Search

Descending vasa recta (DVR) are 15-[micro]m vessels that perfuse the renal medulla. Ouabain has been shown to augment DVR endothelial cytoplasmic [Ca.sup.2+] ([[[Ca.sup.2+]].sub.CYT]) signaling. In this study, we examined the expression of the ouabain-sensitive Na-K-ATPase [alpha]2 subunit in the rat renal vasculature and tested effects of acute ouabain exposure and chronic ouabain treatment on DVR. Immunostaining with antibodies directed against the [alpha]2 subunit verified its expression in both DVR pericytes and endothelium. Acute application of ouabain (100 or 500 nM) augmented the DVR nitric oxide generation stimulated by acetylcholine (ACh; 10 [micro]M). At a concentration of 1 mM, ouabain constricted microperfused DVR, whereas at 100 nM, it was without effect. Acute ouabain (100 nM) did not augment constriction by angiotensin II (0.5 or 10 nM), whereas L-nitroarginine methyl ester-induced contraction of DVR was slightly enhanced. Ouabain-hypertensive (OH) rats were generated by chronic ouabain treatment (30 [micro]g x [kg.sup.-1] x [day.sup.-1], 5 wk). The acute endothelial [[Ca.sup.2+]].sub.CYT] elevation by ouabain (100 nM) was absent in DVR endothelia of OH rats. The [[Ca.sup.2+]].sub.CYT] response to 10 nM ACh was also eliminated, whereas the response to 10 [micro]M ACh was not. The endothelial [[Ca.sup.2+] ]CYT response to bradykinin (100 nM) was significantly attenuated. We conclude that endothelial responses may offset the ability of acute ouabain exposure to enhance DVR vasoconstriction. Chronic exposure to ouabain, in vivo, leads to hypertension and DVR endothelial dysfunction, manifested as reduced [[Ca.sup.2+]].sub.CYT] responses to both ouabain- and endothelium-dependent vasodilators. kidney; medulla; microcirculation; nitric oxide; blood flow

The signaling pathways leading to high NaCl-induced activation of the transcription factor tonicity-responsive enhancer binding protein/osmotic response element binding protein (TonEBP/OREBP) remain incompletely understood. High NaCl has been reported to produce oxidative stress. Reactive oxygen species (ROS), which are a component of oxidative stress, contribute to regulation of transcription factors. The present study was undertaken to test whether the high NaCl-induced increase in ROS contributes to tonicity-dependent activation of TonEBP/OREBP. Human embryonic kidney 293 cells were used as a model. We find that raising NaCl increases ROS, including superoxide. N-acetylcysteine (NAC), an antioxidant, and MnTBAP, an inhibitor of superoxide, reduce high NaCl-induced superoxide activity and suppress both high NaCl-induced increase in TonEBP/OREBP transcriptional activity and high NaCl-induced increase in expression of BGT1mRNA, a transcriptional target of TonEBP/OREBP. Catalase, which decomposes hydrogen peroxide, does not have these effects, whether applied exogenously or overexpressed within the cells. Furthermore, NAC and MnTBAP, but not catalase, blunt high NaCl-induced increase in TonEBP/ OREBP transactivation. [N.sup.G]-monomethyl-L-arginine, a general inhibitor of nitric oxide synthase, has no significant effect on either high NaCl-induced increase in superoxide or TonEBP/OREBP transcriptional activity, suggesting that the effects of ROS do not involve nitric oxide. Ouabain, an inhibitor of Na-K-ATPase, attenuates high NaCl-induced superoxide activity and inhibits TonEBP/OREBP transcriptional activity. We conclude that the high NaCl-induced increase in ROS, including superoxide, contributes to activation of TonEBP/OREBP by increasing its transactivation. superoxides; nitric oxide; ouabain; transactivation; nuclear translocation