Ouabain modulation of endothelial calcium signaling in descending vasa recta

Using fura 2-loaded vessels, we tested whether ouabain modulates endothelial cytoplasmic calcium concentration ([Ca2+]CYT) in rat descending vasa recta (DVR). Over a broad range between 10−10 and 10−4 M, ouabain elicited biphasic peak and plateau [Ca2+]CYT elevations. Blockade of voltage-gated Ca2+ entry with nifedipine did not affect the response to ouabain mitigating against a role for myo-endothelial gap junctions. Reduction of extracellular Na+ concentration ([Na+]o) or Na+/Ca2+ exchanger (NCX) inhibition with SEA-0400 (10−6 M) elevated [Ca2+]CYT, supporting a role for NCX in the setting of basal [Ca2+]CYT. SEA-0400 abolished the [Ca2+]CYT response to ouabain implicating NCX as a mediator. The transient peak phase of [Ca2+]CYT elevation that followed either ouabain or reduction of [Na+]o was abolished by 2-aminoethoxydiphenyl borate (5 × 10−5 M). Cation channel blockade with La3+ (10 μM) or SKF-96365 (10 μM) also attenuated the ouabain-induced [Ca2+]CYT response. Ouabain pretreatment increased the [Ca2+]CYT elevation elicited by bradykinin (10−7 M). We conclude that inhibition of ouabain-sensitive Na+-K+-ATPase enhances DVR endothelial Ca2+ store loading and modulates [Ca2+]CYT signaling through mechanisms that involve NCX, Ca2+ release, and cation channel activation.