Sphingosine 1-phosphate (S1P) induces diverse biological responses in various tissues by activating specific G proteincoupled receptors (S1P 1 -S1P 5 receptors). The biological signaling regulated by S1P 3 receptor has not been fully elucidated because of the lack of an S1P 3 receptor-specific antagonist or agonist. We developed a novel S1P 3 receptor antagonist, 1-(4-chlorophenylhydrazono)-1-(4-chlorophenylamino)-3,3-dimethyl-2-butanone (TY-52156), and show here that the S1P-induced decrease in coronary flow (CF) is mediated by the S1P 3 receptor. In functional studies, TY-52156 showed submicromolar potency and a high degree of selectivity for S1P 3 receptor. TY-52156, but not an S1P 1 receptor antagonist, inhibited the decrease in CF induced by S1P in isolated perfused rat hearts. We further investigated the effect of TY-52156 on both the S1P-induced increase in intracellular calcium ([Ca 2ϩ ] i ) and Rho activation that are responsible for the contraction of human coronary artery smooth muscle cells. TY-52156 inhibited both the S1P-induced increase in [Ca 2ϩ ] i and Rho activation. In contrast, VPC23019 and JTE013 inhibited only the increase in [Ca 2ϩ ] i and Rho activation, respectively. We further confirmed that TY-52156 inhibited FTY-720-induced S1P 3 receptor-mediated bradycardia in vivo. These results clearly show that TY-52156 is both sensitive and useful as an S1P 3 receptor-specific antagonist and reveal that S1P induces vasoconstriction by directly activating S1P 3 receptor and through a subsequent increase in [Ca 2ϩ ] i and Rho activation in vascular smooth muscle cells.Sphingosine 1-phosphate (S1P) is a bioactive lysophospholipid mediator that is mainly released from activated platelets and induces many biological responses, including angiogenesis, vascular development, and cardiovascular function (Siess,

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Accumulation of amyloid-β peptide (Aβ) in the brain is closely associated with cognitive decline in Alzheimer's disease (AD). Stereotaxic infusion of neprilysin-encoding viral vectors into the hippocampus has been shown to decrease Aβ in AD-model mice, but more efficient and global delivery is necessary to treat the broadly distributed burden in AD. Here we developed an adeno-associated virus (AAV) vector capable of providing neuronal gene expression throughout the brains after peripheral administration. A single intracardiac administration of the vector carrying neprilysin gene in AD-model mice elevated neprilysin activity broadly in the brain, and reduced Aβ oligomers, with concurrent alleviation of abnormal learning and memory function and improvement of amyloid burden. The exogenous neprilysin was localized mainly in endosomes, thereby effectively excluding Aβ oligomers from the brain. AAV vector-mediated gene transfer may provide a therapeutic strategy for neurodegenerative diseases, where global transduction of a therapeutic gene into the brain is necessary.

Sphingosine-1-phosphate (S1P) is an intracellular second messenger and an extracellular mediator through endothelial differentiation gene (EDG) receptors, which are a novel class of G-protein-coupled receptors. Although EDG has attracted much attention because of its various roles, no selective agonists or antagonists have yet been developed. This could account for the delay in clarifying the physiological roles of members of the EDG family. Because precise structural information on EDG receptors is not yet available, pharmacophore models were generated based on structural information for S1P using the rational drug design software Catalyst. Novel antagonists, 2-alkylthiazolidine-4-carboxylic acids, were retrieved from a three-dimensional database search using the pharmacophore models, and these showed activity for EDG3. On the basis of their nonphosphoric acid structure, more potent antagonists, 2-(m- or p-heptylphenyl)thiazolidine-4-carboxylic acid, were developed.