Endothelin-A and -B receptors, superoxide, and Ca2+ signaling in afferent arterioles

It is unknown if endothelin-A and -B receptors (ETAR and ETBR) activate the production of superoxide via NAD(P)H oxidase and subsequently stimulate the formation of cyclic adenine diphosphate ribose (cADPR) in afferent arterioles. Vessels were isolated from rat kidney and loaded with fura 2. Endothelin-1 (ET-1) rapidly increased cytosolic Ca2+ concentration ([Ca2+](i)) by 303 nM. The superoxide dismutase mimetic tempol, the NAD(P) H oxidase inhibitor apocynin, and nicotinamide, an inhibitor of ADPR cyclase, diminished the response by similar to 60%. The ETBR agonist sarafotoxin 6c (S6c) increased peak [Ca2+](i) by 117 nM. Subsequent addition of ET-1 in the continued presence of S6c caused an additional [Ca2+] i peak of 225 nM. Neither nicotinamide or 8-bromo-(8-Br) cADPR nor apocynin decreased the [Ca2+] i response to S6c, but inhibited the subsequent [Ca2+] i response to ET-1. The ETBR blockers BQ-788 and A-192621 prevented the S6c [Ca2+] i peak and reduced the ET-1 response by more than one-half, suggesting an ETBR/ETAR interaction. In contrast, the ETAR blocker BQ-123 had no effect on the S6c [Ca2+] i peak and obliterated the subsequent ET-1 response. ET-1 immediately stimulated superoxide formation (measured with TEMPO-9-AC, 68 arbitrary units) that was inhibited 95% by apocynin or diphenyl iodonium. S6c or IRL-1620 increased superoxide by 8% of that caused by subsequent ET-1 addition. We conclude that ETAR activation of afferent arterioles increases the formation of superoxide that accounts for similar to 60% of subsequent Ca2+ signaling. ETBR activation appears to result in only minor increases in superoxide production. Nicotinamide and 8-Br-cADPR results suggest that ET-1 (and primarily ETAR) causes the activation of vascular smooth muscle cell-ADPR cyclase.