title: 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 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.
authors.affiliation: Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7545, USA. sfellner@med.unc.edu
authors.forename: Susan K
authors.initials: SK
authors.lastname: Fellner
authors.forename: William J
authors.initials: WJ
authors.lastname: Arendshorst
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D036541:ADP-ribosyl Cyclase / Q000378:metabolism; D000818:Animals; D001160:Arterioles / Q000502:physiology; D002118:Calcium / Q000502:physiology; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002227:Carbazoles / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology*; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D009569:Nitric Oxide / Q000502:physiology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009599:Nitroprusside / Q000494:pharmacology; D010069:Oxadiazoles / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D011810:Quinoxalines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: C095284:1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one; D002227:Carbazoles; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D020030:Nitric Oxide Donors; D010069:Oxadiazoles; D010455:Peptides; D010726:Phosphodiesterase Inhibitors; D011810:Quinoxalines; C073601:KT 5823; D009599:Nitroprusside; C016276:8-bromocyclic GMP; D009569:Nitric Oxide; D011189:Potassium Chloride; C064719:iberiotoxin; D000242:Cyclic AMP; D017869:Cyclic GMP-Dependent Protein Kinases; D036541:ADP-ribosyl Cyclase; D006152:Cyclic GMP; D002118:Calcium; D015056:1-Methyl-3-isobutylxanthine