The purpose of this proposal is to continue our study of the regulation of tissue kallikrein activity, synthesis and gene expression using both protein chemistry and nucleic acid approaches. Our recent discoveries of kallikrein distribution in new tissue sites and a kallikrein multigene family raises the exciting possibility that kallikrein(s) may play a general role in processing many important precursor proteins into bioactive peptides in addition to kinin formation. Our working hypothesis is that kallikrein gene expression is tissue-specific and differentially regulated by hormones or other physiological factors. Kallikrein may be further regulated posttranslationally by a newly discovered kallikrein binding protein (kallistatin). These findings and our recent development of specific reagents that include pure kallikrein, monoclonal antibodies to kallikrein, kininogen, kinin, kallikrein cDNA, oligonucleotides and kallikrein genomic clones will be used to elucidate the regulation and function of kallikreins. The objective is facilitated by our recent finding of kallikrein induction in estrogen-treated rat pituitary and identification of the synthesis and secretion of kallikrein from a peptide hormone-producing pituitary cell line. The studies will be initiated by purification, characterization and development of monoclonal or polyclonal antibodies to the kallikrein system components (kallikrein, kallistatin, kininogen). These antibodies will be used for quantitation, identification and isolation of the kallikrein components in rat pituitary or pituitary cell lines. Co-localization of the components in the specific cell types of rat pituitary will be determined by immunohistochemistry and/or by cDNA-mRNA in situ hybridization. The regulatory aspects of kallikrein gene expression and enzyme activity will be elucidated using whole animals in vivo and using rat pituitary cell cultures as a model system in vitro. The pathophysiological role of kallikrein will be explored by characterizing kallikrein gene structure, organization and expression in normal and eventually, in hypertensive animal models. The proposal is designed to advance basic knowledge of the function and regulation of tissue kallikrein(s) and their products as the crucial requirement for understanding the role of these enzymes in such disorders as human hypertensive or diabetic disease.
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