Comparison of sphingosine 1-phosphate-induced intracellular signaling pathways in vascular smooth muscles: differential role in vasoconstriction.

Sphingosine 1-phosphate (S1P), a lipid released from activated platelets, influences physiological processes in the cardiovascular system via activation of the endothelial differentiation gene (EDG/S1P) family of 7 transmembrane G protein-coupled receptors. In cultured vascular smooth muscle (VSM) cells, S1P signaling has been shown to stimulate proliferative responses; however, its role in vasoconstriction has not been examined. In the present study, the effects of S1P and EDG/S1P receptor expression were determined in rat VSM from cerebral artery and aorta. S1P induced constriction of cerebral artery, which was partly dependent on activation of p160(ROCK) (Rho-kinase). S1P also induced activation of RhoA in cerebral artery with a similar time course to contraction. In aorta, S1P did not produce a constriction or RhoA activation. In VSM myocytes from cerebral arteries, stimulation with S1P gives rise to a global increase in [Ca2+]i, initially generated via Ca2+ release from the sarcoplasmic reticulum by an inositol 1,4,5-trisphosphate-dependent pathway. In aorta VSM, a small increase in [Ca2+]i was observed after stimulation at higher concentrations of S1P. S1P induced activation of p42/p44(mapk) in aorta and cerebral artery VSM. Subtype-specific S1P receptor antibodies revealed that the expression of S1P3/EDG-3 and S1P2/EDG-5 receptors is 4-fold higher in cerebral artery compared with aorta. S1P(1)/EDG-1 receptor expression was similar in both types of VSM. Therefore, the ability of S1P to act as a vasoactive mediator is dependent on the activation of associated signaling pathways and may vary in different VSM. This differential signaling may be related to the expression of S1P receptor subtypes.

Sphingosine 1-phosphate induces CREB activation in rat cerebral artery via a protein kinase C-mediated inhibition of voltage-gated K+ channels.

Sphingosine 1-phosphate (S1P) is a potential mitogenic stimulus for vascular smooth muscle. S1P promotes an increase in the intracellular calcium concentration ([Ca(2+)](i)) in cerebral arteries, however S1P effects on regulation of gene expression are not known. Activation of the Ca(2+)-dependent transcription factor, cAMP response element-binding protein (CREB), is associated with smooth muscle proliferation. The aim of this study was to examine the Ca(2+)-dependent mechanisms involved in S1P-induced CREB activation in cerebral artery. Western blotting and immunofluorescence with a phospho-CREB antibody were used to detect CREB activation in Sprague-Dawley rat cerebral arteries. Whole-cell patch clamp recording and single cell imaging of [Ca(2+)](i) were performed on freshly isolated cerebral artery myocytes. S1P increased activation of CREB in the nucleus of cerebral arteries. This activation was mediated by Ca(2+)/calmodulin-dependent protein kinase and was dependent on an increase in [Ca(2+)](i) via two mechanisms: (i) intracellular Ca(2+) release via an inositol 1,4,5-trisphosphate (InsP(3))-dependent pathway and (ii) Ca(2+) entry through voltage-dependent Ca(2+) channels (VDCC). Activation of the VDCC occurred through S1P-induced inhibition (approximately 50%) of the voltage-gated potassium (K(+)) current. This inhibition was via a protein kinase C-mediated pathway resulting in tyrosine phosphorylation of at least one isoform of the Kv channel (Kv 1.2). These results demonstrate that S1P can activate the transcription factor CREB through different Ca(2+)-dependent pathways including intracellular Ca(2+) release and inhibition of voltage-gated K(+) channels leading to Ca(2+) influx. Our findings suggest a potential role for S1P in regulation of gene expression in vascular smooth muscle.