Sphingosine differentially inhibits activation of the Na+/H+ exchanger by phorbol esters and growth factors.
The role of protein kinase C in activation of the plasma membrane Na+/H+ exchanger was studied in cultured vascular smooth muscle cells. The basic lipid, sphingosine, was used to block enzymatic activity of protein kinase C. Na+/H+ exchange was activated by phorbol 12-myristate 13-acetate (PMA), diacylglycerols, platelet-derived growth factor (PDGF), thrombin, or by osmotically-induced cell shrinkage. Intracellular pH and Na+/H+ exchange activity were measured using the intracellular pH indicator, 2',7'-bis(carboxyethyl)-5(6) carboxyfluorescein. Acting alone, both crude sphingosine and pure, synthetic C18 D-(+)-erythro-sphingosine raised pHi in a dose-dependent manner (from 6.95 +/- 0.02 to 7.19 +/- 0.09 over 10 min for 10 microM sphingosine). This alkalinization was not due to Na+/H+ exchange as it was not altered by t-butylamiloride (50 microM) nor by replacement of the assay medium with a Na(+)-free solution. Sphingosine-induced alkalinization did not require protein kinase C activity, since it was fully intact in protein kinase C-depleted cells. It was also not due to a detergent action of sphingosine on the cell membrane, since both ionic and non-ionic detergents caused cell acidification. Rather, alkalinization induced by sphingosine appeared to be due to cellular uptake of NH3 groups since N-acetylsphingosine showed no alkalinization. After the initial cell alkalinization, cellular uptake of [3H]sphingosine continued slowly for up to 24 h. The ability of PMA or dioctanoylglycerol to activate Na+/H+ exchange fell to 20% of control after 24 h of sphingosine exposure. At all times, C11 and N-acetylsphingosine failed to block PMA-induced activation of the exchanger. Activation of the Na+/H+ exchanger by sucrose, which does not depend on protein kinase C activity, was unaffected by sphingosine. Activation of Na+/H+ exchange by thrombin and PDGF was partially inhibited by 30 and 20%, respectively. These data indicate that both thrombin and PDGF activate Na+/H+ exchange by pathway(s) that are primarily independent of protein kinase C.
Lowe
J H
JH
Division of Nephrology, University of California, San Francisco 94143.
Huang
C L
CL
Ives
H E
HE
D000818:Animals; D000831:Animals, Newborn; D001692:Biological Transport; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002478:Cells, Cultured; D003902:Detergents / Q000494:pharmacology; D004075:Diglycerides / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D051381:Rats; D017923:Sodium-Hydrogen Exchangers; D013110:Sphingosine / Q000378:metabolism / Q000494:pharmacology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013917:Thrombin / Q000494:pharmacology*
D002352:Carrier Proteins; D003902:Detergents; D004075:Diglycerides; D010982:Platelet-Derived Growth Factor; D017923:Sodium-Hydrogen Exchangers; C044295:1,2-dioctanoylglycerol; D015240:Phorbol 12,13-Dibutyrate; D013917:Thrombin; D013110:Sphingosine; D013755:Tetradecanoylphorbol Acetate