Abstract-Sphingosine 1-phosphate (S1P), a lipid released from activated platelets, influences physiological processes in the cardiovascular system via activation of the endothelial differentiation gene (EDG/S1P) family of 7 transmembrane G protein-coupled receptors. In cultured vascular smooth muscle (VSM) cells, S1P signaling has been shown to stimulate proliferative responses; however, its role in vasoconstriction has not been examined. In the present study, the effects of S1P and EDG/S1P receptor expression were determined in rat VSM from cerebral artery and aorta. S1P induced constriction of cerebral artery, which was partly dependent on activation of p160 ROCK (Rho-kinase). S1P also induced activation of RhoA in cerebral artery with a similar time course to contraction. In aorta, S1P did not produce a constriction or RhoA activation. In VSM myocytes from cerebral arteries, stimulation with S1P gives rise to a global increase in [Ca 2ϩ ] i , initially generated via Ca 2ϩ release from the sarcoplasmic reticulum by an inositol 1,4,5-trisphosphate-dependent pathway. In aorta VSM, a small increase in [Ca 2ϩ ] i was observed after stimulation at higher concentrations of S1P. S1P induced activation of p42/p44 mapk in aorta and cerebral artery VSM. Subtype-specific S1P receptor antibodies revealed that the expression of S1P 3 /EDG-3 and S1P 2 /EDG-5 receptors is 4-fold higher in cerebral artery compared with aorta. S1P 1 /EDG-1 receptor expression was similar in both types of VSM. Therefore, the ability of S1P to act as a vasoactive mediator is dependent on the activation of associated signaling pathways and may vary in different VSM. This differential signaling may be related to the expression of S1P receptor subtypes. Key Words: vascular smooth muscle Ⅲ sphingosine 1-phosphate Ⅲ cerebral artery Ⅲ vasoconstriction S everal studies have now shown that the bioactive sphingolipid, sphingosine 1-phosphate (S1P), is likely to play an important role in regulating cellular processes via activation of specific signal transduction pathways. [1][2][3] In the shortterm, S1P-induced activation of several intracellular signaling pathways occurs, such as an increase in the intracellular calcium concentration ([Ca 2ϩ ] i ), 4 activation of the monomeric GTP-binding protein, p21RhoA (RhoA), 5 and activation of p42/p44 mitogen-activated protein kinase (p42/p44 mapk ). 6,7 Through activation of these signaling pathways, S1P can induce longer term effects, such as proliferation, differentiation, and cell migration. 1 It is now clear that S1P-induced intracellular effects occur predominantly through activation of selective S1P receptors on the plasma membrane. 2 These receptors, belonging to the G protein-coupled receptor superfamily, originally known as EDG (endothelial differentiation gene) receptors. 8 Several isoforms have now been cloned and S1P 1 /EDG-1, 5 S1P 2 / EDG-5, 9 S1P 3 /EDG-3, 10 S1P 4 /EDG-6, 11 and S1P 5 /EDG-8 12 have high affinities for S1P with EC 50 s in the nmol/L range.Recent studies have investigated th...

1 This study examined the action of gabapentin (gabapentin,1-(aminomethyl) 2+ in¯ux were signi®cantly reduced by *25-30% in the presence of gabapentin (25 mM). 4 Gabapentin potently inhibited the peak whole-cell Ca 2+ channel current (I Ba ) in a dosedependent manner with an estimated IC 50 value of 167 nM. Block was incomplete and saturated at a maximal concentration of 25 mM. 5 Inhibition was signi®cantly decreased in the presence of the neutral amino acid L-isoleucine (25 mM) but unaected by application of the GABA B antagonist, saclofen (200 mM), suggesting a direct action on the a 2 d subunit of the Ca 2+ channel. 6 Gabapentin inhibition was voltage-dependent, producing an *7 mV hyperpolarizing shift in current voltage properties and reducing a non-inactivating component of whole-cell current activated at relatively depolarized potentials. 7 The use of speci®c Ca 2+ channel antagonists revealed a mixed pharmacology of the gabapentinsensitive current (N-, L-and P/Q-type), which is dominated by N-type current. 8 The present study is the ®rst to demonstrate that gabapentin directly mediates inhibition of voltage-gated Ca 2+ in¯ux in DRG neurones, providing a potential means for gabapentin to eectively mediate spinal anti-nociception.

1. The effect of intracellular application of the hydrolysis-resistant GTP and GDP analogues, guanosine 5'-O-3-thiotriphosphate (GTP-gamma-S), and guanosine 5'-O-2-thiodiphosphate (GDP-beta-S) has been examined on voltage-activated calcium-channel currents and the ability of the gamma-aminobutyric acid B agonist baclofen to inhibit them, in cultured rat dorsal root ganglion (d.r.g.) neurones. 2. Under control conditions, the calcium-channel current, recorded using the whole-cell patch technique with Ba2+ rather than Ca2+ as the permeant divalent cation, consists of an inactivating and a sustained current. In the presence of 500 microM-GTP-gamma-S included in the patch pipette, the calcium-channel current was activated more slowly and was largely non-inactivating during the 100 ms depolarization voltage step. The effects of GTP-gamma-S were abolished by pre-treatment of cells with pertussis toxin. 3. The calcium-channel current recorded in the presence of 500 microM-GDP-beta-S had a more marked transient component than the control calcium-channel current. The proportion of transient calcium-channel current in the presence of GDP-beta-S was not reduced in Na+-free medium. 4. No statistically significant effects of GTP-gamma-S and GDP-beta-S were observed on the calcium-activated potassium current IK(Ca), the transient outward potassium current activated in Ca2+-free medium, or on the inwardly rectifying current (Ih) activated by hyperpolarization. 5. GTP-gamma-S increased the ability of baclofen to inhibit calcium-channel currents, whereas this was decreased by GDP-beta-S and by pre-treatment of cells with pertussis toxin. The half-maximal effective dose (EC50) for baclofen was 2 microM in the presence of GTP-gamma-S, 15 microM for control and 50 microM in the presence of GDP-beta-S. Comparable results were obtained using a single concentration of the adenosine agonist 2-chloroadenosine (2-CA, 0.05 microM) to inhibit calcium-channel currents; its effect was significantly increased by GTP-gamma-S and reduced by GDP-beta-S. 6. The ability of baclofen to inhibit calcium-channel currents was not affected by 1 microM-forskolin or 50 microM-intracellular cyclic AMP. 7. It is concluded that calcium channels in d.r.g.s are associated with a nucleotide binding protein, and that this mediates the effect of baclofen and 2-CA on calcium-channel currents. The ability of GTP-gamma-S to inhibit the transient component of calcium-channel currents in the absence of agonist may represent a means of differentially regulating calcium-channel activity.

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