CELLULAR FUNCTION OF THE JUXTAGLOMERULAR APPARATUS

哺乳动物肾脏的肾小球体（JGA）是一个解剖学上的器官， 连接远端肾单位致密斑细胞的结构， 肾小球血管极。 已经确定，NaCl的变化 在致密斑细胞的浓度引发一连串事件， 改变传入小动脉张力和肾素分泌。 为了更好 了解不同细胞成分之间可能的相互作用 JGA的这些细胞的内在属性需要知道。 在 第一部分，本建议旨在调查分泌和 血管紧张素II和血管紧张素Ⅱ对传入小动脉细胞的收缩反应 腺苷，这两种药物在水平上发挥深刻的调节作用， 的JGA，并已注意到影响肾小球前血管在一个 协同方式。 此应用程序的第二部分将使用 用于评估mRNA水平的高灵敏度技术 JGA细胞中基因表达的调节。 第一部分的具体目的是描述相互关系的程度。 血管紧张素和腺苷的血管收缩作用的依赖性， 来理解协同作用的机制。 协同程度 将通过审查a）是否以及在多大程度上 血管收缩和抑制肾素分泌产生的 腺苷被血管紧张素II增强并被血管紧张素II抑制 相反，是否以及在多大程度上相同的反应， 腺苷可增强血管紧张素II的生成， 腺苷拮抗剂，和B）是否传入收缩和 增加NaCl对致密斑肾素释放的抑制作用 取决于两种激动剂的可用性。 各机制 协同相互作用将通过检查a）一种可能的 促进各自受体水平上的相互作用，B）a 在产生调节磷酸肌醇中可能的增强作用 代谢物，c）对Ca释放的可能增强作用， 细胞内储存或Ca进入，d）对跨膜离子电流的影响 其增强细胞活化，e）对细胞内循环水平的影响， f）对磷脂酶A依赖性磷脂酶A的产生的影响， 产品. 第2部分的一个具体目标是使用聚合酶链反应（PCR）， 评估单个JGA制剂中的mRNA水平。 在这些研究中，我们将a） 优化监测回收或扩增效率的技术， 应用于单个分离JGA标本的PCR方法的每个步骤，B） 评估一组管家基因的mRNA响应模式， 定义这些mRNA种类对肾素操纵的反应， c）研究血管紧张素II、腺苷和 环核苷酸对单个JGA中的肾素mRNA的作用，以及d）检查环核苷酸对单个JGA中的肾素mRNA的作用。 致密斑细胞中葡萄糖-6-磷酸脱氢酶的表达。 与JGA中的肾素和G6 PDH mRNA水平平行。

The juxtaglomerular apparatus (JGA) of the mammalian kidney is an anatomic structure which connects the macula densa cells of the distal nephron with the glomerular vascular pole. It has been established that changes in NaCl concentration at the macula densa cells initiate a chain of events which alters afferent arteriolar tone and renin secretion. In order to better understand possible interactions between the different cellular components of the JGA the intrinsic properties of these cells need to be known. In its first part this proposal aims at investigating the secretory and contractile responses of afferent arteriolar cells to angiotensin II and adenosine, two agents which exert profound modulating effects at the level of the JGA and which have been noted to affect preglomerular vessels in a synergistic fashion. The second part of this application will utilize a highly sensitive technique for assessment of mRNA levels to study regulation of gene expression in cells of the JGA. The specific aims of part 1 are to describe the extent of the mutual dependency of the vasoconstrictor effects of angiotensin and adenosine and to understand the mechanisms for the synergism. The degree of synergism will be studied by examining a) whether and to what extent the vasoconstriction and the inhibition of renin secretion produced by adenosine is potentiated by angiotensin II and inhibited by angiotensin II antagonists, and conversely, whether and to what extent the same responses produced by angiotensin II are potentiated by adenosine and inhibited by adenosine antagonists, and b) whether the afferent constriction and inhibition of renin release elicited by increased NaCl at the macula densa depend upon the availability of both agonists. The mechanisms of the synergistic interaction will be studied by examining a) a possible facilitating interaction at the level of the respective receptors, b) a possible potentiating effect in generating modulating phosphoinositide metabolites, c) a possible potentiating effect on Ca release from intracellular stores or Ca entry, d) effects on transmembrane ion currents which enhance cell activation, e) effects on intracellular levels of cyclic nucleotides, and f) effects on the generation of phospholipase A dependent products. A specific aims of part 2 are to use the polymerase chain reaction (PCR) to assess mRNA levels in single JGA preparations. In these studies we will a) optimize techniques for monitoring recovery or amplification efficiency at each step of the PCR method applied to single isolated JGA specimen, b) assess the response pattern of mRNA for a set of housekeeping genes and define the response of these mRNA species to manipulations of the renin- angiotensin system, c) study the effect of angiotensin II, adenosine, and cyclic nucleotides on renin mRNA in single JGA, and d) examine the role of glucose-6-phosphate dehydrogenase in macula densa cells by assessing in parallel the levels of renin and G6PDH mRNA in the JGA.