Comparison of responses to rat and human adrenomedullin in the hindlimb vascular bed of the cat

Responses to rat (r) adrenomedullin (ADM) and human (h) ADM were compared in the hindlimb vascular bed of the cat under conditions of controlled blood flow. Intra-arterial injections of rADM and hADM in doses of 0.03–1 nmol caused dose-related decreases in hindlimb perfusion pressure. In terms of relative vasodilator activity, rADM was similar to hADM. The time course of the vasodilator response and the recovery half times (T_1/2) for the vasodilator response to rADM and hADM were not significantly different. Decreases in hindlimb perfusion pressure in response to rADM and hADM were not altered by the calcitonin gene-related peptide receptor antagonist, rCGRP(8–37), at the same time, vasodilator responses to calcitonin gene-related peptide (CGRP) were significantly reduced. The T_1/2 of the vasodilator response to rADM and hADM were significantly greater after administration of the cAMP-selective, type IV phosphodiesterase inhibitor, rolipram. These data demonstrate that decreases in hindlimb perfusion pressure in response to rADM and hADM are similar and that vasodilator responses to rADM are not dependent on the activation of CGRP receptors in the hindlimb vascular bed of the cat. These data further suggest that decreases in hindlimb perfusion pressure in response to rADM are mediated by smooth muscle increases in cAMP levels.     

Expression of adrenomedullin mRNA in adrenocortical tumors and secretion of adrenomedullin by cultured adrenocortical carcinoma cells

Immunoreactive-adrenomedullin concentrations and the expression of adrenomedullin mRNA were studied in the tumor tissues of adrenocortical tumors. Northern blot analysis showed the expression of adrenomedullin mRNA in tumor tissues of adrenocortical tumors, including aldosterone-producing adenomas, cortisol-producing adenomas, a non-functioning adenoma and adrenocortical carcinomas, as well as normal parts of adrenal glands and pheochromocytomas. On the other hand, immunoreactive-adrenomedullin was not detected in about 90% cases of adrenocortical tumors (<0.12 pmol/g wet weight (ww)). Immunoreactive-adrenomedullin concentrations ranged from 0.44 to 198.2 pmol/g ww in tumor tissues of pheochromocytomas and were 9.2 ± 1.2 pmol/g ww (mean ± SD, n = 4) in normal parts of adrenal glands. Adrenomedullin mRNA was expressed in an adrenocortical adenocarcinoma cell line, SW-13 and immunoreactive-adrenomedullin was detected in the culture medium of SW-13 (48.9 ± 1.8 fmol/10⁵ cells/24h, mean ± SEM, n = 4). On the other hand, immunoreactive-adrenomedullin was not detectable in the extract of SW-13 cells (<0.09 fmol/10⁵ cells), suggesting that adrenomedullin was actively secreted from SW-13 cells without long-term storage. These findings indicate that adrenomedullin is produced and secreted, not only by pheochromocytomas, but also by adrenocortical tumors. Undetectable or low levels of immunoreactive-adrenomedullin in the tumor tissues of adrenocortical tumors may be due to very rapid secretion of this peptide soon after the translation from these tumors.     

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.     

Functions of the extracellular histidine residues of receptor activity-modifying proteins vary within adrenomedullin receptors

Receptor activity-modifying protein (RAMP)-2 and -3 chaperone calcitonin receptor-like receptor (CRLR) to the plasma membrane, where together they form heterodimeric adrenomedullin (AM) receptors. We investigated the contributions made by His residues situated in the RAMP extracellular domain to AM receptor trafficking and receptor signaling by co-expressing hCRLR and V5-tagged-hRAMP2 or -3 mutants in which a His residue was substituted with Ala in HEK-293 cells. Flow cytometric analysis revealed that hRAMP2-H71A mediated normal hCRLR surface delivery, but the resultant heterodimers showed significantly diminished [¹²⁵I]AM binding and AM-evoked cAMP production. Expression of hRAMP2-H124A and -H127A impaired surface delivery of hCRLR, which impaired or abolishing AM binding and receptor signaling. Although hRAMP3-H97A mediated full surface delivery of hCRLR, the resultant heterodimers showed impaired AM binding and signaling. Other His residues appeared uninvolved in hCRLR-related functions. Thus, the His residues of hRAMP2 and -3 differentially govern AM receptor function.     

Presence of immunoreactive adrenomedullin in human and bovine milk

We examined by radioimmunoassay the presence of immunoreactive adrenomedullin (ir-AM) in human and bovine milk. Milk samples displaced ¹²⁵I-AM from the AM-antiserum in parallel to the standard curve. RP-HPLC revealed a main immunoreactive peak eluting as synthetic AM. Concentrations in human milk ranged between 140 and 404 pg/mL. In cow, the levels of AM were 73.5 ± 3.8 pg/mL. Bovine milk products had AM levels similar to those found in fresh bovine milk. Human milk had growth promoting activity on the human intestinal cell line Int-407 that could be partially blocked with an anti-AM antibody.     

Intermedin1–53 inhibits rat cardiac fibroblast activation induced by angiotensin II

Intermedin (IMD) is a novel peptide related to calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM). Proteolytic processing of a larger precursor of IMD yields a biologically active C-terminal fragment IMD_1–53. We aimed to observe the cardioprotective antifibrotic effects of IMD_1–53 and its mechanism. Radioimmunoassay and Western blot analysis was used to determine IMD content in angiotensin II (AngII)-treated rat cardiac fibroblasts (CFs). Real-time PCR was used to measure mRNA levels of IMD and the IMD receptor components calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein (RAMP) 1, 2 and 3. AngII was a powerful stimulator of CF activation. It decreased the production and secretion of IMD and increased the mRNA levels of the IMD receptor components CRLR, RAMP2 and RAMP3, but not IMD and RAMP1. Moreover, IMD_1–53 (10^− 8 or 10^− 7 mol/l) exerted a 25% and 45% respective inhibition in [³H]-thymidine incorporation and 16% and 36% respective inhibition in [³H]-proline incorporation in rat CFs incubated with AngII, and the actions of IMD_1–53 could be blocked by CGRP_8–37 and ADM_22–52. Immunofluorescence and Western blot analysis revealed that IMD_1–53 inhibited the increase of alpha-SMA in CFs induced by AngII, and the above effects of IMD_1–53 were similar to or more potent than those of an equivalent dose of ADM. Otherwise, IMD_1–53 resulted in dose-dependent increases of cAMP production in CFs, and co-incubated with H89 blocked the inhibition effect of IMD_1–53 on AngII-induced [³H]-thymidine, [³H]-proline incorporation and alpha-SMA expression. Collectively, these results show that IMD and its receptor components could be involved in an onset of cardiac fibrosis, and like ADM, IMD_1–53 exerts an antifibrotic effect in CFs, and the effect can be mediated by cAMP–PKA pathway and implicated with the ADM and CGRP receptors.     

Significant decrease in plasma midregional proadrenomedullin level in patients with end-stage renal disease after living kidney transplantation

Impaired renal function has been suggested to significantly impact plasma midregional proADM (MR-proADM) level. The aim of this study was to assess whether improvement of renal function after living kidney transplantation has an impact on plasma MR-proADM-like immunoreactive substance (IS) level. Eleven patients with end-stage renal disease (ESRD) who were scheduled to undergo the first living kidney allograft transplantation were enrolled. Plasma MR-proADM-IS levels were measured before and 3, 7, 10, 14, 21, 30, 60 and 90 days after kidney transplantation. Plasma MR-proADM-IS level decreased significantly from day 3 after kidney transplantation compared to before kidney transplantation. A significant negative correlation was observed between creatinine clearance and plasma MR-proADM-IS level from before to 90 days after kidney transplantation (r_s = −0.70, p < 0.0001). These results suggest that recovery of kidney function after kidney transplantation may lead to decrease in plasma MR-proADM level in patients with ESRD, and that plasma MR-proADM level may depend largely on renal function.