SPHINGOSINE INHIBITS VOLTAGE-OPERATED CALCIUM CHANNELS IN GH(4)C(1) CELLS

In the present study we investigated the mechanism of inhibitory actio n of sphingosine (SP) on voltage-activated calcium channels (VOCCs) in pituitary GH(4)C(1) cells. Using the patch-clamp technique in the who le-cell mode, we show that SP inhibits Ba2+ currents (I-Ba) when 0.1 m M BAPTA is included in the patch pipette. However, when the BAPTA conc entration was raised to 1-10 mm, SP was without a significant effect. The effect of SP was apparently not mediated via a kinase, as it was n ot inhibited by staurosporine. By using the double-pulse protocol (to release possible functional inhibition of the VOCCs by G proteins), we observed that G proteins apparently evoked very little functional inh ibition of the VOCCs. Furthermore, including GDP beta S (guanyl-5'-yl thiophosphate) in the patch pipette did not alter the inhibitory effec t of SP on the Ba2+ current, suggesting that SP did not modulate the V OCCs via a G protein-dependent pathway. Single-channel experiments wit h SP in the pipette, and experiments with excised outside-out patches, suggested that SP directly inhibited VOCCs. The main mechanism of act ion was a dose-dependent prolongation of the closed time of the channe ls. The results thus show that SP is a potent inhibitor of VOCCs in GH (4)C(1) cells, and that calcium may be a cofactor in this inhibition.