Endothelium-dependent vasorelaxation independent of nitric oxide and K(+) release in isolated renal arteries of rats

Abstract

1. We investigated whether K(+) can act as an endothelium-derived hyperpolarizing factor (EDHF) in isolated small renal arteries of Wistar-Kyoto rats. 2. Acetylcholine (0.001 - 3 microM) caused relaxations that were abolished by removal of the endothelium. However, acetylcholine-induced relaxations were not affected by the nitric oxide (NO) synthase inhibitor N:(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM), by L-NAME plus the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 1 microM) or by L-NAME plus the cyclo-oxygenase inhibitor indomethacin (10 microM). In rings precontracted with high-K(+)(60 mM) physiological salt solution in the presence of L-NAME, acetylcholine-induced relaxations were abolished. 3. L-NAME-resistant relaxations were abolished by the large-conductance Ca(2+)-activated K(+) channel inhibitor charybdotoxin plus the small-conductance Ca(2+)-activated K(+) channel inhibitor apamin, while the inward rectifier K(+) channel inhibitor Ba(2+) or the gap junction inhibitor 18alpha-glycyrrhetinic acid had no effect. Acetylcholine-induced relaxation was unchanged by ouabain (10 microM) but was partially inhibited by a higher concentration (100 microM). 4. In half of the tissues tested, K(+)(10 mM) itself produced L-NAME-resistant relaxations that were blocked by ouabain (10 microM) and partially reduced by charybdotoxin plus apamin, but not affected by 18alpha-glycyrrhetinic acid or Ba(2+). However, K(+) did not induce relaxations in endothelium-denuded tissues. 5. In conclusion, acetylcholine-induced relaxations in this tissue are largely dependent upon hyperpolarization mechanisms that are initiated in the endothelium but do not depend upon NO release. K(+) release cannot account for endothelium-dependent relaxation and cannot be an EDHF in this artery. However, K(+) itself can initiate endothelium-dependent relaxations via a different pathway from acetylcholine, but the mechanisms of K(+)-induced relaxations remain to be clarified.

Figure 1

Effects of (a) the nitric oxide (NO) synthase inhibitor L-NAME (100 μM), L-NAME plus soluble guanylate cyclase inhibitor ODQ (1 μM) and L-NAME plus cyclo-oxygenase inhibitor indomethacin (10 μM) on acetylcholine (ACh)-induced relaxations (n=4 – 9); and (b) ODQ (1 μM) alone on the NO donor sodium nitroprusside (SNP, 1 μM)-induced relaxations (n=3 – 4) in endothelium-intact small renal arteries from Wistar-Kyoto rats. Relaxations are presented as percentage reductions of the precontractions produced by phenylephrine (1 μM). Data are mean±standard error of the mean (s.e.mean). ^*P<0.001, one-way analysis of the variance (one-way ANOVA) followed by Student's t-test.

Figure 2

Original records showing the relaxations induced by (a) acetylcholine (ACh, 1 nM – 3 μM, as shown by downward arrows) and (b) 10 mM K⁺ (KCl) in tissues treated with high-K⁺ (60 mM) physiological salt solution (PSS), the Na⁺/K⁺-ATPase inhibitor ouabain (10 μM), the inward rectifier K⁺ channel inhibitor Ba²⁺ (50 μM), the gap junction inhibitor 18α-glycyrrhetinic acid (18α-GA, 50 μM), or the large-conductance Ca²⁺-activated K⁺ channel inhibitor charybdotoxin (100 nM) plus the small-conductance Ca²⁺-activated K⁺ channel inhibitor apamin (1 μM) (ChTX+apamin). All experiments were in the presence of L-NAME (100 μM). The first trace in the lower panel shows that 10 mM K⁺-induced relaxations could not be mimicked by equal molar NaCl. (▴) precontractions produced by 1 μM phenylephrine; (Δ) precontraction produced by high-K⁺ PSS.

Figure 3

Effects of (a) high-K⁺ (60 mM), PSS, (b) ouabain (10 and 100 μM), (c) charybdotoxin (ChTX, 100 nM) and charybdotoxin plus apamin (1 μM), and (d) Ba²⁺ (50 μM) or 18α-glycyrrhetinic acid (18α-GA, 50 μM) on acetylcholine (ACh)-induced relaxations in the presence of 100 μM L-NAME. Tissues were precontracted with phenylephrine (1 μM) except for those in high-K⁺ PSS experiments in which tissues were contracted with high-K⁺ PSS. Relaxations are presented as percentage reductions of the precontractions. Data are mean±s.e.mean. (n=3 – 8). ^*P<0.05 vs control, one-way ANOVA followed by Tukey's test.

Figure 4

Effects of ouabain (10 μM), Ba²⁺ (50 μM), 18α-glycyrrhetinic acid (18α-GA, 50 μM) and charybdotoxin (ChTX, 100 nM) plus apamin (1 μM) on relaxations induced by 10 mM K⁺ in L-NAME-pretreated tissues. Open bars are control responses and closed bars are time control (TC) or drug-treated responses. Relaxations are expressed as percentages of 1 μM phenylephrine-induced precontractions, while the negative value means a contraction response. Data are mean±s.e.mean. ^*P<0.05, one-way ANOVA followed by Student's t-test, n=4 – 6.