The long-term objective of this research proposal is to delineate the intracellular cascade of events whereby activation of alpha2- adrenergic receptors on the basolateral surface of the renal proximal tubule cell by specific agonists accelerates the Na+-H+ antiporter located on the luminal membrane of the cell. The current view of this mechanism does not take into account two important factors, namely a) that the receptor and effector proteins may be anatomically located on different membranes of a polarized epithelial cell and b) that suppression of adenylate cyclase activity by alpha2 adrenergic agonists in some cell types can be dissociated from the physiologic response.
The specific aims i n attaining this objective are 1. To determine whether application of alpha2-adrenergic agonists to the basolateral surface of the cell effects Na+-H+ antiport activity at the luminal surface. 2. and 3. To test whether alpha2-adrenergic agonists mediae their effect by hydrolysis of phosphatidylcholine phospholipase D. 4. To examine whether alpha2-adrenergic agonist- induced activation of a phosphatidylcholine phospholipase C is modulated by guanine nucleotide regulatory proteins. 5. To determine whether alpha2-adrenergic agonist suppression of adenylate cyclase activity is a primary indirectly via phosphatidic acid. The experimental model employed is a primary culture of rabbit proximal tubule cells. the principal methodologies used include fluorometric monitoring of Na+-H+ antiport activity; polyacrylamide gel electrophoresis and autoradiography for monitoring [32P] NAD ribosylation of G-proteins by pertussis toxin; determination of phospholipid profiles by measuring inorganic phosphorus, and TLC; determination of fatty acid profiles in individual phospholipids by GLC; analysis of DAG accumulation by two dimensional TLC; activation of PKC by 32P phosphorylation of exogenous histone; measurement of choline accumulation by column chromatography; and verification that the products of phosphatidylcholine hydrolysis accelerate Na+-H+ antiport activity by microinjection and microfluorimetry. The information obtained from this research proposal will be synthesized into an integrated model and will shed new light on the mechanism of alpha2-adrenergic agonist modulation of Na+-H+ antiport in the proximal tubule cell.
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