PD-142893

Nitric oxide (NO)‐deficiency as it occurs during endothelial dysfunction activates the endothelin‐1 (ET‐1) system and increases the expression of receptor activity modifying protein (RAMP)‐1 that acts as a chaperon for calcium‐sensing receptors (CaR) that have recently been identified to improve cardiac function. Here, we hypothesized that ET‐1 increases the cardiac expression of CaR and thereby induces an adaptive type of hypertrophy. Expressions of RAMP‐1, endothelin receptors, and CaR were analyzed by RT‐PCR in left ventricular tissues of L‐NAME‐treated rats. Effects of ET‐1 on CaR expression and cell function (load free cell shortening) were analyzed in adult rat ventricular cardiomyocytes. siRNA directed against CaR and RAMP‐1 was used to investigate a causal relationship. PD142893 and BQ788 were used to dissect the contribution of ETB1, ETB2, and ETA receptors. Non‐specific NO synthase inhibition with L‐Nitro arginine methyl ester (L‐NAME) caused a cardiac upregulation of ETB receptors and CaR suggesting a paracrine effect of ET‐1 on cardiomyocytes. Indeed, ET‐1 induced the expression of CaR in cultured cardiomyocytes. Under these conditions, cardiomyocytes increased cell size (hypertrophy) but maintained normal function. Inhibition of ETA and ETB1 receptors led to ET‐1‐dependent reduction in cell shortening and attenuated up‐regulation of CaR. Down‐regulation of RAMP‐1 reduced CaR responsiveness. In conclusion, ET‐1 causes an adaptive type of hypertrophy by up‐regulation of CaR in cardiomyocytes via ETA and/or ETB1 receptors. J. Cell. Physiol. 232: 2508–2518, 2017. © 2016 Wiley Periodicals, Inc.

We have compared the endothelin receptor subtype affinity (K(D)) and selectivity of four structural classes of antagonists (peptide, sulphonamide-based, carboxylic acid-based, myceric acid-based) in human cardiovascular tissues to determine whether these are predicted by values reported for human cloned receptors. Additionally, affinities (K(B)) for these antagonists, determined in ET-1-mediated vasoconstriction assays in human blood vessels, were used to identify discrepancies between K(B) and K(D) determined in the same tissues.

Competition binding experiments were carried out in sections of human left ventricle, coronary artery and homogenates of saphenous vein to determine K(D) values for structurally different ET(A)-selective (FR139317, BMS 182874, S97-139, sitaxentan, ambrisentan) and mixed (PD142893, Ro462005, bosentan, L-749329, SB209670) antagonists. Schild-derived values of antagonist affinity were obtained in vascular functional studies.

When compared with previously reported data in human cloned endothelin receptors, those antagonists reported to be ET(A)-selective exhibited even greater ET(A) selectivity in human ventricle (BMS 182874, sitaxentan, ambrisentan) that expressed both receptor subtypes. Those antagonists reported to have <100 fold selectivity in cloned receptors (PD142893, Ro-462005, bosentan, SB209670, L-749329) did not distinguish between receptor subtypes in human left ventricle. For antagonists where we determined affinity in vascular functional and binding assays (Ro462005, bosentan, BMS 182874, L-749329, SB209670) there was no correlation between the degree of discrepancy in K(B) and K(D) and structural class.

For an antagonist to retain ET(A)-selectivity in vivo it may be necessary to identify those compounds that have at least 1000 fold ET(A):ET(B) selectivity in in vitro assays.