The endothelin ETA receptor-specific effect of 50-235, a nonpeptide endothelin antagonist

We characterized the endothelin receptor antagonist 27-O-caffeoyl myricerone (50-235), isolated from the bayberry Myrica cerifera, using rat aortic smooth muscle A7r5 cells that express ETA receptors and human Girardi heart cells that express ETB receptors. 50-235 concentration-dependently inhibited 125I-ET-1 binding to A7r5 cells with Ki of 51 +/- 12 nM, while it had no effect on 125I-ET-1 and 125I-ET-3 bindings to Girardi heart cells. Also in affinity cross-linking studies with 125I-ET-1, 50-235 inhibited labeling of a protein of M(r) = 67,000 in A7r5 cells, but did not inhibit labeling of two proteins with M(r) values of 70,000 and 46,000 in Girardi heart cells. Functionally, 50-235 inhibited the ET-1-induced increase in cytosolic free Ca2+ concentration ([Ca2+]i) in a dose-dependent manner (IC50 = 11 +/- 2 nM) in A7r5 cells. On the other hand, this compound had no effect on the basal level of [Ca2+]i and the high K(+)- and bombesin-induced increases in [Ca2+]i in A7r5 cells, nor on the ET-1-induced increase in [Ca2+]i in Girardi heart cells. Also, 50-235 inhibited ET-1-promoted mitogenesis of A7r5 cells. Thus, we conclude that 50-235 is a specific endothelin A receptor antagonist that could be very useful for elucidating the physiological and pathophysiological significance of ET.