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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK036351-05
Application #
3234704
Study Section
General Medicine B Study Section (GMB)
Project Start
1988-07-01
Project End
1995-12-31
Budget Start
1992-01-01
Budget End
1992-12-31
Support Year
5
Fiscal Year
1992
Total Cost
Indirect Cost
Name
State University New York Stony Brook
Department
Type
Schools of Medicine
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
Barnett, R L; Ruffini, L; Hart, D et al. (1994) Mechanism of endothelin activation of phospholipase A2 in rat renal medullary interstitial cells. Am J Physiol 266:F46-56
Friedlaender, M M; Jain, D; Ahmed, Z et al. (1993) Endothelin activation of phospholipase D: dual modulation by protein kinase C and Ca2+. Am J Physiol 264:F845-53
Nord, E P (1993) Renal actions of endothelin. Kidney Int 44:451-63
Barnett, R L; Ruffini, L; Ramsammy, L et al. (1993) Angiotensin-mediated phosphatidylcholine hydrolysis and protein kinase C activation in mesangial cells. Am J Physiol 265:C1100-8
Kraut, J A; Hart, D; Nord, E P (1992) Basolateral Na(+)-independent Cl(-)-HCO3- exchange in primary cultures of rat IMCD cells. Am J Physiol 263:F401-10
Wilkes, B M; Susin, M; Mento, P F et al. (1991) Localization of endothelin-like immunoreactivity in rat kidneys. Am J Physiol 260:F913-20
Wilkes, B M; Ruston, A S; Mento, P et al. (1991) Characterization of endothelin 1 receptor and signal transduction mechanisms in rat medullary interstitial cells. Am J Physiol 260:F579-89
Sidell, N; Verity, M A; Nord, E P (1990) Menthol blocks dihydropyridine-insensitive Ca2+ channels and induces neurite outgrowth in human neuroblastoma cells. J Cell Physiol 142:410-9
Fineman, I; Hart, D; Nord, E P (1990) Intracellular pH regulates Na(+)-independent Cl(-)-base exchange in JTC-12 (proximal tubule) cells. Am J Physiol 258:F883-92
Aboolian, A; Vander Molen, M; Nord, E P (1989) Differential effects of phorbol esters on PGE2 and bradykinin-induced elevation of [Cai2+] in MDCK cells. Am J Physiol 256:F1135-43

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