Sphingosylphosphorylcholine activates an amiloride-insensitive Na+-H+-exchange mechanism in GH4C1 cells.
The effect of sphingosylphosphorylcholine (SphPCho) on the intracellular pH (pHi) in GH4C1 cells was investigated. SphPCho evoked a very slow increase in basal pHi. In cells acidified with nigericin, SphPCho induced a rapid alkalinization of the cells. The effect was inhibited in a Na+-free buffer solution, but was insensitive to ethylisopropyl amiloride, a potent inhibitor of Na+-H+ exchangers (NHE). Reverse transcription and PCR showed that the predominant isoform of the antiport expressed in GH4C1 cells is NHE-1. The rate of alkalinization after stimulation with propionate, and after addition of Na+ to cells acidified with NH4Cl, was enhanced in cells treated with SphPCho. The initial rate of alkalinization after addition of Na+ to acidified cells treated with SphPCho gave an apparent Km value of 15 +/- 2 mM for Na+. The Vmax value was 9 +/- 2 mM H+/min. The effect was insensitive to ouabain, staurosporine and bafilomycin A. However, the SphPCho-evoked alkalinization was abolished in cells treated with 2-deoxy-D-glucose. The effect was not due to the charge of the molecule, as stearylamine increased pHi in Na+-containing and Na+-free buffer. The results show that SphPCho may activate Na+-H+ exchange, and that this effect is mediated via an amiloride-insensitive exchange mechanism.
Törnquist
K
K
Department of Biosciences, University of Helsinki, Finland.
Woodside
M
M
Grinstein
S
S
D000584:Amiloride / Q000494:pharmacology*; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002478:Cells, Cultured; D003847:Deoxyglucose / Q000494:pharmacology; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D018942:Macrolides; D009550:Nigericin / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000494:pharmacology; D010902:Pituitary Gland / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D051381:Rats; D012964:Sodium / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D019311:Staurosporine / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D018942:Macrolides; D017923:Sodium-Hydrogen Exchangers; C005356:sphingosine phosphorylcholine; D010767:Phosphorylcholine; C057620:bafilomycin A; D010042:Ouabain; D000584:Amiloride; D006859:Hydrogen; D003847:Deoxyglucose; D012964:Sodium; D019311:Staurosporine; D013110:Sphingosine; D009550:Nigericin