REGULATION OF MTAL FUNCTION BY 20-HETE, NO, AND CO

低钾血症损害粗升支（TAL）的运输功能，降低尿浓缩能力。由于顶端K通道在TAL中跨顶端膜的K再循环中起重要作用，因此预期顶端K通道的抑制将阻断Na/K/Cl协同转运蛋白的功能并减弱尿浓缩能力。初步结果表明，缺钾饮食动物mTAL中顶端K通道的活性显著低于高钾饮食动物。此外，K-耗竭增加了20-羟基二十碳四烯酸的产生，并且花生四烯酸（AA）的细胞色素P450单加氧酶的抑制增加了顶端70 pS K通道的活性，该通道贡献了顶端膜的80%的K电导。因此，我们将测试的假设，细胞色素p450依赖性代谢产物的AA的增加参与抑制顶端钾电导的TAL在大鼠缺钾饮食。第二个假设是Ca 2+敏感受体导致缺钾饮食动物的TAL顶端钾电导降低。这一假设是基于观察到的细胞外Ca 2+浓度诱导50%的抑制顶端70 pS钾通道显着低于从大鼠的肾小管缺钾饮食比那些高钾饮食。该提议的第三个假设是一氧化氮合酶（NOS）和血红素加氧酶（HO）活性的降低是导致缺钾饮食大鼠TAL中顶端K通道活性降低的原因。初步数据显示，在缺钾饮食的动物肾髓质中，iNOS和HO-2的表达减少。此外，以前的研究表明，NO和CO刺激顶端70 pS钾通道的mTAL。为了实现我们的目标，膜片钳技术，离子敏感染料，生化方法和微灌注技术将被用来评估AA代谢产物，CO和NO对通道活性的影响，以及在mTAL的Na转运。

Hypokalemia impairs the transport function of the thick ascending limb (TAL) and diminishes urinary concentrating ability. Since apical K channels play an important role in K recycling across the apical membrane in the TAL, inhibition of the apical K channels is expected to block the function of the Na/K/Cl cotransporter and to attenuate the urinary concentrating ability. Preliminary results have shown that the activity of apical K channels in the mTAL from animals on a K-deficient diet is significantly lower than those of a high K diet. Moreover, K- depletion increases the production of 20-hydroxyeicosatetraenoic acid and inhibition of cytochrome P450 monoxygenase of arachidonic acid (AA) increases the activity of the apical 70 pS K channels which contribute 80% of K conductance to the apical membrane. Thus, we will test the hypothesis that an increase in cytochrome p450-dependent metabolites of AA is involved in inhibiting the apical K conductance of the TAL in rats on a K-deficient diet. The second hypothesis of Ca2+ sensing receptor is responsible for decreasing the apical K conductance in the TAL from animals on a K-deficient diet. This hypothesis is based on the observation that extracellular Ca2+ concentrations required for inducing a 50% inhibition of the apical 70 pS K channel were significantly lower in the tubules from rats on a K-deficient diet than those on a high K diet. The third hypothesis of the proposal is that a decrease in the activity of nitric oxide synthase (NOS) and heme oxygenase (HO) is responsible for reducing the activity of apical K channels in the TAL from rats on a K- deficient diet. Preliminary data shows that the expression of iNOS and HO-2 decreases in the renal medulla from animals on a K-deficient diet. Moreover, previous studies have shown that NO and CO stimulate the apical 70pS K channels in the mTAL. To achieve our goals, the patch clamp technique, ion-sensitive dye, biochemical approaches and microperfusion techniques will be used to assess effects of AA metabolites, CO and NO on channel activity as well as Na transport in the mTAL.