Protein phosphorylation during activation of Na+/H+ exchange by phorbol esters and by osmotic shrinking. Possible relation to cell pH and volume regulation.
In lymphocytes, the Na+/H+ antiport can be stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA) and by osmotic shrinking. Since TPA acts by stimulating protein kinase C, we undertook experiments to determine if protein phosphorylation also underlies the osmotic stimulation of the antiport. We found that at least one of the membrane polypeptides labeled in cells treated with TPA is also phosphorylated by hypertonic shrinking. In both instances phosphorylation is alkali labile and associated with serine and threonine residues. We tested the possibility that shrinking activates phospholipase C, thereby stimulating protein kinase C through release of diacylglycerol. No decrease in phosphatidylinositol 4,5-bisphosphate levels was detected in hypertonically treated cells. Moreover, the concentrations of inositol phosphates, including inositol trisphosphate, were not altered in shrunken cells. Thus, shrinking does not appear to activate phospholipase C. Whereas TPA induced intracellular redistribution of soluble protein kinase C, no such effect was detected in osmotically activated cells. It was concluded that osmotic stimulation of the Na+/H+ antiport is associated with activation of protein phosphorylation by a kinase that is similar, but not identical to protein kinase C. Experiments in Na+-free or amiloride-containing media indicate that phosphorylation is not a consequence of activation of the antiport.
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006982:Hypertonic Solutions; D066298:In Vitro Techniques; D008297:Male; D008563:Membrane Lipids / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D010704:Phorbols / Q000494:pharmacology*; D010743:Phospholipids / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D017923:Sodium-Hydrogen Exchangers; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013950:Thymus Gland / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
D002352:Carrier Proteins; D006982:Hypertonic Solutions; D008563:Membrane Lipids; D008565:Membrane Proteins; D010704:Phorbols; D010743:Phospholipids; D017923:Sodium-Hydrogen Exchangers; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate