Abstract-Although recent investigations have suggested that a Rho-kinase-mediated Ca 2ϩ sensitization of vascular smooth muscle contraction plays a critical role in the pathogenesis of cerebral and coronary vasospasm, the upstream of this signal transduction has not been elucidated. In addition, the involvement of protein kinase C (PKC) may also be related to cerebral vasospasm. We recently reported that sphingosylphosphorylcholine (SPC), a sphingolipid, induces Rho-kinase-mediated Ca 2ϩ sensitization in pig coronary arteries. The purpose of this present study was to examine the possible mediation of SPC in Ca 2ϩ sensitization of the bovine middle cerebral artery (MCA) and the relation to signal transduction pathways mediated by Rho-kinase and PKC. In intact MCA, SPC induced a concentration-dependent (EC 50 ϭ3.0 mol/L) contraction, without [Ca 2ϩ ] i elevation. In membrane-permeabilized MCA, SPC induced Ca 2ϩ sensitization even in the absence of added GTP, which is required for activation of G-proteins coupled to membrane receptors. The SPC-induced Ca 2ϩ sensitization was blocked by a Rho-kinase inhibitor (Y-27632) and a dominantnegative Rho-kinase, but not by a pseudosubstrate peptide for conventional PKC, which abolished the Ca 2ϩ -independent contraction induced by phorbol ester. In contrast, phorbol ester-induced Ca 2ϩ sensitization was resistant to a Rho-kinase inhibitor and a dominant-negative Rho-kinase. In primary cultured vascular smooth muscle cells, SPC induced the translocation of cytosolic Rho-kinase to the cell membrane. We propose that SPC is a novel messenger for Rho-kinase-mediated Ca 2ϩ sensitization of cerebral arterial smooth muscle and, therefore, may play a pivotal role in the pathogenesis of abnormal contraction of the cerebral artery such as vasospasm. The SPC/Rho-kinase pathway functions independently of the PKC pathway.

Background and Purpose: Rho-kinase (ROK)-mediated Ca²⁺ sensitization of vascular smooth muscle (VSM) contraction plays a pivotal role in cerebral vasospasm (CV). We previously demonstrated that sphingosylphosphorylcholine (SPC) induces Ca²⁺ sensitization through sequential activation of the Src family protein tyrosine kinases (Src-PTKs) and ROK in vitro, and that Ca²⁺ sensitization is inhibited by eicosapentaenoic acid (EPA) through the selective inactivation of Src-PTK. In this study, we examined whether SPC induced CV in vivo, and, if it did, whether EPA would inhibit CV, as induced by SPC or in an in vivo model of subarachnoid hemorrhage (SAH). Methods: Changes in the diameter of the canine basilar artery were investigated by angiography after administering SPC into the cisterna magna. Then, Y27632, a specific Rho-kinase inhibitor, or EPA was injected intracisternally and the effects of both agents were investigated. In another experiment using a single-hemorrhage model, Y27632 or EPA was injected on day 7 after SAH and the changes in the diameter of the canine basilar artery were investigated. Results: At cerebrospinal fluid concentrations of 100 and 300 µmol/l, SPC induced severe vasoconstriction (maximum vasoconstriction by SPC (100 µmol/l): 61.8 ± 8.2%), which was markedly reversed by Y27632 (96.3 ± 4.4%) or EPA (92.6 ± 12.8%). SAH caused severe vasospasm on day 7 (67.6 ± 7.8%), which was significantly blocked by Y27632 (95.5 ± 10.6%) or EPA (90.0 ± 4.4%). Conclusions: SPC is a novel mediator of ROK-induced CV in vivo. The inhibition of CV induced by SPC or after SAH by EPA suggests beneficial roles of EPA in the treatment of CV. Our findings are compatible with the notion that the SPC-ROK pathway may be involved in CV.

Bypass surgery is increasingly performed in patients with complicated aneurysms if sacrifice or temporary occlusion of any major vessel is required. Therefore, de novo aneurysm formation may not be rare in patients with risk factors such as hypertension or multiple aneurysms. Extended follow-up examination is required in such patients.

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