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1 Cell & Molecular physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States
* To whom correspondence should be addressed. E-mail: sfellner{at}med.unc.edu.
It is unknown if endothelin A and B receptors(ETAR, ETBR) activate the production of superoxide via NAD(P)H oxidase and subsequently stimulate the formation of cADPR in afferent arterioles. Vessels were isolated from rat kidney and loaded with fura-2. Endothelin-1 (ET-1) rapidly increased [Ca2+]i by 303nM. The superoxide dismutase mimetic tempol, the NAD(P)H oxidase inhibitor apocynin, and nicotinamide, an inhibitor of ADPR cyclase, diminished the response by about 60%. The ETBR agonist sarafotoxin 6c(S6c) increased peak [Ca2+]i by 117nM. Subsequent addition of ET-1 in the continued presence of S6c caused an additional [Ca2+]i peak of 225nM. Neither nicotinamide or 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 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 9AC, 68 arbitrary units) that was inhibited 95% by apocynin or diphenyl iodonium(DPI). 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 about 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 VSMC-ADPR cyclase.
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