Kohei Shimada

Expression of Endothelin-1, Endothelin-Converting Enzyme, and Endothelin Receptors in Chronic Heart Failure

BACKGROUND Elevated plasma levels of endothelin (ET)-1 have been reported in association with heart diseases, including heart failure. Furthermore, it has been suggested that ET-1 acts as a local autocrine/paracrine factor with biological activities such as vasoconstriction, mitogenesis, and inotropic effects on the heart. This study investigated alterations of ET-1, ET receptor, and endothelin-converting enzyme (ECE) expression in left ventricular myocardium from patients with end-stage heart failure. METHODS AND RESULTS mRNA concentrations of ETA and ETB receptors, prepro-ET-1 (ppET-1), and ECE in left ventricles from nonfailing donors hearts (NF) and from patients with end-stage chronic heart failure (NYHA functional class IV) due to dilated cardiomyopathy (DCM) were compared by use of a competitive reverse transcription-polymerase chain reaction technique. There was no significant difference in mRNA expression for ppET-1, ECE-1, and ETA receptors, whereas a significant reduction of ETB-receptor mRNA was observed in DCM hearts. 125I-labeled ET-1 radioligand binding studies demonstrated a significant downregulation of ETB receptors, whereas ETA-receptor density was increased in membranes from DCM hearts. Phosphoramidon-sensitive ECE activity and immunodetectable amounts of ECE protein in left ventricular membrane preparations did not differ between NF and DCM hearts. Finally, immunoreactive ET-1 concentrations were increased in DCM hearts. CONCLUSIONS The present study demonstrates changes in the ET-receptor expression pattern in favor of the ETA receptor in human end-stage heart failure. Furthermore, activation of the cardiac ET system with increased tissue ET-1 concentrations in the failing myocardium is observed. This is more likely due to decreased clearance than to increased synthesis, because ppET-1 gene expression and ECE activity are unchanged.

Endothelin-Converting Enzyme Expression in the Rat Vascular Injury Model and Human Coronary Atherosclerosis

BACKGROUND Endothelin 1 has been implicated in various human diseases, including atherosclerosis. In this study, we examined the expression and localization of endothelin-converting enzyme-1 (ECE-1), the final key enzyme of endothelin 1 processing, in rat carotid arteries after balloon injury and in human coronary atherosclerotic lesions. METHODS AND RESULTS ECE-1 mRNA levels and ECE activity in rat balloon-injured arteries started to increase between 2 and 5 days after injury. The endothelin 1 content of tissue in injured arteries was concomitantly increased. Immunohistochemical staining located ECE-1 signals in endothelial cells in uninjured arteries, whereas ECE-1 immunoreactivity was detected in neointimal smooth muscle cells in injured arteries 5 to 14 days after balloon denudation. The size of the neointima was effectively reduced by phosphoramidon, an inhibitor of neutral metalloproteases, including ECE-1. In human coronary atherosclerotic lesions, intense ECE-1 immunoreactivity was detected in subsets of cells embedded in atheromatous plaque that correspond to smooth muscle cells and macrophages, as identified by staining for smooth muscle alpha-actin and CD68 surface marker, respectively. CONCLUSIONS The present study ascertained that ECE-1 is expressed in neointimal smooth muscle cells in rat balloon-injured arteries and in both smooth muscle cells and macrophages in human coronary atherosclerotic lesions. Blockade of ECE-1 was effective in reducing neointimal formation after balloon injury. Thus, ECE-1 may contribute to the process of injury-induced neointimal formation and atherosclerosis through the autocrine/paracrine effects of endothelin 1.

Identification and characterization of two isoforms of an endothelin-converting enzyme-1

We report the cloning and sequencing of 5′‐terminal region of a β form of rat ECE‐1 cDNA which is different only in its N‐terminal amino‐acid sequence to the cDNA we have cloned previously (α form [K. Shimada et al. (1994) J. Biol. Chem. 269, 18275–18278]). No significant difference was found in the specific activity and substrate specificity between the two isoforms. The expression level of ECE‐1α mRNA was higher than that of ECE‐1β in various rat cells and tissues, suggesting that the physiologically important isoform is ECE‐1α. The present findings verified the presence of two forms of ECE‐1 over many species, which are created probably through alternative splicing.

Renal endothelin-converting enzyme in rats with congestive heart failure

The expression and immunoreactivity of endothelin-converting enzyme (ECE) were examined in the renal tissue of rats with experimental congestive heart failure (CHF). Reverse transcriptase polymerase chain reaction (RT-PCR) revealed that ECE mRNA was more abundant (about twofold) in the renal medulla than in the cortex. Induction of heart failure caused a significant enhancement in the expression of this key enzyme in the renal cortex of rats with compensated CHF (delta + 28%) and in animals with decompensated heart failure (delta + 58%). An identical trend was also observed in the renal medulla, although these increases were moderate compared to those in the cortex. Similar findings were observed with Western blot techniques applying two monoclonal antibodies to rat ECE (AEC32-236 and AEC27-121). Taken together, these data suggest that upregulation of ECE is an important component in the activated renal ET system in CHF.

Simultaneous Inhibition of PGE2 and PGI2 Signals Is Necessary to Suppress Hyperalgesia in Rat Inflammatory Pain Models

Prostaglandin E2 (PGE2) is well known as a mediator of inflammatory symptoms such as fever, arthritis, and inflammatory pain. In the present study, we evaluated the analgesic effect of our selective PGE2 synthesis inhibitor, compound I, 2-methyl-2-[cis-4-([1-(6-methyl-3-phenylquinolin-2-yl)piperidin-4-yl]carbonyl amino)cyclohexyl] propanoic acid, in rat yeast-induced acute and adjuvant-induced chronic inflammatory pain models. Although this compound suppressed the synthesis of PGE2 selectively, no analgesic effect was shown in both inflammatory pain models. Prostacyclin (PGI2) also plays crucial roles in inflammatory pain, so we evaluated the involvement of PGI2 signaling in rat inflammatory pain models using prostacyclin receptor (IP) antagonist, RO3244019. RO3244019 showed no analgesic effect in inflammatory pain models, but concomitant administration of compound I and RO3244019 showed analgesic effects comparable to celecoxib, a specific cyclooxygenase- (COX-) 2 inhibitor. Furthermore, coadministration of PGE2 receptor 4 (EP4) antagonist, CJ-023423, and RO3244019 also showed an analgesic effect. These findings suggest that both PGE2 signaling, especially through the EP4 receptor, and PGI2 signaling play critical roles in inflammatory pain and concurrent inhibition of both signals is important for suppression of inflammatory hyperalgesia.

Regulation of endothelin-converting enzyme 1 in nephrotic syndrome in rats.

Background: Nephrotic syndrome is characterized by severe proteinuria and sodium and water retention. Although endothelin (ET) 1 can cause natriuresis or antinatriuresis, the role played by ET-1 in proteinuria and in sodium retention due to nephrotic syndrome remains unclear. Methods: We investigated the role played by the ET-1 system in sodium and water retention and in proteinuria in puromycin aminonucleoside induced nephrotic syndrome in rats using microdissected nephron segments, competitive polymerase chain reaction, and Western blot. Results: The expression of prepro ET-1, ET-converting enzyme 1 (ECE-1), and ET A receptor mRNAs, but not ET B receptor mRNA, in the glomeruli was increased in rats with nephrotic syndrome. The cGMP generation in the glomeruli induced by atrial natriuretic peptide and ET-1 was decreased, whereas the ET-3-induced cGMP generation was increased in rats with nephrotic syndrome. ECE-1 mRNA expression was increased not only in the glomeruli, but also in the thick ascending limbs and collecting ducts. The protein expression of ECE-1 was increased in the membrane fraction of the cortex and in the outer and the inner medulla of nephrotic rats. Blockade of ET A and B receptors by bosentan did not inhibit the occurrence of nephrotic syndrome. However, the administration of bosentan increased the urinary sodium excretion. Conclusion: These data suggest that an activated ET-1-ET A receptor pathway in glomeruli and/or an increased ECE-1 mRNA expression in distal segments may participate in sodium and water retention, but not in the occurrence of nephrotic syndrome.