The studies proposed in this application are designed to examine G protein-dependent mechanisms by which transport is regulated in the renal proximal tubule (PT). NaCl and NaHCO3 transport is reciprocally regulated by hormones like angiotensin II (AngII) and a agonists which increase it, and by PTH which decreases it. Receptors for these hormones are coupled by G proteins to enzymes or ion channels which produce their biologic effects. The enzymes and ion channels regulated by G proteins (effectors) may include adenylyl cyclase (AC), phospholipases A2 and C (PLA2 and PLC), and K and Ca channels. Most hormones regulate some, but not all of these effectors. How the specificity of the signalling pathway is determined, how effectors are regulated, and by which G proteins is not known in detail. Hormones such as AngII and PTH activate multiple effectors in the PT, but it is not clear if one G protein activates one or multiple effectors. It is also not clear if a single G protein couples to one or multiple receptors. The studies proposed in this application will address these questions: 1) In situ hybridization studies in sections of rat kidneys using oligonucleotide probes to six G proteins (Gs, Gil-3, Go, and Gz) will determine which G proteins are expressed in the PT. 2) Western blotting of renal cortical basloateral (BLMV) and brush border membranes (BBMV) with G protein-specific antisera will establish the distribution of G proteins on the apical and basolateral membranes of the PT. 3) The effectors (AC, PLA2, PLC, and K and Ca channels), regulated by the G proteins expressed in the PT will be determined by expressing mutant, constitutively active G proteins in PT-like continuous cell lines (OK, BSC-1, or immortalized primary cultures), and measuring the activity of the potential effectors. Purified, preactivated recombinant G proteins will be reconstituted into BLMV and BBMV, and K and Ca channel activity measured. 4) G protein-receptor coupling will be established in BLMV and BBMV using biochemical assays that identify a G protein that is activated by a receptor, or by expressing a mutant G protein with abnormal receptor coupling in a continuous renal cell line. PTH, AngII, and a adrenergic agonists will be studied. 5) The ability of these G protein-dependent signalling systems to regulate transport will be established by measuring Na/H antiporter, Na/3HCO3 cotransporter, and Na,KATPase activity in cells expressing activated G proteins and membrane vesicles containing preactivated, recombinant G proteins. These studies will provide a better understanding of how PT NaCl and NaHCO3 transport is regulated, and how specificity for G protein-dependent signalling is determined.
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