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

Little is known about the effects of nitric oxide (NO) and the cyclic GMP (cGMP)/protein kinase G (PKG) system on Ca(2+) signaling in vascular smooth muscle cells (VSMC) of resistance vessels in general and afferent arterioles in particular. We tested the hypotheses that cGMP-, Ca(2+)-dependent big potassium channels (BK(Ca(2+))) buffer the Ca(2+) response to depolarization by high extracellular KCl and that NO inhibits adenosine diphosphoribose (ADPR) cyclase, thereby reducing the Ca(2+)-induced Ca(2+) release. We isolated rat afferent arterioles, utilizing the magnetized microsphere method, and measured cytosolic Ca(2+) concentration ([Ca(2+)](i)) with fura-2, a preparation in which endothelial cells do not participate in [Ca(2+)](i) responses. KCl (50 mM)-induced depolarization causes an immediate increase in [Ca(2+)](i) of 151 nM. The blockers N(omega)-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 BK(Ca(2+)) activity) do not alter the [Ca(2+)](i) response to KCl, suggesting no discernible endogenous NO production under basal conditions. The NO donor sodium nitroprusside (SNP) reduces the [Ca(2+)](i) response to 77 nM; IBX restores the response to control values. These data show that activation of BK(Ca(2+)) in the presence of NO/cGMP provides a brake on KCl-induced [Ca(2+)](i) responses. Experiments with the inhibitor of cyclic ADPR 8-bromo-cyclic ADPR (8-Br-cADPR) and SNP + downstream inhibitors of PKG and BK(Ca(2+)) 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 muM), 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 [Ca(2+)](i) response to KCl. In summary, we present evidence for effects of the NO/cGMP/PKG system to reduce [Ca(2+)](i), via activation of BK(Ca(2+)) and possibly by inhibition of ADPR cyclase, and to increase [Ca(2+)](i), by a mechanism(s) yet to be defined.