This renewal application seeks to continue studies that use Madin Darby Canine Kidney (MDCK) cells and knockout mice to assess molecular and functional aspects of adrenergic and P2Y (nucleotide, purinergic) receptors. The proposed studies will test hypotheses related to findings that we have obtained in MDCK cells and more recently in mouse primary kidney collecting duct (mpkCCD) cells as well as in P2Y2 knockout mice, which we have recently shown have salt-resistant hypertension and multiple abnormalities in renal function.
The Specific Aims i nvolve studies of: 1) Expression and compartmentation in raft/caveolar microdomains of G protein-coupled receptor/G-proteins/effectors of adrenergic and P2Y receptors in MDCK and mpkCCD cells and assessment of sumoylation (a novel modification that we have recently identified) of caveolins;2) Functional roles of renal P2Y and adrenergic receptors using receptor-knockout mice. The combined use of cultured renal epithelial cells and knockout mice provides a complementary strategy that is designed to reveal new insights regarding the regulation of epithelial cells and renal function by adrenergic and P2Y receptors. The findings may have implications for cardiovascular, renal and other disorders that involve the sympathetic nervous system or in which cell injury leads to release of nucleotides.
This project will assess 2 important classes of proteins, receptors for adrenaline (and adrenaline-like drugs) and for a major cell constituent, ATP. The receptors, which are located on the surface of cells, recognize those molecules in the extracellular environment but then alter intracellular function. Experiments are proposed in: 1) tissue culture cells to assess the organization of the components involved in information transfer across the cell surface and 2) mice engineered to have loss in receptors for ATP or adrenaline with the goal of understanding how the receptors regulate fluid and salt handling, in particular by the kidney.
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