UC San Diego

The lysophospholipid sphingosine 1-phosphate (S1P) has been known for many years to induce cellular changes such as proliferation, survival, migration, and contraction. With the discovery of a family of G-protein coupled receptors that use S1P as their ligand, the fundamental mechanism by which S1P signals cellular responses has been clarified. S1P has been shown to protect cardiomyocytes from death induced by stresses, including hypoxia, and addition of S1P to isolated perfused hearts limits damage induced by ischemia reperfusion. It is not known, however, whether S1P actually serves as a physiological regulator of cardiomyocyte survival in vivo. The studies presented here used S1P receptor knockout mice to ask whether activation of S1P receptors protects the heart from in vivo ischemia reperfusion injury. Following brief arterial occlusion and reperfusion, myocardial infarction develops in WT mice, and to the same extent in mice lacking either S1P₂ or S1P₃ receptors. In contrast, in S1P₂,₃ receptor double knockout hearts, infarct size was significantly increased. Ischemia reperfusion leads to activation of MAP kinases and this was not altered in the S1P₂,₃ receptor double knockout heart. Akt activation, however, was significantly decreased in the S1P₂,₃ receptor double knockout, but not in either of the single S1P₂ or S1P₃ receptor knockouts, correlating loss of Akt activation with enlarged infarct size. Thus S1P released during in vivo ischemia reperfusion protects the heart through combined effects on S1P₂ and S1P₃ receptors and Akt activation. We subsequently analyzed S1P-mediated signaling pathways in isolated adult mouse ventricular myocytes. S1P activates Akt and MAP kinases downstream of S1P₂ and S1P₃ receptors, and these responses do not occur when only the S1P₁ receptor remains or when it is selectively activated. However the S1P₁ receptor is present in S1P₂,₃ receptor knockout mice and can inhibit isoproterenol-stimulated cAMP accumulation. Subsequent cell fractionation studies showed that the S1P₁ receptor is localized to caveolae which are also enriched for adenylyl cylase. Thus we propose that localization of the S1P₁ receptor in caveolae enables it to inhibit adenlyly cylase while precluding it from activating Akt or MAP kinases, although all of these responses are regulated through coupling to Gi. Conversely, localization of S1P₂ and S1P₃ receptors outside of caveolae prevent these receptors from accessing adenylyl cylase while allowing them to access the upstream activators of Akt and MAP kinases. Thus localization of S1P receptors is a critical factor in conferring their selectivity for downstream signaling pathways