Magnocellular axons in passage through the median eminence release vasopressin

Vasopressin (arginine vasopressin, AVP) is present in two types of nerve fibres in the median eminence (ME). First, it is found in nerve terminals that originate in the parvicellular neurones of the hypothalamic paraventricular nucleus (PVN) and abut on the pericapillary space surrounding the fenestrated capillaries of the primary pituitary portal plexus in the external zone (EZ) of the ME. These neurones also synthesize corticotropin-releasing factor (CRF), which acts synergetically with vasopressin to stimulate release of adrenocorticotropin (ACTH) from the pituitary gland (see ref. 7). Second, vasopressinergic axons of the magnocellular neurosecretory system pass through the internal zone (IZ) of the ME to terminate in the neurohaemal contact zone of the neurohypophysis. The involvement of vasopressinergic magnocellular neurones in the control of ACTH secretion is much debated. Of particular interest in this context is the origin of the vasopressin found in pituitary portal blood. Although it has been demonstrated that vasopressin and CRF are present in the same neurosecretory granules of EZ fibres, parallel determinations of vasopressin and CRF in pituitary portal blood have shown alterations of the concentration of vasopressin without a concomitant change in that of CRF. Such a dissociation suggests that either differential release of vasopressin and CRF can occur from a single population of nerve endings, or there are fibres in the pituitary-stalk ME which release vasopressin but not CRF. Here we present evidence for the latter. Our results indicate that stimuli causing depolarization of the axonal membrane in vitro elicit release of vasopressin from nerve fibres in the external and internal zones of the ME.     

Functional corticotropin releasing factor receptors in the primate peripheral sympathetic nervous system

Corticotropin releasing factor (CRF) is a key hormone in the integrated response to stress, acting both as the major regulator of pituitary adrenocorticotropic hormone (ACTH) release and as a neuropeptide in the brain. The actions of CRF are mediated by specific plasma membrane receptors in the anterior pituitary gland and in discrete brain areas including the cerebral cortex and several regions related to the limbic system. In addition to the pituitary and central actions of CRF, systemic administration of the peptide in the rat, dog, monkey and man causes hypotension and tachycardia because of a decrease in peripheral vascular resistance. These observations, in conjunction with the finding of immunoreactive and bioactive CRF in peripheral tissues, suggest that the peptide is locally released in tissues to act as a neurotransmitter or paracrine hormone. As CRF is present in the adrenal medulla and the peptide is known to modulate the central activity of the autonomic nervous system, we investigated the possibility that CRF is involved in the regulation of the peripheral autonomic nervous system. Such an action of CRF is supported by our demonstration of specific CRF receptors in the monkey adrenal medulla and sympathetic ganglia. In the adrenal medulla, these receptors are coupled to adenylate cyclase and can stimulate the secretion of catecholamines and Met-enkephalin.     

Modulation of stress-induced ACTH release by corticotropin-releasing factor, catecholamines and vasopressin

The stress-induced release of ACTH is believed to involve the activation of several humoral and neural pathways, including corticotropin-releasing factor (CRF), catecholamines and vasopressin. The essential role of CRF was supported by our observation that immunoneutralization of this releasing factor significantly lowers plasma ACTH levels of ether-stressed rats. However, the presence of a small but measurable residual ACTH secretion suggested the possible involvement of factors other than CRF in the stress response. We report here that pretreatment with a vasopressin antagonist decreases the plasma ACTH levels of ether-stressed rats in later (10-20 min), but not earlier (0-10 min), phases of ether stress. The ganglionic blocker chlorisondamine, inhibits ACTH release during both phases of the response to ether by 40-60% when used alone, and by 100% when administered with anti-CRF antibody. These results support a role of CRF, catecholamines and vasopressin in mediating ACTH release by ether stress.     

Co-localization of corticotropin-releasing factor and vasopressin in median eminence neurosecretory vesicles

Vasopressin (VP) potentiates the effect of corticotropin-releasing factor (CRF) on the secretion of adrenocorticotropic hormone (ACTH) from anterior pituitary cells in vitro, and both CRF and VP have been found in portal blood. These data support the hypothesis that VP acts synergistically with CRF to cause the secretion of ACTH in vivo but the origin of the CRF and VP, and the physiology of their release, have not been precisely defined. Parvocellular cell bodies in the paraventricular nucleus (PVN) which project to the external zone of the median eminence can be stained for both CRF and VP after adrenalectomy, and there is light microscopic immunocytochemical evidence that neurophysin (NP) may be located within some of the CRF-containing axons. Electron microscopic immunocytochemical studies have demonstrated the presence of CRF, VP and its 'carrier' protein, VP-associated neurophysin (NP-VP) in 100-nm neurosecretory vesicles (NSVs) in axons terminating near the portal capillary plexus in the external zone of the median eminence. If these peptides are extensively co-localized in the same NSVs in the median eminence, then coordinate secretion of CRF and VP in vivo is obligatory, at least in some physiological circumstances. We demonstrate in this report, using post-embedding electron microscopic immunocytochemistry on serial ultrathin sections, that CRF, VP and NP-VP are contained not only in the same axons and terminals, but in the same 100-nm NSVs in the median eminence of both normal and adrenalectomized rats. In addition, in the normal rat median eminence 44% of the CRF-positive axons and terminals stained strongly for VP and NP-VP, whereas in the adrenalectomized rat virtually all the CRF-positive structures in the median eminence showed strong staining for VP and NP-VP, indicating a transformation of one subpopulation of CRF-positive axons and terminals by adrenalectomy.     

Cloning and characterization of the cDNAs for human and rat corticotropin releasing factor-binding proteins.

Corticotropin-releasing factor (CRF), is a potent stimulator of synthesis and secretion of preopiomelanocortin-derived peptides. Although CRF concentrations in the human peripheral circulation are normally low, they increase throughout pregnancy and fall rapidly after parturition. Maternal plasma CRF probably originates from the placenta, which responds to the bioactive peptide and produces the peptide and its messenger RNA. Even though CRF concentrations in late gestational maternal plasma are similar to those in rat hypothalamic portal blood and to those that can stimulate release of adrenocorticotropic hormone (ACTH) in vitro, maternal plasma ACTH concentrations increase only slightly with advancing gestation and remain within the normal range. Several groups have now reported the existence of a CRF-binding protein in human plasma which inactivates CRF and which has been proposed to prevent inappropriate pituitary-adrenal stimulation in pregnancy. The binding protein was recently purified from human plasma. We have now isolated and partially sequenced the binding protein, allowing us to clone and characterize its complementary DNA from human liver and rat brain. Expression of the cDNAs for human and rat binding protein in COS7 cells showed that these proteins bind CRF with the same affinity as the native human protein. Both rat and human recombinant binding proteins inhibit CRF binding to a CRF antibody and inhibit CRF-induced ACTH release by pituitary cells in vitro.     

Evidence for local stimulation of ACTH secretion by corticotropin-releasing factor in human placenta

The hypothalamic-pituitary-adrenocortical axis is activated in pregnancy and parturition. Levels of immunoreactive corticotrophin releasing factor (irCRF), immunoreactive adrenocorticotropic hormone (irACTH) and cortisol concentrations in maternal plasma are elevated throughout gestation, increase further during labour and fall precipitously after parturition. The placenta contains biologically active CRF and ACTH and it has been suggested that the placenta produces these peptides during pregnancy. Here we show that irCRF is located in the cytotrophoblast cells of placenta collected at term. Using a monolayer primary culture of human placental cells we have found that CRF stimulates secretion of peptides containing the ACTH sequence in the placenta in a dose-dependent manner, as it does in the pituitary. This effect is reversed by a CRF antagonist and is mimicked by dibutyryl cyclic AMP and forskolin. Glucocorticoids, which suppress the secretion of pituitary ACTH, were found to have no influence on release of irACTH by the placenta. Oxytocin and prostaglandins stimulate irACTH and irCRF secretion from cultured placental cells and the irACTH-releasing activity of two prostaglandins is partially reversed by a CRF antagonist. Thus CRF may be involved in the paracrine regulation of placental irACTH secretion.     

Somatostatin selectively inhibits noradrenaline release from hypothalamic neurones

Somatostatin in a hypothalamic peptide hormone which inhibits growth hormone release from the anterior pituitary. However, biochemical and morphological investigations have revealed that somatostatin is located not only in the hypothalamus but also in other brain areas (for example the cerebral cortex) where it occurs and in nerve cell bodies and fibres from which it can be released in a Ca2+-dependent manner. It has therefore been suggested that the neuropeptide may have functions in the central nervous system other than its effect on growth hormone release; one possible action is that of a neuromodulator. Therefore, hypothalamic and cerebral cortical slices of the rat were used to examine whether somatostatin modifies the electrically or CaCl2-evoked release of tritiated monoamines from monoaminergic neurones. it is reported here that somatostatin inhibits 3H-noradrenaline release from the hypothalamus (but not from the cerebral cortex) but does not affect the release of 3H-dopamine and 3H-serotonin.     

Corticotropin releasing factor induction of leukocyte-derived immunoreactive ACTH and endorphins

Human peripheral leukocytes infected by virus or treated with endotoxin will, like unstimulated mouse spleen macrophages, synthesize immunoreactive corticotrophin (ir-ACTH) and endorphins. The ir-ACTH produced appears to be identical with authentic ACTH, while enough of the material has been produced in hypophysectomized mice infected with virus to demonstrate a steroidogenic response. Because the production of ACTH by in vivo pituitary cells and by leukocytes is suppressed by dexamethasone both in vitro and in vitro, suggesting that the production of ACTH and endorphins by leukocytes is indeed controlled, we have investigated the effects of corticotropin releasing-factor (CRF), which is known to regulate the pituitary production of both ACTH and beta-endorphin. We now report that the production of ACTH and endorphins by leukocytes is indeed induced by synthetic CRF and, in turn, suppressed by dexamethasone, suggesting that, as in pituitary cells, the proopiomelanocortin (POMC) gene may be expressed and similarly controlled in leukocytes.     

Absence of oestradiol concentration in cell nuclei of LHRH-immunoreactive neurones

Oestrogen, acting in both the brain and pituitary, has a critical role in regulating the reproductive cycle in most mammals. In the brain, oestrogen regulates the release of luteinizing hormone-releasing hormone (LHRH) partly through a mechanism that is blocked by inhibitors of DNA-dependent RNA synthesis or protein synthesis. The distributions of oestrogen-concentrating neurones and of LHRH neurones overlap. The present study was undertaken to determine whether genomic effects of oestrogen mediated by nuclear oestradiol concentration include a direct effect on LHRH-containing neurones. During extensive studies in which the immunocytochemical method for localizing LHRH neurones was optimized and made compatible with the autoradiographic method for detecting oestrogen-concentrating neurones, doubly-labelled cells were very rarely seen. This suggests that genomic regulatory effects of oestrogen which depend on nuclear retention are not exerted directly on most LHRH neurones, but rather must be mediated by another class of neurones.     

Corticotropin releasing activity of the new CRF is potentiated several times by vasopressin

Initially the hypothalamic factor responsible for the release of corticotropin (CRF), was thought to be a simple peptide. More recent work has led to the conclusion that CRF is a multifactorial complex. In 1979 we proposed that vasopressin, much disputed as a CRF candidate, was a major constituent of the complex, interacting with a potentiating the CRF activity of the other component(s). The recent characterization of a 41 residue ovine hypothalamic peptide capable of releasing adrenocorticotropic hormone (ACTH) in a dose-related manner has allowed us to compare its CRF bioactivity with that of vasopressin and simple extracts of the hypothalamus, and to investigate any interaction it may have with vasopressin and other hypothalamic factors in the release of ACTH. We report here that the new CRF is more potent than vasopressin in releasing ACTH. When given simultaneously with vasopressin a fourfold potentiation of CRF activity with steep dose-response characteristics were observed. It also potentiated vasopressin-free hypothalamic extracts, suggesting that a new CRF does not account for all the nonvasopressin portion of the CRF complex.     

Changes in hypothalamic preproenkephalin A mRNA following stress and opiate withdrawal

The median eminence of the pituitary is rich in opioid receptors, and exogenous opioids have major effects on the release of adrenocorticotropic hormone (ACTH), luteinizing hormone (LH), prolactin, growth hormone (GH) and thyrotropin. Stress results in similar changes in anterior pituitary hormone secretion. Enkephalin immunoreactivity has been reported in the medial parvocellular neurons of the hypothalamic paraventricular nucleus which project to the median eminence, the site where hypothalamic releasing factors are secreted into the portal blood and thence to the anterior pituitary gland. The endocrine response to stressful stimuli might therefore, at least in part, be mediated through the activation of hypothalamic enkephalinergic neurons. We show that two stressful stimuli, opiate withdrawal and intraperitoneal injection of hypertonic saline, both result in very rapid and marked increases in enkephalin mRNA in the parvocellular paraventricular nucleus. The activation of hypothalamic enkephalin neurons may be important in the neuroendocrine response to stress.     

Reciprocal changes in corticotropin-releasing factor (CRF)-like immunoreactivity and CRF receptors in cerebral cortex of Alzheimer's disease

Alzheimer's disease is a progressive degenerative disease of the nervous system characterized neuropathologically by the presence of senile plaques and neurofibrillary tangles in amygdala, hippocampus and neocortex. Dysfunction and death of basal forebrain cholinergic neurones projecting to forebrain targets are associated with marked decreases in cholinergic markers, including the activity of choline acetyltransferase (ChAT). Although cortical levels of somatostatin and somatostatin receptors are reduced in Alzheimer's, no consistent changes have been reported in other neuropeptide systems. We have now examined in control and Alzheimer's brain tissues pre- and postsynaptic markers of corticotropin-releasing factor (CRF), a hypothalamic peptide regulating pituitary-adrenocortical secretion which also seems to act as a neurotransmitter in the central nervous system (CNS). We have found that in Alzheimer's, the concentrations of CRF-like immunoreactivity (CRF-IR) are reduced and that there are reciprocal increases in CRF receptor binding in affected cortical areas. These changes are significantly correlated with decrements in ChAT activity. Our results strongly support a neurotransmitter role for CRF in brain and demonstrate, for the first time, a modulation of CNS CRF receptors associated with altered CRF content. These observations further suggest a possible role for CRF in the pathophysiology of the dementia. Future therapies directed at increasing CRF levels in brain may prove useful for treatment.     

Regulation of pro-opiomelanocortin biosynthesis and processing by transplantation immunity

It is more than thirty years since Billingham and Medawar showed that adrenocorticotrophic hormone (ACTH) and cortisol can prolong the survival of skin allografts. It has since become clear that glucocorticoid hormones are critically involved in the regulation of immunity. The level of glucocorticoids secreted in response to antigenic challenge corresponds to the magnitude of the immune response and in general reaches immunosuppressive levels. Interestingly, not all immune responses enhance ACTH and glucocorticoid hormone production. In transplantation immunity, the reverse seems to be true: circulating glucocorticoid levels at the time of skin graft rejection are lower than control levels. Because beta-endorphin and ACTH originate from the same prohormone, pro-opiomelanocortin (POMC), and are closely related in their tissue-specific processing and coordinate release, we have investigated the role of pituitary beta-endorphin in transplantation immunity. We report here that POMC biosynthesis and processing in the pars intermedia, but not in the anterior pituitary, can be regulated by T cell-specific factors secreted in animals undergoing transplantation immunity.     

Characterization of single pacemaker channels in cardiac sino-atrial node cells

Normal pacemaking in the mammalian heart is driven by spontaneously active cells located in the sino-atrial (SA) node. The rate of firing of these cells and the modulation of this rate by catecholamines are controlled by if, an inward Na- and K-current that turns on at voltages more negative than -40 mV. The 'pacemaker' current if is also present in other types of cell where its ability to produce and modulate a depolarizing process may be useful. For example, in vertebrate photoreceptors if drives the depolarization that terminates the light-induced hyperpolarization. Currents similar to if are also found in hippocampal neurones and DRG neurones. The present report shows for the first time that the opening of single if-channels of low conductance (1 pS) can be resolved using a modification of the patch-clamp technique on isolated SA-node cells. Modulation of if by adrenaline is shown to be mediated by an increase in the probability of channel opening, whereas the single-channel amplitude remains unchanged.     

Evidence that the insulin secretagogue, beta-cell-tropin, is ACTH22-39

The pituitary neurointermediate lobe of genetically obese (ob/ob) mice contains a hormone which stimulates insulin release and which cross-reacts with a -COOH-terminal ACTH antiserum, suggesting that it is related to corticotropin-like intermediate lobe peptide (CLIP), the 18-39 fragment of ACTH. The hormone, which we have called beta-cell-tropin, has been shown to be present in the plasma of the ob/ob mouse and to potentiate glucose induced insulin secretion. We have now shown that ACTH22-39 prepared by tryptic digestion of human synthetic CLIP behaves similarly on Biogel chromatography and on reverse-phase HPLC to the naturally occurring beta-cell-tropin. Furthermore, beta-cell-tropin and ACTH22-39 have indistinguishable antigenic and insulin releasing properties.     

Dopaminergic stimulation of prolactin release

Prolactin (PRL) secretion from anterior pituitary is believed to be under tonic inhibitory control of dopamine (DA) released from the tubero-infundibular dopaminergic neurones into the hypophysial portal blood. Inhibition of PRL release by DA seems to be mediated by sereospecific DA receptors located in PRL cells. Apomorphine and various ergot alkaloids such as bromocryptine mimic the inhibitory effect of DA both in vivo and in vitro, presumably by a direct agonist action on these 'inhibitory' receptors. We now report that PRL secretion in primary cultures of rat pituitary cells can be stimulated by DA when concentrations a thousand times lower than those required for inhibition are used. Secretion rates above basal release can also be induced by apomorphine and bromocryptine when the 'inhibitory' receptors are blocked with certain DA receptor antagonists.     

Auto-inhibition of brain histamine release mediated by a novel class (H3) of histamine receptor

Although histaminergic neurones have not yet been histochemically visualized, there is little doubt that histamine (HA) has a neurotransmitter role in the invertebrate and mammalian central nervous system. For example, a combination of biochemical, electrophysiological and lesion studies in rats have shown that histamine is synthesized in and released from a discrete set of neurones ascending through the lateral hypothalamic area and widely projecting in the telencephalon. Histamine acts on target cells in mammalian brain via stimulation of two classes of receptor (H1 and H2) previously characterized in peripheral organs and probably uses Ca2+ and cyclic AMP, respectively, as second messengers. It is well established that several neurotransmitters affect neuronal activity in the central nervous system through stimulation not only of postsynaptic receptors, but also of receptors located presynaptically which often display distinct pharmacological specificity and by which they may control their own release. Such 'autoreceptors' have been demonstrated (or postulated) in the case of noradrenaline, dopamine, serotonin, acetylcholine and gamma-aminobutyric acid (GABA) neurones but have never been demonstrated for histamine. We show here that histamine inhibits its own release from depolarized slices of rat cerebral cortex, an action apparently mediated by a class of receptor (H3) pharmacologically distinct from those previously characterized, that is, the H1 and H2 receptors.     

精氨酸加压素免疫阳性神经元在中菊头蝠(Rhinolophus affinis)脑中的分布

精氨酸加压素(arginine vasopressin,AVP)属于垂体后叶激素家族,它与神经内分泌的调节、心血管功能的调节、血压调节、学习和记忆以及生殖等生理功能密切相关.AVP在不同哺乳动物中枢神经系统内的分布方式和传输路径存在明显差异.本实验采用免疫组织化学ABC法系统观察了AVP免疫阳性神经元和免疫阳性神经纤维在中菊头蝠(Rhinolophus affinis)脑中的形态特征和分布特点.结果显示AVP免疫阳性神经元和免疫阳性神经纤维明显可见于中菊头蝠下丘脑室旁核、视上核、视交叉上核、下丘脑外侧区、正中隆起和垂体后叶,其细胞形态与其它哺乳动物相应结构的细胞特征类似,提示AVP神经元在哺乳动物下丘脑中的分布具有高度的保守性,AVP在中菊头蝠下丘脑可能有着与其它哺乳动物类似的功能.室周视前区、终纹床核和前脑外侧束也有少量AVP免疫阳性(immunoreactive arginine-vasopressin,AVP-ir)神经元分布,而在海马、隔核、杏仁核和侧间隔等边缘核团没有发现与大鼠等哺乳动物相应结构类似的分布,这可能与蝙蝠的视觉退化有关.