The primary goal of this proposal is to determine how the plasma membrane (Na,K)-ATPase is regulated in friend virus transformed murine erythroleukemia cells. The secondary goals are to investigate other Na+ and Ca++ fluxes in plasma membranes from these cells. Friend cells may be grown indefinitely in suspension but undergo an apparently normal erythropoesis when treated with dimethyl sulfoxide (DMSO) or a variety of other agents. The interest in cation fluxes stems from the observation that ouabain (which specifically inhibits the (Na,K) ATPase from the outside of the cell) induced differentiation in some Friend cell lines and acts synergistically with other inducers to accelerate commitment in other cell lines. From studies of cation fluxes during the past two years we have developed a scheme to explain the early events essential in Frient cell commitment: 1) the inducer inhibits the (Na,K)ATPase 2) Cytoplasmic Na+ rises 3) Ca++ influx is accelerated probably via a Na+/Ca++ antiport and 4) Ca++ signals a series of events essential for commitment. The mechanism by which Ca++ acts is obscure but it may turn out to be a general signal for diferentiation in a variety of developing cells. While studying the plasma membranes from these cells we discovered a membrane bound kinase which specifically and stoichiometrically phosphorylates a threonine residue on the Alpha subunit of the (Na,K)ATPase. The Alpha subunit is also phosphorylated on a threonine residue in vivo and preliminary results suggest that the level of phosphorylation decreases with committment. The obvious implication that this kinase may be a mechanism for regulating the (Na,K)ATPase in a variety of cells in supported by our recent discovery of a similar (Na,K)ATPase specific kinase in plams membranes from shark rectal glan (a hormonally sensitive ion secreting tissue). Thus, initial efforts will focus on characterizing the kinase, determining whether it alters pumping by the (Na,K)ATPase, and determining how the kinase is controlled in vivo. It is hoped that this research will lead to an understanding of hormonal regulation of the (Na,K)ATPase and may also provide some understanding of how cells are maintained in a transformed state.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM036133-04
Application #
3289679
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1985-05-01
Project End
1989-01-31
Budget Start
1988-02-01
Budget End
1989-01-31
Support Year
4
Fiscal Year
1988
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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