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

低钾血症损害粗大升肢(TAL)的运输功能，降低尿浓缩能力。由于心尖K通道在TAL根尖膜钾循环中起重要作用，抑制心尖K通道有望阻断Na/K/Cl共转运体的功能，削弱尿液浓缩能力。初步结果表明，低钾饮食动物mTAL的尖端钾通道活性显著低于高钾饮食动物。此外，钾缺乏增加了20-羟基二十碳四烯酸的产生，抑制花生四烯酸细胞色素P450单加氧酶增加了顶端70pS钾通道的活性，该通道贡献了顶膜80%的钾电导。因此，我们将检验这一假设，即AA依赖细胞色素P450的代谢物的增加参与了抑制缺钾饮食大鼠TAL顶端K电导的过程。钙感受器的第二个假说是导致缺钾动物TAL顶端钾电导降低的原因。这一假说是基于这样的观察，即低钾饮食大鼠肾小管上皮细胞外钙离子浓度显著低于高钾饮食大鼠肾小管上皮细胞70pS钾通道50%抑制所需的细胞外钙离子浓度。提出的第三个假设是，一氧化氮合酶(NOS)和血红素氧合酶(HO)活性的降低是缺钾饮食大鼠TAL心尖钾通道活性降低的原因。初步数据显示，缺钾饮食动物肾髓质中iNOS和HO-2的表达减少。此外，以前的研究表明，NO和CO刺激mTAL顶端70pS的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、生化方法和微灌流技术来评估AA代谢物、CO和NO对mTAL通道活性和Na转运的影响。

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.