CALMODULIN KINASE II AND ION TRANSPORT IN THE KIDNEY

钙调蛋白激酶 II 和肾脏中的离子转运

• 批准号: 3464890
• 负责人: ROCHELLE M HANLEY
• 金额: $ 4.4万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-01 至 1993-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3464890
• 关键词: SDS polyacrylamide gel electrophoresis; angiotensin II; autoradiography; basolateral membrane; brush border membrane; calcium flux; calmodulin dependent protein kinase; dopamine; enzyme activity; hormone regulation /control mechanism; ion transport; laboratory rabbit; laboratory rat; membrane transport proteins; northern blottings; phosphorylation; radiotracer; renal tubular transport; renal tubule

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.

钙调蛋白依赖性蛋白激酶 II) (CaM-KII) 是一种广泛应用的 具有广泛底物特异性的分布式酶。 最近的研究来自 我们的实验室已证明 CaM-KII 在细胞中的存在 兔肾近曲小管。 α 亚基 mRNA 的存在 CaM-KII 通过 Northern 杂交分析可视化 通过 Western 免疫印迹分析，使用适当的分子大小 CaM-KII 抗肽抗体和 CaM-KII 蛋白激酶活性 本身就成立了。 该蛋白激酶激活的效果 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效果的实验 CaM-KII 对肾刷状缘和基底外侧电解质转运的影响 肾近端膜和蛋白激酶的调节 曲管。 将特别强调以下效果： CaM-KII 在介导氢离子、碳酸氢根和 钠在肾近曲小管中的转运。 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。 最近的 其他人的研究也提出了 CaM-KII 调节的可能性 钠依赖性碳酸氢盐协同转运的活性 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可能的影响 肾小管功能尚未研究。 此外，迄今为止还没有研究表明 检查了肾近曲小管中这种蛋白激酶的调节。 这 目前的提案采用了一些新开发的技术，避免了一些 先前研究中所涉及的实验困难 CaM-KII 的生物学效应和调节。 研究CaM-KII对肾电解质转运、肾刷的影响 边界（BBM）和基底外侧通过暂时打开 通过暴露于含有 ATP、Mg2+、Ca2+ 的低渗溶液中形成囊泡， 钙调蛋白和 CaM-KII。 Na+-H+交换器的活性，Na+依赖性 葡萄糖转运蛋白，以及 BBM 中除 Na+- 外的 Na+PO'4 转运蛋白 BLM 中的 HCO3 协同转运蛋白和 Na+-K+ ATP 酶将使用以下方法进行测量 既定方法。 这种实验方法已被采用 之前研究过 PKA 和 PKC 的影响，应该提供新的 有关 CaM-KII 在离子控制中的调节作用的信息 在肾近曲小管中运输。 CaM-KII 可能参与 通过激素调节近曲小管功能，其信号 转导途径与细胞内钙的变化有关 水平。 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 50 Kda 亚基的自身磷酸化和激活，以及 Northern 杂交和狭缝印迹法检测 CaM-KII mRNA 的表达 使用 cDNA 对大鼠脑 CaM-KII 活性的 α 亚基进行分析 悬浮的近曲小管的提取物也将被 使用特定的肽底物进行测定。 这些实验应该 提供有关该蛋白激酶在 肾近曲小管。