Abstract
Sphingosine-1-phosphate (SPP) has attracted much attention as a possible
second messenger controlling cell proliferation and motility and
as an intracellular Ca(2+)-releasing agent. Here, we present evidence
that SPP activates a G protein-coupled receptor in the plasma membrane
of various cells, leading to increase in cytoplasmic Ca2+ concentration
(Ca2+i), inhibition of adenylyl cyclase, and opening of G protein-regulated
potassium channels. In human enbryonic kidney (HEK) cells, SPP potently
(EC50, 2 nM) and rapidly increased Ca2+i in a pertussis toxin-sensitive
manner. Pertussis toxin-sensitive increase in Ca2+i was also observed
with sphingosylphosphorylcholine (EC50, 460 nM), whereas other sphingolipids,
including ceramide-1-phosphate, N-palmitoyl-sphingosine, psychosine,
and D-erythro-sphingosine at micromolar concentrations did not or
only marginally increased Ca2+i. Furthermore, SPP inhibited forskolin-stimulated
cAMP accumulation in HEK cells and increased binding of guanosine
5'3-O-(thio) triphosphate to HEK cell membranes. Rapid Ca2+i responses
were also observed in human transitional bladder carcinoma (J82)
cells, monkey COS-1 cells, mouse NIH 3T3 cells, Chinese hamster ovary
(CHO-K1) cells, and rat C6 glioma cells, whereas human HL-60 leukemia
cells and human erythroleukemia cells failed to respond to SPP. In
guinea pig atrial myocytes, SPP activated Gi protein-regulated inwardly
rectifying potassium channels. Activation of these channels occurred
strictly when SPP was applied at the extracellular face of atrial
myocyte plasma membrane as measured in cell-attached and inside-out
patch clamp current recordings. We conclude that SPP, in addition
to its proposed direct action on intracellular Ca2+ stores, interacts
with a high affinity Gi protein-coupled receptor in the plasma membrane
of apparently many different cell types.
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