Sphingosine kinase regulates voltage operated calcium channels in GH4C1 rat pituitary cells

We have previously shown that sphingosine inhibits depolarisation-induced calcium influx through voltage-operated calcium channels (VOCCs) in GH4C1 cells, whereas sphingosine-1-phosphate (S1P) does not. In the present study we investigated whether sphingosine kinase modulates VOCC activity in GH4C1 cells by removing inhibitory sphingosine. Sphingosine and the structurally similar sphingosine kinase inhibitor dimethylsphingosine (DMS) both rapidly attenuated the calcium influx evoked by depolarisation. The inhibitory effect declined over time to a greater extent in cells treated with sphingosine than in cells treated with DMS, indicating that sphingosine is being metabolised more rapidly. When the specific sphingosine kinase inhibitor 2-(p-Hydroxyanilino)-4-(p-chlorophenyl) thiazole (SKi) was added to the cells after depolarisation there was likewise a reduction of the calcium response. This inhibitory effect was slow and reached a plateau about 3 min after application. In contrast, the sphingosine-mediated inhibition was immediate, suggesting that the SKi-induced inhibition was due to build-up of cellular sphingosine. In experiments on cells overexpressing sphingosine kinase, the inhibitory effect of sphingosine was reversed faster than in control cells. The effect was not due to the produced S1P, since S1P did not have any effect on VOCCs even at concentrations as high as 50 μM. In patch-clamp experiments the calcium entry through VOCCs was attenuated in GH4C1 cells overexpressing a kinase-dead sphingosine kinase, compared with cells overexpressing the wild type sphingosine kinase. In addition, in cells treated with SKi the calcium entry through VOCCs was attenuated compared with control cells. Our results provide compelling evidence that sphingosine kinase regulates the function of voltage-operated calcium channels in GH4C1 cells, not through its catalytic product, but by removal of the substrate sphingosine.