SPHINGOSYLPHOSPHORYLCHOLINE ACTIVATES AN AMILORIDE-INSENSITIVE NA-H+-EXCHANGE MECHANISM IN GH(4)C(1) CELLS()

The effect of sphingosylphosphorylcholine (SphPCho) on the intracellul ar pH (pH(i)) in GH(4)C(1) cells was investigated. SphPCho evoked a ve ry slow increase in basal pH(i). In cells acidified with nigericin, Sp hPCho induced a rapid alkalinization of the cells. The effect was inhi bited in a Na+-free buffer solution, but was insensitive to ethylisopr opyl amiloride, a potent inhibitor of Na+-H+ exchangers (NHE). Reverse transcription and PCR showed that the predominant isoform of the anti port expressed in GH(4)C(1) 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 cel ls treated with SphPCho gave an apparent K-m value of 15+/-2 mM for Na +. The V-max 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 pH, in Na+-containing and Na+-free buffer. The results show that SphPCho mall activate Na+-H+ exchange, and that this effect is me diated via an amiloride-insensitive exchange mechanism.