Vasopressin-induced nitric oxide production in rat inner medullary collecting duct is dependent on V2 receptor activation of the phosphoinositide pathway

We previously reported that arginine vasopressin ( AVP) stimulates the production of nitric oxide ( NO) in inner medullary collecting duct ( IMCD) via activation of V2 receptors ( V2R) and the mobilization of intracellular Ca2+. The aim of this study was to determine the pathway( s) through which this response is mediated. IMCDs were dissected from male SpragueDawley rats and intracellular Ca2+ concentration ([Ca2+](i)) and NO production were measured using a fluorescence imaging system. AVP ( 100 nmol/l) produced a rapid increase [ Ca2+](i) of 381 +/- 78 nmol/l that was followed by a significant increase of NO production ( 166 +/- 61%). The specific nonpeptide V2R antagonist OPC31260 ( 1 mu M), but not the V1R antagonist OPC21268 ( 1 mu M), inhibited the increase in [ Ca2+](i) ( up to 91 +/- 5%) and abolished the NO response to AVP. Both the phospholipase C inhibitor U73112 ( 3 mu M) and the inositol ( 1,4,5) tri- phosphate 3 receptor blocker 2- APB ( 75 mu M) reduced the peak [ Ca2+](i) response to AVP ( by 65 +/- 9 and 59 +/- 15%, respectively) and abolished the NO response. Although forskolin ( 100 mu M; an activator of adenylyl cyclase) elicited a moderate increase in [ Ca2+](i), neither preincubation with the adenylyl cyclase inhibitor 2'- 5'- dideoxyadenosine ( 50 mu M) nor the protein kinase A ( PKA) inhibitor PKA14- 22 ( 100 mu M) significantly inhibited peak [ Ca2+](i) in response to AVP. IMCD [ Ca2+](i) responses to AVP were reduced by 72 +/- 8% when incubated in Ca2+- free media and could be completely abolished by preincubation with the Ca-2+- ATPase inhibitor thapsigargin. We conclude that AVP- induced NO production in IMCD is dependent on V2R activation of the phosphoinositide pathway and the mobilization of Ca2+ from both intracellular and extracellular pools.