Substance P regulates the vasodilator activity of calcitonin gene-related peptide

The 37-amino-acid calcitonin gene-related peptide (CGRP) occurs as a result of alternative processing of mRNA from the calcitonin gene. The potency of CGRP as a vasodilator and the occurrence of the peptide in nerves associated with blood vessels suggest an important role for CGRP in the regulation of blood flow. The finding that CGRP induces protracted vasodilatation when administered extra-vascularly, to mimic release from nerves, has led us to investigate how the vasodilator activity of CGRP is controlled in vivo. CGRP is often co-localized with substance P in C-fibre nerves. Here, we demonstrate that injection of CGRP with substance P into human skin converts the long-lasting vasodilatation induced by CGRP into a transient response. Experiments in animals reveal that the phenomenon is dependent on the action of proteases from mast cells stimulated by substance P. The results reveal a new regulatory interaction between two neuropeptides and provide evidence for an in vivo role for mast cell proteases.     

Arterial dilations in response to calcitonin gene-related peptide involve activation of K+ channels

Calcitonin gene-related peptide (CGRP) is a 37-amino-acid peptide produced by alternative processing of messenger RNA from the calcitonin gene. CGRP is one of the most potent vasodilators known. It occurs in and is released from perivascular nerves and has been detected in the blood stream, suggesting that it is important in the control of blood flow. The mechanism by which it dilates arteries is not known. Here, we report that arterial dilations in response to CGRP are partially reversed by blockers of the ATP-sensitive potassium channel (K(ATP)), glibenclamide and barium. We also show that CGRP hyperpolarizes arterial smooth muscle and that blockers of K(ATP) channels reverse this hyperpolarization. Finally, we show that CGRP opens single K+ channels in patches on single smooth muscle cells from the same arteries. We propose that activation of K(ATP) channels underlies a substantial part of the relaxation produced by CGRP.     

Calcitonin gene-related peptide acts as a novel vasodilator neurotransmitter in mesenteric resistance vessels of the rat

Systemic blood pressure is controlled by changes in the resistance of the peripheral vascular bed for example in the mesenteric blood vessels. The tone of peripheral blood vessels is primarily maintained by sympathetic vasoconstrictor nerves. Although vasodilator innervation has been identified in certain isolated elastic arteries, it is not known whether vasodilator nerves contribute to the regulation of the peripheral resistance vessels. We present pharmacological evidence for the existence of nonadrenergic, noncholinergic (NANC) vasodilator nerves in the mesenteric resistance vessel of the rat and that the resistance is controlled by not only sympathetic vasoconstrictor nerves but also NANC vasodilator nerves. We also show that the neurogenic vasodilation was selectively abolished by depleting endogenous calcitonin gene-related peptide (CGRP), a potent vasodilator neuropeptide, from perivascular nerves. This indicates that CGRP is a novel vasodilator neurotransmitter and may play a role in control of the total peripheral resistance of systemic circulation through a local reflex mechanism.     

Pancreatic amylin and calcitonin gene-related peptide cause resistance to insulin in skeletal muscle in vitro

Insulin resistance occurs in a variety of conditions, including diabetes, obesity and essential hypertension, but its underlying molecular mechanisms are unclear. In type 2 (non-insulin-dependent) diabetes mellitus, it is insulin-resistance in skeletal muscle, the chief site of insulin-mediated glucose disposal in humans, that predominantly accounts for the low rates of glucose clearance from the blood, and hence for impaired glucose tolerance. Human type 2 diabetes is characterized by a decrease in non-oxidative glucose storage (muscle glycogen synthesis), and by the deposition of amyloid in the islets of Langerhans. Amylin is a 37-amino-acid peptide which is a major component of islet amyloid and has structural similarity to human calcitonin gene-related peptide-2 (CGRP-2; ref. 8). CGRP is a neuropeptide which may be involved in motor activity in skeletal muscle. We now report that human pancreatic amylin and rat CGRP-1 are potent inhibitors of both basal and insulin-stimulated rates of glycogen synthesis in stripped rat soleus muscle in vitro. These results may provide a basis for a new understanding of the molecular mechanisms that cause insulin resistance in skeletal muscle.     

mRNA sequence for human cardiodilatin-atrial natriuretic factor precursor and regulation of precursor mRNA in rat atria

The mammalian cardiac atrium has recently been shown to contain numerous peptides that exert marked effects on kidney function and vascular resistance. With one exception, the peptides have potent natriuretic and diuretic activities, and sequence similarities suggest that they may be derived from a common atrial natriuretic factor (ANF) precursor. The exception, cardiodilatin (CDD), differs from the ANF peptides in possessing potent vasorelaxant activity, but not natriuretic and diuretic activities. Here we report the cloning and sequence analysis of the cDNA for the human precursor protein (preproCDD-ANF) containing both the CDD and ANF sequences. The CDD sequence represents the N-terminal sequence preceded directly by a signal peptide, while the ANF sequence is present at the C-terminal end of the protein. Using hybridization analysis, we further show that the amount of rat preproCDD-ANF mRNA, which is synthesized selectively in the atria but not the ventricles, markedly decreases on water deprivation, suggesting that the water-electrolyte balance may be an important factor in the regulation of the expression of the preproCDD-ANF gene.     

AVP对VSMC脂质过氧化的影响及CGRP、SP的调节作用

目的：研究AVP对VSMC脂质过氧化的影响及CGRP、SP的调节作用。方法：以培养的大鼠血管平滑肌细胞（VSMC）为模型,动态观察了精氨酸加压素（AVP）对VSMC脂质过氧化的影响,及降钙素基因相关肽（CGRP）、P物质（SP）对AVP作用的调节,旨在探讨它们在高血压病发病中的意义。结果：（1）10－7M的AVP作用后,VSMP的丙二醛（MDA）含量明显高于对照组（P＜0．01）;（2）10－7M的CGRP、SP分别与10－7M的AVP共同作用后,VSMP的MDA含量均减少,与AVP对照组比较,差异非常显著（P＜0．01）。结论：AVP参与高血压病的发病与其致VSMC的脂质过氧化损伤有关,CGRP、SP对其有拮抗作用。     

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.     

Expression of dopaminergic phenotypes in the mouse olfactory bulb induced by the calcitonin gene-related peptide

In the olfactory bulb, tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of catecholamines, is expressed after birth when the axons of olfactory epithelial neurons have made synapses in the bulb. It has been suggested that expression of TH is regulated trans-synaptically because on deafferentation of the bulb there is a marked decrease in the contents of TH, dopamine and 3,4-dihydroxyphenylacetic acid, which, however, return to normal levels after regeneration of the primary afferents. To date the molecular signalling involved in this trans-synaptic induction has not yet been characterized; I have therefore studied the expression of dopaminergic properties (presence of TH and dopamine uptake) in dissociated cell cultures from embryonic mouse olfactory bulb. I report that the number of dopaminergic cells increases fivefold when olfactory bulb neurons are co-cultured with olfactory epithelial neurons and that soluble factors, rather than cell interactions, mediate this effect. The dopaminergic-inducing factor is the calcitonin gene-related peptide (CGRP) which is present in chemosensory neurons of the olfactory epithelium and when added at nanomolar concentrations to olfactory bulb cultures mimics the effect of olfactory epithelial neurons. Significantly the induction of dopaminergic phenotypes brought about by olfactory epithelial neurons is abolished by an antiserum to CGRP. These observations show that CGRP is involved in the differentiation of dopaminergic olfactory bulb neurons.     

Calcitonin gene-related peptide regulates calcium current in heart muscle

The influx of Ca2+ due to the transmembrane calcium current, ICa, has a fundamental role in cardiac pacemaker activity, in the action potential plateau and in excitation-contraction coupling. Both sympathetic and parasympathetic neurotransmitters can modulate ICa. Recent studies indicate that in both the cardiovascular and the central nervous systems, nerve varicosities exist that contain a novel non-adrenergic, non-cholinergic peptide--calcitonin gene-related peptide (CGRP). Although CGRP is known to exert strong positive inotropic and chronotropic effects, as well as to cause vasodilation, very little is known about the ionic mechanisms of these effects. Here we report that CGRP dramatically increases ICa in single heart cells. Although this CGRP-induced increase in ICa resembles the effect of beta-adrenergic agonists, our results demonstrate some significant differences between the effects of CGRP and these agonists: (1) the increase due to CGRP cannot be blocked by beta-adrenergic antagonists; (2) the CGRP-induced effect is transient; and, (3) CGRP can inhibit isoproterenol-stimulated ICa. Our results provide the first electrophysiological evidence that CGRP can significantly modulate ICa in the heart, and suggest a new additional mechanism for the neurogenic control of cardiac function.     

Structural identification of beta- and gamma-human atrial natriuretic polypeptides

Atrial natriuretic polypeptides (ANPs) of varying chain length have been identified recently in human and rat atrial tissue. Their potent natriuretic-diuretic activities indicate their key role in the regulation of extracellular fluid volume and electrolyte balance. Furthermore, human and rat cDNAs encoding their precursor have been cloned and identified. Natriuretic-diuretic activity in human atrial extract comprises three distinct components (alpha, relative molecular mass (Mr) approximately 3,000; beta, Mr approximately 6,000; gamma, Mr approximately 13,000). However, only the 3,000-Mr peptide, alpha-human atrial polypeptide (alpha-hANP), comprising 28 amino acids, has so far been identified. We report here the purification and sequence analysis of two novel hANPs of higher Mr, beta- and gamma-hANP, both of which exhibit natriuretic and hypotensive activity. gamma-hANP, composed of 126 amino acids, carries the alpha-hANP sequence at its carboxy terminus. The identification of gamma-hANP reveals that the peptide, being the largest form of hANP, is processed directly from a 151-residue precursor by removal of a 26-residue signal peptide. In contrast, beta-hANP (56 residues) comprises an anti-parallel dimer of alpha-hANP; such a dimeric peptide possessing bioactivity has never been found in the tissue as an endogenous entity.     

Vanilloid receptors on sensory nerves mediate the vasodilator action of anandamide.

The endogenous cannabinoid receptor agonist anandamide is a powerful vasodilator of isolated vascular preparations, but its mechanism of action is unclear. Here we show that the vasodilator response to anandamide in isolated arteries is capsaicin-sensitive and accompanied by release of calcitonin-gene-related peptide (CGRP). The selective CGRP-receptor antagonist 8-37 CGRP, but not the cannabinoid CB1 receptor blocker SR141716A, inhibited the vasodilator effect of anandamide. Other endogenous (2-arachidonylglycerol, palmitylethanolamide) and synthetic (HU 210, WIN 55,212-2, CP 55,940) CB1 and CB2 receptor agonists could not mimic the action of anandamide. The selective 'vanilloid receptor' antagonist capsazepine inhibited anandamide-induced vasodilation and release of CGRP. In patch-clamp experiments on cells expressing the cloned vanilloid receptor (VR1), anandamide induced a capsazepine-sensitive current in whole cells and isolated membrane patches. Our results indicate that anandamide induces vasodilation by activating vanilloid receptors on perivascular sensory nerves and causing release of CGRP. The vanilloid receptor may thus be another molecular target for endogenous anandamide, besides cannabinoid receptors, in the nervous and cardiovascular systems.     

Human urotensin-II is a potent vasoconstrictor and agonist for the orphan receptor GPR14.

Urotensin-II (U-II) is a vasoactive 'somatostatin-like' cyclic peptide which was originally isolated from fish spinal cords, and which has recently been cloned from man. Here we describe the identification of an orphan human G-protein-coupled receptor homologous to rat GPR14 and expressed predominantly in cardiovascular tissue, which functions as a U-II receptor. Goby and human U-II bind to recombinant human GPR14 with high affinity, and the binding is functionally coupled to calcium mobilization. Human U-II is found within both vascular and cardiac tissue (including coronary atheroma) and effectively constricts isolated arteries from non-human primates. The potency of vasoconstriction of U-II is an order of magnitude greater than that of endothelin-1, making human U-II the most potent mammalian vasoconstrictor identified so far. In vivo, human U-II markedly increases total peripheral resistance in anaesthetized non-human primates, a response associated with profound cardiac contractile dysfunction. Furthermore, as U-II immunoreactivity is also found within central nervous system and endocrine tissues, it may have additional activities.     

Purification, sequencing and synthesis of natriuretic and vasoactive rat atrial peptide

Mammalian atria contain potent natriuretic and diuretic substances which exist in high- and low-molecular-weight forms and which appear to be associated with atrium-specific granules. The natriuretic effect of atrial extract is largely accountable for by its renal haemodynamic effects; atrial extracts also antagonize hormone- and non-hormone-induced contraction of the isolated rabbit aorta and isolated rat kidney vasculature. We have completely purified a low-molecular-weight natriuretic and vasoactive substance from rat atria and characterized it as a 24-amino acid peptide. Synthetic peptide, produced by solid-phase synthesis, mimics biological effects of crude atrial extract and purified peptide; its activity is enhanced by slow oxidation, suggesting a disulphide (Cys 4-Cys 20) configuration for the native peptide. If secreted into blood, this atrial natriuretic peptide (' auriculin B') could be a novel peptide hormone of considerable importance to renal and cardiovascular homeostasis.     

Coffee contains potent opiate receptor binding activity

Opiate receptor-active peptide fragments (exorphins) have been identified recently in casein and gluten hydrolysates, and morphine has been found in bovine and human milk. To determine whether similar peptides or alkaloids occur in other foodstuffs, we have screened potential sources using a rat brain homogenate assay to detect opiate receptor activity. We report here that instant coffee powders from a variety of manufacturers compete with tritiated naloxone for binding to opiate receptors in the rat brain membrane preparations, with no significant difference between normal and decaffeinated coffee. The receptor binding activity resembles that seen with opiate antagonists, in that there was no change in the half-maximal effective dose (ED50) in the presence of 100 mM Na+; on bioassay, the activity was similarly shown to be antagonistic and specific for opiate-induced inhibition of twitch. Preliminary characterization of the activity reveals that it has a molecular weight (MW) in the range 1,000-3,500, is heat-stable, ether-extractable, not modified by enzymatic digestion with papain, and clearly separable from caffeine and morphine on TLC. As its concentration in an average cup of coffee is five times the ED50, these data suggest that drinking coffee may be followed by effects mediated via opiate receptors, as well as effects of caffeine.     

Immunocytochemical localization in rat brain of a prolactin release-inhibiting sequence of gonadotropin-releasing hormone prohormone

The structure of a precursor protein for gonadotropin-releasing hormone (GnRH) of relative molecular mass 10,000 has recently been deduced from cloned complementary DNA sequences derived from human placental messenger RNA. The 56-amino-acid peptide representing residues 14-69 of this prohormone exhibits potent inhibition of prolactin secretion. To investigate whether the same prohormone is synthesized in mammalian brain and describe the anatomical distribution of the prolactin-inhibiting region of this molecule, we have generated antiserum to a synthetic peptide containing residues 40-53 of the human placental precursor. We report here that a substance recognized by this antibody is present in GnRH-containing neurones of the rat brain and appears to coexist with GnRH in secretory granules of nerve terminals in the median eminence. These results indicate homology between hypothalamic and placental prohormones for GnRH and are consistent with the suggestion elsewhere in this issue that a prolactin-inhibiting factor (PIF) is generated from this prohormone and cosecreted with GnRH by nerve terminals in the median eminence.     

Identification of receptors for neuromedin U and its role in feeding

Neuromedin U (NMU) is a neuropeptide with potent activity on smooth muscle which was isolated first from porcine spinal cord and later from other species. It is widely distributed in the gut and central nervous system. Peripheral activities of NMU include stimulation of smooth muscle, increase of blood pressure, alteration of ion transport in the gut, control of local blood flow and regulation of adrenocortical function. An NMU receptor has not been molecularly identified. Here we show that the previously described orphan G-protein-coupled receptor FM-3 (ref. 15) and a newly discovered one (FM-4) are cognate receptors for NMU. FM-3, designated NMU1R, is abundantly expressed in peripheral tissues whereas FM-4, designated NMU2R, is expressed in specific regions of the brain. NMU is expressed in the ventromedial hypothalamus in the rat brain, and its level is significantly reduced following fasting. Intracerebroventricular administration of NMU markedly suppresses food intake in rats. These findings provide a molecular basis for the biochemical activities of NMU and may indicate that NMU is involved in the central control of feeding.     

Isolation of a cDNA clone encoding rat insulin-like growth factor-II precursor

Insulin-like growth factor-I (IGF-I) and IGF-II are mitogenic polypeptides of relative molecular mass (Mr) approximately 7,500 isolated from human plasma each containing four peptide domains in a single chain and identical at more than 60% of their amino acid loci. The B- and A-domains of the IGFs are approximately 40% identical to the B- and A-chains of human insulin. IGF-I and IGF-II have similar in vitro biological activities and receptor reactivity, but are immunologically distinct. IGF-I appears to mediate the effects of growth hormone on cartilage to promote skeletal growth whereas IGF-II may have a special role in fetal development and in the central nervous system. To investigate the in vivo role of IGF-II, we have studied IGF-II biosynthesis in the BRL-3A rat liver cell line. BRL-3A cells synthesize and secrete a 7,484 Mr protein 93% identical to human IGF-II and representing rat IGF-II (rIGF-II). Rat IGF-II is synthesized as a approximately 22,000 Mr prepro-rIGF-II (ref. 12) from 12 S poly(A)+mRNA. In addition, approximately 20,000 Mr pro-rIGF-II has been identified in lysates of biosynthetically labelled intact BRL-3A cells. We report here the isolation of an almost complete cDNA clone for rIGF-II. Our results indicate that pro-rIGF-II is synthesized as a 156 amino acid peptide precursor (17,619 Mr) containing mature rIGF-II 1-67 at its amino-terminus and an 89-residue carboxy-terminal peptide extension.     

gamma-Interferon induced by S. aureus protein A augments natural killing and ADCC

Staphylococcus aureus produces a protein identified as protein A (SpA) with a molecular weight of 41,000 (ref. 1) which binds to the Fc region of many types of mammalian IgG2, activates complement, blocks opsonins and is both chemotactic and mitogenic. SpA has been used for various immunological purposes including removal of immune complexes, arming effector cells with antibody, distinguishing between antibody-dependent cell-mediated cytotoxicity (ADCC) and natural killing (NK), and exerting anti-tumor activity. We have now demonstrated that SpA induces the production of gamma interferon (IFN-gamma) in nonadherent, T lymphocyte-depleted (E-), Fc receptor-bearing (FcR+) human peripheral blood lymphocytes. Our data also suggest that IFN-gamma is a potent stimulator of both NK and ADCC activity.