The amino terminus of receptor activity modifying proteins is a critical determinant of glycosylation state and ligand binding of calcitonin receptor-like receptor.
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The calcitonin receptor-like receptor (CRLR) can function as either a receptor for calcitonin gene-related peptide (CGRP) or for adrenomedullin (ADM), depending upon the coexpression of a novel family of single transmembrane proteins, which we have called receptor activity modifying proteins or RAMPs. RAMPs 1, 2, and 3 transport CRLR to the plasma membrane with similar efficiencies, however RAMP1 presents CRLR as a terminally glycosylated, mature glycoprotein and a CGRP receptor, whereas RAMPs 2 and 3 present CRLR as an immature, core glycosylated ADM receptor. Characterization of the RAMP2/CRLR and RAMP3/CRLR receptors in HEK293T cells by radioligand binding (125I-ADM as radioligand), functional assay (cAMP measurement), or biochemical analysis (SDS-polyacrylamide gel electrophoresis) revealed them to be indistinguishable, even though RAMPs 2 and 3 share only 30% identity. Chimeric proteins were created with the transmembrane and cytosolic portions of RAMP1 associated with the amino terminus of RAMP2 (RAMP2/1) and vice versa (RAMP1/2). Coexpression of RAMP2/1 with CRLR formed a core glycosylated ADM receptor, whereas the RAMP1/2 chimera generated both core glycosylated and mature forms of CRLR and enabled both ADM and CGRP receptor binding. Hence, the glycosylation state of CRLR appears to correlate with its pharmacology. (+info)
Reduced adrenomedullin expression in gastric mucosa of portal hypertensive rats after ethanol-induced injury.
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OBJECTIVE: To determine the expression and localization of adrenomedullin (AM) and its receptor (AM-R) in portal hypertensive (PHT) gastric mucosa after intragastric ethanol administration. SUMMARY BACKGROUND DATA: The repair of gastric mucosal injury requires reestablishment of the microvascular network. The authors previously demonstrated impaired angiogenesis of PHT gastric mucosa after ethanol-induced injury. Because AM, a potent vasodilatory peptide, is also a novel growth and angiogenic factor, the authors hypothesized that AM is involved in the impaired repair of PHT gastric mucosa and its microvasculature after damage. METHODS: Either PHT or sham-operated rats received intragastrically 100% ethanol, and the stomachs were excised at 1, 6, and 24 hours later. Expression and localization of AM and AM-R mRNA were examined by competitive reverse transcription-polymerase chain reaction and in situ hybridization. AM protein expression was examined by Western blot analysis. RESULTS: One hour after ethanol administration, AM mRNA expression in PHT gastric mucosa was significantly decreased by 81%, especially in the superficial mucosa, compared with the gastric mucosa in sham-operated rats. The significant decrease lasted for 24 hours. AM protein expression was significantly decreased by 43% compared with the sham-operated gastric mucosa. Although AM-R mRNA expression in both groups was significantly increased 1 hour after ethanol administration and lasted for 24 hours compared with baseline, there were no differences between the two groups. CONCLUSIONS: The expression of AM in PHT gastric mucosa after ethanol-induced injury is significantly decreased compared with controls. This finding could explain one mechanism for the impaired angiogenesis after injury of PHT gastric mucosa. (+info)
Bioactivity and interactions of adrenomedullin and brain natriuretic peptide in patients with heart failure.
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Plasma concentrations of the recently discovered hormones adrenomedullin (ADM), from vascular tissue, and brain natriuretic peptide (BNP), secreted by myocardium, are elevated in patients with heart failure. We tested the hypotheses that short-term increments in circulating levels of these hormones, within the pathophysiological range, would have biological effects and that the 2 hormone systems interact. Eight patients with heart failure (left ventricular ejection fractions <35%) received 4-hour infusions of BNP (3.0 pmol. kg(-1). min(-1)) alone, ADM (2.7 pmol. kg(-1). min(-1) and 5.4 pmol. kg(-1). min(-1) for 2 hours each) alone, ADM and BNP combined, and placebo. BNP and ADM infusions raised plasma levels of the respective peptide within the pathophysiological range. Arterial blood pressure fell (P<0.05) with all peptide infusions, but cardiac output was unchanged. Heart rate increased with ADM and combined infusions (P<0.01). Sodium excretion rose (P<0.05), and creatinine clearance was sustained during both BNP and combined infusions. Urine volume increased in response to BNP alone (P=0.02). Despite a >2-fold increase in plasma renin with both ADM and combined infusions (P<0.05), plasma aldosterone remained lower than time-matched placebo levels. Plasma noradrenaline was increased by combined, BNP, and higher dose ADM infusions (P<0.05). ADM suppressed plasma cGMP (P<0.05) and inhibited the plasma cGMP response to BNP (P<0.05). The vascular hormones ADM and BNP, produced by myocardium, at plasma concentrations within the pathophysiological range have hemodynamic, renal, and hormonal effects and measurable interactions in patients with heart failure. (+info)
Adrenomedullin in monocytes and macrophages: possible involvement of macrophage-derived adrenomedullin in atherogenesis.
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Macrophages secrete a variety of growth factors, cytokines and vasoactive peptides, which are related to the progression of atherosclerosis. Adrenomedullin (ADM) is a potent vasodilator peptide and inhibits proliferation and migration of vascular smooth muscle cells. In this study, we investigated the production and secretion of ADM by monocytes and macrophages by Northern blot analysis, RIA and immunocytochemistry. Northern blot analysis showed that ADM mRNA was expressed in human monocytes obtained from peripheral blood and monocyte-derived macrophages. The expression level of ADM mRNA in monocyte-derived macrophages was about five times higher than that in monocytes. Treatment with lipopolysaccharide (100 ng/ml) for 24 h increased ADM mRNA expression levels in both monocytes and monocyte-derived macrophages. The levels of immunoreactive ADM in the media of monocyte-derived macrophages were about three times higher than that of monocytes (0. 718+/-0.046 fmol/24 h/10(5) cells, n=8 compared with 0.259+/-0.018 fmol/24 h/10(5) cells, n=8; mean+/-S.E.M., P<0.01). The secretion was also increased by treatment with lipopolysaccharide. Immunocytochemistry showed positive ADM immunostaining in macrophages in atherosclerotic lesions of human aorta obtained at autopsy. ADM secreted from activated macrophages may play an inhibitory role in atherogenesis. (+info)
Adrenomedullin and atrial natriuretic peptide concentrations in normal pregnancy and pre-eclampsia.
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Adrenomedullin (AM) is a peptide that elicits a long-lasting vasorelaxant activity, while atrial natriuretic peptide (ANP) has also been shown to be a potent vasodilatory agent. To clarify the possible role of AM and ANP in the physiology of pregnancy and pathophysiology of pre-eclampsia, we measured plasma concentrations of these peptides in non-pregnant women, normal pregnant women and women with pre-eclampsia. A gradual increase in plasma AM was observed as pregnancy progressed. The plasma AM concentrations during the second trimester (12.7 +/- 1.4 fmol/ml) were significantly elevated, in comparison with the non-pregnant follicular phase (6.4 +/- 0.61 fmol/ml), luteal phase (6.0 +/- 0.49 fmol/ml), and the first trimester (6.5 +/- 0.8 fmol/ml). The plasma AM concentrations of the third trimester (21.5 +/- 1.4 fmol/ml) were significantly elevated when compared with those of the second trimester (P < 0.05). Northern blot analysis confirmed the expression of the AM mRNA transcript (1.6 kb) in third trimester placentas. In comparison with those observed at term (25.3 +/- 4.5 fmol/ml), the plasma concentrations were significantly reduced post-partum (6.4 +/- 0.6 fmol/ml). In the third trimester, plasma AM concentrations did not differ significantly between women with pre-eclampsia (17.2 +/- 2.3 fmol/ml) and normal pregnant women. In contrast, the plasma ANP concentrations in pre-eclampsia (39.5 +/- 7. 1 pg/ml) were significantly elevated when compared with those of the normal third trimester (14.4 +/- 1.4 pg/ml) (P < 0.05). ANP concentrations were reasonably constant throughout the pregnancy. (+info)
Circulating adrenomedullin is increased after heart transplantation.
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OBJECTIVE: Adrenomedullin (ADM), secreted by the failing human heart, is a newly discovered potent endogenous vasorelaxing and natriuretic peptide that may play a role in cardiorenal regulation. No data are available on ADM in heart-transplant recipients (Htx) and the aim of this study was to determine the short- and long-term responses of ADM after heart transplantation. METHODS: Circulating ADM and its relationship with parameters of cardiovascular hemodynamics, humoral factors and renal function were determined in normal subjects and Htx early (1, 2, 4, 8, 15 and 30 days) and late (32 +/- 16 months) after transplantation. Additionally, ADM was obtained in matched hypertensive and renal-transplant patients (n = 9 in each group). RESULTS: Plasma ADM, elevated in heart failure patients, further increased transiently at day 1 after transplantation (from 37.9 +/- 15.9 to 125.8 +/- 15.3 pmol/l, P < 0.01) and, although decreasing thereafter, remained elevated until the 30th day after transplantation (52.1 +/- 25.2 pmol/l). Late after transplantation. ADM concentrations were still increased compared to normal values (31.3 +/- 5.3 vs. 19.4 +/- 2.7 pmol/l, P < 0.001). ADM positively correlated with endothelin, atrial natriuretic peptide (ANP) and cyclosporine. ADM was also correlated with increased diastolic (r = 0.68, P < 0.04) and systolic (r = 0.66, P < 0.05) blood pressure in late Htx. No relationship was observed between ADM and left ventricular mass index, aldosterone and creatinine. ADM elevation was similar in hypertensive, renal-transplant patients and in Htx. CONCLUSIONS: Circulating ADM is increased after heart transplantation, in relation to hypertension, endothelin, cyclosporine and ANP. In view of ADM's biological properties, these results might suggest a compensatory role for ADM against further development of vasoconstriction and fluid retention states after heart transplantation. (+info)
Influence of CGRP (8-37), but not adrenomedullin (22-52), on the haemodynamic responses to lipopolysaccharide in conscious rats.
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1. The functional involvement of the vasodilator peptides, adrenomedullin (ADM) and calcitonin gene-related peptide (CGRP), in the haemodynamic sequelae of continuous infusion of lipopolysaccharide (LPS) was assessed in conscious, male, Long Evans rats, by the use of peptide antagonists. 2. It was demonstrated that ADM (22-52) at a dose of 500 nmol kg-1 h-1 caused significant inhibition of the effects of ADM (1 nmol kg-1), without affecting responses to CGRP (0.1 or 1 nmol kg-1). 3. Even when the regional vasodilator responses to LPS infusion were enhanced (by pre-treatment with dexamethasone and the endothelin antagonist, SB 209670, or by pretreatment with SB 209670 and the AT1-receptor antagonist, losartan), ADM (22-52) had no significant cardiovascular effects. In contrast, the CGRP1-receptor antagonist, CGRP (8-37), caused small, but significant, inhibitions of the hypotensive and renal and mesenteric vasodilator effects of LPS, but only 6 h after onset of infusion in the presence of dexamethasone and SB 209670. 4. The results indicate that, in this model of endotoxaemia, the marked regional vasodilatations seen in the presence of dexamethasone and SB 209670 do not involve ADM, but do involve CGRP, albeit only to a small extent. (+info)
Cyclic AMP-dependent synthesis and release of adrenomedullin and proadrenomedullin N-terminal 20 peptide in cultured bovine adrenal chromaffin cells.
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Adrenomedullin and proadrenomedullin N-terminal 20 peptide are peptides with multiple physiological functions and are most abundant in adrenal medulla. We studied whether the cAMP-dependent pathway is involved in the regulation of synthesis and release of adrenomedullin and proadrenomedullin N-terminal 20 peptide in cultured bovine adrenal chromaffin cells. Exposure of the cells to dibutyryl cAMP (dbcAMP) increased a progressive accumulation of immunoreactive-adrenomedullin and immunoreactive-proadrenomedullin N-terminal 20 peptide in the extracellular medium, while reciprocally decreasing their cellular content in a time-dependent manner. The decrease of levels of both peptides in the cells was much greater in extent than the increase of the peptides in the medium. H89, an inhibitor of cAMP-dependent protein kinase attenuated these changes, induced by dbcAMP. The resulting changes by dbcAMP and H89 were similar to those of chromogranin B, a marker peptide of chromaffin granule. Northern blot analysis showed that the mRNA encoding these peptides, detected as a band of 1.6 kb, was decreased by the treatment with dbcAMP. The effect of dbcAMP on mRNA was attenuated by H89, and was reversible as the decreased mRNA level caused by dbcAMP could be returned to control levels by culturing cells after removal of dbcAMP. These results suggest that the cAMP-dependent protein kinase pathway stimulates the release of adrenomedullin and proadrenomedullin N-terminal 20 peptide, whereas it lowers synthesis of these peptides via the reduction of their transcript level. (+info)