CALMODULIN KINASE II AND ION TRANSPORT IN THE KIDNEY

钙-钙调蛋白依赖性蛋白激酶II（CaM-KII）是一种广泛存在于 具有广泛底物特异性的分布式酶。 最近的研究来自 我们的实验室已经证明了CaM-KII存在于细胞中， 兔肾近端小管 α亚基mRNA的存在， 通过北方杂交分析可视化的CaM-KII， 通过Western免疫印迹分析，使用合适的分子大小， 抗CaM-KII的抗肽抗体和CaM-KII蛋白激酶活性 本身成立。 这种蛋白激酶的激活作用 对肾近曲小管电解质转运的影响尚不明确。 本提案描述了旨在研究以下因素影响的实验 CaM-KII对肾刷状缘和基底外侧电解质转运的影响 膜和蛋白激酶的调节在肾近端 曲小管 特别强调的是， CaM-KII对介导氢离子、碳酸氢根和 钠转运在肾近端小管。 这样做的理由是 来自最近的研究表明，CaM-KII的磷酸化 抑制溶解和重构的Na +-H+交换剂。 最近 其他人的研究也提出了CaM-KII调节 钠依赖性碳酸氢盐共转运活性除了 近曲小管基底膜Na~+-H~+交换转运 细胞 这种蛋白激酶对其他近端细胞的可能影响 还没有研究小管的功能。 此外，迄今为止， 研究了这种蛋白激酶在肾近端小管中的调节。 的 本建议采用了一些新开发的技术， 的研究中所涉及的先前的实验困难， 生物学效应和CaM-KII的调节。 为了研究CaM-KII对肾脏电解质转运的影响，肾刷 边界（BBM）和基底外侧通过短暂打开 通过暴露于含有ATP、Mg2+、Ca2+的低渗溶液， 钙调素和CaM-KII。 Na~+-H~+交换器活性，Na~+依赖性 葡萄糖转运蛋白和Na + PO4转运蛋白，除了Na +- BLM中的HCO3协同转运蛋白和Na +-K + ATP酶将使用 建立方法。 这种实验性的方法， 以前研究PKA和PKC的作用，应该提供新的 关于CaM-KII在离子控制中的调节作用的信息 在肾近端小管中的转运。 CaM-KII可能参与 通过激素调节近端小管功能， 转导途径与细胞内钙离子的变化有关 程度. 因此，利用分离的近曲小管，我们将 检查多巴胺或血管紧张素II治疗对 肾CaM-KII的50 Kda亚基的自身磷酸化和活化，以及 用北方杂交和狭缝印迹法检测CaM-KII mRNA的表达 使用cDNA分析大鼠脑CaM-KII活性的α亚基， 从悬浮的近曲小管中提取的提取物也将 使用特定的肽底物测定。 这些实验应该 提供了新的信息，这种蛋白激酶的调节， 肾近曲小管

Calcium-calmodulin dependent protein kinase II) (CaM-KII) is a widely distributed enzyme with a broad substrate specificity. Recent studies from our laboratory have demonstrated the presence of CaM-KII in cells of the rabbit renal proximal tubule. The presence of MRNA for alpha subunit of CaM-KII visualized by Northern hybridization analysis a protein of appropriate molecular size by Western immunoblot analysis using an antipeptide antibody against CaM-KII, and CaM-KII protein kinase activity itself were established. The effect of activation of this protein kinase on electrolyte transport in the renal proximal tubule is not well defined. The present proposal describes experiments designed to study the effect of CaM-KII on electrolyte transport in renal brush border and basolateral membranes and the regulation of the protein kinase in the renal proximal convoluted tubule. Particular emphasis will be placed on the effect of CaM-KII on transport systems which mediate hydrogen ion, bicarbonate, and sodium transport in the renal proximal tubule. The rationale for this is derived from recent studies indicating that phosphorylation by CaM-KII inhibits the solubilized and reconstituted Na + -H+ exchanger. Recent studies by others have also raised the possibility that CaM-KII regulates the activity of sodium dependent bicarbonate co-transport in addition to Na+-H+ exchange transport in the basolateral membrane of proximal tubule cells. The possible effects of this protein kinase on other proximal tubule functions have not been studied. Moreover, no studies to date have examined regulation of this protein kinase in renal proximal tubules. The present proposal employs some newly developed techniques which obviate some of the prior experimental difficulties involved in the study of the biologic effects and the regulation of CaM-KII. To study the effect of CaM-KII on renal electrolyte transport, renal brush border (BBM) and basolateral accomplished through transiently opening the vesicles by exposure to hypotonic solutions containing ATP, Mg2+,Ca2+, calmodulin, and CaM-KII. The activities of Na+-H+ exchanger, Na+-dependent glucose transporter, and the Na+PO'4 transporter in BBM in addition to Na+- HCO3 co-transporter and Na+-K+ ATPase in BLM will be measured using established methods. This experimental approach, which has been employed previously to study the effects of PKA and PKC, should provide new information on the regulatory role of CaM-KII in the control of ion transport in the renal proximal tubule. CaM-KII may be involved in regulation of proximal tubule function by hormones whose signal transduction pathways are linked to changes in intracellular calcium levels. Therefore, utilizing isolated proximal convoluted tubules, we will examine the effect of dopamine or angiotensin II treatment on the autophosphorylation and activation of 50 Kda subunit of renal CaM-KII, and the expression of CaM-KII MRNA by Northern hybridization and slot blot analyses using a CDNA to the alpha subunit of rat brain CaM-KII activity in extracts from the suspended proximal convoluted tubules will also be assayed using specific peptide substrates. These experiments should provide new information on the regulation of this protein kinase in the renal proximal convoluted tubule.