Background and purpose: Sphingosine 1-phosphate (S1P) selectively and potently constricts isolated cerebral arteries, but this response has not been pharmacologically characterized. Experimental approach: The receptor subtype(s) involved in S1P-induced cerebrovascular constriction were characterized using genetic (S1P 2 and S1P 3 receptor null mice) and pharmacological tools (phospho-FTY720, a S1P 1/3/4/5 receptor agonist; SEW2871, a S1P 1 receptor agonist, JTE-013, a S1P 2 receptor antagonist, VPC23019, a S1P 1/3 receptor antagonist). Isolated basilar or peripheral (femoral, mesenteric resistance) arteries, from either rat or mouse, were studied in a wire myograph. Key results: S1P concentration-dependently constricted basilar artery in rat, wild-type (WT) and S1P 2 null mice, but barely affected vascular tone in S1P 3 null mice. Vasoconstriction to U46619 (a thromboxane analogue) or to endothelin-1 did not differ between WT, S1P 2 and S1P 3 null mice. JTE-013 inhibited not only S1P-induced vasoconstriction, but also KCl-, U46619-and endothelin-1-induced constriction. This effect was observed in WT as well as in S1P 2 null mice. VPC23019 increased the concentration-dependent vasoconstriction to S1P in both rat and mouse basilar arteries with intact endothelium, but not in rat basilar artery without endothelium. Phospho-FTY720 concentration-dependently constricted rat basilar arteries, but not femoral or mesenteric resistance arteries, while SEW2871 did not induce any response in the same arteries. Conclusions and implications: S1P constricts cerebral arteries through S1P 3 receptors. The purported S1P 2 receptor antagonist JTE-013 does not appear to be selective, at least in rodents. Enhancement of S1P-induced contraction by VPC23019 might be related to blockade of S1P 1 receptors and NO generation.

Rho-kinase is a serine threonine kinase that increases vasomotor tone via its effects on both endothelium and smooth muscle. Rho-kinase inhibition reduces cerebral infarct size in wild type, but not endothelial nitric oxide synthase deficient (eNOS À/À ) mice. The mechanism may be related to Rho-kinase activation under hypoxic/ischemic conditions and impaired vasodilation because of downregulation of eNOS activity. To further implicate Rho-kinase in impaired vascular relaxation during hypoxia/ischemia, we exposed isolated vessels from rat and mouse to 60 mins of hypoxia, and showed that hypoxia reversibly abolished acetylcholine-induced eNOS-dependent relaxation, and that Rho-kinase inhibitor hydroxyfasudil partially preserved this relaxation during hypoxia. We, therefore, hypothesized that if hypoxia-induced Rho-kinase activation acutely impairs vasodilation in ischemic cortex, in vivo, then Rho-kinase inhibitors would acutely augment cerebral blood flow (CBF) as a mechanism by which they reduce infarct size. To test this, we studied the acute cerebral hemodynamic effects of Rho-kinase inhibitors in ischemic core and penumbra during distal middle cerebral artery occlusion (dMCAO) in wild-type and eNOS À/À mice using laser speckle flowmetry. When administered 60 mins before or immediately after dMCAO, Rho-kinase inhibitors hydroxyfasudil and Y-27632 reduced the area of severely ischemic cortex. However, hydroxyfasudil did not reduce the area of CBF deficit in eNOS À/À mice, suggesting that its effect on CBF within the ischemic cortex is primarily endothelium-dependent, and not mediated by its direct vasodilator effect on vascular smooth muscle. Our results suggest that Rho-kinase negatively regulates eNOS activity in acutely ischemic brain, thereby worsening the CBF deficit. Therefore, rapid nontranscriptional upregulation of eNOS activity by small molecule inhibitors of Rho-kinase may be a viable therapeutic approach in acute stroke.

Voltage-dependent calcium channels (VDCCs) play a pivotal role in normal excitation-contraction coupling in cardiac myocytes. These channels can be modulated through activation of ␤-adrenergic receptors (␤-ARs), which leads to an increase in calcium current (I Ca-L ) density through cardiac Ca v 1 channels as a result of phosphorylation by cAMP-dependent protein kinase A. Changes in I Ca-L density and kinetics in heart failure often occur in the absence of changes in Ca v 1 channel expression, arguing for the importance of posttranslational modification of these channels in heart disease. The precise molecular mechanisms that govern the regulation of VDCCs and their cell surface localization remain unknown. Our data show that sustained ␤-AR activation induces internalization of a cardiac macromolecular complex involving VDCC and ␤-arrestin 1 (␤-Arr1) into clathrincoated vesicles. Pretreatment of myocytes with pertussis toxin prevents the internalization of VDCCs, suggesting that G i/o mediates this response. A peptide that selectively disrupts the interaction between Ca V 1.2 and ␤-Arr1 and tyrosine kinase inhibitors readily prevent agonist-induced VDCC internalization. These observations suggest that VDCC trafficking is mediated by G protein switching to G i of the ␤-AR, which plays a prominent role in various cardiac pathologies associated with a hyperadrenergic state, such as hypertrophy and heart failure. Regulation of voltage-dependent calcium channels (VDCCs)3 plays a pivotal role in excitation-contraction coupling in cardiac myocytes. During the action potential upstroke, membrane depolarization causes the opening of VDCCs, encoded by the pore-forming ␣ 1 subunit, Ca v 1.2 (1). Ca 2ϩ entry through VDCCs triggers the release of Ca 2ϩ from the sarcoplasmic reticulum via ryanodine receptors. Although the regulation of VDCCs in the heart has been extensively studied, key molecular mechanisms underlying channel function, trafficking, membrane targeting, retention, and internalization remain unknown. Activation of the ␤-AR, a G protein-coupled receptor (GPCR), leads to positive inotropic effects mediated by phosphorylation of the VDCC via cAMP-dependent protein kinase A (2). This, however, is a transient phenomenon since persistent activation of the receptor causes its subsequent phosphorylation by GPCR kinases (GRKs) (3), causing the ␤-AR to become a target for arrestin (4), which mediates the recruitment of the receptor into clathrin-coated vesicles (5).In addition to decreasing single channel permeability, persistent membrane depolarization can regulate the number of Ca v 1.2 channels at the plasma membrane. For example, sustained KCl-induced depolarization of rat cortical neurons effectively decreases Ca v 1.2 channel activity (6). Ca v 1.2 channels have been proposed to contain a membrane-targeting domain within their calmodulin (CaM)-binding domain in the C terminus (7). Pitt and colleagues (8) showed that Ca 2ϩ -CaM interaction with this domain accelerated the rate of trafficking of Ca v 1.2 channels to ...