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

1 The Ca2+ entry pathway activated by sphingosine-1-phosphate (S1P) was exa mined in primary cultured vascular endothelial cells dispersed from human u mbilical 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 incr ease in [Ca2+](i). When Ca2+, was absent from the bathing solution, no SIP- induced changes in [Ca2+], were observed. Tert-butylhydroquinone (BHQ), an inhibitor of Ca2+ pumps in endoplasmic reticulum, and histamine induced a t ransient, elevation of [Ca2+](i) in HUVECS. 3. Pretreatment of HUVECs with 100 ng ml(-1)pertussis toxin (PTX) for 15 h almost abolished the SIP effect on [Ca2+](i) and reduced the histamine effe ct to 40% of the control. The BHQ-induced elevation of [Ca2+](i) was insens itive to PTX. 4. When whole-cell membrane currents were recorded using the amphotericin B -perforated-patch clamp technique while monitoring [Ca2+](i), application o f SIP induced a tiny inward current (I-S1P) which was followed by the eleva tion of [Ca2+](i). I-S1P reversed at +20.0 +/- 2.7 mV under these experimen tal 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 intrac ellular 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-sensitiv e NSC channel acts as a Ca2+ entry pathway in endothelium.