Sphingosine differentially inhibits activation of the Na+/H+ exchanger by phorbol esters and growth factors

John H N Lowe, Chou Long Huang, Harlan E. Ives

Research output: Contribution to journalArticle

28 Scopus citations

Abstract

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 pH(i), in a dose-dependent manner (from 6.95 ± 0.02 to 7.19 ± 0.09 over 10 min for 10 μM sphingosine). This alkalinization was not due to Na+/H+ exchange as it was not altered by t-butylamiloride (50 μM) 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 ty, 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.

Original languageEnglish (US)
Pages (from-to)7188-7194
Number of pages7
JournalJournal of Biological Chemistry
Volume265
Issue number13
StatePublished - 1990

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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