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.

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Abstract-Hypercholesterolemia is a major risk factor involved in abnormal cardiovascular events. Rho-kinase-mediated Ca 2ϩ sensitization of vascular smooth muscle (VSM) plays a critical role in vasospasm and hypertension. We recently identified sphingosylphosphorylcholine (SPC) and Src family tyrosine kinase (Src-TK) as upstream mediators for the Rho-kinase-mediated Ca 2ϩ sensitization. Here we report the strong linkage between cholesterol and the Ca 2ϩ sensitization of VSM mediated by a novel SPC/Src-TK/Rho-kinase pathway in both humans and rabbits. The extent of the sensitization correlated well with the total cholesterol or low-density lipoprotein cholesterol levels in serum.However, an inverse correlation with the serum level of high-density lipoprotein cholesterol was observed, and a correlation with other cardiovascular risk factors was nil. When cholesterol-lowering therapy was given to patients and rabbits with hypercholesterolemia, the SPC-induced contractions diminished. Depletion of VSM cholesterol by ␤-cyclodextrin resulted in a loss of membrane caveolin-1, a marker of cholesterol-enriched lipid raft, and inhibited the SPC-induced Ca 2ϩ sensitization and translocation of Rho-kinase from cytosol to the cell membrane. Vasocontractions induced by membrane depolarization and by an adrenergic agonist were cholesterol-independent. Our data support the previously unreported concept that cholesterol potentiates the Ca 2ϩ sensitization of VSM mediated by a SPC/Src-TK/ Rho-kinase pathway, and are also compatible with a role for cholesterol-enriched membrane microdomain, a lipid raft. This process may play an important role in the development of abnormal vascular contractions in patients with hypercholesterolemia. ( Key Words: Ca 2ϩ sensitization Ⅲ contraction Ⅲ membrane lipid raft Ⅲ Rho-kinase Ⅲ sphingosylphosphorylcholine Ⅲ vascular smooth muscle A bnormal vascular contraction as a result of Ca 2ϩ sensitization of vascular smooth muscles (VSMs) has attracted attention as a cause of hypertension and vasospasm. 1 Kureishi et al showed that constitutively active Rho-kinase applied to the cytosol of permeabilized VSM induced Ca 2ϩ sensitization and increased myosin light chain phosphorylation through a mechanism that is independent of a Ca 2ϩ -dependent myosin light chain kinase pathway. 2 In addition, the Rho-kinase blocker Y27632 cures hypertension in rats without affecting normal blood pressure, 3 and it also inhibits vasospasm of coronary arteries in a porcine model. 4 However, the mechanism by which Rho-kinase-mediated Ca 2ϩ sensitization of VSM is triggered in cardiovascular diseases has not been evident.Hyperlipidemia is a major risk factor for abnormal cardiovascular events. Evidence for this is provided by the J-shaped curve showing a strong relationship between serum cholesterol levels and the relative risk for coronary disease 5-7 (see also Figure I in the online data supplement, available at http://circres.ahajournals.org). Atheromatous plaques may be involved in cardiovascular events, 8,9 but the ext...

Hepatic artery vascular responsiveness to altered portal blood flow is blunted in cirrhotic livers.