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.     

Adrenomedullin is anti-apoptotic in osteoblasts through CGRP1 receptors and MEK-ERK pathway

Adrenomedullin (ADM) has been shown to mediate multifunctional responses in cell culture and animal system such as regulation of growth and apoptosis. ADM stimulates the proliferation of osteoblasts in vitro and promotes bone growth in vivo. The ability of ADM to influence osteoblastic cell number through inhibition of apoptosis has not yet been studied. To address this question we have investigated its effect on the apoptosis of serum-deprived osteoblastic cells using mouse MC3T3-E1 cells which express both ADM and ADM receptors. Treatment with ADM significantly blunted apoptosis, evaluated by caspase-3 activity, DNA fragmentation quantification and annexin V-FITC labeling. This effect was abolished by the subtype-1 CGRP receptor antagonist, CGRP(8-37). Both ADM and its specific receptor antagonist, the (22-52) ADM fragment exhibited a similar anti-apoptotic effect. Thus, our data suggest that ADM exerts anti-apoptotic effects through CGRP1 receptors. This was substantiated by a similar protective effect of CGRP on MC3T3-E1 cells apoptosis. Accordingly, neutralization of endogenous ADM by a specific antibody enhanced apoptosis. Finally, the selective inhibitor of MAPK kinase (MEK), PD98059, abolished the apoptosis protective effect of ADM and prevented ADM activation of ERK1/2. These data show that ADM acts as a survival factor in osteoblastic cells via a CGRP1 receptor-MEK-ERK pathway, which provides further understanding on the physiological function of ADM in osteoblasts.     

Distribution, functional role, and signaling mechanism of adrenomedullin receptors in the rat adrenal gland

Adrenomedullin (ADM) is a hypotensive peptide, highly expressed in the mammalian adrenal medulla, which belongs to a peptide superfamily including calcitonin gene-related peptide (CGRP) and amylin. Quantitative autoradiography demonstrated the presence of abundant [¹²⁵I]ADM binding sites in both zona glomerulosa (ZG) and adrenal medulla. ADM binding was selectively displaced by ADM(22–52), a putative ADM-receptor antagonist, and CGRP(8–37), a ligand that preferentially antagonizes the CGRP1-receptor subtype. ADM concentration-dependently inhibited K⁺-induced aldosterone secretion of dispersed rat ZG cells, without affecting basal hormone production. Both ADM(22–52) and CGRP(8–37) reversed the ADM effect in a concentration-dependent manner. ADM counteracted the aldosterone secretagogue action of the voltage-gated Ca²⁺-channel activator BAYK-8644, and blocked K⁺- and BAYK-8644-evoked rise in the intracellular Ca²⁺ concentration of dispersed ZG cells. ADM concentration-dependently raised basal catecholamine (epinephrine and norepinephrine) release by rat adrenomedullary fragments, and again the response was blocked by both ADM(22–52) and CGRP(8–37). ADM increased cyclic-AMP release by adrenal-medulla fragments, but not capsule-ZG preparations, and the catecholamine response to ADM was abolished by the PKA inhibitor H-89. Collectively, the present findings allow us to draw the following conclusions: (1) ADM modulates rat adrenal secretion, acting through ADM(22–52)-sensitive CGRP1 receptors, which are coupled with different signaling mechanisms in the cortex and medulla; (2) ADM selectively inhibits agonist-stimulated aldosterone secretion, through a mechanism probably involving the blockade of the Ca²⁺ channel-mediated Ca²⁺ influx; (3) ADM raises catecholamine secretion, through the activation of the adenylate cyclase/PKA signaling pathway.     

Intermedin 1-53 in central nervous system elevates arterial blood pressure in rats

Intermedin (IMD) is a novel member of the calcitonin/calcitonin gene-related peptide (CGRP) family identified from human and other vertebrate tissues. Preprointermedin can generate various mature peptides by proteolytic cleavage. Amino acid sequence analysis showed cleavage sites located between two basic amino acids at Arg93-Arg94 resulting in the production of prepro-IMD(95-147), namely IMD(1-53). The present study was designed to determine the effects of the IMD(1-53) fragment in the central nervous system (CNS) on mean arterial blood pressure and heart rate in normal rats and its possible mechanism. Rats were given doses of adrenomedullin (ADM) or IMD(1-53), intracerebroventricularly or intravenously, respectively, with continuous blood pressure and heart rate monitoring for 45min. Analysis with CGRP receptor antagonist CGRP(8-37), ADM receptor antagonist ADM(22-52), and anti-prepro-IMD antibody showed that 0.1, 0.5, and 1.0 nmol/kg IMD(1-53), caused a dose-dependent elevation in blood pressure, which was more prominent than the increase with equivalent IMD(1-47) or ADM. As well, IMD(1-53) caused a persistent increase in heart rate. The CNS action of IMD(1-53) could be blocked by ADM(22-52), CGRP(8-37), or prepro-IMD antibody. In contrast to the CNS action, intravenous administration of IMD(1-53) induced a depressor effect. These results suggest that IMD(1-53) is an important regulatory factor in mean arterial blood pressure and heart rate through its central and peripheral bioaction.     

Adrenomedullin inhibits neurotransmission of calcitonin gene-related peptide (CGRP)-containing vasodilator nerves in rat mesenteric resistance arteries.

We have reported that the rat mesenteric resistance artery has innervation of calcitonin gene-related peptide (CGRP)-containing vasodilator nerves (CGRPergic nerves). We also demonstrated that adrenomedullin (AM) causes mesenteric vasodilation through activation of CGRP receptors. The present study was designed to examine the effect of AM on neurotransmission of CGRPergic nerves in rat mesenteric arteries. In preconstricted preparations without endothelium, periarterial nerve stimulation (PNS, 1 and 2 Hz) induced a frequency-dependent vasodilation. A bolus injection of CGRP (10 pmol) into the perfusate also caused a vasodilation. AM (0.1 to 10 nM) concentration-dependently caused 40% to 60% inhibition of the PNS-induced vasodilation, but AM did not attenuate vasodilation induced by exogenous CGRP injection. The inhibitory effect of AM (10 nM) on PNS-induced vasodilation was further potentiated by CGRP [8-37] (CGRP receptor antagonist, 50 nM), which attenuated the vasodilator response to the CGRP injection. Combined perfusion of AM [22-52] (AM receptor antagonist, 10 to 100 nM) resulted in further inhibition of PNS-induced neurogenic vasodilation without affecting the vasodilator response to the CGRP injection. CGRP [8-37] but not AM [22-52] antagonized vasodilation induced by AM perfusion. These findings suggest that AM presynaptically inhibits neurotransmission of CGRPergic nerves, probably decreasing CGRP release, via receptors different from CGRP receptors.     

Digestive motor effects and vascular actions of CGRP in dog are expressed by different receptor subtypes

Calcitonin gene-related peptide (CGRP) is a 37 AA peptide localized in blood vessels and nerves of the GI tract. Activation of CGRP receptors (subtypes 1 or 2) usually induces vasodilation and/or muscle relaxation, but its effects in dog and on gastroduodenal motility are still unclear. This study looked for the effect of CGRP and the antagonist CGRP8-37, specific for CGRP type 1 receptor, 1) on GI motility (interdigestive and postprandial), and 2) on hemodynamy, in conscious dogs. During the interdigestive period, the infusion of CGRP1-37 (200 pmol/kg/h) or CGRP8-37 (2000 pmol/kg/h) did not modify the duration of the migrating motor complex nor the release nor the motor action of plasma motilin. The gastric emptying of a solid meal (15 g meat/kg) was reduced by the administration of CGRP1-37 (AUC: 2196 +/- 288.6 versus 3618 +/- 288.4 with saline or T12: 78 +/- 7.3 versus 50 +/- 4.3 min; P < 0.01) and this effect was reversed by the antagonist CGRP8-37. CGRP1-37 significantly (P < 0. 01) diminished arterial pressures (118 +/- 1.6/64 +/- 1.4 vs. 125 +/- 1.4/75 +/- 1.2 mmHg with saline) and accelerated the basal cardiac rhythm (110 +/- 1.4 versus 83 +/- 1.6 beats/min). However, CGRP8-37 failed to block the cardiovascular effects of CGRP1-37. In dog, CGRP could influence digestive motility by slowing the gastric emptying of a meal through an action on CGRP-1 receptors. Hemodynamic effects of CGRP were not blocked by CGRP8-37 and seem therefore mediated by CGRP-2 receptor subtype.     

Antiinflammatory activity of adrenomedullin in the acetic acid peritonitis in rats

The antiinflammatory effect of ADM was studied in different models of inflammation and compared to the one of CGRP. Peptides were active against acetic acid-induced peritonitis in the rats. ADM and CGRP exerted the antiinflammatory effect at different doses, 400 and 20 ng/kg respectively, but with different efficacy (ADM >CGRP). This effect was blocked by pretreatment with CGRP (8-37) fragment or with L-NAME. No antiinflammatory activity was evidenced against serotonin- or carrageenin-induced rat paw edema. Our data suggest that ADM exerts antiinflammatory activity in the model characterized by a vascular component. This effect involves CGRP receptors and appears to be mediated by nitric oxide system.     

Adrenomedullin receptors: pharmacological features and possible pathophysiological roles

Three receptor activity modifying proteins (RAMPs) chaperone calcitonin-like receptor (CLR) to the cell surface. RAMP2 enables CLR to form an adrenomedullin (AM)-specific receptor that is sensitive to AM-(22-52) (AM(1) receptor). RAMP3 enables CLR to form an AM receptor sensitive to both calcitonin gene-related peptide (CGRP)-(8-37) and AM-(22-52) (AM(2) receptor), though rat and mouse AM(2) receptors show a clear preference for CGRP alpha-(8-37) over AM-(22-52). RAMP1 enables CRL to form the CGRP-(8-37)-sensitive CGRP(1) receptor, which can also be activated by higher concentrations of AM. Here we review the available information on the pharmacological features and possible pathophysiological roles of the aforementioned AM receptors.     

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.     

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.     

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.     

The effect of adrenomedullin on the L-type calcium current in myocytes from septic shock rats: signaling pathway

Adrenomedullin (ADM) is upregulated in cardiac tissue under various pathophysiological conditions, particularly in septic shock. The intracellular mechanisms involved in the effect of ADM on adult rat ventricular myocytes are still to be elucidated. Ventricular myocytes were isolated from adult rats 4 h after an intraperitoneal injection of lipopolysaccharide (LPS, 10 mg/kg). Membrane potential and L-type calcium current (I(Ca,L)) were determined using whole cell patch-clamp methods. APD in LPS group was significantly shorter than control values (time to 50% repolarization: LPS, 169 +/- 2 ms; control, 257 +/- 2 ms, P < 0.05; time to 90% repolarization: LPS, 220 +/- 2 ms; control, 305 +/- 2 ms, P < 0.05). I(Ca,L) density was significantly reduced in myocytes from the LPS group (-3.2 +/- 0.8 pA/pF) compared with that of control myocytes (-6.7 +/- 0.3 pA/pF, P < 0.05). The ADM antagonist ADM-(22-52) reversed the shortened APD and abolished the reduction of I(Ca,L) in shock myocytes. In myocytes from control rats, incubating with ADM for 1 h induced a marked decrease in peak I(Ca,L) density. This effect was reversed by ADM-(22-52). The G(i) protein inhibitor, pertussis toxin (PTX), the protein kinase A (PKA) inhibitor, KT-5720, and the specific cyclooxygenase 2 (COX-2) inhibitor, nimesulide, reversed the LPS-induced reduction in peak I(Ca,L). The results suggest a COX-2-involved PKA-dependent switch from G(s) coupled to PTX-sensitive G(i) coupling by ADM in adult rat ventricular myocytes. The present study delineates the intracellular pathways involved in ADM-mediated effects on I(Ca,L) in adult rat ventricular myocytes and also suggests a role of ADM in sepsis.     

Two new peptides to improve post-operative gastric ileus in dog

Peptides can influence gastrointestinal motility, and from data obtained earlier in rats, we hypothesized that MTL-RP/Ghrelin, as well as CGRP receptor antagonist 8-37, could improve gastric post-operative ileus in dog. Dogs submitted to laparotomy were perfused with or saline or CGRP 8-37 or MTL-RP/Ghrelin on days 1-4 post-operatively while gastric emptying was estimated by measuring the postprandial increase in plasma acetaminophen ingested with a meal. As expected, in saline-treated animals the gastric emptying function was impaired post-operatively. The total amount of acetaminophen emptied (AUC over 150 min) on post-operative days 1-4 reached respectively 31+/-5%, 65+/-8%, 60+/-8% and 62+/-8% of the normal emptying capacity. CGRP antagonist increased the total emptying of acetaminophen to 52+/-5% on day 1, 95+/-2% on day 2 and 103+/-3% (P<0.05) on day 3. The delayed emptying of acetaminophen seen post-operatively in saline-treated animals could be completely reversed by MTL-RP/Ghrelin (P<0.01) whether it was given at 100 microg/kg on day 2 (102+/-7% of the normal emptying capacity), 4 microg/kg on day 3 (106+/-7%) or 20 microg/kg on day 4 (132+/-8%). As found earlier in rodents, CGRP receptor antagonist 8-37 as well as MTL-RP/Ghrelin are potent prokinetics to improve post-operative gastric ileus in dog.     

Inhibition of periarterial nerve stimulation-induced vasodilation of the mesenteric arterial bed by CGRP (8-37) and CGRP receptor desensitization

We provide the first functional evidence that calcitonin gene-related peptide (8-37) induces a direct vasoconstriction and reversibly antagonizes vasodilation of the mesenteric arterial bed induced by calcitonin gene-related peptide (CGRP) suggesting that CGRP (8-37) is a competitive antagonist of vascular CGRP receptors. Vasodilation induced by periarterial nerve stimulation was inhibited both by CGRP (8-37) and by desensitization of CGRP receptors. These results further support the evidence that the periarterial nerve stimulation-induced nonadrenergic noncholinergic vasodilation of the mesenteric vasculature is mediated by endogenous CGRP and its receptors.     

CGRP 8-37 enhances lipopolysaccharide-induced acute lung injury and regulating aquaporin 1 and 5 expressions in rats

Calcitonin gene-related peptide (CGRP) has been shown to play important roles in biological functions. However, there is very little evidence on the value of CGRP in lipopolysaccharide (LPS)-induced acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Therefore, this study aimed to investigate the role of CGRP in LPS-induced ALI in rats. In the experiment, Sprague-Dawley (SD) rats were randomized into control, an antagonist of α-calcitonin gene-related peptide receptor (CGRP8-37), LPS groups, and CGRP8-37 + LPS groups. ALI model was prepared through retrograde injection of LPS (10 mg/kg). At 6 and 12 h, bronchoalveolar lavage was performed and used to assess total cell count and levels of tumor necrosis factor-α, interleukin-1β, -6, and -10 by enzyme-linked immunosorbent assay (ELISA). Lung tissue was collected for assessing wet-to-dry (W/D) ratio, hematoxylin and eosin staining. Aquaporin (AQP)-1 and -5 expressions in lung tissues were detected by quantitative PCR and Western blot. The results showed that histological injury, total cell count, and W/D ratio significantly reduced in LPS group after 6 h. The levels of inflammatory cytokines in CGRP8-37 + LPS-treated rats were higher than that in LPS-treated rats (all, P < 0.001). Real-time RT-PCR analysis showed that levels of AQP-1 in rats from CGRP8-37 + LPS group was lower than that in LPS-treated rats (P = 0.005 and P < 0.001). Western blotting analysis showed that AQP-1 protein levels at 6 h significantly decreased in CGRP8-37 + LPS rats. Together, our data suggest that CGRP antagonists, CGRP8-37 could enhance ALI induced by LPS in the rat model, and regulate the expression levels of AQP-1 and AQP-5 by affecting inflammatory cytokines. Thereby, regulating endogenous CGRP may be a potential treatment for ALI/ARDS.     

Peripheral adrenomedullin inhibits gastric emptying through CGRP8-37 -sensitive receptors and prostaglandins pathways in rats

The effects of intravenous (iv) adrenomedullin (AM) on gastric emptying were investigated in conscious rats. AM induced a maximal 50% inhibition of gastric emptying at a dose of 1.2 nmol/kg. AM was about two-fold less potent than alpha-calcitonin gene-related peptide (alpha-CGRP), which induced a similar 50% maximal inhibition of gastric emptying at 0.6 nmol/kg. Delayed gastric emptying induced by i.v. AM and alpha-CGRP was prevented by peripheral injection of the selective CGRP1 antagonist, CGRP8-37, and by pretreatment with indomethacin, while not altered by blockade of the sympathetic nervous system with propranolol. These data indicate that peripheral AM inhibits gastric emptying through the interaction with CGRP8-37 -sensitive receptors, likely CGRP1 receptors, and the recruitment of prostaglandin-dependent mechanisms.     

Role of capsaicin-sensitive afferents and sensory neuropeptides in endotoxin-induced airway inflammation and consequent bronchial hyperreactivity in the mouse

Substance P (SP) and calcitonin gene-related peptide (CGRP) released from capsaicin-sensitive afferents induce neurogenic inflammation via NK(1), NK(2) and CGRP1 receptor activation. This study examines the role of capsaicin-sensitive fibres and sensory neuropeptides in endotoxin-induced airway inflammation and consequent bronchial hyperreactivity with functional, morphological and biochemical techniques in mice. Carbachol-induced bronchoconstriction was measured with whole body plethysmography 24 h after intranasal lipopolysaccharide administration. SP and CGRP were determined with radioimmunoassay, myeloperoxidase activity with spectrophotometry, interleukin-1beta with ELISA and histopathological changes with semiquantitative scoring from lung samples. Treatments with resiniferatoxin for selective destruction of capsaicin-sensitive afferents, NK(1) antagonist SR 140333, NK(2) antagonist SR 48968, their combination, or CGRP1 receptor antagonist CGRP(8-37) were performed. Lipopolysaccharide significantly increased lung SP and CGRP concentrations, which was prevented by resiniferatoxin pretreatment. Resiniferatoxin-desensitization markedly enhanced inflammation, but decreased bronchoconstriction. CGRP(8-37) or combination of SR 140333 and SR 48968 diminished neutrophil accumulation, MPO levels and IL-1beta production, airway hyperresponsiveness was inhibited only by SR 48968. This is the first evidence that capsaicin-sensitive afferents exert a protective role in endotoxin-induced airway inflammation, but contribute to increased bronchoconstriction. Activation of CGRP1 receptors or NK(1)+NK(2) receptors participate in granulocyte accumulation, but NK(2) receptors play predominant role in enhanced airway resistance.     

Comparative affinities of adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) for [125I] AM and [125I] CGRP specific binding sites in porcine tissues.

We have investigated the binding characteristics of rat [125I] adrenomedullin (AM) and human [125I] calcitonin gene-related peptide (CGRP) to membranes prepared from a number of porcine tissues including atrium, ventricle, lung, spleen, liver, renal cortex and medulla. These membranes displayed specific, high affinity binding for [125I] rat AM and [125I] human CGRP. Porcine lung displayed the highest density of binding sites for radiolabeled AM and CGRP followed by porcine renal cortex. Competition experiments performed with [125I] rat AM indicated that the rank order of potencies of various peptides for inhibiting [125I] rat AM binding to various tissues were rat AM > or = human AM > or = human AM(22-52) > h alpha CGRP > or = h alpha CGRP(8-37) > sCT except spleen, atrium, renal cortex and renal medulla where rAM and hAM were 20-300 fold more potent than hAM (22-52). When the same experiments were performed using [125I] h alpha CGRP as the radioligand, the rank order potencies for various peptides were rAM = hAM > h alpha CGRP > h alpha CGRP(8-37) in most of the tissues except in spleen and liver where h alpha CGRP was the most potent ligand. In lung, h alpha CGRP was almost as potent as rAM and hAM in displacing [125I] h alpha CGRP binding. These data suggest the existence of distinct CGRP and AM specific binding sites in contrast to previous reports that showed that both peptides interact differently in rat tissues.     

Hypertension induced by nitric oxide synthase inhibitor increases responsiveness of ventricular myocardium and aorta of rat tissue to adrenomedullin stimulation in vitro

In this work, we aimed to observe the changes in adrenomedullin (ADM) and its receptor-calcitonin receptor-like receptor (CL), receptor activity-modifying protein (RAMP) 1, RAMP2 and RAMP3-in cardiac ventricles and aortas of hypertensive rats, and the responsiveness of injured cardiovascular tissue to ADM, then to illustrate the protective mechanism of ADM on the cardiovascular system. Male SD rats were subjected to treatment with chronic N(G)-nitro-L-arginine (L-NNA), an inhibitor of nitric oxide synthase. The ADM contents and cAMP production in myocardia and aortas were measured by RIA. The mRNA levels of ADM, CL, and RAMP1-3 were determined by RT-PCR. L-NNA induced severe hypertension and cardiomegaly. The ir-ADM content in plasma, ventricles and aortas in L-NNA-treated animals increased by 80%, 72% and 57% (all p<0.01), respectively. Furthermore, mRNA levels of ADM, CL, RAMP2 and RAMP3 were elevated by 91%, 33%, 50% and 72.5% (all p<0.01), respectively, in ventricles and by 95%, 177%, 74.7% and 85% (all p<0.01), respectively, in aortas. mRNA level of RAMP1 was elevated by 129% (p<0.01) in aortas but no significant difference in ventricles. The elevated mRNA levels of RAMP2 and RAMP3 were positively correlated with that of ADM in hypertrophic ventricles (r=0.633 and 0.828, p<0.01, respectively) and the elevated mRNA levels of CL, RAMP2 and RAMP3 were positively correlated with that of ADM in aortas (r=0.941, 0.943 and 0.736, all p<0.01, respectively). The response of ventricular myocardia and aortas to ADM administration potentiated, and the production of cAMP was increased by 41% and 68% (both p<0.01), respectively. ADM-stimulated cAMP generation in ventricular myocardia and aortas was blocked by administration of both ADM22-52, the specific antagonist of ADM receptor, and CGRP8-37, the antagonist of the CGRP1 receptor. The results showed an increased in cardiovascular ADM generation and an up-regulation of the gene expression of ADM and its receptor-CL, RAMP1-3 during hypertension, augmented responsiveness of ventricular myocardia and aortas of hypertensive rats to ADM, suggesting that these receptors may play a role in the cardiovascular adaptation in response to sub-chronic NO-inhibition.