S1P, a lipid released from activated platelets, influences physiological processes via activation of the endothelial differentiation gene (EDG) family of G protein-coupled receptors. The potential role of S1P in vasoconstriction has not been examined. The aim of this study was to determine the S1P-induced intracellular signalling events in rat aorta (Ao) and cerebral artery (CA) that may contribute to vasoconstriction. Freshly dispersed CA and Ao smooth muscle cells were isolated by enzymatic digestion from humanely killed rats and loaded with fura-2. Fluorescence Ca²⁺ imaging was carried out using a CCD camera. RhoA activation was assessed using a GST-rhotekin ‘pull-down’ assay which binds to GTP bound (i.e. active) RhoA. Following isolation of GST-rhotekin, proteins were separated using SDS polyacrylamide gel electrophoresis and subjected to immunoblotting with an anti-RhoA antibody. Results are means ± S.E.M.

Transient intracellular Ca²⁺ increases were induced by 1 mM S1P in dispersed cells from CA (mean peak amplitude, 0.45 ± 0.01 ratio units, n = 109). Stimulation of Ao cells with S1P produced only a small Ca²⁺ increase (maximum 30 mM S1P, 0.13 ± 0.03 ratio units, n = 32). In both smooth muscle types, 100 nM ET-1 evoked similar transient Ca²⁺ increases. Pre-incubation with either thapsigargin or the phospholipase C inhibitor, U73122, almost completely abolished the S1P-induced Ca²⁺ increases evoked by 1 mM S1P in CA cells. In CA rings, S1P stimulation resulted in a sustained rise in tension and was inhibited by addition of 10 mM Y27632, a rho-kinase inhibitor. Peak S1P-induced contraction was 35 ± 5 % (n = 5) of the ET-1-induced contraction. In Ao rings no change in tension was observed following incubation with S1P. RhoA was activated by S1P in a time course compatible with a role in the S1P-induced contraction of CA. Following 5 min stimulation with S1P, the proportion of GTP-RhoA compared with total RhoA protein increased 4.2 ± 0.6-fold (n = 3) in isolated CA. S1P did not activate RhoA in isolated Ao. Immunoblots of homogenates from CA and Ao stained with specific antibodies revealed that EDG-1 receptor expression was similar in Ao and CA. EDG-3 receptor expression was 4.4 ± 0.2-fold (n = 4) greater and EDG-5 receptor expression 4.2 ± 0.8-fold (n = 4) greater in CA than in Ao.

In conclusion, S1P can activate intracellular signalling pathways compatible with its potential role as a vasoconstrictor. These pathways are not activated in all smooth muscle types, which may correlate with the differential expression of EDG receptor subtypes.

This work was supported by The Wellcome Trust.

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