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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL033552-04
Application #
3345537
Study Section
(SRC)
Project Start
1984-09-30
Project End
1989-09-29
Budget Start
1987-09-30
Budget End
1988-09-29
Support Year
4
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Medical University of South Carolina
Department
Type
Schools of Medicine
DUNS #
183710748
City
Charleston
State
SC
Country
United States
Zip Code
29425
Wang, J; Chao, J; Chao, L (1991) Purification and characterization of recombinant tissue kallikrein from Escherichia coli and yeast. Biochem J 276 ( Pt 1):63-71
Chen, L M; Chao, L; Mayfield, R K et al. (1990) Differential interactions of human kallikrein-binding protein and alpha 1-antitrypsin with human tissue kallikrein. Biochem J 267:79-84
Murray, S R; Chao, J; Lin, F K et al. (1990) Kallikrein multigene families and the regulation of their expression. J Cardiovasc Pharmacol 15 Suppl 6:S7-16
Simson, J A; Currie, M G; Chao, L et al. (1989) Co-localization of a kallikrein-like serine protease (arginine esterase A) and atrial natriuretic peptide in rat atrium. J Histochem Cytochem 37:1913-7
Lu, H S; Lin, F K; Chao, L et al. (1989) Human urinary kallikrein. Complete amino acid sequence and sites of glycosylation. Int J Pept Protein Res 33:237-49
Woodley-Miller, C; Chao, J; Chao, L (1989) Restriction fragment length polymorphisms mapped in spontaneously hypertensive rats using kallikrein probes. J Hypertens 7:865-71
Chao, L; Chen, Y P; Woodley-Miller, C et al. (1989) Structural analysis of a rat renal kallikrein gene. Adv Exp Med Biol 247A:73-80
Xiong, W; Chao, L; Chao, J (1989) Renal kallikrein mRNA localization by in situ hybridization. Kidney Int 35:1324-9
Shai, S Y; Woodley-Miller, C; Chao, J et al. (1989) Characterization of genes encoding rat tonin and a kallikrein-like serine protease. Biochemistry 28:5334-43
Chao, S; Chao, L; Chao, J (1989) Sex dimorphism and inflammatory regulation of T-kininogen and T-kininogenase. Biochim Biophys Acta 991:477-83

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