Spontaneous spreading depolarizations are frequent after various forms of human brain injury such as ischemic or hemorrhagic stroke and trauma, and worsen the outcome. We have recently shown that supply-demand mismatch transients trigger spreading depolarizations in ischemic stroke. Here, we examined the mechanisms triggering recurrent spreading depolarization events for many days after subarachnoid hemorrhage. Despite large volumes of subarachnoid hemorrhage induced by cisternal injection of fresh arterial blood in rodents, electrophysiological recordings did not detect a single spreading depolarization for up to 72 h after subarachnoid hemorrhage. Cortical susceptibility to spreading depolarization, measured by direct electrical stimulation or topical KCl application, was suppressed after subarachnoid hemorrhage. Focal cerebral ischemia experimentally induced after subarachnoid hemorrhage revealed a biphasic change in the propensity to develop peri-infarct spreading depolarizations. Frequency of peri-infarct spreading depolarizations decreased at 12 h, increased at 72 h and normalized at 7 days after subarachnoid hemorrhage compared with sham controls. However, ischemic tissue and neurological outcomes were significantly worse after subarachnoid hemorrhage even when peri-infarct spreading depolarization frequency was reduced. Laser speckle flowmetry implicated cerebrovascular hemodynamic mechanisms worsening the outcome. Altogether, our data suggest that cerebral ischemia is required for spreading depolarizations to be triggered after subarachnoid hemorrhage, which then creates a vicious cycle leading to the delayed cerebral ischemia syndrome.

Sphingosine-1-phosphate (S1P) has been shown to modulate intracellular Ca(2+) through both G protein-coupled receptors and intracellular second messenger pathways. The precise mechanism by which S1P activates store-operated calcium entry (SOCE) in vascular smooth muscle cells (VSMCs) has not been fully characterized. Because sphingolipids and Ca(2+) modulate proliferation and constriction in VSMCs, characterizing the connection between S1P and SOCE may provide novel therapeutic targets for vascular diseases. We found that S1P triggered STIM1 puncta formation and SOCE in VSMCs. S1P-activated SOCE was inhibited by 2-aminoethoxydiphenyl borate (2-APB), diethylstilbestrol (DES), and gadolinium (Gd(3+)). SOCE was observed in VSMCs lacking either S1P(2) or S1P(3) receptors, suggesting that S1P acts via multiple signaling pathways. Indeed, both extracellular and intracellular S1P application increased the total internal reflection fluorescence signal in VSMCs cells transfected with STIM1-yellow fluorescent protein in a 2-APB-sensitive manner. These data, and the fact that 2-APB, DES, and Gd(3+) all inhibited S1P-induced cerebral artery constriction, suggest that SOCE modulates S1P-induced vasoconstriction in vivo. Finally, S1P-induced SOCE was larger in proliferative than in contractile VSMCs, correlating with increases in STIM1, Orai1, S1P(1), and S1P(3) receptor mRNA. These data demonstrate that S1P can act through both receptors and a novel intracellular pathway to activate SOCE. Because S1P-induced SOCE contributes to vessel constriction and is increased in proliferative VSMCs, it is likely that S1P/SOCE signaling in proliferative VSMCs may play a role in vascular dysfunction such as atherosclerosis and diabetes.

ObjectiveTo characterize signaling pathways by which sphingosine‐1‐phosphate (S1P) modulates vascular tone.MethodsThe role of store‐operated calcium entry (SOCE) in S1P‐induced Ca2+ entry in vascular smooth muscle cells (VSMCs) and vessel constriction was assessed using ratiometric Ca2+ measurements, quantitative RT‐PCR and isolated arteries.ResultsIn VSMCs, S1P triggered STIM1 puncta formation, and SOCE activated by S1P was inhibited by 2‐aminoethoxydiphenylborate (2‐APB), diethylstilbestrol and gadolinium. In VSMCs prepared from S1P3 receptor knockout mice, the initial calcium rise triggered by S1P was abolished, but SOCE was still present suggesting that S1P acts via multiple signaling pathways. S1P‐induced SOCE was larger in proliferative than in contractile VSMCs, correlating with increases in STIM1, Orai1, S1P1 and S1P3 receptor mRNA. STIM1, S1P1 and S1P3 receptor mRNA expression were larger in freshly isolated brain vessels than in aorta. These data, and the fact that 2‐APB inhibits S1P‐induced cerebral artery constriction, suggest that SOCE modulates S1P‐induced vasoconstriction in vivo.ConclusionThese data support a role for SOCE in S1P induced calcium entry and vessel constriction. The relationship between increased S1P signaling and SOCE in proliferative VSMCs suggests a role of S1P in disorders associated with vascular proliferation, such as atherosclerosis or vasospasm.