The proposed studies are directed at elucidating and characterizing, at the cellular level, the angiotensin system associated with nervous system pathways. Our finding of high affinity binding sites in both cultured rat brain cells and on C6 glioma indicate that the brain angiotensin system may involve both neuronal and glial functions. Further, our evidence indicates that the neural angiotensin II receptor (AII/R) is regulated differently from the receptor present in non-neural tissues. The focus of this proposal will be on the identification, characterization and mechanisms of regulation of the angiotensin II receptor on isolated cultured rat brain cells. Primary cultures of oligodendrocytes, astrocytes and neurons will be obtained from fetal and neonatal rat brain tissue. Specific objectives include: (1) to develop highly enriched cultures of Sprague-Dawley (S-D) rat brain neurons, astrocytes and oligodendrocytes in defined culture media; (2) to identify high affinity angiotensin binding sites on the isolated cells and to characterize these sites as to whether they represent receptors or degarding enzymes; (3) to study the regulation and expression of these sites/receptors in isolated cell cultures and in co-cultures; (4) to determine the influence of steroid hormones, peptides and modifiers of neuronal activity on expression of AII/R; (5) to define which isolated cells synthesize angiotensinogen and mechanisms of regulation of the biosynthesis of this prohormone; and (6) to extend these studies into a characterization of the expression of high affinity binding sites on brain cells from the spontaneously hypertensive (SHR) rat strain. Cells will be identified with cell-specific markers for glia and neurons. Receptors and binding sites will be studied through the use of ((125)I)-angiotensin II and iodinated analogs in both direct binding studies on intact cells and membranes and by receptor autoradiography. The results of the proposed investigation will provide information concerning the distribution, function and regulation of the nervous system angiotensin pathway and the relationship of all cellular sites to the development of hypertension in the spontaneously hypertensive rat.
