Contributions of nitric oxide, EDHF, and EETs to endothelium-dependent relaxation in renal afferent arterioles

Background. Acetylcholine-induced endothelium-dependent relaxation in the renal afferent arteriole has been ascribed to nitric oxide, but the role of endothelium-derived hyperpolarizing factors (EDHFs) and 14,15-epoxyeicosatrienoic acid (14,15-EET) are unclear. Methods. Single afferent arterioles were dissected from kidney of normal rabbits and microperfused in vitro at 60 mm Hg. Vessels were preconstricted submaximally with norepinephrine (10(-8) mol/L). Relaxation was assessed following cumulative addition of ACh (10(-9) to 10(-4) mol/L) alone, or in the presence of indomethacin (to inhibit cyclooxygenase), N-w-nitro-L-arginine (L-NNA) (to inhibit nitric oxide synthase), methylene blue (to inhibit soluble guanylate cyclase), or a combination of L-NNA + methylene blue. To assess contributions by EDHF, studies were repeated with either apamin + charybdotoxin [to block Ca2+ -activated K+ channels (K-Ca)] or with 40 mmol/L KCl. To asses the role of 14,15-EET, relaxations were evaluated in the presence of its competitive inhibitor 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE). Results. Relaxation by acetylcholine was abolished following endothelial denudation. It was unaffected by indomethacin but was inhibited 54%+/-5% (P<0.001) by L-NNA, 57%+/-5% by methylene blue, and 60%+/-4% by the combination of L-NNA plus methylene blue. Relaxation was inhibited further by KCl (80%+/-6%) or by apamin + charybdotoxin (96%+/-2%). 14,15-EEZE, alone, inhibited acetylcholine-induced relaxation by 29%+/-3%, and by 80%+/-5% in the presence of L-NNA. Conclusion. Acetylcholine-induced afferent arteriolar relaxation depends strongly on both nitric oxice, acting via soluble guanylate cyclase, and on an EDHF, likely 14,15-EET, acting via K-Ca.