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1 Medicine, Division of Nephrology, University of Maryland School of Medicine, Baltiomre, Maryland, United States
2 Physiology, University of Maryland School of Medicine, Baltmore, Maryland, United States
* To whom correspondence should be addressed. E-mail: tpallone{at}medicine.umaryland.edu.
Using fura-2 loaded vessels, we tested whether ouabain modulates endothelial cytoplasmic calcium [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+ ([Na+]O) or NCX inhibition with SEA0400 (10-6 M) elevated [Ca2+]CYT, supporting a role for Na+/Ca2+ exchanger (NCX) in the setting of basal [Ca2+]CYT. SEA0400 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 (2-APB, 5 x 10-5 M). Cation channel blockade with La3+ (10-5 M) or SKF96365 (10-5 M) 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.
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