Kenji Kuwasako

Adrenomedullin: a possible autocrine or paracrine inhibitor of hypertrophy of cardiomyocytes.

Adrenomedullin (AM), a potent vasodilator peptide, exists in the cardiac ventricle; however, the role of AM in the ventricular tissue remains unknown. In the present study, we investigated the production and secretion of AM in cultured neonatal rat cardiomyocytes, and we examined the effect of AM on de novo protein synthesis in these cells by measuring [14C]phenylalanine incorporation. The cardiomyocytes cultured with serum-free media secreted AM into the media in a time-dependent manner at the rate of 12.2+/-0.5 fmol/10(5) cells/48 hours (mean+/-SEM). Angiotensin II (1 micromol/L) or 10% fetal bovine serum significantly (P<.01) increased the AM secretion by 115% and 305%, respectively. In addition, Northern blot analysis of total RNA extracted from the myocytes disclosed the expression of prepro-AM mRNA of 1.6 kb. Synthetic AM at 1 micromol/L significantly reduced the 10(-6) mol/L angiotensin II- and 10% fetal bovine serum-stimulated [14C]phenylalanine incorporation into the cells, by 16% (P<.05) and 20% (P<.01), respectively. The inhibitory effect of AM on the angiotensin II-stimulated [14C]phenylalanine incorporation was abolished dose-dependently by a calcitonin gene-related peptide receptor antagonist, CGRP(8-37). Furthermore, blockade of the action of endogenous AM by either 10(-6) mol/L CGRP(8-37) or anti-AM monoclonal antibody significantly enhanced the basal and 10(-6) mol/L angiotensin II-stimulated [14C]phenylalanine incorporation. In summary, cultured neonatal rat cardiomyocytes produce and secrete AM, and the secreted AM inhibits the protein synthesis of these cells. Thus, AM may act on cardiomyocytes as an autocrine or a paracrine factor modulating the cardiac growth.

Visualization of the calcitonin receptor-like receptor and its receptor activity-modifying proteins during internalization and recycling.

Expression of the calcitonin receptor-like receptor (CRLR) and its receptor activity modifying proteins (RAMPs) can produce calcitonin gene-related peptide (CGRP) receptors (CRLR/RAMP1) and adrenomedullin (AM) receptors (CRLR/RAMP2 or -3). A chimera of the CRLR and green fluorescent protein (CRLR-GFP) was used to study receptor localization and trafficking in stably transduced HEK 293 cells, with or without co-transfection of RAMPs. CRLR-GFP failed to generate responses to CGRP or AM without RAMPs. Furthermore, CRLR-GFP was not found in the plasma membrane and its localization was unchanged after agonist exposure. When stably coexpressed with RAMPs, CRLR-GFP appeared on the cell surface and was fully active in intracellular cAMP production and calcium mobilization. Agonist-mediated internalization of CRLR-GFP was observed in RAMP1/CGRP or AM, RAMP2/AM, and RAMP3/AM, which occurred with similar kinetics, indicating the existence of ligand-specific regulation of CRLR internalization by RAMPs. This internalization was strongly inhibited by hypertonic medium (0.45m sucrose) and paralleled localization of rhodamine-labeled transferrin, suggesting that CRLR endocytosis occurred predominantly through a clathrin-dependent pathway. A significant proportion of CRLR was targeted to lysosomes upon binding of the ligands, and recycling of the internalized CRLR was not efficient. In HEK 293 cells stably expressing CRLR-GFP and Myc-RAMPs, these rhodamine-labeled RAMPs were co-localized with CRLR-GFP in the presence and absence of the ligands. Thus, the CRLR is endocytosed together with RAMPs via clathrin-coated vesicles, and both the internalized molecules are targeted to the degradative pathway.

An autocrine or a paracrine role of adrenomedullin in modulating cardiac fibroblast growth.

Objective: The aim of the present study was to determine the role of adrenomedullin (AM) in cardiac fibroblasts. Methods: The production and secretion of AM were examined in cultured neonatal rat cardiac fibroblasts, and the effects of AM on proliferation and protein synthesis of these cells were assessed by [3H]thymidine and [3H]phenylalanine incorporation, respectively. Results: Cultured cardiac fibroblasts secreted AM into the medium time-dependently at a rate of 20.3±3.0 fmol/5×104 cells/48 h, mean±S.D. Northern blot analysis showed expression of preproAM mRNA of 1.6 kb in these cells. In addition, 10−6 mol/l of angiotensin II (Ang II) and endothelin-1 (ET-1) significantly increased the AM secretion by 55 and 48%, respectively. Synthetic AM significantly reduced 10−6 mol/l Ang II- or 10−7 mol/l ET-1-stimulated [3H]thymidine and [3H]phenylalanine incorporation in a dose-dependent manner, and these effects were attenuated by a calcitonin gene-related peptide (CGRP) type 1 receptor antagonist, CGRP(8-37). Synthetic AM also had a dose-dependent stimulatory effect on cAMP accumulation in these cells, which was significantly attenuated by CGRP(8-37). A cAMP analogue, 8-bromo-cAMP, mimicked the AM effects, inhibiting the Ang II-stimulated [3H]thymidine and [3H]phenylalanine incorporation. Blockage of the effect of endogenous AM by anti-AM monoclonal antibody not only significantly reduced the basal level of intracellular cAMP, but also enhanced the [3H]thymidine and [3H]phenylalanine incorporation into the cells. Conclusions: Cultured neonatal rat cardiac fibroblasts produce and secrete AM, and the secreted AM may inhibit proliferation and protein synthesis of these cells. AM may exert these inhibitory effects partly by elevating intracellular cAMP. It is suggested that AM has an important role in modulating the growth of cardiac fibroblasts in an autocrine or a paracrine manner.

The RAMP2/CRLR complex is a functional adrenomedullin receptor in human endothelial and vascular smooth muscle cells

Adrenomedullin, a potently hypotensive peptide isolated from human pheochromocytoma, is known to elicit a rise in cAMP levels within mammalian endothelial and smooth muscle cells. Until now, however, little has been known about the adrenomedullin receptor. Recently, a group called receptor activity‐modifying proteins that complex with the calcitonin receptor‐like receptor, and thereby regulate its transport and ligand specificity, were identified. Here we show that mRNA for both the calcitonin receptor‐like receptor and the receptor activity‐modifying protein 2, but not the receptor activity‐modifying protein 1 or receptor activity‐modifying protein 3, are expressed in human endothelial and vascular smooth muscle cells. We also found that adrenomedullin increased cAMP levels in HeLa EBNA and 293 EBNA cells, expressing both the receptor activity‐modifying protein 2 and calcitonin receptor‐like receptor proteins. Thus, the receptor activity‐modifying protein 2/calcitonin receptor‐like receptor complex apparently serves as a functional adrenomedullin receptor in human endothelial and vascular smooth muscle cells.

Purification and characterization of PAMP-12 (PAMP(9-20)) in porcine adrenal medulla as a major endogenous biologically active peptide

Proadrenomedullin N‐terminal 20 peptide (PAMP‐20) is a potent hypotensive peptide processed from the adrenomedullin (AM) precursor. We developed a specific radioimmunoassay which recognizes the C‐terminal region of PAMP‐20. Using this radioimmunoassay, the distribution of immunoreactive (ir‐) PAMP was determined in porcine tissues. High concentrations of ir‐PAMP were observed in the adrenal medulla and in the atrium, and these values were comparable to the corresponding concentrations of ir‐AM. The concentration of ir‐PAMP was almost the same as that of ir‐AM in the kidney, while ir‐PAMP was significantly lower than ir‐AM in the ventricle, lung, and aorta. Reversed‐phase high performance liquid chromatography in each porcine tissue sample revealed that two major peaks of ir‐PAMP existed: one emerged at a position identical to that of authentic porcine PAMP‐20; the other unknown peak was eluted earlier. The unknown peptide was purified to homogeneity from porcine adrenal medulla, and its complete amino acid sequence was determined. This peptide was found to be PAMP[9–20] with a C‐terminal amide structure, and was named PAMP‐12. Intravenous injections of PAMP‐12 in anesthetized rats showed a significant hypotensive effect in a dose‐dependent fashion, and the effect was comparable to that of PAMP‐20. These data indicate that PAMP‐12, a major component of ir‐PAMP, is processed from the AM precursor, as is PAMP‐20, and may participate in cardiovascular control.

Plasma adrenomedullin in patients with primary aldosteronism.

Adrenomedullin (AM) is a novel hypotensive peptide originally isolated from the pheochromocytoma tissue of humans. To examine the pathophysiological role of AM in primary aldosteronism (PA), the plasma concentration of AM in patients with PA was measured with a specific radioimmunoassay and compared to that in age- and sex-matched healthy normotensive subjects. In addition, the concentrations of AM as well as catecholamines in the plasma from both the adrenal vein and the inferior vena cava (IVC) were measured to determine whether or not the circulating AM in these PA patients is supplied from the adrenal medulla, which contains a much higher concentration of AM than any other human tissue does. The plasma concentration of AM in the PA patients (4.57 +/- 0.32 fmol/mL, n = 6) was significantly (P < .01) higher than that in the healthy subjects (3.06 +/- 0.20 fmol/mL, n = 12). A significant positive correlation (r = 0.62, P < .01) was observed between the mean blood pressure and the plasma AM level. The AM concentration in plasma from the adrenal vein was almost the same level as that from the IVC although the concentrations of both epinephrine and norepinephrine in the adrenal vein were much higher than those in the IVC. Therefore, it seems unlikely that the plasma AM in the PA patients is mainly supplied from the adrenal medulla. Judging from the potent hypotensive activity of AM, the present findings suggest that AM participates in defense mechanisms acting against the elevation of blood pressure in the patients with PA.

Shared and separate functions of the RAMP-based adrenomedullin receptors

Adrenomedullin (AM) is a novel hypotensive peptide that exerts a variety of strongly protective effects against multiorgan damage. AM-specific receptors were first identified as heterodimers composed of calcitonin-receptor-like receptor (CLR), a G protein coupled receptor, and one of two receptor activity-modifying proteins (RAMP2 or RAMP3), which are accessory proteins containing a single transmembrane domain. RAMPs are required for the surface delivery of CLR and the determination of its phenotype. CLR/RAMP2 (AM₁ receptor) is more highly AM-specific than CLR/RAMP3 (AM₂ receptor). Although there have been no reports showing differences in intracellular signaling via the two AM receptors, in vitro studies have shed light on their distinct trafficking and functionality. In addition, the tissue distributions of RAMP2 and RAMP3 differ, and their gene expression is differentially altered under pathophysiological conditions, which is suggestive of the separate roles played by AM₁ and AM₂ receptors in vivo. Both AM and the AM₁ receptor, but not the AM₂ receptor, are crucial for the development of the fetal cardiovascular system and are able to effectively protect against various vascular diseases. However, AM₂ receptors reportedly play an important role in maintaining a normal body weight in old age and may be involved in immune function. In this review article, we focus on the shared and separate functions of the AM receptor subtypes and also discuss the potential for related drug discovery. In addition, we mention their possible function as receptors for AM2 (or intermedin), an AM-related peptide whose biological functions are similar to those of AM.

Secretion of proadrenomedullin N-terminal 20 peptide from cultured neonatal rat cardiac cells.

Proadrenomedullin N-terminal 20 peptide (PAMP) is generated from post-transcriptional enzymatic processing of a 185-amino acid precursor for adrenomedullin (AM), a potent vasodilator peptide. We have reported that AM is secreted from cultured neonatal rat cardiac myocytes and fibroblasts, and that secreted AM modulates the growth of these cells; however, it is unknown whether or not the cardiac cells produce PAMP. In this study, we examined the production of PAMP in cultured neonatal rat cardiac myocytes and fibroblasts. Both the cardiac myocytes and fibroblasts cultured with serum-free media secreted PAMP time-dependently at rates of 5.7+/-0.9 fmol/10(5) cells/40 h and 8.4+/-0.7 fmol/5x10(4) cells/48 h (mean+/-SD), respectively. Reverse-phase high performance liquid chromatography showed that immunoreactive PAMP secreted from these cells was identical to PAMP[1-20], a whole active molecule. PAMP and AM secretions were significantly (P<0.01) stimulated by 10(-6) mol/L angiotensin II (Ang II) and 10% fetal bovine serum (FBS) in myocytes and fibroblasts, whereas the ratio of PAMP to AM secretion in the myocytes was smaller than that of the fibroblasts. These results suggest that PAMP is secreted along with AM from rat cardiac myocytes and fibroblasts, and the secretion is augmented by the growth-promoting stimuli of Ang II and FBS for these cells.

Increased plasma proadrenomedullin N-terminal 20 peptide in patients with essential hypertension

The novel hypotensive peptide, proadrenomedullin N-terminal 20 peptide (PAMP), is processed from the adrenomedullin precursor. Recently, we identified PAMP-12 [PAMP(9-20)] from the porcine adrenal medulla as a major endogenous and biologically active peptide. Using a new, sensitive radioimmunoassay which recognizes the C-terminal region of PAMP-20 [PAMP(1-20)], we investigated the role of PAMP in patients with essential hypertension who had normal renal function, and whether PAMP-12 is present in humans. The mean PAMP plasma concentration, like that of adrenomedullin, was significantly higher in hypertensive [1·51 fmol/mL, standard error of the mean (SEM) 0·09 fmol/mL] than normotensive participants (1·08 fmol/mL, SEM 0·05). The increase in plasma PAMP concentration in patients with organ damage accompanied by hypertension was significantly higher than that in patients without organ damage. The PAMP concentration had a significant positive correlation with mean blood pressure and adrenomedullin concentration. The immunoreactive PAMP in human tissue and plasma was characterized by reverse-phase high-performance liquid chromatography. PAMP-12, as well as PAMP-20, was abundant in the phaeochromocytoma tissue. These findings suggest that PAMP plays some pathophysiological role against the development of essential hypertension.

Adrenomedullin treatment reduces intestinal inflammation and maintains epithelial barrier function in mice administered dextran sulphate sodium.

Hyperactivation and hyperpermeability of the intestinal epithelium is a hallmark of IBD. AM has been shown to reduce the severity of colitis in the acetic acid and TNBS‐induced colitis model, however the mechanism of the therapeutic effect of AM against the colitis has not been clarified. Here, we show that the protective capability of AM is associated with suppression of inflammation and maintenance of the intestinal epithelial barrier function. In the DSS‐induced colitis model, intra‐rectal AM‐treated mice showed a reduction in loss of body weight and severity of colitis. AM‐treatment suppressed phosphorylation of STAT1 and STAT3 in the colonic epithelium, and altered the cytokine balance in the intestinal T cells, with lower levels of IFN‐γ and TNF‐α but higher levels of TGF‐β. Expression of the epithelial intercellular junctions such as tight and adherence junctions were sustained in the AM‐treated mice. In contrast, the epithelial junctions were down‐regulated in the control mice, leading to loss of epithelial barrier integrity and enhanced permeability. Collectively, these data indicate a broad spectrum of AM‐induced effects with respect to protection against DSS‐induced colitis, and suggest a potential therapeutic value of this treatment for IBD.