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.