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

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Abstract
In order to study the in vivo function of the phosphatidylinositol transfer protein beta (PI-TPbeta), mouse NIH3T3 fibroblasts were transfected with cDNA encoding mouse PI-TPbeta. Two stable cell lines were isolated (SPIbeta2 and SPIbeta8) in which the levels of PI-TPbeta were increased 16- and 11-fold respectively. The doubling time of the SPIbeta cells was about 1.7 times that of the wild-type (wt) cells. Because PI-TPbeta expresses transfer activity towards sphingomyelin (SM) in vitro, the SM metabolism of the overexpressors was investigated. By measuring the incorporation of [methyl-(3)H]choline chloride in SM and phosphatidylcholine (PtdCho), it was shown that the rate of de novo SM and PtdCho synthesis was similar in transfected and wt cells. We also determined the ability of the cells to resynthesize SM from ceramide produced in the plasma membrane by the action of bacterial sphingomyelinase (bSMase). In these experiments the cells were labelled to equilibrium (60 h) with [(3)H]choline. At relatively low bSMase concentrations (50 munits/ml), 50% of [(3)H]SM in wt NIH3T3 cells was degraded, whereas the levels of [(3)H]SM in SPIbeta cells appeared to be unaffected. Since the release of [(3)H]choline phosphate into the medium was comparable for both wt NIH3T3 and SPIbeta cells, these results strongly suggest that breakdown of SM in SPIbeta cells was masked by rapid resynthesis of SM from the ceramide formed. By increasing the bSMase concentrations to 200 munits/ml, a 50% decrease in the level of [(3)H]SM in SPIbeta 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 SPIbeta cells than in 50% [(3)H]SM-depleted wt NIH3T3 cells. After 6 h of recovery about 50% of the resynthesized SM in the SPIbeta cells was available for a second hydrolysis by bSMase. When monensin was present during the recovery period, the resynthesis of SM in bSMase-treated SPIbeta cells was not affected. However, under these conditions 100% of the resynthesized SM was available for hydrolysis. On the basis of these results we propose that, under conditions where ceramide is formed in the plasma membrane, PI-TPbeta plays an important role in restoring the steady-state levels of SM.

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