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Ouabain modulation of endothelial calcium signaling in descending vasa recta

¹Division of Nephrology, Department of Medicine, and ²Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland

Submitted 12 August 2005 ; accepted in final form 27 March 2006

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 x 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.

kidney; medulla; fura 2; SEA-0400; bradykinin; 2-aminoethoxydiphenyl borate

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