Klein J D, O'Neill W C
Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322.
J Biol Chem. 1990 Dec 25;265(36):22238-42.
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.
为了确定缓激肽如何调节钠-钾-2氯共转运体，我们对从牛主动脉培养的内皮细胞进行了钾离子内流和[3H]布美他尼结合的同步测量。[3H]布美他尼表现出饱和结合，且可被低浓度的未标记布美他尼取代。结合需要所有三种转运离子，高浓度的氯离子会抑制结合，这与布美他尼与转运体的第二个氯离子位点结合一致。在最大条件下（100 mM钠、30 mM钾、30 mM氯）对结合进行Scatchard分析，结果显示存在一类结合位点，其结合常数为112 nM，密度为22 fmol/cm2或约122,000个位点/细胞。在生理离子浓度下，以布美他尼敏感的钾离子内流来衡量的钠-钾-2氯共转运体受到缓激肽的刺激，增加了118±30%（p<0.01）。刺激作用会因钾离子浓度升高或细胞外氯离子浓度降低而受到抑制，在布美他尼最大结合所需的条件下未观察到这种刺激作用。在含有10 mM钾和130 mM氯的培养基中，对示踪剂[3H]布美他尼的结合及其对钾离子内流的抑制作用进行同步测量，结果显示共转运体的周转数为293±68 s-1，缓激肽作用后增加到687±105 s-1（p<0.001）。这种刺激作用下细胞体积没有变化，细胞内钠浓度仅增加了5.6 mM。缓激肽还增加了共转运体对布美他尼的亲和力，钾离子内流抑制常数Ki从464±46 nM降至219±19 nM（p<0.005）即可表明这一点。我们的结果表明，[3H]布美他尼可用于定量主动脉内皮细胞中的钠-钾-2氯共转运体位点，并确定共转运体的调节机制。缓激肽对主动脉内皮细胞中共转运体的刺激作用是由于现有转运体活性增加，而非转运体数量增加。这一点，再加上对布美他尼亲和力的增加，强烈表明共转运体结构正在响应缓激肽发生变化。
