A novel function of sphingosine‐1‐phosphate to activate a non‐selective cation channel in human endothelial cells

The Ca2+ entry pathway activated by sphingosine-1-phosphate (S1P) was examined in primary cultured vascular endothelial cells dispersed from human umbilical vein (HUVECs) by measuring intracellular Ca2+ concentration ([Ca2+]i), whole-cell membrane currents and single channel activity. 2. Application of S1P to HUVECs induced a slowly developing, sustained increase in [Ca2+]i. When Ca2+ was absent from the bathing solution, no S1P-induced changes in [Ca2+]i were observed. Tert-butylhydroquinone (BHQ), an inhibitor of Ca2+ pumps in endoplasmic reticulum, and histamine induced a transient elevation of [Ca2+]i in HUVECs. 3. Pretreatment of HUVECs with 100 ng x ml(-1) pertussis toxin (PTX) for 15 h almost abolished the S1P effect on [Ca2+]i and reduced the histamine effect to 40% of the control. The BHQ-induced elevation of [Ca2+]i was insensitive to PTX. 4. When whole-cell membrane currents were recorded using the amphotericin B-perforated-patch clamp technique while monitoring [Ca2+]i, application of S1P induced a tiny inward current (I(S1P)) which was followed by the elevation of [Ca2+]i. I(S1P) reversed at +20.0 +/- 2.7 mV under these experimental conditions. 5. When S1P was included in the pipette solution in the excised inside-out patch clamp configuration, single channel activity with a conductance of 17 pS was activated. This channel activity depended on the presence of intracellular GTP. 6. In summary, these results show that S1P has a novel effect in mammalian cardiovascular endothelium to activate a non-selective cation (NSC) channel in a GTP-dependent manner via a PTX-sensitive G-protein. This S1P-sensitive NSC channel acts as a Ca2+ entry pathway in endothelium.