The objective of the proposed study is to investigate the potential role of the brain tonin-angiotensin system in blood pressure regulation with new biochemical and immunological tools. Tonin is an enzyme of the serine protease family which acts on a natural protein substrate, angiotensinogen, on the tetradecapeptide renin substrate, and on angiotensin I to form angiotensin II directly. This enzyme has attracted attention in the field of hypertension because of its putative involvement in the generation of angiotensin II, the most powerful mammalian vasoconstrictor. Studies using animal models have implicated tonin in hypertension under certain experimental conditions. In order to gain more insight into the regulation of the tonin-angiotensin system, we have initiated work on tonin monoclonal antibody production and characterization. We have also purified mRNA from several tissue sources and identified the primary translation product of rat tonin. A rat genomic library and a cDNA library have been constructed. From the cDNA library we have recently identified a group of putative tonin clones by immunoscreening based on tonin expression. Two major new tools, hydridoma technology and recombinant DNA technology, will be used to study the basic biochemical aspects of brain tonin. We will generate a large collection of monoclonal antibodies to rat tonin and angiotensin II. These monoclonal antibodies will be used to develop highly specific radioimmunoassays and enzyme linked immunosorbant assays for brain tonin and angiotensin II quantitation. In addition the monoclonal antibodies will be used to identify tonin, tonin precursors and tonin degradation products in brain extracts by immunoprecipitation and Western blot analysis. We will investigate the effect of genetic background, age, hormones and beta-adrenergic agonist and antagonist on the levels of tonin and angiotensin in rat brain and cerebrolspinal fluids. At the nucleic acid level we will continue our work on tonin RNA translatior and processing as well as cDNA and gene cloning. Tonin cDNA will be used to probe tonin mRNA levels by Northern blotting. Special attention will be focused on tonin gene organization and expression in hypertensive rat strains to explore the molecular basis of blood pressure regulation in the central nervous system. The overall goal is to provide a comprehansive understanding of the brain tonin-angiotensin system at DNA, RNA and protein levels.
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