Sphingosine inhibits voltage-operated calcium channels in GH4C1 cells.
In the present study we investigated the mechanism of inhibitory action of sphingosine (SP) on voltage-activated calcium channels (VOCCs) in pituitary GH4C1 cells. Using the patch-clamp technique in the whole-cell mode, we show that SP inhibits Ba2+ currents (IBa) when 0.1 mM BAPTA is included in the patch pipette. However, when the BAPTA concentration 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 not 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 inhibition of the VOCCs. Furthermore, including GDPbetaS (guanyl-5'-yl thiophosphate) in the patch pipette did not alter the inhibitory effect of SP on the Ba2+ current, suggesting that SP did not modulate the VOCCs via a G protein-dependent pathway. Single-channel experiments with SP in the pipette, and experiments with excised outside-out patches, suggested that SP directly inhibited VOCCs. The main mechanism of action was a dose-dependent prolongation of the closed time of the channels. The results thus show that SP is a potent inhibitor of VOCCs in GH4C1 cells, and that calcium may be a cofactor in this inhibition.
Titievsky
A
A
Department of Biosciences, Division of Animal Physiology, University of Helsinki, Helsinki, and the Department of Biology, Abo Akademi University, Turku, Finland.
Titievskaya
I
I
Pasternack
M
M
Kaila
K
K
Törnquist
K
K
D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D015220:Calcium Channels / Q000502:physiology*; D002460:Cell Line; D015640:Ion Channel Gating; D018408:Patch-Clamp Techniques; D010902:Pituitary Gland / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D013110:Sphingosine / Q000494:pharmacology*
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D013110:Sphingosine