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

Abstract

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 Sprague-Dawley 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 μM), but not the V1R antagonist OPC21268 (1 μM), inhibited the increase in [Ca2+]i (up to 91 ± 5%) and abolished the NO response to AVP. Both the phospholipase C inhibitor U73112 (3 μM) and the inositol ( 1 , 4 , 5 ) tri-phosphate 3 receptor blocker 2-APB (75 μM) reduced the peak [Ca2+]i response to AVP (by 65 ± 9 and 59 ± 15%, respectively) and abolished the NO response. Although forskolin (100 μM; an activator of adenylyl cyclase) elicited a moderate increase in [Ca2+]i, neither preincubation with the adenylyl cyclase inhibitor 2′-5′-dideoxyadenosine (50 μM) nor the protein kinase A (PKA) inhibitor PKA14-22 (100 μM) 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 Ca2+-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.