The present study was designed to deterrnine whether arginine vasopressin (AVP) stimulates NO production in the epithelial collecting duct cells of the inner medulla (IMCDs) and if this is mediated through Ca2(+) signaling. Thin tissue layers containing IMCDs were dissected from Sprague-Dawley rats. Intracellular Ca2+ concentration ([Ca2+](f) )and NO production were measured in IMCDs by a fluorescence imaging system with the use of fura 2-AM and the cell-permeable form of the NO-sensitive dye 4,5-diaminofluorescein (DAF-2), respectively. AVP (100 nmol/L) produced a rapid peak increase in [Ca2+](i) of 320 +/- 70 nmol/L within a few seconds and a sustained increase of 120 +/- 62 nmol/L. The peak increase in [Ca2+](i) was followed by a significant increase of NO production (34 +/- 7 U). This was similar to that produced by 20 mumol/L of the NO donor DETA-NONOate (42 +/-11 U). The NO scavenger carboxy-PTIO (100 mumol/L) or depletion of [Ca2+](i) by preincubation with 5 mumol/L of the Ca2+-ATPase inhibitor thapsigargin in Ca2+-free buffer abolished the NO response to AVP. We conclude that AVP mobilizes Ca2+ to produce NO in IMCDs.