Increase of corticotropin-releasing factor staining in rat paraventricular nucleus neurones by depletion of hypothalamic adrenaline

In response to stress, adrenocorticotropic hormone (ACTH) is released by corticotrophs in the anterior pituitary under the control of several central and peripheral factors including corticotropin-releasing factor (CRF), which was recently isolated from the brain and sequenced. Immunocytochemical studies have shown that most of the CRF-containing cell bodies that project to the median eminence are present in the hypothalamic paraventricular nucleus (PVN). A dense PNMT(phenylethanolamine-N-methyltransferase)-containing fibre network was also observed in the same region--PNMT is the final enzyme in the biosynthesis of adrenaline and has been demonstrated in the brain. In the present study we found an association of adrenergic nerve fibres and CRF neurones by immunohistochemistry using antisera to PNMT and CRF. To examine the functional significance of the adrenergic projection to the PVN, we blocked the synthesis of adrenaline using a specific inhibitor of PNMT. The depletion of adrenaline resulted in an increase in CRF immunoreactivity. The present results suggest that, as well as catecholamines which regulate ACTH release at the anterior pituitary level via a beta 2-adrenergic receptor mechanism, central catecholamines (mainly adrenaline) also affect ACTH release through their action on CRF cells. Peripheral catecholamines seem to have a direct stimulatory effect on the pituitary corticotroph cells, whereas the present findings suggest that central adrenaline-containing neurones have an inhibitory role in the physiological response to 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.     

Evidence for opiate receptors on pituicytes

A hypothalamo-neurohypophyseal enkephalinergic pathway has been described and the pars nervosa of the rat pituitary contains enkephalin-like material which may coexist in vasopressin and oxytocin terminals. At the level of the pars nervosa itself, stereospecific opiate receptors with properties very similar to those of brain receptors have been described, and opiates have been shown to inhibit the release of both vasopressin and oxytocin. The location of the opiate receptors involved has been presumed to be pre-terminal on the neurosecretory fibres. Using an autoradiographic technique to visualize opiate receptors, however, we now report that destruction of the neurosecretory fibres following pituitary stalk section does not result in a significant change in the neural lobe opiate receptor population. This suggests that the opiate receptors within the neural lobe may be present on pituicytes rather than on neurosecretory fibres.     

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.     

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.     

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.     

Relaxin affects the release of oxytocin and vasopressin from the neurohypophysis

The hormone relaxin has recently been shown to inhibit not only uterine muscle contraction, but also the release of oxytocin into the plasma. Intravenous injection of porcine relaxin in anaesthetized lactating rats inhibits milk ejection and injection of relaxin into the cerebral ventricles disturbs the pattern of the milk ejection reflex. Recent experiments performed in vivo indicate that relaxin might act not only in the uterus, but also in the hypothalamus and possibly in the neurohypophysis. We tested this hypothesis in vitro by studying the effect of relaxin on hormone release from isolated neural lobes of the pituitary and isolated neurosecretory nerve endings of the neurohypophysis from the rat. We report here that relaxin has a dual effect on neurohypophysial hormone secretion. Under basal conditions, vasopressin and oxytocin release was inhibited by relaxin but, when the nerve endings were depolarized, vasopressin and oxytocin secretion was potentiated. We also found that relaxin acts at a stage before the increase in cytoplasmic free Ca2+ that is necessary for inducing hormone release, possibly by gating the calcium channel.     

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.     

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.     

赤点石斑鱼脑垂体超微结构的初步研究

 应用透射电镜技术观察不同时期的性成熟赤点石斑鱼（Epinephelus akaara）脑垂体的超微结构。结果表明,性成熟赤点石斑鱼脑垂体近椭圆形,由神经垂体和腺垂体两部分组成。神经垂体主要由神经纤维、脑垂体细胞和微血管组成。其中神经分泌纤维存在A(A1、A2)和B型神经分泌纤维。腺垂体中外侧部（PPD）腺细胞由嗜酸性的GH细胞和嗜碱性的TSH细胞及GTH细胞3类细胞构成。在不同的时期,GTH细胞内的分泌颗粒明显不同。     

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.     

Inhibin beta in central neural pathways involved in the control of oxytocin secretion

Inhibin (I) a gonadal hormone glycoprotein which suppresses follicle-stimulating hormone (FSH) secretion from the anterior pituitary, is a heterodimer consisting of an alpha subunit and one of two distinct beta subunits. S1 nuclease analysis has revealed that RNAs encoding all three subunits (alpha, beta A and beta B) are expressed in rat brain. We report here on the localization, and a potential function, of inhibin beta in the rat brain. A cell group centred in the nucleus of the solitary tract (NTS), a major recipient of visceral sensory information, was stained immunohistochemically with antisera against synthetic fragments of I beta, but not I alpha. The distribution of I beta-stained fibres is consistent with known NTS projections, and includes a prominent projection to oxytocinergic aspects of the magnocellular neurosecretory system.     

法国鹌鹑脑垂体神经叶的超微结构研究

用电子显微镜技术,对法国鹌鹑脑垂体神经叶的超微结构进行了研究,观察结果表明,脑垂体神经叶由无髓神经纤维及末梢、垂体细胞,室管膜细胞、毛细血管、结缔组织构成,无髓神经纤维末梢内含许多神经分泌颗粒,大多终止于有孔型毛细血管的基膜外,这为分泌物质放入血提供了有利条件,神经叶中漏斗隐有面有一层室管膜细胞,室管膜细胞基部与神经末梢紧密相接,此外胞质中可见微吞饮小泡,因此认为,室管膜细胞可能参与社会分泌物质的释放。     

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.     

Oxytocin-immunoreactive terminals synapse on oxytocin neurones in the supraoptic nucleus

The neuropeptide oxytocin, synthesized by magnocellular neurones in the hypothalamus, is well known for its peripheral action during lactation and parturition after its release into the bloodstream from axons in the neurohypophysis. However, it may also be released centrally to control the activity of oxytocinergic neurones themselves. Oxytocin release has been measured from isolated magnocellular nuclei in vitro and in the cerebrospinal fluid. When injected into the third ventricle, the peptide increases the basal firing rate of oxytocinergic neurones as well as the frequency and amplitude of the bursts of action potentials they normally show before each reflex milk ejection. Oxytocin also excites magnocellular neurones when applied microiontophoretically. I now report that immunocytochemical staining reveals synapses in the supraoptic nucleus of the hypothalamus where both the pre- and postsynaptic elements contain oxytocin. These oxytocinergic synapses, impinging on their own neurones, may represent the anatomical basis for the hypothalamic release of this peptide and for the facilitatory action on its own secretion.     

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.     

Inhibition of the firing of vasopressin neurons by atriopeptin

Atriopeptin, the atrial natriuretic peptide, is a circulating hormone that is released from the atria of mammalian hearts in response to volume expansion and acts upon the kidneys, adrenal glands and vasculature to regulate fluid and electrolyte homeostasis. Atriopeptin is also present in the brain of the rat. Atriopeptin immunoreactive cell bodies and fibres are found in many areas known to be involved in the central regulation of the cardiovascular system, suggesting that it may be a neuromediator in the central control of fluid and electrolyte balance. The paraventricular nucleus of the hypothalamus, which contains the cell bodies of neurons that secrete vasopressin from the posterior pituitary gland, receives a dense innervation from atriopeptin-like immunoreactive fibres. We have studied the effect of atriopeptin on the electrical activity of single neurons in the paraventricular nucleus of anaesthetized rats and found that atriopeptin is a potent inhibitor of putative vasopressin neurons. Atriopeptin, which has systemic actions that oppose those of vasopressin, may act as a neuromodulator in the brain to prevent vasopressin secretion.     

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.     

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.