Sphingosine 1-phosphate increases an intracellular Ca(2+) concentration via S1P3 receptor in cultured vascular smooth muscle cells.
OBJECTIVE
We investigated the effect of sphingosine 1-phosphate (S1P) on intracellular Ca(2+) dynamics in rat vascular smooth muscle cells (VSMCs).


METHODS
Intracellular Ca(2+) concentration ([Ca(2+) ]i) was determined using a fluorescence dye fura-2/AM. Small interfering RNAs (siRNA) were transfected into VSMCs to deplete the expression of S1P2 and S1P3 receptors.


RESULTS
S1P induced a rapid and transient elevation in [Ca(2+) ]i, which was maximal 1 min after the stimulation, followed by a sustained increase. When extracellular Ca(2+) was removed, a decrease in resting level and a small and transient increase in [Ca(2+) ]i by S1P stimulation were observed. siRNA targeted for the S1P3 receptor almost completely inhibited the S1P-induced increase in [Ca(2+) ]i. The rapid and transient increase in [Ca(2+) ]i was significantly inhibited by diltiazem at a high concentration. Pertussis toxin and a phospholipase C (PLC) inhibitor inhibited the S1P-induced increase in [Ca(2+) ]i regardless of the presence of extracellular Ca(2+) . Furthermore, S1P activated store-operated and receptor-operated Ca(2+) entry.


CONCLUSIONS
These results suggest that S1P increases [Ca(2+) ]i via the S1P3 receptor by inducing an influx of extracellular Ca(2+) partially through the voltage-dependent Ca(2+) channels, as well as by mobilizing Ca(2+) from its intracellular stores. S1P3 receptor-coupled Gi/o protein and PLC activation mediate the mechanisms.
Department of Pharmacological Sciences, School of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido, Japan.
Kazumi
K
Fujii
Takuji
T
Machida
Kenji
K
Iizuka
Masahiko
M
Hirafuji
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000502:physiology; D002478:Cells, Cultured; D000068876:Fingolimod Hydrochloride; D008246:Lysophospholipids / Q000494:pharmacology*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D011409:Propylene Glycols / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D049349:Receptors, Lysosphingolipid / Q000502:physiology*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology
D020746:Calcium Channels, L-Type; D008246:Lysophospholipids; D011409:Propylene Glycols; D049349:Receptors, Lysosphingolipid; C060506:sphingosine 1-phosphate; D000068876:Fingolimod Hydrochloride; D013110:Sphingosine; D002118:Calcium
L-type voltage-dependent Ca2+ channel; S1P3 receptor; intracellular Ca2+ concentration; sphingosine 1-phosphate; vascular smooth muscle cells