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.     

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.     

Mitotic EBNA-positive lymphocytes in peripheral blood during infectious mononucleosis

Infectious mononucleosis (IM) is usually a benign lymphoproliferative disease caused by Epstein-Barr virus (EBV). Although EBV induces a state of continuous proliferation in infected B lymphocytes in vitro, the most prominent lymphoproliferation during IM is of activated, or atypical, T lymphocytes presumably responding to the virus or virus-infected cells. However, EBV genome-carrying cells are known to be circulating during IM, as cultured peripheral blood leukocytes from patients with the disease give rise to continuous lymphoblastoid cell lines, each cell of which contains the EBV genome and expresses the EBV determined nuclear antigen (EBNA). The proposal that EBV-infected cells in IM blood are not endowed with enhanced growth potential but are merely latently infected is supported by demonstrations that cells infected in vivo enter a viral replicative cycle when placed in vitro and that most cell lines derived from cultured lymphocytes of IM patients are infected by virus released in vitro. However the cells could also be capable of proliferation in vivo, since virus production and transformation are not mutually exclusive properties of EBV-transformed cells. Recently, EBNA has been detected in a very small fraction of peripheral blood lymphocytes of IM patients after T cells were first removed and this has been interpreted to indicate that cell transformation occurs in vivo during IM. The isolation of colonies of EBNA-positive cells from IM blood leukocytes cultures in soft agar suggests that at least some infected cells are capable of direct outgrowth into transformed cells. We report here direct evidence that circulating EBV-infected cells exhibit increased growth properties during IM.     

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.     

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.     

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.     

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.     

Immunoreactive beta-endorphin in sheep ovary

Although the ovarian production of sex steroids is of obvious physiological importance, recent studies suggest that peptides such as oxytocin, relaxin and inhibin are also synthesized in the ovary. We report here the presence of immunoreactive (ir) beta-endorphin, ir-adrenocorticotropic hormone (ACTH) and presumptive high molecular weight forms of both in extracts of sheep ovary, consistent with ovarian production from a common precursor. Our findings suggest that beta-endorphin and ACTH are produced and secreted by the follicular cells, and that their production may be related to the oestrous cycle.     

Corticotropin-releasing factor is a potent inhibitor of sexual receptivity in the female rat

Corticotropin-releasing factor (CRF), the recently characterized and synthesized 41-amino acid polypeptide isolated from ovine hypothalami, has been shown to be a potent stimulator of adenohypophyseal beta-endorphin and corticotropin (ACTH) secretion both in vitro and in vivo. In common with other regulatory peptides, CRF has also been demonstrated to possess extra-hypophysiotropic roles. Indeed, intracerebroventricularly (i.c.v.) administered CRF elicits several endocrine and behavioural responses compatible with the concept that this peptide could be a key signal in coordinating the organism's endocrine and behavioural responses to stressful and other adaptive stimuli. We now provide the first evidence for neurally placed CRF in the control of a specific hormone-dependent behavioural response and unequivocally demonstrate an extremely potent suppressive effect of CRF on sexual behaviour in the female rat when microinfused into the arcuate-ventromedial area of the hypothalamus (ARC-VMH) and the mesencephalic central grey (MCG).     

瓦氏黄颡鱼脑垂体组织学和组织化学研究

对不同季节瓦氏黄颡鱼成鱼脑垂体进行组织学和组织化学研究.结果表明:瓦氏黄颡鱼脑垂体为背腹型腺体.神经垂体居中,前腺垂体位于垂体背面前部和后缘,由PRL细胞和ACTH细胞组成;中腺垂体位于垂体中部及背面后部,包括GH细胞、GTH细胞和TSH细胞,后腺垂体位于垂体腹面后方,组织学研究仅见一种分泌细胞.前腺垂体ACTH细胞形态、中腺垂体GTH细胞形态和数量在繁殖前后有明显变化,与生殖活动密切相关.     

Different antiviral spectra of human macrophage interferon activities

The antiviral activity of alpha-interferon (IFN-alpha) produced by human leukocytes in response to viral infection has been considered to be independent of the virus which induced its production. Recently, however, IFN-alpha has been found to include at least eight different subtypes, as indicated by measurement of antigenic variability, DNA hybridization and amino acid sequencing. We considered the possibility that interferon heterogeneity may play a part in enhancing host antiviral defence and now present data suggesting that purified human macrophages, when exposed to different viruses, produce interferons having differing spectra of antiviral activity. These findings may provide a functional correlation for IFN-alpha heterogeneity.     

Most of the coding region of rat ACTH beta--LPH precursor gene lacks intervening sequences

The peptide hormones ACTH, beta-endorphin, alpha- and beta-melanotropin(MSH) and possibly gamma-MSH are synthesized in the pituitary gland by the processing of a 32,000-molecular weight (MW) polypeptide called proopiomelanocortin (POMC). The existence of a further precursor (pre form) to POMC containing an additional N-terminal 'leader' peptide has been suggested by analysis of the in vitro translation products of poly(A)-containing RNA from AtT-20 cells, a mouse ACTH-producing cell line of pituitary origin. Nakanishi et al. cloned and sequenced a cDNA copy of the bovine prePOMC mRNA. This sequence confirmed the known structure of the carboxyl half of POMC and revealed the presence of a new MSH-like moiety, gamma-MSH, within the 16,000-MW amino half of the precursor (16K fragment). Recent experiments have suggested that this peptide may act in synergy with ACTH to increase corticosterone and aldosterone production in vivo and in vitro. We have now isolated from a rat genomic DNA library a segment of a DNA encoding most of POMC, using as probe a mouse 144-base pair cloned cDNA fragment encoding beta-MSH and beta-endorphin. The cloned rat gene is one of two (or more) closely related POMC genes. The DNA sequence obtained shows that the cloned POMC gene is not interrupted by any intervening sequence (IVS) between the codon for amino acid 19 and the presumptive poly(A) addition site. This region of POMC encodes all the biologically active peptides mentioned above. The DNA sequence encoding the putative gamma-MSH and the coding sequence that precedes it are highly conserved between rat and cow. This may indicate an as yet unrecognized biological function(s) for the NH2-terminal portion of the 16K fragment.     

Binding sites for growth hormone releasing factor on rat anterior pituitary cells

Growth hormone releasing factors (GRFs) have been isolated from human pancreatic tumours (hGRF) and rat hypothalamus (rhGRF). The response to GRF at the pituitary level can be modulated by other factors, including glucocorticoids, thyroid hormones, somatostatin and other neuropeptides and somatomedins. Glucocorticoids enhance GRF-induced growth hormone (GH) secretion in primary cultures of rat anterior pituitary cells, and the synthetic glucocorticoid dexamethasone has recently been shown to increase the amounts of GH released in freely moving rats in response to submaximal doses of intravenous GRF. To investigate whether somatotroph sensitivity to GRF is modulated at its receptor level, we have developed a radioreceptor assay using an iodinated analogue of hGRF as radioligand. We report here that the relative binding affinities of rGRF, hGRF and the two analogues are correlated with their in vitro biological potencies. Further, the number of GRF binding sites is drastically decreased in cells deprived of glucocorticoids either in vivo or in vitro.     

Circulating human pituitary pro-gamma-melanotropin enhances the adrenal response to ACTH

The amino-terminal region of the common corticotropin/beta-lipotrophin (beta-LPH) precursor has been identified in the AtT-20 mouse tumor cells as a glycopeptide with an apparent molecular weight of 16,000 (the '16K fragment'). A third melanotropin core sequence or gamma-MSH similar to that found in ACTH and beta-LPH was predicted to occur in this glycopeptide from the complementary DNA sequence of mRNA isolated from bovine pituitary intermediate tissue. Recently, the mouse of 16K fragment has been found to have a small but significant potentiation on the corticosteroidogenesis elicited by ACTH in a static cell system, an effect that could be enhanced when the glycopeptide was pretreated with trypsin. This synergism could also be mimicked by synthetic gamma-MSH peptides in vitro and in vivo. We report here the potentiating properties of a naturally occurring human pro-gamma-MSH glycopeptide on the ACTH-induced steroidogenic response of isolated perfused rat and human adrenocortical cells.     

Effect of recombinant soluble CD4 in rhesus monkeys infected with simian immunodeficiency virus of macaques

The CD4 molecule is a high-affinity cell-surface receptor for the human immunodeficiency virus (HIV-1) and a soluble truncated form of CD4 produced by recombinant DNA technology is a potent inhibitor of HIV-1 replication and HIV-1-induced cell fusion in vitro. Rhesus monkeys infected with the simian immunodeficiency virus of macaques (SIVMAC), a virus closely related to HIV-1, develop an AIDS-like syndrome, and so provide an important model for the evaluation of potential AIDS therapies. We have assessed the therapeutic effect of recombinant soluble CD4 in SIVMAC-infected rhesus monkeys. Virus was readily isolated from peripheral blood lymphocytes and bone marrow cells of these animals before starting treatment with soluble CD4, but became difficult to isolate soon after treatment had begun. Moreover the diminished growth of both granulocyte-macrophage and erythrocyte progenitor colonies from the bone marrow of these monkeys rose to normal levels during treatment. These findings indicate that soluble CD4 could prove valuable in the treatment of AIDS.     

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.     

Rapid on/off cycling of cytokine production by virus-specific CD8+ T cells.

CD8-positive T cells protect the body against viral pathogens by two important mechanisms: production of antiviral cytokines and lysis of infected cells. Cytokine production can have both local and systemic consequences, whereas cytolytic activity is limited to infected cells that are in direct contact with T cells. Here we analyse activated CD8-positive T cells from mice infected with lymphocytic choriomeningitis virus and find that cytokines are not produced ex vivo in the absence of peptide stimulation, but that they are rapidly generated after T cells encounter viral peptides bound to the major histocompatibility complex. Remarkably, cytokine production ceases immediately upon dissociation of the T cells from their targets and resumes when antigenic contact is restored. In contrast to the 'on/off/on' cycling of cytokines, the pore-forming cytotoxic protein perforin is constitutively maintained. Our results indicate that there is differential expression of effector molecules according to whether the antiviral product is secreted (like cytokines) or stored inside the cell (like perforin). The ability to turn cytokines on and off while maintaining intracellular stores of perforin shows the versatility of the cellular immune response and provides a mechanism for maintaining effective immune surveillance while reducing systemic immunopathology.     

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.