SPHINGOSINE DERIVATIVES INHIBIT DEPOLARIZATION-EVOKED CALCIUM-ENTRY IN RAT GH(4)C(1) CELLS

Several investigations have suggested that sphingosine (SP) derivative s are potent inhibitors of protein kinase C. In GH(4)C(1) cells, prote in kinase C is a potent modulator of voltage-operated calcium channels (VOCCs). The aim of the present study was to investigate whether SP d erivatives could modify calcium entry via VOCCs. Using fura-2-loaded c ells and Ca-45(2+) flux studies, we show that several SPs potently and rapidly inhibit depolarization-evoked calcium entry in a dose-depende nt manner. The effect was not due to an enhanced efflux of calcium fro m the cells, as the depolarization-evoked entry of Ba2+ was inhibited by the SPs. A similar inhibition was observed with 1,2-dioctanoylglyce rol, an activator of sphingomyelinase in GH(3) cells. Phorbol myristat e acetate and 1-oleyl-2-acetylglycerol had only a modest inhibitory ef fect. Furthermore, whole cell patch-clamp experiments showed that sphi ngosinephosphorylcholine (SPC) potently attenuated calcium entry via V OCCs. In experiments using cells grown on coverslips, we found that th e inhibitory effect of SPC on calcium entry was reversible. The additi on of sphingomyelinase or hexanoyl ceramide, a cell-permeable ceramide , only modestly inhibited the depolarization-evoked entry of calcium, whereas arachidonic acid and phosphatidic acid had no effect. The SP m etabolite sphingosine-1-phosphate had no effect on the entry of calciu m. The results suggest that the effects of the SPs were probably not t he result of a conversion to ceramide or of the production of other li pid second messengers. In cells with down-regulated protein kinase C, SPC, SP, and 1,2-dioctanoylglycerol inhibited depolarization-evoked ca lcium entry, suggesting that the inhibition was independent of an acti on mediated via protein kinase C. The SPs per se did not induce any ch anges in intracellular free calcium, and they did not inhibit the TRH- evoked release of sequestered calcium in the cells. However, TRH-evoke d calcium entry was inhibited. The results suggest that SPs are potent ial regulators of calcium entry mediated by VOCCs GH(4)C(1) cells.