Sphingosine inhibits phorbol 12-myristate 13-acetate-, but not serum-induced, activation of Na+/H+ exchange in mammalian cells.
Addition of serum to quiescent mammalian cells in culture initiates a series of events which culminates in DNA replication and cell division. One of the earliest events in this sequence of events is activation of Na+/H+ exchange, which can result in an increase in intracellular pH (pHin). The regulation of this change in activity is not known. Since treatment of 3T3 cells with activators of protein kinase C (kinase C) can result in an increased pHin, it has been hypothesized that serum stimulation of kinase C is responsible for activation of Na+/H+ exchange. Recently, sphingolipids have been discovered to inhibit kinase C both in vitro and in vivo. Therefore, we undertook the present study to ask whether or not inhibition of kinase C using sphingolipids prevents mitogen-induced alkalinization in 3T3 cells. Our results indicate that activators of kinase C stimulate Na+/H+ exchange in normal human fibroblasts (BoGi), but not in mouse embryo (3T3) cells. Addition of serum to BoGi cells, on top of saturating doses of phorbol 12-myristate 13-acetate (PMA), results in a further cytoplasmic alkalinization. Furthermore, sphingosine prevents the PMA-induced increase in pHin in BoGi cells, and phosphorylation of an 80 kDa protein in 3T3 cells, but not the serum-induced alkalinization in either BoGi or 3T3 cells. These data indicate that activation of kinase C does not participate in the physiological activation of Na+/H+ exchange in human fibroblasts or mouse embryo cells by serum.
Gillies
R J
RJ
Department of Biochemistry, Colorado State University, Ft. Collins 80523.
Martinez
R
R
Sneider
J M
JM
Hoyer
P B
PB
D000818:Animals; D001790:Blood Physiological Phenomena; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D004075:Diglycerides / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers; D013110:Sphingosine / Q000494:pharmacology*; D013755:Tetradecanoylphorbol Acetate / Q000037:antagonists & inhibitors / Q000494:pharmacology*
D002352:Carrier Proteins; D004075:Diglycerides; D017923:Sodium-Hydrogen Exchangers; C044295:1,2-dioctanoylglycerol; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D013110:Sphingosine; D013755:Tetradecanoylphorbol Acetate