The objective of the proposed research is to elucidate the cellular mechanism by which vasopressin (VP) regulates tubular luminal membranes of mammalian kidney in health and disease. The major focus will be on elucidation of biochemical mechanism by which cyclic AMP (cAMP), generated in response to VP, regulates function of luminal plasma membranes (""""""""post cAMP steps""""""""). Studies will be conducted on two segments of tubules both regulated by VP via cAMP, but with different functional responses of luminal membranes: on collecting tubules and ducts and on ascending limb of Henle's loop. The specific objectives include: 1. To determine the localization, subcellular distribution and regulatory properties of the major enzymes and modulators of the cAMP-dependent protein phosphorylation system. We will study in particular: a) isoenzymes of cAMP-dependent protein kinases, b) protein phosphatases and c) non-enzymatic protein modulators. 2. To examine VP-regulated phosphorylations of endogenous substrates in intact tubules. Proteins and/or glycoproteins which are phosphorylated in response to increases in cAMP will be identified. Specifically, we will examine the relationship of cAMP-dependent endogenous phosphorylations to components of cytoskeleton, namely microtubules and microfilaments, calcium-calmodulin dependent proteins, and to components of luminal plasma membranes of collecting tubules and ducts. 3. To identify basic biochemical components of luminal plasma membranes of collecting tubules and ducts, we will use radiolabeled non-penetrating covalent chemical probes. Further, we will determine specific biochemical changes elicited by VP via cAMP in these membranes. 4. To study the """"""""post cAMP"""""""" components in animal models of both hyporesponsiveness and hyperresponsiveness to VP. Nephrogenic diabetes insipidus and a model of hyperresponsiveness to VP induced by administration of chlorpropamide will be studied to elucidate the pathogenesis of these dysfunctions. The studies will be conducted on renal tubules microdissected from normal rats, rabbits and mice or from animals with hereditary or drug-induced urinary concentrating defects.
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