Many questions still remain concerning both proximal tubule (PT) renin angiotensin system (RAS) intracellular configuration and mechanisms of its effects. This application uses two lines of immortalized rat PT cells (IRPTC) developed in this application's first cycle to define PT RAS paracrine, autocrine and intracrine effects. It is hypothesized that Ang II and other angiotensins produced locally within PT influence salt and water reabsorption by modulating transporters via specific intracellular signaling mechanisms and intranuclear mechanisms as yet incompletely deduced. Ang II appears to reduce cAMP and stimulate Ca2+ exchanger activity; to increase intracellular calcium via PKC and stimulate Na+/Ca++ transport; and to increase phospholipase A2, stimulating hydrolysis of phosphatidylcholine to lysophosphatidylcholine and fatty acid, influencing sodium transport. Such Ang II effects may be carried out both via surface and intracellular receptors in PT, as suggested by recent evidence from the applicant's laboratory that IRPTC have nuclear Ang II receptors.
Specific Aims are to define: I: Intracellular configuration and spatial arrangement of RAS in IRPTC. While all RAS components are clearly demonstrated in PT (and in both lines of IRPTC), actual intracellular localization is incompletely known. Intracellular production, localization and trafficking of RAS components will be defined by immunoelectron microscopy, confocal microscopy, subcellular fractionation plus molecular and cellular techniques. II: Mechanisms of Ang II effect on solute transport in PT. Ang II exhibits a biphasic influence on water and salt transport. The influence of Ang II on transporter regulation will be defined, using Na+/H+ exchanger as a prototype in parallel studies encompassing both molecular biology and cell physiology. Ang II-responsive elements will be sought; effects of Ang II on mRNA and protein regulation of NHEs will be delineated [e.g., mRNA steady state level, stability and localization; immunostaining intensity and pattern [e.g., with trafficking and immunoassay]; and cell signaling and physiology will be defined. III: Ang II and its autocrine and intracrine effects, as these affect PT functions such as growth and repair, and protein processing, hypothesizing that Ang II modulates cell growth and repair. The functional role of Ang II in IRPTCs will be defined by blocking or stimulating RAS components to dissect autocrine effects. IRPTCs have intracellular Ang II receptors; thus, possible intracellular processing of angiotensinogen (renin substrate) will be studied.
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