Interaction between brain natriuretic peptide and atrial natriuretic peptide in caecal circular smooth muscle cells

Guinea pig caecal circular smooth muscle cells were used to determine whether brain natriuretic peptide (BNP) can inhibit the contractile response produced by cholecystokinin-octapeptide (CCK-8). In addition, we examined the effect of an inhibitor of cAMP-dependent protein kinase, an inhibitor of particulate or soluble guanylate cyclase, an atrial natriuretic peptide (ANP) antagonist (ANP 1-11), and selective receptor protection on the BNP-induced relaxation of these muscle cells. The effect of BNP on cAMP formation was also examined. BNP inhibited the contractile response produced by CCK-8 in a dose-response manner, with an IC50 value of 8.5 nM, and stimulated the production of cAMP. The inhibitor of cAMP-dependent protein kinase and the inhibitor of soluble guanylate cyclase significantly inhibited the relaxation produced by BNP. In contrast, the inhibitor of particulate guanylate cyclase did not have any significant effect on the relaxation produced by BNP. ANP 1-11 significantly but partially inhibited the relaxation produced by BNP. The muscle cells where CCK-8 and ANP binding sites were protected completely preserved the inhibitory response to ANP, but partially preserved the inhibitory response to BNP. The muscle cells where CCK-8 and BNP binding sites were protected completely preserved the inhibitory response to both ANP and BNP. This study demonstrates that BNP induces relaxation of these muscle cells via both ANP binding sites coupled to soluble guanylate cyclase and distinct BNP binding sites coupled to adenylate cyclase.     

Direct inhibitory effect of calcitonin gene-related peptide and atrial natriuretic peptide on gastric smooth muscle cells via different mechanisms.

Smooth muscle cells isolated from the gastric muscle layers of the guinea pig were used to determine whether calcitonin gene-related peptide (CGRP) and atrial natriuretic peptide (ANP) can inhibit the contractile response produced by 10(-6) M carbachol by exerting a direct action on muscle cells. In addition, the inhibitory effect of 2', 5'-dideoxyadenosine, an inhibitor of adenylate cyclase, on the CGRP-induced or ANP-induced relaxation of gastric smooth muscle cells were examined. CGRP and ANP inhibited the contractile response produced by carbachol in a dose-dependent manner, and the values of IC50 were 3 nM and 2 nM, respectively. 2',5'-dideoxyadenosine significantly inhibited the relaxation produced by CGRP. On the other hand, 2',5'-dideoxyadenosine did not have any significant effect on the relaxation produced by ANP. These results demonstrate the difference between intracellular mechanism responsible for gastric smooth muscle relaxation by CGRP and the mechanism responsible for muscle relaxation by ANP, and strongly suggest that the action of CGRP is mediated by adenosine 3',5'-cyclic monophosphate.     

Expression of the calcitonin receptor, calcitonin receptor-like receptor, and receptor activity modifying proteins during osteoclast differentiation

The expressions of the calcitonin receptor (CTR), the calcitonin receptor-like receptor (CLR), the receptor activity-modifying proteins (RAMP) 1-3, and of the receptor component protein (RCP) have been studied in mouse bone marrow macrophages (BMM) during osteoclast differentiation, induced by treatment with M-CSF and RANKL. Analyses of mRNA showed that CLR and RAMP1-3, but not CTR, were expressed in M-CSF stimulated BMM. RANKL gradually increased CTR mRNA, transiently enhanced CLR and transiently decreased RAMP1 mRNA, but did not affect RAMP2, RAMP3, or RCP mRNA. However, RANKL did not affect protein levels of CLR or RAMP1-3 as assessed by Western blots or FACS analyses, whereas immunocytochemistry showed enhanced CTR protein. Analyses of cAMP production showed that BMM cells expressed functional receptors for calcitonin gene-related peptide (CGRP), amylin, adrenomedullin, and intermedin, but not for calcitonin and calcitonin receptor stimulating peptide (CRSP), but that RANKL induced the expression of receptors for calcitonin and CRSP as well. Calcitonin, CGRP, amylin, adrenomedullin, intermedin, and CRSP all down regulated the CTR mRNA, but none of the peptides caused any effects on the expression of CLR or any of the RAMPs. Our data show that BMM cells express receptors for CGRP, amylin, adrenomedullin, and intermedin and that RANKL induces the formation of receptors for calcitonin and CRSP in these cells. We also show, for the first time, that the CTR is not only down regulated by signaling through the CTR but also by the peptides signaling through CLR/RAMPs.     

The effect of adrenomedullin,amylin fragment 8-37 and calcitonin gene-related peptide on contractile force,heart rate and coronary perfusion pressure in isolated rat hearts

The effect of human adrenomedullin, human amylin fragment 8-37 (amylin 8-37) and rat calcitonin gene-related peptide (CGRP) on contractile force, heart rate and coronary perfusion pressure has been investigated in the isolated perfused rat hearts. Adrenomedullin (2x10(-10), 2x10(-9) and 2x10(-8) M) produced a significant decrease in contractile force and perfusion pressure, but only the peptide caused a decline in heart rate at the highest dose. Amylin (10(-9), 10(-8) and 10(-7) M) significantly increased and then decreased contractile force. Two doses of amylin (10(-8) and 10(-7) M) induced a significant increase in heart rate, however amylin did not change perfusion pressure in all the doses used. Rat alpha CGRP (10(-8), 10(-7) and 10(-6) M) evoked a slight decline in contractile force following a significant increase in contractile force induced by the peptide. CGRP in all the doses raised heart rate and lowered perfusion pressure. Our results suggest that adrenomedullin has negative inotropic, negative chronotropic and coronary vasodilator actions. Amylin produces a biphasic inotropic effect and evokes a positive chronotropy. CGRP causes positive inotropic, positive chronotropic and vasodilatory effects in isolated rat hearts.     

降钙素基因相关肽家族受体及其受体活性修饰蛋白

降钙素基因相关肽家族是一类多功能的激素家族 ,参与人体的多种生物学功能 ,与多种疾病有关。降钙素基因相关肽受体包括降钙素受体 (CTR)和降钙素受体样受体 (CRLR) ,CTR可以独自与降钙素结合 ,而CRLR必须与一组称作受体活性修饰蛋白 (RAMPs)的蛋白质共同作用才能发挥生物学功能。综述CTR的研究概况及CRLR与RAMPs相互作用的机制和表达调控 ,以期为人们设计新型药物提供参考。     

Effect of calcitonin gene-related peptide on the neuroeffector mechanism of sympathetic nerve terminals in rat vas deferens

In order to evaluate the mode of action of calcitonin gene-related peptide (CGRP) on the neuroeffector mechanism of peripheral sympathetic nerve fibers, the effects of CGRP were tested on the electrical stimulated and the non-stimulated preparations of the isolated rat vas deferens. The contractile responses, which were mediated predominantly by activation of postganglionic noradrenergic nerve fibers, were dose-dependently inhibited by CGRP in concentrations ranging from 0.1 to 10 nM. The inhibitory response produced by CGRP in high concentrations (greater than 2 nM) usually returned to the control level at 20-30 min and were rarely tachyphylactic. The inhibitory action of CGRP was not modified by pretreatment with 10(-7) M propranolol or 10(-7) M atropine. Contractions produced by exogenous norepinephrine (NE) and 5-hydroxytryptamine (5-HT) in unstimulated preparations were not affected by pretreatment with CGRP in a low concentration (less than 2 nM). On the other hand, the contractions were slightly reduced 1 min after pretreatment with CGRP in high concentrations (greater than 5 nM), which recovered in 15 min after constant flow washout. High concentrations of CGRP also caused a concentration-dependent relaxation on the precontracted preparations produced by high potassium (60 mM K+) solution. These results suggest that CGRP in high concentrations (greater than 5 nM) may have a non-specific inhibitory action on the postsynaptic plasma membrane of the smooth muscle cell and a postulated CGRP receptor exists presynaptically in the rat vas deferens and that CGRP may inhibit the release of NE during adrenergic nerve stimulation.     

Intermedin, a novel calcitonin family peptide that exists in teleosts as well as in mammals: a comparison with other calcitonin/intermedin family peptides in vertebrates

Endocrine regulation in vertebrates is critical for the adaptation and regulation of homeostasis. The G protein-coupled receptor (GPCR) signaling transduction system represents one of the most ancient forms of cell surface signaling. Recently, comparative sequence analysis has aided in the identification and pairing of a variety of ligand/GPCR signaling systems. Among the ligands of type II GPCRs, the calcitonin family peptides including calcitonin, alpha-calcitonin gene-related peptide (alphaCGRP), betaCGRP, adrenomedullin, and amylin are among the best studied hormones, and the founding member, calcitonin, was originally identified and isolated from teleosts. This unique group of peptides shares a conserved tertiary structure with an N-terminal disulfide-bridged ring. In mammals, these peptides signal through two closely related type II GPCRs and three unique receptor activity-modifying proteins. Recently, based on the analysis of multiple vertebrate genomes, we identified a novel calcitonin/CGRP family peptide named intermedin. Here we show that in humans the five paralogous family genes, calcitonin, CGRP, amylin, adrenomedullin, and intermedin, evolved before the emergence of modern vertebrates, and that teleost genomes carry multiple copies of these co-evolved hormone genes. Sequence comparison showed that each of these genes is highly conserved in different vertebrates and that multiple copies of these peptides in teleosts could be derived from ancient genome duplication and/or lineage-specific intragenic duplications. The present article provides an overview of the calcitonin/intermedin family peptides found in teleost and mammalian genomes, and describes their putative functions. In addition, we demonstrate that one of the intermedin orthologs deduced from the pufferfish (Fugu rubripes) genome shares a conserved signaling activity with mammalian intermedin. The combined results indicate that the physiology associated with each of these family peptides likely evolved during early vertebrate evolution and diverged to serve select physiological functions in different vertebrates.     

Deletions of genes encoding calcitonin/alpha-CGRP, amylin and calcitonin receptor have given new and unexpected insights into the function of calcitonin receptors and calcitonin receptor-like receptors in bone

It has been suggested that skeletal nerves fibers may play important roles in neuro-osteogenic interactions. This view is partly based upon information obtained from immunohistochemical studies, chemical and surgical denervation experiments and clinical observations in patients with stroke and spinal cord injury, indicating the presence of a network of nerve fibers in the skeleton and that defective signalling in skeletal nerve fibers affects remodelling of bone. This view is also supported by data showing that functional receptors for signalling molecules in skeletal nerve fibers are expressed in bone cells and that activation of these receptors leads to profound effects on bone forming osteoblasts and bone resorbing osteoclasts. Convincing evidence for a role of neuronal signalling in bone metabolism has been provided by gene deletion approaches in which it has been shown that leptin-sensitive and neuropeptide Y-sensitive receptors in hypothalamus are important for bone remodelling in mice. Recently, gene deletion experiments have shown that calcitonin gene-related peptide (CGRP), one of the neuropeptides present in skeletal nerve fibers, is an important physiological regulator of bone formation at the level of osteoblast activity. CGRP belongs to the calcitonin (CT) family of peptides also including CT, amylin and adrenomedullin, as well as the recently described intermedin and calcitonin receptor-stimulating peptide. These peptides utilize two seven transmembrane G protein-coupled receptors - the calcitonin receptor (CTR) and the calcitonin receptor- like receptor (CRLR) - which can dimerize with three different single transmembrane proteins, making up the RAMP family. Associations between RAMPs and either CTR or CRLR give rise to seven distinct, molecularly characterized, receptors for CT, CGRP, amylin and adrenomedullin. Deletions of the genes for ligands in the CT family of peptides and for one of the receptors have revealed unexpected findings that have changed our view on the role of these peptides in bone remodelling. It was anticipated that deletions of the CT/alpha-CGRP and CTR genes would lead to bone loss, since CT has been shown to inhibit bone resorption in vitro and in vivo and has been used to treat patients with excessive bone resorption. Surprisingly, it was found that CT/alpha-CGRP-/- and CTR+/- mice have increased bone mass due to increased bone formation. Mice with deletion of the amylin gene, however, exhibited bone loss due to enhanced bone resorption. Selective deletion of the alpha-CGRP gene also leads to bone loss, but due to decreased bone formation. Thus, our understanding of the role of the CT family of peptides has been changed dramatically and much more data have to be gained before we fully understand the roles these peptides have in bone biology.     

Calcitonin gene-related peptide stimulates cyclic AMP formation in rat aortic smooth muscle cells

In rat aortic smooth muscle cells in culture, calcitonin gene-related peptide stimulated cAMP formation in a dose-dependent manner, half-maximally effective at 0.5 to 1 nM. There was no effect on formation of cGMP, which was increased 300-fold in the same experiments by atriopeptin or sodium nitroprusside. The vasodilator effect of CGRP in rat aorta requires an intact endothelium, indicating that increase in vascular smooth muscle cAMP is not in itself sufficient to bring about relaxation. cAMP is probably a mediator of CGRP action in vascular smooth muscle.     

Novel calcitonin-(8-32)-sensitive adrenomedullin receptors derived from co-expression of calcitonin receptor with receptor activity-modifying proteins

We tested whether heterodimers comprised of calcitonin (CT) receptor lacking the 16-amino acid insert in intracellular domain 1 (CTR(I1-)) and receptor activity-modifying protein (RAMP) can function not only as calcitonin gene-related peptide (CGRP) receptors but also as adrenomedullin (AM) receptors. Whether transfected alone or together with RAMP, human (h)CTR(I1-) appeared mainly at the surface of HEK-293 cells. Expression of CTR(I1-) alone led to significant increases in cAMP in response to hCGRP or hAM, though both peptides remained about 100-fold less potent than hCT. However, the apparent potency of AM, like that of CGRP, approached that of CT when CTR(I1-) was co-expressed with RAMP. CGRP- or AM-evoked cAMP production was strongly inhibited by salmon CT-(8-32), a selective amylin receptor antagonist, but not by hCGRP-(8-37) or hAM-(22-52), antagonists of CGRP and AM receptors, respectively. Moreover, the inhibitory effects of CT-(8-32) were much stronger in cells co-expressing CTR(I1-) and RAMP than in cells expressing CTR(I1-) alone. Co-expression of CTR(I1-) with RAMP thus appears to produce functional CT-(8-32)-sensitive AM receptors.     

Mesenteric arterial relaxation to calcitonin gene-related peptide is increased during pregnancy and by sex steroid hormones

The present study investigated whether pregnancy and circulatory ovarian hormones increase the sensitivity of the mesenteric artery to calcitonin gene-related peptide (CGRP)-induced relaxation and possible mechanisms involved in this process. Mesenteric arteries from young adult male rats or female rats (during estrous cycle, after ovariectomy, at Day 20 of gestation, or Postpartum Day 2) were isolated, and the responsiveness of the vessels to CGRP was examined with a small vessel myograph. The CGRP (10(-10) to 10(-7) M) produced a concentration-dependent relaxation of norepinephrine-induced contractions in mesenteric arteries of all groups. Arterial relaxation sensitivity to CGRP was significantly (P < 0.05) greater in female rats compared with male rats. Pregnancy increased the sensitivity to CGRP significantly (P < 0.05) compared to ovariectomized and Postpartum Day 2 rats. In pregnant rats, CGRP-receptor antagonist, CGRP(8-37), inhibited the relaxation responses produced by CGRP. The CGRP-induced relaxation was not affected by N(G)-nitro-l-arginine methyl ester (nitric oxide inhibitor, 10(-4) M) but was significantly (P < 0.05) attenuated by an inhibitor of guanylate cyclase (1H-[1 , 2 , 4 ]oxadizaolo[4 , 3 -a]quinoxalin-1-one, 10(-5) M). Relaxation responses of CGRP on mesenteric arteries were blocked (P < 0.05) by a cAMP-dependent protein kinase A inhibitor, Rp-cAMPs (10(-5) M). The CGRP-induced vasorelaxation was significantly (P < 0.05) attenuated by calcium-dependent (tetraethylammonium, 10(-3) M), but not ATP-sensitive (glybenclamide, 10(-5) M), potassium channel blocker. Therefore, the results of the present study suggest that mesenteric vascular sensitivity to CGRP is higher during pregnancy and that cAMP, cGMP, and calcium-dependent potassium channels appear to be involved. Therefore, we propose that CGRP-mediated vasodilation may be important to maintain vascular adaptations during pregnancy.     

Adrenomedullin: receptor and signal transduction

Adrenomedullin is a vascular tissue peptide and a member of the calcitonin family of peptides, which includes calcitonin, calcitonin-gene-related peptide (CGRP) and amylin. Its many biological actions are mediated via CGRP type 1 (CGRP(1)) receptors and by specific adrenomedullin receptors. Although the pharmacology of these receptors is distinct, they are both represented in molecular terms by the type II family G-protein-coupled receptor, calcitonin-receptor-like receptor (CRLR). The specificity here is defined by co-expression of receptor-activity-modifying proteins (RAMPs). CGRP(1) receptors are represented by CRLR and RAMP1, and specific adrenomedullin receptors by CRLR and RAMP2 or 3. Here we discuss how CRLR/RAMP2 relates to adrenomedullin binding, pharmacology and pathophysiology, and how chemical cross-linking of receptor-ligand complexes in tissue relates to that in CRLR/RAMP2-expressing cells. CRLR, like other type II family G-protein-coupled receptors, signals via G(s) and adenylate cyclase activation. We demonstrated that adrenomedullin signalling in cell lines expressing specific adrenomedullin receptors followed this expected pattern.     

Phenotypic changes of adrenomedullin receptor components, RAMP2, and CRLR mRNA expression in cultured rat vascular smooth muscle cells.

Adrenomedullin is known to inhibit cell proliferation in cultured rat vascular smooth muscle cells, through a cAMP-dependent process. The calcitonin receptor-like receptor could function as an adrenomedullin receptor when co-expressed with receptor activity-modifying protein 2. To determine whether vascular adrenomedullin receptor components, the calcitonin receptor-like receptor and the receptor activity-modifying protein 2, phenotypically change during in vitro culture conditions, we examined the expression of adrenomedullin receptor components, adrenomedullin-induced cAMP production, and the inhibition of cell proliferation in culture rat vascular smooth muscle cells during serial passages. The results demonstrated that the receptor activity-modifying protein 2 and calcitonin receptor-like receptor mRNAs increased in a passage-dependent manner in rat vascular smooth muscle cells. Furthermore, the responses of both the elevation of cAMP and the inhibition of cell proliferation became larger in vascular smooth muscle cells with an increasing number of passages. The results suggest that the increase in functional AM receptor during phenotypic change may in part contribute to the development of vascular lesions, such as in atherosclerosis.     

Receptor autoradiography as mean to explore the possible functional relevance of neuropeptides: focus on new agonists and antagonists to study natriuretic peptides, neuropeptide Y and calcitonin gene-related peptides

Over the past 20 years, receptor autoradiography has proven most useful to provide clues as to the role of various families of peptides expressed in the brain. Early on, we used this method to investigate the possible roles of various brain peptides. Natriuretic peptide (NP), neuropeptide Y (NPY) and calcitonin (CT) peptide families are widely distributed in the peripheral and central nervous system and induced multiple biological effects by activating plasma membrane receptor proteins. The NP family includes atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). The NPY family is composed of at least three peptides NPY, peptide YY (PYY) and the pancreatic polypeptides (PPs). The CT family includes CT, calcitonin gene-related peptide (CGRP), amylin (AMY), adrenomedullin (AM) and two newly isolated peptides, intermedin and calcitonin receptor-stimulating peptide (CRSP). Using quantitative receptor autoradiography as well as selective agonists and antagonists for each peptide family, in vivo and in vitro assays revealed complex pharmacological responses and radioligand binding profile. The existence of heterogeneous populations of NP, NPY and CT/CGRP receptors has been confirmed by cloning. Three NP receptors have been cloned. One is a single-transmembrane clearance receptor (NPR-C) while the other two known as CG-A (or NPR-A) and CG-B (or NPR-B) are coupled to guanylate cyclase. Five NPY receptors have been cloned designated as Y(1), Y(2), Y(4), Y(5) and y(6). All NPY receptors belong to the seven-transmembrane G-protein coupled receptors family (GPCRs; subfamily type I). CGRP, AMY and AM receptors are complexes which include a GPCR (the CT receptor or CTR and calcitonin receptor-like receptor or CRLR) and a single-transmembrane domain protein known as receptor-activity-modifying-proteins (RAMPs) as well as an intracellular protein named receptor-component-protein (RCP). We review here tools that are currently available in order to target each NP, NPY and CT/CGRP receptor subtype and establish their respective pathophysiological relevance.     

Amylin increases cyclic AMP formation in L6 myocytes through calcitonin gene-related peptide receptors.

The cellular function of amylin is investigated in L6 myocytes, a rat skeletal muscle cell line. Both rat amylin and human amylin-amide acutely cause a dose-dependent increase in cyclic AMP formation in L6 myocytes. 100 nM amylin stimulates intracellular cyclic AMP concentrations 12-fold, whereas human amylin-amide at this concentration causes only a 2-fold increase. Up to 10 mM human amylin has no effect on cyclic AMP levels. Rat calcitonin gene-related peptide (CGRP) is more potent than amylin, causing a 60-fold increase over basal at 1 nM, with an EC50 value of 0.2 nM. The CGRP receptor antagonist, human CGRP8-37 (hCGRP8-37), completely blocks the stimulatory effect of both rat amylin and human amylin-amide on cyclic AMP production. [125I]CGRP binds specifically to a membrane fraction prepared from L6 [125I]CGRP with a Ki of 0.9 nM, while rat amylin also displaces [125I]CGRP with a Ki of 91 nM. Specific binding of [125I]CGRP to plasma membranes of rat liver and brain is also displaced by rat amylin with Ki values of 35 nM and 37 nM, respectively. In contrast, specific binding of [125I]amylin to numerous cells and tissues, under similar conditions, can not be demonstrated. These results suggest that the cellular effects and physiological actions of amylin may be mediated through receptors for CGRP.     

Proadrenomedullin NH(2)-terminal 20 peptide (PAMP) and adrenomedullin bind to teratocarcinoma cells

Proadrenomedullin NH(2-)terminal 20 peptide (PAMP) and adrenomedullin (ADM) bind to teratocarcinoma cells. The effects of PAMP and ADM on teratocarcinoma cells were investigated. (125)I-PAMP bound to PA1 cells with moderate affinity (K(d) = 110 nM) to a single class of sites (B(max) = 110 000/cell). Specific (125)I-PAMP binding was inhibited by PAMP (IC(50) of 100 nM) but not ADM, calcitonin gene-related peptide (CGRP), or amylin. Specific (125)I-ADM binding was inhibited with high affinity by ADM, CGRP, and CGRP(8-37) (IC(50) values of 10, 10, and 15 nM respectively) but not PAMP or amylin. ADM elevated cAMP (ED(50) value of 100 nM), whereas PAMP had no effect on basal cAMP but inhibited the increase in cAMP caused by 10 nM ADM. Also, the increase in cAMP caused by ADM was inhibited CGRP(8-37), suggesting that ADM is binding to CGRP receptors. ADM (100 nM) stimulated transiently c-fos mRNA, whereas PAMP (1000 nM) had little effect; however, PAMP inhibited the increase in c-fos mRNA caused by ADM. ADM stimulated [(3)H]thymidine uptake into PA1 cells, whereas PAMP inhibited the increase in thymidine uptake caused by ADM. These results indicate that ADM and PAMP are both biologically active in teratocarcinoma cells.     

Activation of particulate guanylate cyclase by adrenomedullin in cultured SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells

We investigated the effects of adrenomedullin (ADM) on cGMP production in cultured SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. ADM increased cGMP accumulation in a time- and concentration- dependent manner. The peptide increased cGMP formation in the transformed cells by 405-fold as compared to 1. 6-fold in primary cultured CISM cells. The basal cGMP concentrations in both cell types were comparable. In addition, ADM increased cAMP accumulation in SV-CISM-2 cells and in primary cultured cells by 18. 9- and 5.8-fold, respectively. The ADM receptor antagonist, ADM(26-52), but not the atrial natriuretic peptide (ANP) receptor antagonist, anantin, inhibited ADM-induced cGMP formation. The phorbol ester, phorbol 12, 13-dibutyrate (PDBu), which inhibits particulate guanylate cyclases in smooth muscle, blocked ADM-stimulated cGMP accumulation. In contrast, inhibitors of the soluble guanylate cyclases, such as LY83583 and ODQ, and inhibitors of the nitric oxide cascade had little effect on ADM-stimulated cGMP production. The stimulatory effect of ADM on cGMP formation is due to activation of the guanylate cyclase system and not to a much reduced phosphodiesterase activity. ADM stimulated guanylate cyclase activity in membrane fractions isolated from SV-CISM-2 cells in a concentration-dependent manner with EC(50) value of 72 nM. Pertussis toxin, an activator of the G-protein, Gi, inhibited ADM-stimulated cGMP accumulation, whereas cholera toxin, a stimulator of the Gs G-protein and subsequently cAMP accumulation, had little effect. Pretreatment of the plasma membrane fraction with Gialpha antibody attenuated ADM-stimulated guanylate cyclase activity by 75%. We conclude that ADM increases intracellular cGMP levels in SV-CISM-2 cells through activation of the ADM receptor and subsequent stimulation of a Gi-mediated membrane-bound guanylate cyclase.     

Responses to human CGRP,ADM, and PAMP in human thymic arteries

Responses to human CGRP, adrenomedullin (ADM), and proadrenomedullin NH2-terminal 20 peptide (PAMP) were studied in small human thymic arteries. CGRP, ADM, and PAMP produced concentration-dependent vasodilator responses in arteries preconstricted with the thromboxane mimic U-46619. Responses to ADM and PAMP were attenuated, whereas responses to CGRP were not altered by endothelial denudation. Inhibitors of nitric oxide synthase and guanylyl cyclase attenuated responses to ADM and PAMP but not to CGRP. The CGRP1 receptor antagonist CGRP(8-37) attenuated responses to CGRP and ADM but not to PAMP. Responses to CGRP were reduced by SQ-22536 and Rp-cAMPS, inhibitors of adenylyl cyclase and PKA. These data suggest that responses to CGRP and ADM are mediated by CGRP(8-37)-sensitive receptors and that the endothelial ADM receptor induces vasodilation by a nitric oxide-guanylyl cyclase mechanism, whereas a smooth muscle CGRP receptor signals by a cAMP-dependent mechanism. A different endothelial receptor recognizes PAMP and signals by a nitric oxide-dependent mechanism.     

Sodium nitrite, a potent relaxant of rat stomach fundus: in vitro evidence

The effects of sodium nitrite (0.1, 1, 10 mM) on mechanical activity of isolated rat stomach fundus muscle and the influence of guanylate cyclase activity inhibitor (methylene blue) and channel inhibitors (tetrodotoxin, charybdotoxin, apamin) were studied. Nitrite evoked dose-dependent relaxation in the longitudinal and circular muscle layers. The lowest effective concentration of sodium nitrite was 0.1 mM, which is comparable with the NOAEL (no observed adverse effect level). Tetrodotoxin (1 microM) markedly inhibited electrically induced contraction and rebound relaxation, but did not influence the nitrite-induced relaxation. Charybdotoxin (100 nM) decreased the relaxation evoked by 10 mM nitrite to 52.3 and 65.7% of control reaction in the circular and longitudinal muscle layer, respectively. Apamin (100 nM) did not influence the nitrite-induced relaxation. Methylene blue (10 microM) decreased relaxation induced by nitrite in the longitudinal and circular muscle layer, respectively, to 66.7 and 54.3% of the response to 1 mM nitrite alone. Relaxation induced by nitrite was decreased in the presence of L-cysteine (5 mM), and in the circular and longitudinal muscle layer reached 29.6 and 23.1%, respectively, of the response to 1 mM nitrite alone. We conclude that the relaxing effect of nitrite on gastric fundus results from its direct action on smooth muscle cells and probably the enteric nervous system is not involved in this action. The nitrite-elicited relaxation depends on activation of guanylate cyclase and high conductance Ca2+-activated potassium channels; however, activation of potassium channels might be a part of or might act in parallel with the mechanism involving the cyclic GMP system. Effects of nitrite observed in the presence of L-cysteine suggest that nitrosothiols are not responsible for nitrite-evoked activation of guanylate cyclase.