Calcitonin gene-related peptide and hypertension

Capsaicin-sensitive sensory nerves participate in the regulation of cardiovascular functions both in the normal state and the pathophysiology of hypertension through the actions of potent vasodilator neuropeptides, including calcitonin gene-related peptide (CGRP). CGRP, a very potent vasodilator, is the predominant neurotransmitter in capsaicin-sensitive sensory nerves, and plays an important role in the initiation, progression and maintenance of hypertension via: (1) the alterations in its synthesis and release and/or in vascular sensitivity response to it; (2) interactions with pro-hypertensive systems, including renin-angiotensin-aldosterone system, sympathetic nervous system and endothelin system; and (3) anti-hypertrophy and anti-proliferation of vascular smooth muscle cells. The decrease in CGRP synthesis and release contributes to the elevated blood pressure, as shown in the spontaneously hypertensive rats, alpha-CGRP knockout mice, Dahl-salt or phenol-induced hypertensive rats. In contrast, the increase in CGRP levels or the enhancement of vascular sensitivity response to CGRP plays a beneficial compensatory depressor role in the development of hypertension, as shown in deoxycorticosterone-salt, sub-total nephrectomy-salt, N(omega)-nitro-L-arginine methyl ester or two-kidney, one-clip models of hypertension in rats. We found that rutaecarpine causes a sustained depressor action by stimulation of CGRP synthesis and release via activation of vanilloid receptor subtype 1 (VR1) in hypertensive rats, which reveals the therapeutic implications of VR1 agonists for treatment of hypertension.     

Preserved expression of GLUT4 prevents enhanced agonist-induced vascular reactivity and MYPT1 phosphorylation in hypertensive mouse aorta

We previously showed that GLUT4 expression is decreased in arterial smooth muscle of deoxycorticosterone acetate (DOCA)-salt hypertensive rats and that GLUT4-knockout mice have enhanced arterial reactivity. Therefore, we hypothesized that increased GLUT4 expression in vascular smooth muscle in vivo would prevent enhanced arterial reactivity and possibly reduce blood pressure in DOCA-salt hypertensive mice. Adult wild-type (WT) and GLUT4 transgenic (TG) mice were subjected to DOCA-salt hypertension with uninephrectomy or underwent uninephrectomy and remained normotensive. GLUT4 expression was increased more than twofold in the aortas of GLUT4 TG mice compared with WT aortas. Eight weeks after implantation of the DOCA pellets, GLUT4 expression decreased by 75% in aortas of WT hypertensive mice, but not in GLUT4 TG hypertensive aortas. Systolic blood pressure was significantly and similarly increased in WT and GLUT4 TG DOCA-salt mice compared with their respective sham-treated controls (159 vs. 111 mmHg). Responsiveness to the contractile agonist 5-HT was significantly increased in aortic rings from WT DOCA-salt mice but remained normal in GLUT4 TG DOCA mice. Phosphorylation of the myosin phosphatase targeting subunit MYPT1 was significantly enhanced in aortas of WT DOCA-salt mice, and this increase was prevented in GLUT4 TG mice. MYPT1 phosphorylation was also increased in nonhypertensive GLUT4-knockout mice. Myosin phosphatase, a major negative regulator of calcium sensitivity, is itself negatively regulated by phosphorylation of MYPT1. Therefore, our results show that preservation of GLUT4 expression prevents enhanced arterial reactivity in hypertension, possibly via effects on myosin phosphatase activity.     

Cloning and transcriptional analysis of the mouse receptor activity modifying protein-1 gene promoter

Background  

Receptor activity modifying protein-1 (RAMP-1) is a single transmembrane-domain protein required for the functional expression of calcitonin gene-related peptide (CGRP) receptors. To date, little is known about the molecular mechanism(s) that activate/inhibit RAMP-1 gene expression. Such mechanism(s) are likely to play a major role in modulating the responsiveness of tissues to CGRP.     

Increased expression in calcitonin-like receptor induced by aldosterone in cerebral arteries from spontaneously hypertensive rats does not correlate with functional role of CGRP receptor

OBJECTIVE: To analyze the effect of aldosterone on the expression of calcitonin gene-related peptide (CGRP) receptor components, calcitonin-like receptor (CL receptor) and receptor activity modifying protein 1 (RAMP1), as well as the effect of this mineralocorticoid on CGRP-mediated vasodilation in middle cerebral arteries from Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). RESULTS: CGRP 0.1 nM-0.1 microM induced a concentration-dependent relaxation that was nitric oxide independent and higher in SHR middle cerebral arteries. CL receptor and RAMP1 expression were similar in both strains. The relaxation to CGRP was not modified by aldosterone 1 microM in either strain, although aldosterone 1 microM increased expression of CL receptor without modifying RAMP1 in segments from SHR rats. CONCLUSIONS: CGRP elicits greater vasodilation in middle cerebral arteries from SHR than WKY rats, that is nitric oxide independent, and by mechanism independent of CGRP receptor components expression. Although aldosterone increases the expression of CL receptor in SHR, it does not alter vasodilation to CGRP, since RAMP1 expression is not increased. These results indicate that the increase in CL receptor, without an increase in RAMP1, does not correlate with changes in functional role of the CGRP receptor.     

Novel regulation of adrenomedullin receptor by PDGF: role of receptor activity modifying protein-3

Receptor activity modifying protein-3(RAMP-3) has been shown to complex with the calcitonin receptor-likereceptor, establishing a functional receptor for adrenomedullin (AM).AM exhibits potent antiproliferative and antimigratory effects on ratmesangial cells (RMCs). In this study we investigated the effect ofplatelet-derived growth factor (PDGF) on RAMP-3 expression in RMCs. Weshow here that PDGF-BB stimulates RAMP-3 mRNA expression in aconcentration-dependent manner. Pretreatment with actinomycin-D and-amanitin demonstrates that this effect is independent of new RNAsynthesis. Furthermore, PDGF increased the half-life of RAMP-3 mRNAfrom 66.5 to 331.6 min. Using selective inhibitors, our results alsoindicate that the increase in RAMP-3 mRNA is mitogen-activated proteinkinase (MAPK) kinase (MEK)/MAPK and p38 MAPK dependent. PDGF alsocaused a corresponding elevation in membrane-associated RAMP-3 protein. Associated with this increase, PDGF pretreatment led to a significantly higher AM-mediated adenylate cyclase activity, suggesting a functional consequence for the PDGF-induced increase in RAMP-3 expression. Takentogether, these data identify PDGF-dependent regulation of RAMP-3expression as a possible mechanism for modulating the responsiveness ofthe mesangial cell to AM.

     

Intermedin is a calcitonin/calcitonin gene-related peptide family peptide acting through the calcitonin receptor-like receptor/receptor activity-modifying protein receptor complexes

Calcitonin, calcitonin gene-related peptide (CGRP), adrenomedullin (ADM), and amylin belong to a unique group of peptide hormones important for homeostasis in diverse tissues. Calcitonin is essential for calcium balance, whereas CGRP and ADM are important for neurotransmission and cardiovascular and respiratory regulation. Based on phylogenetic analysis, we identified intermedin as a novel member of the calcitonin/CGRP peptide family. Analysis of intermedin expression indicated that intermedin is expressed primarily in the pituitary and gastrointestinal tract. Intermedin increased cAMP production in SK-N-MC and L6 cells expressing endogenous CGRP receptors and competed with labeled CGRP for binding to its receptors in these cells. In addition, treatment of 293T cells expressing recombinant calcitonin receptor-like receptor (CRLR) and one of the three receptor activity-modifying proteins (RAMPs) showed that a CRLR/RAMP receptor complex is required for intermedin signaling. In contrast to CGRP and ADM, which exhibited a preferential stimulation of CRLR when co-expressed with RAMP1 and RAMP2 or RAMP3, respectively, intermedin represents a nonselective agonist for the RAMP coreceptors. In vivo studies demonstrated that intermedin treatment led to blood pressure reduction in both normal and spontaneously hypertensive rats via interactions with the CRLR/RAMP receptor complexes. Furthermore, in vivo treatment in mice with intermedin led to suppression of gastric emptying activity and food intake. Thus, identification of intermedin as a novel member of the calcitonin/CGRP peptide family capable of signaling through CRLR/RAMP receptor complexes provides an additional player in the regulation of peripheral tissues by CRLR and will allow development of new therapeutic agents for pathologies associated with diverse vascular and gastrointestinal disorders.     

Role of calcitonin gene-related peptide in the phenol-induced neurogenic hypertension in rats

Previous investigations have demonstrated that capsaicin-sensitive sensory nerves are involved in the development of hypertension in some rat models of hypertension. To determine the role played by calcitonin gene-related peptide (CGRP; the predominant neurotransmitter in capsaicin-sensitive sensory nerves) in a rat model of neurogenic hypertension, in which hypertension was induced by injecting 50 microl of 10% phenol in the lower pole of the left kidney, systolic blood pressure (SBP) was monitored by the tail-cuff method throughout the experiment. Fifteen days after injection of phenol, mean arterial pressure (MAP), concentrations of CGRP in the plasma, the expression of CGRP mRNA in dorsal root ganglia (DRG) and CGRP content in laminae I and II of the spinal cord were measured. SBP was significantly increased 5 days after the intrarenal injection of phenol (164+/-7 mm Hg, p<0.01). At the end of experiment, blood pressure (BP) was significantly elevated in the phenol-injected rats compared with the controls (SBP: 187+/-6 vs. 122+/-4 mm Hg, p<0.01; MAP: 157.56+/-3.02 vs. 103.80+/-2.04 mm Hg, p<0.01). Treatment with capsaicin, which selectively depletes neurotransmitters from the capsaicin-sensitive nerves, failed to enhance the development of hypertensive responses to the intrarenal injection of phenol. Intravenous administration of CGRP(8-37), the specific CGRP receptor antagonist, also failed to increase the already elevated MAP. The expression of CGRP mRNA (both alpha- and beta-CGRP isoforms), the content of CGRP in laminae I and II of the dorsal horn of the spinal cord and the concentration of CGRP in the plasma was decreased in the rats treated with phenol. These results suggest that CGRP does not play a counterregulatory role in the phenol-induced hypertensive rats, and support the hypothesis that reduction of CGRP (alpha and beta isoforms) could contribute to a blood pressure elevation in this setting.     

Cerebral pressure-flow relations in hypertensive elderly humans: transfer gain in different frequency domains.

The dynamics of the cerebral vascular response to blood pressure changes in hypertensive humans is poorly understood. Because cerebral blood flow is dependent on adequate perfusion pressure, it is important to understand the effect of hypertension on the transfer of pressure to flow in the cerebrovascular system of elderly people. Therefore, we examined the effect of spontaneous and induced blood pressure changes on beat-to-beat and within-beat cerebral blood flow in three groups of elderly people: normotensive, controlled hypertensive, and uncontrolled hypertensive subjects. Cerebral blood flow velocity (transcranial Doppler), blood pressure (Finapres), heart rate, and end-tidal CO(2) were measured during the transition from a sit to stand position. Transfer function gains relating blood pressure to cerebral blood flow velocity were assessed during steady-state sitting and standing. Cerebral blood flow regulation was preserved in all three groups by using changes in cerebrovascular resistance, transfer function gains, and the autoregulatory index as indexes of cerebral autoregulation. Hypertensive subjects demonstrated better attenuation of cerebral blood flow fluctuations in response to blood pressure changes both within the beat (i.e., lower gain at the cardiac frequency) and in the low-frequency range (autoregulatory, 0.03-0.07 Hz). Despite a better pressure autoregulatory response, hypertensive subjects demonstrated reduced reactivity to CO(2). Thus otherwise healthy hypertensive elderly subjects, whether controlled or uncontrolled with antihypertensive medication, retain the ability to maintain cerebral blood flow in the face of acute changes in perfusion pressure. Pressure regulation of cerebral blood flow is unrelated to cerebrovascular reactivity to CO(2).     

Cardiovascular effects of exogenous adrenomedullin and CGRP in Ramp and Calcrl deficient mice

Adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) are potent vasodilator peptides and serve as ligands for the G-protein coupled receptor (GPCR) calcitonin receptor-like receptor (CLR/Calcrl). Three GPCR accessory proteins called receptor activity-modifying proteins (RAMPs) modify the ligand binding affinity of the receptor such that the CLR/RAMP1 heterodimer preferably binds CGRP, while CLR/RAMP2 and CLR/RAMP3 have a stronger affinity for AM. Here we determine the contribution of each of the three RAMPs to blood pressure control in response to exogenous AM and CGRP by measuring the blood pressure of mice with genetic reduction or deletion of the receptor components. Thus, the cardiovascular response of Ramp1^−/−, Ramp2^+/−, Ramp3^−/−, Ramp1^−/−/Ramp3^−/− double-knockout (dKO), and Calcrl^+/− mice to AM and CGRP were compared to wildtype mice. While under anesthesia, Ramp1^−/− male mice had significantly higher basal blood pressure than wildtype males; a difference which was not present in female mice. Additionally, anesthetized Ramp1^−/−, Ramp3^−/−, and Calcrl^+/− male mice exhibited significantly higher basal blood pressure than females of the same genotype. The hypotensive response to intravenously injected AM was greatly attenuated in Ramp1^−/− mice, and to a lesser extent in Ramp3^−/− and Calcrl^+/− mice. However, Ramp1^−/−/Ramp3^−/− dKO mice retained some hypotensive response to AM. These results suggest that the hypotensive effect of AM is primarily mediated through the CLR/RAMP1 heterodimer, but that AM signaling via CLR/RAMP2 and CLR/RAMP3 also contributes to some hypotensive action. On the other hand, CGRP’s hypotensive activity seems to be predominantly through the CLR/RAMP1 heterodimer. With this knowledge, therapeutic AM or CGRP peptides could be designed to cause less hypotension while maintaining canonical receptor-RAMP mediated signaling.     

Adrenomedullin 2 (AM2)/intermedin is a more potent activator of hypothalamic oxytocin-secreting neurons than AM possibly through an unidentified receptor in rats

Central administration of either adrenomedullin 2 (AM2) or adrenomedullin (AM) activates hypothalamic oxytocin (OXT)-secreting neurons in rats. We compared AM2 with AM, given intracerebroventricularly (icv), across multiple measures: (1) plasma OXT levels in conscious rats; (2) blood pressure, heart rate and circulating catecholamine levels in urethane-anesthetized rats; and (3) the expression of the c-fos gene in the supraoptic (SON) and the paraventricular nuclei (PVN). We also tested the effects of the AM receptor antagonist, AM(22-52) and calcitonin gene-related peptide (CGRP) antagonist, CGRP(8-37) on these measures. Plasma OXT levels at 10 min after icv injection of AM (1 nmol/rat) were increased (compared with vehicle), but OXT levels after AM2 (1 nmol/rat) were nearly double the levels seen after AM injection. OXT levels remained elevated at 30 min. Pretreatment with AM(22-52) (27 nmol/rat) and CGRP(8-37) (3 nmol/rat), nearly abolished the increase in plasma OXT level after AM injection, but partially blocked OXT level changes due to AM2. Increases in blood pressure, heart rate and circulating catecholamines were all greater in response to central AM2 than to AM at the same dose. In situ hybridization histochemistry showed that both AM2 and AM induced expression of the c-fos gene in the SON and the PVN, but AM(22-52)+CGRP(8-37) could only nearly abolish the effects of centrally administered AM. These results suggest that the more potent central effects of AM2 and only partial blockade by AM/CGRP receptor antagonists may result from its action on an additional, as yet unidentified, specific receptor in the central nervous system.     

A review of the biological properties and clinical implications of adrenomedullin and proadrenomedullin N-terminal 20 peptide (PAMP), hypotensive and vasodilating peptides.

Adrenomedullin (AM), identified from pheochromocytoma and having 52 amino acids, elicits a long-lasting vasodilatation and diuresis. AM is mainly mediated by the intracellular adenylate cyclase coupled with cyclic adenosine monophosphate (cAMP) and nitric oxide (NO) -cyclic guanosine monophosphate (cGMP) pathway through its specific receptor. The calcitonin receptor-like receptor (CLCR) and receptor-activity modifying protein (RAMP) 2 or RAMP3 models have been proposed as the candidate receptor. AM is produced mainly in cardiovascular tissues in response to stimuli such as shear stress and stretch, hormonal factors and cytokines. Recently established AM knockout mice lines revealed that AM is essential for development of vitelline vessels of embryo. Plasma AM levels elevate in cardiovascular diseases such as heart failure, hypertension and septic shock, where AM may play protective roles through its characteristic biological activities. Human AM gene delivery improves hypertension, renal function, cardiac hypertrophy and nephrosclerosis in the hypertensive rats. AM decreases cardiac preload and afterload and improves cardiac contractility and diuresis in patients with heart failure and hypertension. Advances in gene engineering and receptor studies may contribute to further understandings of biological implication and therapeutic availability of AM.     

Deficiency of RAMP1 Attenuates Antigen-Induced Airway Hyperresponsiveness in Mice

Asthma is a chronic inflammatory disease affecting the lung, characterized by breathing difficulty during an attack following exposure to an environmental trigger. Calcitonin gene-related peptide (CGRP) is a neuropeptide that may have a pathological role in asthma. The CGRP receptor is comprised of two components, which include the G-protein coupled receptor, calcitonin receptor-like receptor (CLR), and receptor activity-modifying protein 1 (RAMP1). RAMPs, including RAMP1, mediate ligand specificity in addition to aiding in the localization of receptors to the cell surface. Since there has been some controversy regarding the effect of CGRP on asthma, we sought to determine the effect of CGRP signaling ablation in an animal model of asthma. Using gene-targeting techniques, we generated mice deficient for RAMP1 by excising exon 3. After determining that these mice are viable and overtly normal, we sensitized the animals to ovalbumin prior to assessing airway resistance and inflammation after methacholine challenge. We found that mice lacking RAMP1 had reduced airway resistance and inflammation compared to wildtype animals. Additionally, we found that a 50% reduction of CLR, the G-protein receptor component of the CGRP receptor, also ameliorated airway resistance and inflammation in this model of allergic asthma. Interestingly, the loss of CLR from the smooth muscle cells did not alter the airway resistance, indicating that CGRP does not act directly on the smooth muscle cells to drive airway hyperresponsiveness. Together, these data indicate that signaling through RAMP1 and CLR plays a role in mediating asthma pathology. Since RAMP1 and CLR interact to form a receptor for CGRP, our data indicate that aberrant CGRP signaling, perhaps on lung endothelial and inflammatory cells, contributes to asthma pathophysiology. Finally, since RAMP-receptor interfaces are pharmacologically tractable, it may be possible to develop compounds targeting the RAMP1/CLR interface to assist in the treatment of asthma.     

Effects of alpha calcitonin gene-related peptide in human bronchial smooth muscle and pulmonary artery

Although airway and pulmonary vessel tone are regulated predominantly by cholinergic and adrenergic impulses, biologically active peptides such as calcitonin gene-related peptide (CGRP) may significantly influence human smooth muscle tone in normal and pathophysiological states. In the present study, the expression of CGRP and its receptor CGRPR-1 and the biological effect of the peptide were investigated in human airways and pulmonary arteries. Immunohistochemistry revealed the presence of CGRP in human airway nerves and neuro-epithelial cells, whereas the receptor was found in epithelial cells and smooth muscle myocytes of the bronchi and in pulmonary artery endothelium. On precontracted bronchi (3-4 mm in diameter) alpha-CGRP (0.01-10 nM) caused a concentration-dependent contraction on epithelium-denuded bronchi, whereas no significant effect was recorded in bronchi with intact epithelium. In pulmonary arteries (2-6 mm in diameter), alpha-CGRP caused a concentration-dependent relaxation of endothelium intact and denuded vessels. Pre-treatment with indomethacin, but not with l-NAME, prevented the relaxation induced by alpha-CGRP in pulmonary arteries suggesting that prostaglandins but not nitric oxide (NO) are involved in the intracellular signal transduction pathway. The effects induced by alpha-CGRP in bronchi and vessels were prevented by application of the antagonist CGRP((8-37)). In summary, the present studies examined the biological function of CGRP in human airways and demonstrated a constrictory effect of CGRP only in epithelium-denuded airway smooth muscle indicating an alteration of CGRP airway effects in respiratory tract pathological states with damaged epithelium such as chronic obstructive pulmonary disease or bronchial asthma.     

Calcitonin gene-related peptide-evoked sustained tachycardia in calcitonin receptor-like receptor transgenic mice is mediated by sympathetic activity

Calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are potent vasodilators and exert positive chronotropic and inotropic effects on the heart. Receptors for CGRP and AM are calcitonin receptor-like receptor (CLR)/receptor-activity-modifying protein (RAMP) 1 and CLR/RAMP2 heterodimers, respectively. The present study was designed to delineate distinct cardiovascular effects of CGRP and AM. Thus a V5-tagged rat CLR was expressed in transgenic mice in the vascular musculature, a recognized target of CGRP. Interestingly, basal arterial pressure and heart rate were indistinguishable in transgenic mice and in control littermates. Moreover, intravenous injection of 2 nmol/kg CGRP, unlike 2 nmol/kg AM, decreased arterial pressure equally by 18 +/- 5 mmHg in transgenic and control animals. But the concomitant increase in heart rate evoked by CGRP was 3.7 times higher in transgenic mice than in control animals. The effects of CGRP in transgenic and control mice, different from a decrease in arterial pressure in response to 20 nmol/kg AM, were suppressed by 2 micromol/kg of the CGRP antagonist CGRP(8-37). Propranolol, in contrast to hexamethonium, blocked the CGRP-evoked increase in heart rate in both transgenic and control animals. This was consistent with the immunohistochemical localization of the V5-tagged CLR in the superior cervical ganglion of transgenic mice. In conclusion, hypotension evoked by CGRP in transgenic and control mice was comparable and CGRP was more potent than AM. Unexpectedly, the CLR/RAMP CGRP receptor overexpressed in postganglionic sympathetic neurons of transgenic mice enhanced the positive chronotropic action of systemic CGRP.     

Alteration of airway neuropeptide expression and development of airway hyperresponsiveness following respiratory syncytial virus infection

The mechanisms by which respiratory syncytial virus (RSV) infection causes airway hyperresponsiveness (AHR) are not fully established. We hypothesized that RSV infection may alter the expression of airway sensory neuropeptides, thereby contributing to the development of altered airway function. BALB/c mice were infected with RSV followed by assessment of airway function, inflammation, and sensory neuropeptide expression. After RSV infection, mice developed significant airway inflammation associated with increased airway resistance to inhaled methacholine and increased tracheal smooth muscle responsiveness to electrical field stimulation. In these animals, substance P expression was markedly increased, whereas calcitonin gene-related peptide (CGRP) expression was decreased in airway tissue. Prophylactic treatment with Sendide, a highly selective antagonist of the neurokinin-1 receptor, or CGRP, but not the CGRP antagonist CGRP(8-37), inhibited the development of airway inflammation and AHR in RSV-infected animals. Therapeutic treatment with CGRP, but not CGRP(8-37) or Sendide, abolished AHR in RSV-infected animals despite increased substance P levels and previously established airway inflammation. These data suggest that RSV-induced airway dysfunction is, at least in part, due to an imbalance in sensory neuropeptide expression in the airways. Restoration of this balance may be beneficial for the treatment of RSV-mediated airway dysfunction.     

Adrenomedullin release in the rat mesenteric resistance artery

Adrenomedullin (AM) is a potent vasodilator peptide whose major source is the vascular wall. In the present study, the mechanism of release of AM was investigated in the rat mesenteric resistance artery. The isolated mesenteric vascular bed was perfused with Krebs solution at a constant flow rate (5 ml/min) and AM in the perfusate was measured by a highly sensitive enzyme immunoassay (Immunoenzymometric assay; IEMA) method. In preparations without endothelium, spontaneous release of AM was detected in the perfusate (68.7+/-5.8 fmol/ml, n=45). Periarterial nerve stimulation (PNS, 4 and 8 Hz) caused 11.4+/-3.9% (4 Hz) and 9.1+/-3.5% (8 Hz) decreases in the spontaneous release of AM. Removal of Ca2+ from the medium did not affect the spontaneous AM release, but abolished the PNS-induced inhibition of spontaneous AM release. Perfusion of 10nM calcitonin gene-related peptide (CGRP) or 0.1 microM capsaicin (inducer of CGRP release) inhibited significantly the spontaneous AM release. PNS (8 Hz)-induced inhibition of spontaneous AM release was antagonized by CGRP(8-37) (CGRP receptor antagonist). These results suggest that AM is mainly released from vascular smooth muscle cells of the rat mesenteric artery and endogenous or exogenous CGRP inhibits AM release.     

Intermedin/adrenomedullin-2 acts within central nervous system to elevate blood pressure and inhibit food and water intake

Intermedin (IMD)/adrenomedullin-2 (AM2) is a novel peptide that was independently discovered by two groups. The 47-amino acid peptide is homologous to adrenomedullin (AM) and can activate both the AM and calcitonin gene-related peptide (CGRP) receptors. IMD should therefore have actions similar to those of AM and CGRP. Indeed, like AM and CGRP, intravenous administration of IMD decreased blood pressure in rats and mice. We demonstrate here that immunoreactive IMD is present in plasma as well as heart, lung, stomach, kidney, pituitary, and brain. Because IMD is present in brain and both AM and CGRP have potent central nervous system (CNS) effects, we examined the ability of IMD within brain to regulate blood pressure and ingestive behaviors. Administration of IMD into the lateral cerebroventricle of rats caused significant, long-lasting elevations in mean arterial pressure and heart rate. These elevations are similar to the effects of CGRP and significantly greater than the effects of AM. IMD-induced elevations in mean arterial pressure were inhibited by intravenous administration of phentolamine, indicating that IMD activates the sympathetic nervous system. Intracerebroventricular administration of IMD also inhibited food and water intake in sated and in food- and water-restricted animals. The effects on feeding are likely related to activation of the CGRP receptor and are independent of the effects on water intake, which are likely through the AM receptor. Our data indicate that IMD has potent actions within the CNS that may be a result of the combined activation of both AM and CGRP receptors.     

Progesterone upregulates calcitonin gene-related peptide and adrenomedullin receptor components and cyclic adenosine 3'5'-monophosphate generation in Eker rat uterine smooth muscle cell line

Calcitonin gene-related peptide (CGRP) and adrenomedullin (AM), two potent smooth-muscle relaxants, have been shown to cause uterine relaxation. Both CGRP- and AM-binding sites in the uterus increase during pregnancy and decrease at labor and postpartum. These changes in binding sites appear to be related to the changes in calcitonin receptor-like receptor (CRLR), receptor activity-modified protein 1 (RAMP1), RAMP2, and RAMP3 mRNA levels. It is not clear, however, whether the changes in the receptor components occur in the myometrial cells and whether the steroid hormones can directly alter these receptor components in the muscle cells. In addition, the mechanism of CGRP and AM signaling in the rat myometrium is not well understood. Therefore, we examined the mRNA expression of CGRP- and AM-receptor components, G protein Galphas, CGRP, and AM stimulation of cAMP and cGMP, and the effects of progesterone on these parameters in the Eker rat uterine myometrial smooth-muscle cell line (ELT3). ELT3 cells expressed CGRP- and AM-receptor components CRLR, RAMP1, RAMP2, and RAMP3. Expression of CRLR and RAMP1 mRNA increased with progesterone treatment and decreased with estradiol-17beta treatment. However, RAMP2 and RAMP3 mRNA expressions were unaltered by both progesterone and estradiol. Progesterone increased (P<0.05) Galphas expression and augmented CGRP- and AM-induced increases in cAMP levels. In uterine smooth-muscle cells, the antagonist to Galphas protein NF449 decreased basal as well as CGRP- and AM-stimulated cAMP levels. None of the cell treatments affected cyclic GMP production. Our results suggest that the progesterone-stimulated increases in CGRP and AM receptors, Galphas protein levels, and cAMP generation in the myometrial cells may be responsible for increased uterine relaxation sensitivity to CGRP and AM during pregnancy.     

Molecular and functional characterization of adrenomedullin receptors in pufferfish

The receptors for the calcitonin gene-related peptide (CGRP)/adrenomedullin (AM) family peptides were characterized in the mefugu Takifugu obscurus, a euryhaline fugu species very close to Takifugu rubripes, which has as many as five adrenomedullin genes (AM1-5). CGRP and AM share a G protein-coupled core receptor called calcitonin receptor-like receptor (CLR), and the specificity of the CLR is determined by the interaction with receptor activity-modifying proteins (RAMPs). Through database mining, three CLRs (CLR1-3) and five RAMPs (RAMP1-5) were identified, and all of them were cloned by RT-PCR and characterized by functional expression in COS7 cells in every possible combination of CLR-RAMP. The following combinations generated cAMP in response to physiological concentrations of CGRP, AM1 (an ortholog of mammalian AM), AM2, and AM5: CLR1-RAMP1/4 (CGRP), CLR1-RAMP2/3/5 (AM1), CLR2-RAMP2 (AM1), CLR1-RAMP3 (AM2), and CLR1-RAMP3 (AM5). Their expressions were found by Northern blot analysis to be tissue specific and salinity dependent. For example, CLR1-RAMP5 and CLR1-RAMP2 are expressed specifically in the gill and kidney, respectively, suggesting their involvement in osmoregulation. Furthermore, relatively high levels of CLRs and RAMPs were found in the spleen and ovary, suggesting roles in the immune and female reproductive systems. Immunohistochemistry revealed that AM receptors of the following types are expressed in the locations, indicated in brackets, of the mefugu gill and kidney: CLR1-RAMP5 (interlamellar vessels), CLR2-RAMP2 (pillar cells), and CLR1-RAMP2 (apical side of renal proximal tubule cells).