The mechanism by which mammalian cells regulate sodium, potassium, calcium and pH in response to hormones and other environmental factors has been extensively studied at the physiological level but is poorly understood at the biochemical level. The long term goal of this grant has been to understand the biochemical mechanism by which cation transport across the plasma membrane of mammalian cells is regulated by purifying the transport systems and regulatory systems to homogeneity. The two systems which have been the focus of this grant in the past few years and which will continue to be the focus are the (Na,K) ATPase, the enzyme primarily responsible for establishing cation gradients in most cells and an extracellullar ATP receptor which rapidly abolishes plasma membrane cation gradients. The study of the (Na,K)ATPase will focus on the mechanism by which expression of a novel """"""""ouabain resistance gene"""""""" results in a totally ouabain insensitive (Na,K)ATPase in the plasma membrane of green monkey CV-1 fibroblasts. This gene was isolated by the ability to confer ouabain resistance on green monkey fibroblasts upon transfection of genomic DNA from murine fibroblasts. A 6.5 kbase fragment of murine genomic DNA has cloned into a plasmid and shown to confer ouabain resistance in recipients cells. The gene has been partially sequenced and the predicted coding region has no homology to the alpha or beta subunits of the (Na,K)ATPase. The goal of this grant is to complete the gene sequence, isolate a cDNA clone, verify that expression of the cDNA clone also confers ouabain resistance, obtain antibodies against the expressed protein and determine the function of the gene product. The physiological relevance of this gene is not understood but possible functions include 1) the ability to confer tissue specific resistance to circulating inhibitors of the pump, 2) the ability to alter cation transport stoichiometries in specific tissues. Since the (Na,K)ATPase is almost always defined as the ouabain sensitive component of cation transport or ATPase activity the completely ouabain resistant form of the pump found in cells expressing the ouabain resistance gene could have easily been overlooked in past studies. A second goal is to isolate the extracellular receptor for ATP which regulates plasma membrane sodium, calcium and potassium fluxes. Extracellular ATP is much more effective than any other hormone or growth regulator in stimulating plasma membrane cation fluxes in Friend erythroleukemia cells, murine T cells and rat parotid cells. Although there are reports that extracellular ATP is a membrane permeabilizing agent in some cells, this is clearly not so in the cells listed above: small organic anions do not become permeant. A 50% maximal effect is observed at 10 uM ATP4- and it appears likely that ATP4- which is sorted at very high concentration (>100 mM) in many secretory vesicles is acting locally as a hormone in synergy with the other hormones and/or transmitter with which it is stored.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM036133-07
Application #
3289681
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1985-05-01
Project End
1994-01-31
Budget Start
1991-02-01
Budget End
1992-01-31
Support Year
7
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Tufts University
Department
Type
Schools of Medicine
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02111
Soltoff, S P; McMillian, M K; Talamo, B R et al. (1993) Blockade of ATP binding site of P2 purinoceptors in rat parotid acinar cells by isothiocyanate compounds. Biochem Pharmacol 45:1936-40
Soltoff, S P; McMillian, M K; Talamo, B R (1992) ATP activates a cation-permeable pathway in rat parotid acinar cells. Am J Physiol 262:C934-40
Soltoff, S P; McMillian, M K; Cragoe Jr, E J et al. (1990) Effects of extracellular ATP on ion transport systems and [Ca2+]i in rat parotid acinar cells. Comparison with the muscarinic agonist carbachol. J Gen Physiol 95:319-46
Soltoff, S P; McMillian, M K; Cantley, L C et al. (1989) Effects of muscarinic, alpha-adrenergic, and substance P agonists and ionomycin on ion transport mechanisms in the rat parotid acinar cell. The dependence of ion transport on intracellular calcium. J Gen Physiol 93:285-319
Soltoff, S P; McMillian, M K; Talamo, B R (1989) Coomassie Brilliant Blue G is a more potent antagonist of P2 purinergic responses than Reactive Blue 2 (Cibacron Blue 3GA) in rat parotid acinar cells. Biochem Biophys Res Commun 165:1279-85
Soltoff, S P; Cantley, L C (1988) Mitogens and ion fluxes. Annu Rev Physiol 50:207-23
Emanuel, J R; Schulz, J; Zhou, X M et al. (1988) Expression of an ouabain-resistant Na,K-ATPase in CV-1 cells after transfection with a cDNA encoding the rat Na,K-ATPase alpha 1 subunit. J Biol Chem 263:7726-33
Schulz, J T; Cantley, L C (1988) CV-1 cell recipients of the mouse ouabain resistance gene express a ouabain-insensitive Na,K-ATPase after growth in cardioactive steroids. J Biol Chem 263:624-32
Fleischman, L F; Cantley, L (1988) Cell cycle dependence of inositol phosphate levels in neuroblastoma cells. Am J Physiol 255:C531-5
McMillian, M K; Soltoff, S P; Lechleiter, J D et al. (1988) Extracellular ATP increases free cytosolic calcium in rat parotid acinar cells. Differences from phospholipase C-linked receptor agonists. Biochem J 255:291-300

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