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

低钾血症损害了厚升肢（TAL）的运输功能，降低了尿的浓缩能力。由于根尖钾通道在TAL中通过根尖膜的钾循环中起着重要作用，抑制根尖钾通道有望阻断Na/K/Cl共转运体的功能，并减弱尿浓缩能力。初步结果表明，缺钾饲粮动物的金属tal顶端钾通道活性显著低于高钾饲粮动物。此外，钾的消耗增加了20-羟基二碳四烯酸的产生，而花生四烯酸（AA）细胞色素P450单加氧酶的抑制增加了顶端70 pS钾通道的活性，这些通道为顶端膜贡献了80%的钾电导。因此，我们将验证一个假设，即在缺钾饮食的大鼠中，AA的细胞色素p450依赖性代谢物的增加参与抑制TAL的顶端钾传导。钙离子感应受体的第二个假设是负责降低顶端钾传导从缺钾饮食动物TAL。这一假设是基于观察到，细胞外Ca2+浓度需要诱导顶端70ps钾通道50%的抑制，在小管中，钾缺乏饮食的大鼠明显低于高钾饮食的大鼠。该建议的第三个假设是，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中顶端钾通道活性降低的原因。初步数据显示，缺钾动物肾髓质中iNOS和HO-2的表达降低。此外，先前的研究表明，NO和CO刺激了金属顶端70pS K通道。为了实现我们的目标，我们将使用膜片钳技术、离子敏感染料、生化方法和微灌注技术来评估AA代谢物、CO和NO对金属中通道活性和Na运输的影响。

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.