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J. Biol. Chem., Vol. 265, Issue 36, 22238-22242, Dec, 1990

Effect of bradykinin on Na-K-2Cl cotransport and bumetanide binding in aortic endothelial cells

JD Klein and WC O'Neill
Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322.

Simultaneous measurements of potassium influx and binding of [3H]bumetanide were performed in endothelial cells cultured from bovine aortas to determine how bradykinin regulates Na-K-2Cl cotransport. [3H]Bumetanide displayed saturable binding and was displaced by low concentrations of unlabeled bumetanide. All three transported ions were required for binding and high concentrations of chloride inhibited binding, consistent with binding of bumetanide to the second chloride site of the transporter. Scatchard analysis of binding under maximal conditions (100 mM sodium, 30 mM potassium, 30 mM chloride) revealed a single class of binding sites with a binding constant of 112 nM and a density of 22 fmol/cm2 or approximately 122,000 sites/cells. Na-K-2Cl cotransport, measured as bumetanide-sensitive potassium influx, was stimulated 118 +/- 30% by bradykinin (p less than 0.01) at physiologic ion concentrations. Stimulation was inhibited by increased potassium or decreased external chloride concentrations and was not seen in conditions required for maximal binding of bumetanide. Simultaneous measurement of the binding of tracer [3H]bumetanide and its inhibition of potassium influx in medium containing 10 mM potassium and 130 mM chloride revealed a turnover number for the cotransporter of 293 +/- 68 s-1 which increased to 687 +/- 105 s-1 with bradykinin (p less than 0.001). There was no change in cell volume and only a 5.6 mM increase in intracellular sodium concentration associated with this stimulation. Bradykinin also increased the affinity of the cotransporter for bumetanide as indicated by a decrease in the Ki for potassium influx from 464 +/- 46 nM to 219 +/- 19 nM (p less than 0.005). Our results show that [3H]bumetanide can be used to quantitate Na-K-2Cl cotransporter sites in aortic endothelial cells and to determine the mechanism by which cotransport is regulated. The stimulation of cotransport in aortic endothelial cells by bradykinin is due to an increase in the activity of existing transporters rather than to an increase in the number of transporters. This, together with the increased affinity for bumetanide, strongly suggests that a change in cotransporter structure is occurring in response to bradykinin.
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