CELLULAR INTERACTION OF RENIN AND ITS SUBSTRATE

Recent experiments, including our own, suggest that local generation of angiotensin II is an important mechanism in the regulation of the vascular smooth muscle tension. It appears that renin containing cells also contain angiotensin I and II. To test the cellular mechanisms underlying this process, I propose to test the hypothesis that renin containing cells internalize native renin substrate by receptor mediated endocytosis. In preliminary experiments, I found that renin containing cells internalize synthetic renin substrate. In the proposed experiments, native renin substrate will be isolated and appropriate experimental probes developed to study its fate in renin containing cells. Established neuroblastoma cell lines known to contain renin and juxtaglomerular cells will be utilized to test whether these cells generate angiotensin intracellularly by internalizing renin substrate by receptor-mediated endocytosis. The mechanisms controlling the internalization process will be explored using a variety of techniques (receptor binding studies, inhibitors of binding, inhibitors of internalization). The cellular fate of the internalized renin substrate will also be examined and immunofluorescent techniques will be used to determine whether renin substrate is stored in renin containing granules. It is also proposed to examine whether renin containing cells synthesize renin substrate and if so, to determine the factors controlling this synthesis. Specifically, studies with inhibitors of protein synthesis and stimuli known to increase hepatic synthesis of renin substrate will be performed. Finally, utilizing a hindlimb preparation perfused under in vitro conditions it will be determined whether renin in the peripheral vasculature utilizes renin substrate to generate angiotension in order to control vascular tone. In addition, whether peripheral vascular renin is influenced by stimuli that are activated during changes in extracellular fluid volume and blood pressure will be determined. The overall objective is to study the cellular mechanisms controlling the interaction of renin substrate and renin containing cells and the potential relationship of these processes to blood pressure control.