Evidence that genetic variation in 5-HT transporter expression is linked to changes in 5-HT2A receptor function.

Variability in expression of the 5-HT transporter (5-HTT) gene in the human population has been associated with a range of behavioural phenotypes. The underlying mechanisms are unclear but may involve changes in 5-HT receptor levels and/or signalling. The present study used a novel 5-HTT overexpressing transgenic mouse to test the hypothesis that variability in 5-HTT expression may alter 5-HT(2A) receptor function. In wildtype mice, the 5-HT(2) receptor agonist DOI increased regional brain mRNA expression of two immediate early genes (c-fos and Arc), and induced head twitches, and both effects were abolished by pre-treatment with the 5-HT(2A) receptor antagonist MDL 100907. In 5-HTT overexpressing mice, DOI induced a greater increase in both c-fos and Arc mRNA expression in cortical brain regions, and more head twitches, compared to wildtype mice. Autoradiographic and in situ hybridisation experiments showed that 5-HT(2A) receptor binding sites and 5-HT(2A) receptor mRNA did not differ between transgenic and wildtype mice. Finally, the transgenic mice had lower regional brain 5-HT levels compared to wildtype mice. This depletion of 5-HT may underpin the increase in 5-HT(2A) receptor function because in wildtype mice 5-HT depletion using the 5-HT synthesis inhibitor, p-chlorophenylalanine, enhanced the head twitch response to DOI. These data demonstrate that elevated 5-HTT expression is accompanied by increased 5-HT(2A) receptor function, an effect possibly mediated by decreased availability of synaptic 5-HT. Variation in levels of 5-HTT expression may therefore be a source of variability in 5-HT(2A) receptor function, which may be an important modifier of 5-HTT-linked phenotypes.

Overexpression of the human VPAC2 receptor in the suprachiasmatic nucleus alters the circadian phenotype of mice.

The neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) belong to a superfamily of structurally related peptide hormones that includes glucagon, glucagon-like peptides, secretin, and growth hormone-releasing hormone. Microinjection of VIP or PACAP into the rodent suprachiasmatic nucleus (SCN) phase shifts the circadian pacemaker and VIP antagonists, and antisense oligodeoxynucleotides have been shown to disrupt circadian function. VIP and PACAP have equal potency as agonists of the VPAC(2) receptor (VPAC(2)R), which is expressed abundantly in the SCN, in a circadian manner. To determine whether manipulating the level of expression of the VPAC(2)R can influence the control of the circadian clock, we have created transgenic mice overexpressing the human VPAC(2)R gene from a yeast artificial chromosome (YAC) construct. The YAC was modified by a strategy using homologous recombination to introduce (i) the HA epitope tag sequence (from influenza virus hemagglutinin) at the carboxyl terminus of the VPAC(2)R protein, (ii) the lacZ reporter gene, and (iii) a conditional centromere, enabling YAC DNA to be amplified in culture in the presence of galactose. High levels of lacZ expression were detected in the SCN, habenula, pancreas, and testis of the transgenic mice, with lower levels in the olfactory bulb and various hypothalamic areas. Transgenic mice resynchronized more quickly than wild-type controls to an advance of 8 h in the light-dark (LD) cycle and exhibited a significantly shorter circadian period in constant darkness (DD). These data suggest that the VPAC(2)R can influence the rhythmicity and photic entrainment of the circadian clock.

Circadian changes in PACAP type 1 (PAC1) receptor mRNA in the rat suprachiasmatic and supraoptic nuclei.

A receptor for pituitary adenylate cyclase activating polypeptide (PACAP), denoted as PAC1, is expressed in the suprachiasmatic nuclei (SCN). Since the circadian clock demonstrates phase-dependent sensitivity to PACAP, we have used in situ hybridization histochemistry to examine whether PAC1 mRNA is differentially expressed in the rat SCN across the 24-h cycle. There was a significant variation in PAC1 mRNA within the SCN and supraoptic nuclei during the light-dark cycle and in constant darkness, with peaks at the middle of both the real and subjective day and night; no significant variation was observed in the cingulate cortex. The results suggest that the phase-dependent actions of PACAP on the clock may involve phase-specific changes in the availability of PAC1 receptors within the SCN.

Expression of PACAP, and PACAP type 1 (PAC1) receptor mRNA during development of the mouse embryo.

Pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) have been reported to have a number of neurotrophic effects. We have examined the expression of mRNA for PACAP and PACAP type 1 (PAC1) receptor in the mouse embryo by in situ hybridization and the effects of PACAP and VIP on the growth of mouse embryos in vitro. Although we were unable to detect gross effects of either peptide on the growth rates of embryos maintained in culture, mRNAs for both PAC1 receptor and PACAP peptide were present in the nervous system from day 9.5 of embryonic development. PAC1 receptor mRNA was most abundant in the neural tube and the rhombencephalon and was present also in the dorsal root and trigeminal ganglia and the sympathetic chain. The distribution of mRNA for the PACAP peptide overlapped in part with that of the receptor, but was more extensively distributed in the rhombencephalon and in the developing hypothalamus. Within the neural tube, PAC1 receptor mRNA was located in the roof and floor plates, while the distribution of PACAP peptide mRNA was more complex, being located in two columns of cells in the ventromedial neural tube (consistent with the position of developing autonomic motor neurons) and in cells in the dorsolateral neural tube. These data are concordant with a role for PACAP or a related peptide in neural development.

Circadian changes in the expression of vasoactive intestinal peptide 2 receptor mRNA in the rat suprachiasmatic nuclei.

The suprachiasmatic nuclei (SCN) in the hypothalamus function as the primary circadian pacemaker. A receptor for vasoactive intestinal peptide (VIP), denoted as VIP2, is abundantly expressed in the SCN. Since the rodent circadian clock demonstrates phase-dependent sensitivity to exogenous VIP, we investigated the possibility that VIP2 receptor mRNA is differentially expressed in the SCN across the 24 h cycle. To establish whether VIP2 receptor mRNA levels change across the 12:12 h light-dark (LD) cycle (lights on designated as Zeitgeber time (ZT)O), rats were killed at ZT 0, 2, 6, 10, 12, 14, 18 and 22. To determine if variation in this mRNA occurs in the absence of LD entrainment cues, lights were not turned on at the time of transition from dark to light (designated as CT O); the animals in this group were killed in constant darkness (DD) at CT 0, 2, 6, 10, 12, 14, 18 and 22. In situ hybridization histochemistry indicated no variations in VIP2 receptor mRNA in the cingulate cortex under either LD or DD conditions. There was, however, significant variation in the expression of VIP2 receptor mRNA within the SCN during the LD cycle, with one peak at ZT 6 and at ZT 22. A comparable biphasic pattern of mRNA expression was observed in DD animals with peaks at CT 10 and another at CT 22. The results suggest that the phase-dependent actions of VIP on the clock may involve phase-specific changes in the availability of VIP2 receptor within the SCN.

Celsr1, a neural-specific gene encoding an unusual seven-pass transmembrane receptor, maps to mouse chromosome 15 and human chromosome 22qter.

We have identified Celsr1, a gene that encodes a developmentally regulated vertebrate seven-pass transmembrane protein. The extracellular domain of Celsr1 contains two regions each with homology to distinct classes of well-characterized motifs found in the extra-cellular domains of many cell surface molecules. The most N-terminal region contains a block of contiguous cadherin repeats, and C-terminal to this is a region containing seven epidermal growth factor-like repeats interrupted by two laminin A G-type repeats. Celsr1 is unique in that it contains this combination of repeats coupled to a seven-pass transmembrane domain. As part of the characterization of the Celsr1 gene, we have determined its chromosomal map location in both mouse and human. The European Collaborative Interspecific Backcross (EUCIB) and BXD recombinant inbred strains were used for mapping Celsr1 cDNA clones in the mouse, and fluorescence in situ hybridization was used to map human Celsr1 cosmid clones on metaphase chromosomes. We report that Celsr1 maps to proximal mouse Chromosome 15 and human chromosome 22qter, a region of conserved synteny. Reverse transcriptase-polymerase chain reaction analysis and in situ hybridization were used to determine the spatial restriction of Celsr1 transcripts in adult and embryonic mice. The results presented here extend our previous finding of expression of the Celsr1 receptor in the embryo and show that expression continues into adult life when expression in the brain is localized principally in the ependymal cell layer, choroid plexus, and the area postrema.

Endopeptidase EC 3.4.24.15 presence in the rat median eminence and hypophysial portal blood and its modulation of the luteinizing hormone surge.

The endopeptidase EC 3.4.24.15 (EP24.15) is a zinc metalloendopeptidase that is widely distributed in a variety of tissues, including the testes, pituitary and the central nervous system. Among its numerous roles in metabolizing and processing biologically-active peptides, the enzyme degrades gonadotropin-releasing hormone (GnRH) by cleaving the central Tyr5-Gly6 bond. The aim of the present studies was to determine whether EP24.15 can modulate the concentrations of GnRH within the hypothalamo-hypophysial portal blood and thereby play a physiological role in reproduction. Our data suggest the presence of immunoreactive EP24.15 in the perivascular space of the median eminence and that this enzyme is secreted into portal blood. We have also shown a physiological role for this enzyme in that an inhibition of its activity with a specific inhibitor augmented the steroid-induced LH increase in ovariectomized rats. The present results suggest that secretory and post-secretory mechanisms are important in shaping the GnRH signal from the central nervous system; GnRH metabolism by EP24.15 may be one such mechanism.

Expression of pituitary adenylate cyclase activating polypeptide receptors in the early mouse embryo as assessed by reverse transcription polymerase chain reaction and in situ hybridisation.

We have examined the expression of mRNA for the pituitary adenylate cyclase activating polypeptide (PACAP) receptor in the mouse from day 9.5 of embryonic development onwards by reverse transcription polymerase chain reaction (RT-PCR) and by in situ hybridisation. PACAP receptor mRNAs were detected by PCR in the earliest developmental stage examined and thereafter the levels of mRNA for all receptors increased during development. Wholemount in situ hybridisation first showed the expression of the PACAP receptor in the neural tube of 9.5 day old embryos, in later embryos heaviest expression of the PACAP receptor was found in the developing rhombencephalon (day 10.5 and 11.5). These results are consistent with a role for PACAP or a related peptide in the early development of the central nervous system.

The distribution of vasoactive intestinal peptide2 receptor messenger RNA in the rat brain and pituitary gland as assessed by in situ hybridization.

The distribution of rat vasoactive intestinal peptide2 (VIP2) receptor messenger RNA in the brain and the pituitary gland was examined by in situ hybridization and by ribonuclease protection assay. labelled cells were found chiefly in the suprachiasmatic nucleus, the central nucleus of the amygdala and the thalamus (the lateral geniculate nucleus, and the paraventricular, mediodorsal and ventral nuclei of the thalamus). The distribution of the VIP2 receptor overlaps only in part with that of the VIP1 receptor, for example in the hippocampus, where VIP2 receptor messenger RNA was found in the pyramidal cells of the CA1-CA3 subfields and in the granule cells of the dentate gyrus. Small numbers of neurons containing high concentrations of VIP2 receptor messenger RNA were present in the brainstem in the principal sensory trigeminal nucleus and in the substantia gelatinosa of the spinal cord, suggesting a role for the VIP2 receptor in the processing of sensory information. The presence of the VIP2 receptor in the suprachiasmatic nucleus suggests that it is this receptor subtype which is involved in the control of circadian rhythms.

The expression of the calcitonin receptor gene in the brain and pituitary gland of the rat.

The distribution of rat calcitonin receptor mRNA in the brain and pituitary gland were examined by in situ hybridization. Labelled cells were found in the nucleus accumbens, the preoptic and medial preoptic areas, the paraventricular nucleus of the hypothalamus, and the arcuate nucleus; a smaller number of cells were found in the brainstem and olfactory bulb. No strongly labelled cells were found in the pituitary gland.

The VIP2 receptor: molecular characterisation of a cDNA encoding a novel receptor for vasoactive intestinal peptide.

We have cloned and sequenced a cDNA (RPR4) encoding a new member of the secretin/calcitonin/parathyroid hormone (PTH) receptor family. RPR4 was identified by PCR of rat pituitary cDNA, and a full-length clone was isolated from a rat olfactory bulb cDNA library. When RPR4 was functionally expressed in COS 7 cells, cyclic adenosine monophosphate (cAMP) production was stimulated by vasoactive intestinal peptide (VIP), pituitary adenylate cyclase activating peptides (PACAP-38 and PACAP-27) and helodermin, with equal potency. Peptide histidine isoleucine (PHI) and rat growth hormone releasing hormone (rGHRH) also stimulated cAMP production at lower potency. This suggests that RPR4 encodes a novel VIP receptor which we have designated the VIP2 receptor. In situ hybridisation showed that mRNA for this receptor was present mainly in the thalamus, hippocampus and in the suprachiasmatic nucleus.

Effect of adrenalectomy and dexamethasone on neuropeptide content of dorsal root ganglia in the rat.

Neuropeptides, including substance P (SP), calcitonin gene-related peptide (CGRP) and somatostatin (SS) in dorsal root ganglia (DRG) may play a role in neurogenic inflammation and pain transmission. Adrenal corticosteroids regulate neuropeptide synthesis in some areas of the CNS and may modulate neurogenic inflammation and sensory perception. We have investigated the effects of adrenalectomy and dexamethasone (0.2 mg/kg/day) treatment on neuropeptide content of rat cervical DRG using specific and sensitive radioimmunoassays. In control animals, a differential distribution of neuropeptide was found; SP and CGRP content increased from C4 to C7 in contrast to SS content, which decreased from C4 to C7. Ten days following adrenalectomy, the mean SS content of cervical DRG decreased significantly to 79.6 +/- 4.5% of sham-operated controls. In contrast, SP and CGRP content increased significantly 10 days after adrenalectomy to 134.6 +/- 6.9% and 132.0 +/- 11.6% of sham-operated controls, respectively. The effects of adrenalectomy on CGRP and SS were reversed by administration of dexamethasone. These results suggest that glucocorticoids affect the neuropeptide content of DRG in the adult rat.

Hypothalamic release of atrial natriuretic factor and beta-endorphin into rat hypophysial portal plasma: relationship to oestrous cycle and effects of hypophysectomy.

The release of beta-endorphin and atrial natriuretic factor (ANF) into hypophysial portal plasma was investigated in male and female Wistar rats. The principal aim of the study was to investigate the possible role of beta-endorphin and ANF in the hypothalamic control of LH and prolactin secretion. In male rats, anaesthetized with urethane, the concentrations of beta-endorphin in portal blood collected immediately after hypophysectomy were within the same range as those in peripheral plasma. Furthermore, electrical stimulation of the median eminence did not increase the portal plasma concentrations of beta-endorphin. In female rats, anaesthetized with alphaxalone, the portal plasma concentrations in long-term (6-8 weeks) or acutely hypophysectomized rats were significantly greater than those in peripheral plasma. In acutely hypophysectomized female rats the concentrations and contents of beta-endorphin in portal plasma collected at 10.00-11.30 h of pro-oestrus were significantly (approximately sixfold) greater than at dioestrus or at 20.00-21.00 h of pro-oestrus, but these changes were not consistently seen in all experiments. In female rats in which the pituitary gland was not removed for portal blood collection, portal plasma contents of ANF remained unchanged throughout the day of pro-oestrus, suggesting that it is unlikely that ANF is involved in the spontaneous LH or prolactin surge. The effects of ovarian steroids on the secretion of hypothalamic ANF and beta-endorphin were determined by measuring the portal plasma concentration of ANF and beta-endorphin on the morning of presumptive pro-oestrus in rats ovariectomized 24 h previously and injected with either oil or oestradial benzoate (OB). Portal plasma contents of ANF were significantly lower in OB- compared with oil-treated rats, suggesting that oestradiol inhibits ANF release into rat hypophysial portal plasma. In contrast, there were no significant between-group differences in the content or concentration of beta-endorphin in portal plasma. Thus, the increased beta-endorphin in the portal plasma of some of the intact animals during the morning of pro-oestrus is not due to the preovulatory surge of oestradiol-17 beta. The output of beta-endorphin into portal blood in long-term hypophysectomized rats was lower than in dioestrous or pro-oestrous rats in which the pituitary gland was removed immediately before portal blood collection.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of corticosterone on the secretion of corticotrophin-releasing factor, arginine vasopressin and oxytocin into hypophysial portal blood in long-term hypophysectomized rats.

To investigate the feedback effects of corticosterone on the secretion of corticotrophin-releasing factor-41 (CRF-41), oxytocin and arginine vasopressin (AVP), hypophysial portal vessel blood was collected from control (intact) and long-term (6-8 weeks) hypophysectomized rats. In preliminary experiments in rats anaesthetized with urethane, long-term hypophysectomy resulted in a significant increase in the secretion of oxytocin and AVP; the hypothalamic contents of oxytocin and AVP were also increased in comparison with pituitary-intact rats. In long-term hypophysectomized rats anaesthetized with sodium pentobarbitone, but not with urethane, the output of CRF-41 into portal blood was increased twofold in comparison with that in control rats. In long-term hypophysectomized rats anaesthetized with pentobarbitone, the i.v. infusion of corticosterone (7.2 nmol/min) for a 2 h period of portal blood collection did not alter the secretion of CRF-41, oxytocin or AVP into portal blood; however, the secretion of CRF-41 and, to a lesser extent, AVP was significantly reduced in hypophysectomized rats by continuous corticosterone replacement, by a pellet of corticosterone implanted s.c. for 5 days before portal blood collection. These results confirm that the secretion of CRF-41 is differently affected by the anaesthetics urethane and pentobarbitone, and in long-term hypophysectomized rats show (i) that there were no apparent feedback effects of corticosterone infusion over a 2 h period on the secretion of any of the peptides studied, (ii) that late delayed feedback effects of continuous administration of corticosterone are mediated by a reduction in CRF-41 and AVP output, and (iii) that corticosterone has no effects on oxytocin secretion into portal blood.

Corticotrophin-releasing factor-41, vasopressin and oxytocin release into hypophysial portal blood in the rat: effects of electrical stimulation of the hypothalamus, amygdala and hippocampus.

The role of the paraventricular nuclei (PVN), amygdala and hippocampus in the control of the hypothalamic-pituitary-adrenal axis has been studied by determining the effect of electrical stimulation of the PVN, amygdala and hippocampus on the release of corticotrophin-releasing hormone (CRF-41) and arginine vasopressin (AVP) into hypophysial portal blood and ACTH and corticosterone into peripheral blood. Adult female Wistar rats were anaesthetized with sodium pentobarbitone and stimulation was carried out through previously implanted bipolar, glass-insulated platinum electrodes. Hypophysial portal blood was collected 30 min before and 30 min during the application of the stimulus which consisted of trains (30 s on and 30 s off) of biphasic rectangular pulses with a frequency of 50 Hz, pulse width 1 ms and amplitude 1 mA. Bilateral stimulation of the PVN increased while unilateral stimulation of the amygdala decreased the release of CRF-41 into hypophysial portal blood. The threefold increase in release of CRF-41 induced by PVN stimulation correlated with a marked increase in peripheral plasma concentrations of ACTH and corticosterone. Stimulation of the hippocampus had no significant effect on CRF-41 release, and stimulation of each of the three brain regions had no effect on AVP release into portal blood. These findings were extended in a second study to compare the effects of unilateral bipolar electrical stimulation of the PVN and of the supraoptic nucleus (SON) on the release of CRF-41, AVP and oxytocin. This study was carried out on adult male rats, anaesthetized with sodium pentobarbitone, in which the stimulus was applied through previously implanted concentric stainless-steel electrodes.(ABSTRACT TRUNCATED AT 250 WORDS)

Steroid control of central neuronal interactions and function.

Steroids have potent actions on the brain which can be categorized as; (i) fast (approximately ms-s), (ii) intermediate (h-days), (iii) long-term reversible (days-weeks) and (iv) long-term irreversible. Here attention is focussed on the intermediate and long-term reversible effects of steroids with emphasis on glucocorticoids and oestrogen. Glucocorticoid negative feedback is generally classified as fast, delayed and long-term. Fast negative feedback would appear to depend mainly on a reduction in pituitary responsiveness to corticotrophin releasing factor-41 (CRF-41) and possibly arginine vasopressin (AVP). Delayed feedback is mediated by reduced AVP release into hypophysial portal blood and blockade of the ACTH response to CRF-41. Long-term negative feedback is a consequence of reduced CRF-41 and AVP release into portal blood. Lesion and electrical stimulation studies pinpoint the paraventricular nuclei as the main site at which glucocorticoids act to control ACTH release. Oestrogen at physiologically low plasma concentrations inhibits gonadotrophin secretion. At physiologically high plasma concentrations, such as those that occur during the preovulatory surge, oestradiol-17 beta stimulates the biosynthesis of LHRH mRNA and LHRH and the release of LHRH into hypophysial portal blood. Oestradiol also increases pituitary responsiveness to LHRH. The action of oestrogen on LHRH neurons is probably mediated by interneurons and may involve disinhibition; this view is supported by our in situ hybridization studies which show that oestrogen, in its positive feedback mode, significantly reduces the synthesis of proopiomelanocortin mRNA in arcuate neurons which when active are likely to inhibit LHRH neurons. The mechanism of action of oestrogen on the pituitary gland is not yet established, but clues from the action of the priming effect of LHRH suggests that oestrogen may potentiate phosphoinositide second messenger cascades. LHRH priming involves the synthesis of a 70 kDa protein the N-terminus of which is identical to an oestrogen-induced protein in the ventromedial hypothalamic nucleus involved in lordosis, and to that of phospholipase C alpha. Attention is drawn to the remarkable economy of the system by which a single steroid, oestrogen, has effects on the brain and pituitary gland which result in a co-ordinated sequence of amplifier cascades which lead first to the ovulatory surge of luteinizing hormone and then to mating behaviour, both of which are obviously essential for continuation of the species.(ABSTRACT TRUNCATED AT 400 WORDS)

Corticotrophin-releasing peptides in rat hypophysial portal blood after paraventricular lesions: a marked reduction in the concentration of corticotrophin-releasing factor-41, but no change in vasopressin.

Previous data show that corticotrophin-releasing factor-41 (CRF-41), arginine vasopressin (AVP) and oxytocin are released into hypophysial portal blood. It has been presumed that the CRF-41 originates mainly from parvicellular neurones of the paraventricular nuclei (PVN); however, AVP and oxytocin could also be derived as a consequence of preterminal release from magnocellular projections to the neurohypophysis. The latter has been suggested to be the case for AVP as assessed by studies of the median eminence in vitro. Here we have investigated the source of CRF-41, AVP and oxytocin in hypophysial portal blood of adult male Wistar rats 8-10 days after surgical lesioning of the PVN. In PVN-lesioned animals the output of CRF-41 into hypophysial portal blood was reduced by about 90%, and that of oxytocin by about 40%: however, the output of AVP into portal blood was reduced only by about 10%. The release of AVP into portal blood increased after adrenalectomy; this increased release could be returned to normal by treatment with dexamethasone. No change of AVP release occurred after adrenalectomy in animals in which the PVN had been lesioned. These results show (i) that most of the CRF-41 released into hypophysial portal blood is derived from the PVN, (ii) that in PVN-lesioned animals AVP and oxytocin release remains at near normal or 60% of normal respectively, suggesting that a substantial amount of both neuropeptides in portal blood is derived as a consequence of preterminal release from supraoptic nuclei projections in the median eminence, and (iii) that glucocorticoid feedback inhibition of AVP release is exerted at the level of the PVN.

Release of oxytocin but not corticotrophin-releasing factor-41 into rat hypophysial portal vessel blood can be made opiate dependent.

The effects of morphine dependence and abrupt opiate withdrawal on the release of oxytocin and corticotrophin-releasing factor-41 (CRF-41) into hypophysial portal vessel blood in rats anaesthetized with urethane were investigated. Adult female Sprague-Dawley rats were made dependent upon morphine by intracerebroventricular infusion of morphine for 5 days; abrupt opiate withdrawal was induced by injection of the opiate antagonist naloxone. The basal concentrations of oxytocin in portal or peripheral plasma from morphine-dependent rats did not differ significantly from those in control, vehicle-infused rats. In rats in which the pituitary gland was not removed after stalk section, the i.v. injection of naloxone hydrochloride (5 mg/kg) resulted in a large and sustained increase in the concentration of oxytocin in both portal and peripheral plasma in control and morphine-dependent rats. The i.v. injection of naloxone resulted in a threefold increase in the secretion of oxytocin into portal blood in acutely hypophysectomized rats infused with morphine, but did not alter oxytocin secretion in vehicle-infused hypophysectomized rats. The concentration of oxytocin in peripheral plasma in both vehicle- and morphine-infused hypophysectomized rats was at the limit of detection of the assay and was unchanged by the administration of naloxone. There were no significant differences in the secretion of CRF-41 into portal blood in vehicle- or morphine-infused hypophysectomized rats either before or after the administration of naloxone. These data show that, as for oxytocin release from the neurohypophysis into the systemic circulation, the mechanisms which regulate oxytocin release into the portal vessel blood can also be made morphine dependent. The lack of effect of morphine or naloxone on the release of CRF-41 or other stress neuro-hormones suggests that the effect of opiate dependence and withdrawal is selective for oxytocin and is not simply a non-specific response to 'stress'.

Atrial Natriuretic Factor is Released into Hypophysial Portal Blood: Direct Evidence that Atrial Natriuretic Factor may be a Neurohormone Involved in Hypothalamic Pituitary Control.

Atrial natriuretic factor (ANF) is produced in atriomyocytes (1, 2), released as a 28 amino-acid peptide into the systemic circulation and probably plays an important role in fluid and electrolyte balance (3, 4). The facts that immunoreactive ANF (ir-ANF) is also present in nerve terminals in the external layer of the median eminence (ME) (5), ir-ANF can be released from the hypothalamus in vitro by potassium depolarization (6) and specific ANF binding sites are present at a high concentration in rat pituitary tissue (7) suggest that ANF may be involved in hypothalamic-pituitary regulation. We report here that ir-ANF concentrations in hypophysial portal blood are about two to four times greater than in peripheral plasma from hypophysectomized as well as pituitary-intact adult female rats. These results show for the first time that ir-ANF is secreted from the hypothalamus into the hypophysial portal circulation at a concentration ( approximately 10(-9) M) consistent with a role for ANF as a hypothalamic-pituitary regulator or modulator.