CALMODULIN KINASE II AND ION TRANSPORT IN THE KIDNEY

钙调素依赖的蛋白激酶II(CaM-KII)是一种广泛的 分布的酶具有广泛的底物专一性。最近来自 我们的实验室已经证实了CaM-KII在人卵巢癌细胞中的存在。 兔肾近端小管。核糖核酸α亚基的存在 CaM-KII蛋白的Northern杂交分析 用Western免疫印迹分析合适的分子大小 抗CaM-KII抗肽抗体及CaM-KII蛋白激酶活性 它本身就已经确立了。该蛋白激酶的激活作用 关于电解质在肾近端小管中的转运，目前还没有明确的定义。 本提案描述了旨在研究以下效应的实验 CaM-KII对肾刷缘和基底外侧电解质转运的影响 肾近端的膜与蛋白激酶的调节 曲折的小管。我们将特别强调 CAM-KII关于调节氢离子、碳酸氢盐和 钠在肾近端小管中的转运。这样做的理由是 源于最近的研究表明，CaM-KII的磷酸化 抑制溶解和重组的Na+-H+交换器。近期 其他人的研究也提出了一种可能性，即Cam-Kii调节 钠依赖的碳酸氢盐共转运活性 近曲小管基底外侧膜的Na+-H+交换转运 细胞。该蛋白激酶对其他近端的可能影响 小管功能尚未被研究过。此外，到目前为止，还没有研究表明 研究了这种蛋白激酶在肾近端小管中的调节。这个 本提案采用了一些新开发的技术，从而避免了一些 在过去的实验研究中涉及哪些困难 CaM-KII的生物学效应及其调控。 研究CaM-KII对肾脏电解质转运、肾刷功能的影响 边界(BBM)和基侧通过瞬时打开 通过暴露在含有ATP，Mg2+，Ca2+， 钙调素和CaM-Kii。Na~+依赖的Na~+-H~+交换活性 葡萄糖转运蛋白和Na+PO‘4转运蛋白。 BLM中的HCO3共转运体和Na+-K+ATPase将用 既定的方法。这种实验性的方法，已经被采用 以往研究PKA和PKC的影响，应提供新的 关于CaM-KII在离子调控中的调节作用的信息 在肾近端小管的转运。Cam-Kii可能参与了 激素对近曲小管功能的调节作用 信号转导通路与细胞内钙离子的变化有关 级别。因此，利用分离的近端曲管，我们将 观察多巴胺或血管紧张素II治疗对心脏功能的影响 肾脏CaM-KII 50KDA亚基的自动磷酸化和激活，以及 Northern杂交和狭缝印迹法检测CaM-KII基因的表达 大鼠脑CaM-KIIα亚基活性的CDNA分析 悬浮的近端曲管的提取物也将被 使用特定的多肽底物进行检测。这些实验应该 提供有关该蛋白激酶在细胞内的调控的新信息 肾近端曲管。

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.