Abstract

The lysosphingolipids sphingosine-1-phosphate (SPP) and sphingosylphosphorylcholine (SPPC) reportedly increase free cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in a variety of cell types, apparently by activating G protein-coupled plasma membrane receptors. We investigated whether and how sphingolipids modulate Ca²⁺ homeostasis in the insulinoma cell line RINm5F. The addition of SPPC and glucopsychosine (GPS) did not affect basal [Ca²⁺]_i but inhibited the KCl (30 mm)-induced increase in [Ca²⁺]_i in a pertussis toxin-insensitive and concentration-dependent manner (EC₅₀ ∼ 5 μm). Similar inhibitory effects were observed with dihydro-SPPC and psychosine, whereas SPP and variousN-acylated sphingolipids (at 10 μm each) had little or no effect on the KCl-induced [Ca²⁺]_i increase. Because in RINm5F cells the primary pathway for depolarization-induced [Ca²⁺]_i increase are L-type Ca²⁺ channels, we studied whether sphingolipids reduceL-type Ca²⁺ current (I_Ca.L). When added to the bath, GPS and SPPC, but not SPP (10 μmeach), rapidly reduced maximal I_Ca.L by ∼35%, similar to the α₂-adrenoceptor agonist clonidine (30 μm). However, when applied internally, GPS had no effect on I_Ca.L. When the electrode solution contained the stable GDP analog guanosine-5′-O-(2-thio)diphosphate (1 and 10 mm), the inhibitory effect of GPS was abolished. In conclusion, a novel cellular action of lysosphingolipids is observed in RINm5F cells (i.e., a guanine nucleotide-sensitive inhibition ofL-type Ca²⁺ currents). The pharmacological profile of this inhibition is unique and unlike any known lysosphingolipid receptor-mediated action.