Rapid replenishment of sphingomyelin in the plasma membrane upon degradation by sphingomyelinase in NIH3T3 cells overexpressing the phosphatidylinositol transfer protein beta

In order to study the in vivo function of the phosphatidylinositol transfer protein beta (PI-TP beta), mouse NIH3T3 fibroblasts were transfected with cDNA encoding mouse PI-TP beta. Two stable cell lines were isolated (SPI be ta 2 and SPI beta 8) in which the levels of PI-TP beta were increased 16- a nd 11-fold respectively. The doubling time of the SPI beta cells was about 1.7 times that of the wild-type (wt) cells. Because PI-TP beta expresses tr ansfer activity towards sphingomyelin (SM) in vitro, the SM metabolism of t he over-expressors was investigated. By measuring the incorporation of [met hyl-H-3]choline chloride in SM and phosphatidylcholine (PtdCho), it was sho wn that the rate of de novo SM and PtdCho synthesis was similar in transfec ted and wt cells. We also determined the ability of the cells to resynthesi ze SM from ceramide produced in the plasma membrane by the action of bacter ial sphingomyelinase (bSMase). In these experiments the cells were labelled to equilibrium (60 h) with [H-3]choline. At relatively low bSMase concentr ations (50 munits/ml), 50 % of [H-3]SM in wt NIH3T3 cells was degraded, whe reas the levels of [H-3]SM in SPI beta cells appeared to be unaffected. Sin ce the release of [H-3]choline phosphate into the medium was comparable for both wt NIH3T3 and SPI beta cells, these results strongly suggest that bre akdown of SM in SPI beta cells was masked by rapid resynthesis of SM from t he ceramide formed. By increasing the bSMase concentrations to 200 munits/m l, a 50 % decrease in the level of [H-3]SM in SPI beta cells was attained. During a recovery period of 6 h (in the absence of bSMase) the resynthesis of SM was found to be much more pronounced in these SPI beta cells than in 50 % [H-3]SM-depleted wt NIH3T3 cells. After 6 h of recovery about 50% of t he resynthesized SM in the SPI beta cells was available for a second hydrol ysis by bSMase. When monensin was present during the recovery period, the r esynthesis of SM in bSMase-treated SPI beta cells was not affected. However , under these conditions 100 % of the resynthesized SM was available for hy drolysis. On the basis of these results we propose that, under conditions w here ceramide is formed in the plasma membrane, PI-TP beta plays an importa nt role in restoring the steady-state levels of SM.