Complex interactions of NO/cGMP/PKG systems on Ca2+signaling in afferent arteriolar vascular smooth muscle

Abstract

Little is known about the effects of nitric oxide (NO) and the cyclic GMP (cGMP)/protein kinase G (PKG) system on Ca2+signaling in vascular smooth muscle cells (VSMC) of resistance vessels in general and afferent arterioles in particular. We tested the hypotheses that cGMP-, Ca2+-dependent big potassium channels (BKCa2+) buffer the Ca2+response to depolarization by high extracellular KCl and that NO inhibits adenosine diphosphoribose (ADPR) cyclase, thereby reducing the Ca2+-induced Ca2+release. We isolated rat afferent arterioles, utilizing the magnetized microsphere method, and measured cytosolic Ca2+concentration ([Ca2+]i) with fura-2, a preparation in which endothelial cells do not participate in [Ca2+]iresponses. KCl (50 mM)-induced depolarization causes an immediate increase in [Ca2+]iof 151 nM. The blockers Nω-nitro-l-arginine methyl ester (of nitric oxide synthase), 1,2,4-oxodiazolo-[4,3- a]quinoxalin-1-one (ODQ, of guanylyl cyclase), KT-5823 (of PKG activation), and iberiotoxin (IBX, of BKCa2+activity) do not alter the [Ca2+]iresponse to KCl, suggesting no discernible endogenous NO production under basal conditions. The NO donor sodium nitroprusside (SNP) reduces the [Ca2+]iresponse to 77 nM; IBX restores the response to control values. These data show that activation of BKCa2+in the presence of NO/cGMP provides a brake on KCl-induced [Ca2+]iresponses. Experiments with the inhibitor of cyclic ADPR 8-bromo-cyclic ADPR (8-Br-cADPR) and SNP + downstream inhibitors of PKG and BKCa2+suggest that NO inhibits ADPR cyclase in intact arterioles. When we pretreat afferent arterioles with 8-bromoguanosine 3′,5′-cyclic monophosphate (8-Br-cGMP; 10 μM), the response to KCl is 143 nM. However, in the presence of both IBX and 8-Br-cGMP, we observe a surprising doubling of the [Ca2+]iresponse to KCl. In summary, we present evidence for effects of the NO/cGMP/PKG system to reduce [Ca2+]i, via activation of BKCa2+and possibly by inhibition of ADPR cyclase, and to increase [Ca2+]i, by a mechanism(s) yet to be defined.