in 24 h and 2 h for for 8 days respectively. In situ hybridization studies confirmed significant increases (P<0.05) in V1b receptor mRNA levels after 8 and 14 days repeated immobilization (63 ± 19% and 83 ± 10%) or i.p. hypertonic saline injection (110 ± 13% and 73 ± 20%). In response to acute stress, V1b receptor mRNA increased by 77 ± 5% (3.7 Kb band) after 4 h immobilization for 1 h, whereas both bands were reduced by 49 ± 5% and 45 ± 5%, 4 h after a single i.p. hypertonic saline injection. The decrease in V1b receptor mRNA following a single i.p. hypertonic saline injection was prevented by pretreatment with a V1 receptor antagonist, suggesting that increased VP secretion may account for this effect. In spite of the decrease in V1 b receptor mRNA following i.p. hypertonic saline injection, VP binding in pituitary membrane rich fractions, and VP-stimulated inositol phosphate formation in quartered hemipituitaries were increased by 24 and 39%, respectively. V1b receptor mRNA levels were unchanged or decreased following prolonged osmotic stimulation. These studies suggest that increased V1b receptor mRNA levels contribute to the VP receptor upregulation observed during repeated immobilization and i.p. hypertonic saline injection, whereas the lack of parallelism between V1b receptor mRNA and VP binding indicates that regulation of steady-state levels of V1b receptor mRNA is not a primary determinant in the control of pituitary VP receptor concentration during stress.     

Pituitary Hormone Gene Expression in Male Golden Hamsters: Interactions Between Photoperiod and Testosterone

Daylength regulates neuroendocrine function in male golden hamsters. Exposure to short days triggers gonadal regression and decreases serum luteinizing hormone (LH), prolactin and testosterone concentrations. Inhibitory photoperiods also amplify the negative feedback actions of androgens upon gonadotropin secretion. To examine whether these changes arise from altered adenohypophyseal gene expression, we measured the abundance of the messenger ribonucleic acids (mRNAs) encoding β-LH, prolactin and proopiomelanocortin in anterior pituitaries of male golden hamsters which were either left intact, castrated, castrated and implanted with testosterone, or pinealectomized and maintained in either long (14 h light/10 h dark) or short (5 h light/19 h dark) days. Short days caused testicular atrophy in intact male hamsters and reduced serum LH in intact and castrated, testosterone-replaced hamsters. The relative abundance of β-LH mRNA was suppressed by exposure to short days only in castrated hamsters. Serum prolactin was decreased by short days regardless of circulating testosterone concentrations. Prolactin mRNA abundance was decreased by short days in all pineal-intact groups. Castration reduced proopiomelanocortin mRNA abundance in long days and testosterone replacement reversed this effect. In the presence of testosterone, photoperiod influenced serum LH concentrations without altering hypophyseal abundance of β-LH mRNA. In contrast, photoperiodic influences on prolactin secretion were correlated with alterations in steady-state mRNA abundance.     

Immunohistochemical Distribution of Oestrogen Receptor and Luteinizing Hormone B Subunit in the Ovine Pituitary Gland During Foetal Development

The presence of oestrogen receptor in the developing hypothalamo-hypophyseal system is an essential prerequisite for the development of sex-steroid feedback on gonadotrophin secretion. We have used dual immunocytochemistry to examine the ontogeny and regional distribution of oestrogen receptor and LH β subunit in the ovine pituitary gland during foetal development. At day 65 gestation (term=145 days) oestrogen receptor and LH β immunopositive cells are found in a small region at the base of the anterior pituitary gland, and also in a band immediately adjacent to the neurointermediate lobe. By day 100 gestation there was a significant increase in the number of immunopositive LH β cells accounting for around 12% of the total cell population, and these were widely distributed throughout the anterior pituitary gland. There was also a significant increase in the proportion of gonadotrophs which contain oestrogen receptor compared with day 65. By day 130 gestation the percentage of LH containing cells had declined to around 7% of the total population, but the proportion which also contained oestrogen receptor remained the same. There were no differences in the numbers or distribution of cells containing LH or oestrogen receptors between male and female foetuses, at any age. These data describing a parallel change in the number of oestrogen receptors and LH β containing cells in the pituitary gland throughout gestation suggest that the development of pituitary sex-steroid feedback is not solely dependent on changes in the numbers of oestrogen receptor containing cells alone.     

Advent and Recent Advances in Research on the Role of Pituitary Adenylate Cyclase-activating Polypeptide (PACAP) in the Regulation of Gonadotropic Hormone Secretion of Female Rats

PACAP (ADCYAP1) was isolated from ovine hypothalami. PACAP activates three distinct receptor types: G-protein coupled PAC1, VPAC1, and VPAC2 with seven transmembrane domains. Eight splice variants of PAC1 receptor are described. A part of the hypothalamic PACAP is released into the hypophyseal portal circulation. Both hypothalamic and pituitary PACAP are involved in the dynamic control of gonadotropic hormone secretion. In female rats, PACAP in the paraventricular nucleus is upregulated in the morning and pituitary PACAP is upregulated in the late evening of the proestrus stage of the reproductive cycle. PACAP mRNA peak in the hypothalamic PVN precedes the LHRH release into the portal circulation. It is supposed that PACAP peak is evoked by the elevated estrogen on proestrous morning. At the beginning of the so-called critical period of the same day, PACAP level starts to decline allowing LHRH release into the portal circulation, resulting in the LH surge that evokes ovulation. Just before the critical period, icv-administered exogenous PACAP blocks the LH surge and ovulation. The blocking effect of PACAP is mediated through CRF and endogenous opioids. The effect of the pituitary-born PACAP depends on the intracellular cross-talk between PACAP and LHRH.