The aim of these studies is to examine the hormonal regulation of phosphoinositide metabolism in rat hepatocytes and other cells. In liver, alpha-1 catecholamines and vasopressin activate glycogen phosphorylase secondary to an elevation of intracellular calcium and diacylglycerol. The rise in calcium is secondary to formation of inositol 1,4,5 trisphosphate derived from phospholipase C-induced breakdown of phosphatidylinositol 4,5- bisphosphate (PIP2) as is diacylglycerol. It is now clear that phospholipase C activity is regulated by guanine nucleotides. This project focuses on the role of guanine nucleotides in hormone induced breakdown of phosphatidyl-inositol 4,5- bisphosphate. Our long-term goal is isolation of the membrane bound phospholipase C responsible for breakdown of phosphoinositides. The requirement for guanine nucleotides suggests that a guanine nucleotide binding protein, tentatively called Np, may be involved in coupling of hormone-receptor complexes to phospholipase C. Phospholipase C activity will be examined using tritiated PIP2. We will attempt to label Np with GTP analogues and find a toxin that will ADP-ribosylate it, with the eventual aim of isolating this putative guanine nucleotide binding protein. Investigations will continue on the regulation by agonists that stimulate polyphosphoinositide breakdown, of high affinity GTPase activity in hepatocytes and other cell types. We intend to establish the identity of the guanine nucleotide binding protein involved. The inhibition by vasopressin of hepatocyte (Ca2+ Mg2+) ATPase activity will be further investigated in an attempt to elucidate the link, if any, between this effect and phosphoinositide breakdown. This project is primarily a metabolic investigation using biochemical techniques. The studies are done in experimental animals and should provide significant insights into various diseases of metabolism and especially diabetes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK037004-04
Application #
3235638
Study Section
Metabolism Study Section (MET)
Project Start
1985-09-01
Project End
1991-12-31
Budget Start
1989-01-01
Budget End
1989-12-31
Support Year
4
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Tennessee Health Science Center
Department
Type
Schools of Medicine
DUNS #
941884009
City
Memphis
State
TN
Country
United States
Zip Code
38163
Claro, E; Sarri, E; Picatoste, F (1995) Measurement of phospholipase C activity in brain membranes. Methods Mol Biol 41:177-88
Myles, M E; Fain, J N (1994) Carbachol, but not norepinephrine, NMDA, ionomycin, ouabain, or phorbol myristate acetate, increases inositol 1,3,4,5-tetrakisphosphate accumulation in rat brain cortical slices. J Neurochem 62:2333-9
Salles, J; Wallace, M A; Fain, J N (1993) Differential effects of alkylating agents on the multiple muscarinic receptor subtypes linked to activation of phospholipase C by carbachol in rat brain cortical membranes. J Pharmacol Exp Ther 264:521-9
Salles, J; Wallace, M A; Fain, J N (1993) Modulation of the phospholipase C activity in rat brain cortical membranes by simultaneous activation of distinct monoaminergic and cholinergic muscarinic receptors. Brain Res Mol Brain Res 20:111-7
Wallace, M A; Claro, E (1993) Transmembrane signaling through phospholipase C in human cortical membranes. Neurochem Res 18:139-45
Claro, E; Fain, J N; Picatoste, F (1993) Noradrenaline stimulation unbalances the phosphoinositide cycle in rat cerebral cortical slices. J Neurochem 60:2078-86
Claro, E; Wallace, M A; Fain, J N (1992) Concerted CMP-dependent [3H]inositol labeling of phosphoinositides and agonist activation of phospholipase C in rat brain cortical membranes. J Neurochem 58:2155-61
Llahi, S; Fain, J N (1992) Alpha 1-adrenergic receptor-mediated activation of phospholipase D in rat cerebral cortex. J Biol Chem 267:3679-85
Pittner, R A; Fain, J N (1992) Ethanol is a potent stimulator of phosphatidylcholine breakdown in cultured rat hepatocytes. Biochim Biophys Acta 1133:316-20
Llahi, S; Claro, E; Fain, J N (1992) Quisqualate-stimulated phosphatidylinositol breakdown in rat cerebellar membranes. J Neurochem 58:714-21

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