Sphingolipids are formed via the metabolism of sphingomyelin, a constituent of the plasma membrane, or by de novo synthesis. Enzymatic pathways result in the formation of several different lipid mediators, which are known to have important roles in many cellular processes, including proliferation, apoptosis and migration. Several studies now suggest that these sphingolipid mediators, including ceramide, ceramide 1-phosphate and sphingosine 1-phosphate (S1P), are likely to have an integral role in inflammation. This can involve, for example, activation of pro-inflammatory transcription factors in different cell types and induction of cyclooxygenase-2, leading to production of pro-inflammatory prostaglandins. The mode of action of each sphingolipid is different. Increased ceramide production leads to the formation of ceramide-rich areas of the membrane, which may assemble signalling complexes, whereas S1P acts via high-affinity G-protein-coupled S1P receptors on the plasma membrane. Recent studies have demonstrated that in vitro effects of sphingolipids on inflammation can translate into in vivo models. This review will highlight the areas of research where sphingolipids are involved in inflammation and the mechanisms of action of each mediator. In addition, the therapeutic potential of drugs that alter sphingolipid actions will be examined with reference to disease states, such as asthma and inflammatory bowel disease, which involve important inflammatory components. A significant body of research now indicates that sphingolipids are intimately involved in the inflammatory process and recent studies have demonstrated that these lipids, together with associated enzymes and receptors, can provide effective drug targets for the treatment of pathological inflammation.

The purpose of this study was to identify guanine nucleotide-binding proteins (G proteins) MgCl2/1 mM dithiothreitol/1 mM phenylmethylsulfonyl fluoride/20 ,tM GTP (buffer A), disrupted in a glass homogenizer, and centrifuged at 640 x g for 15 min, and the supernatant was decanted. The residue was twice resuspended in 70 ml of buffer A and recentrifuged. The combined supernatants containing the cell membrane fragments were centrifuged at 186,000 x g for 90 min, and the pellet was resuspended in 50 ml of buffer A containing 2% octyl glucoside, homogenized, and gently shaken for 45 min. The solution was centrifuged at 142,000 x g for 90 min, and the supematant was filtered through a 0.45-,tm Minisart cellulose acetate filter (Sartorius). The solution (-0.4 mg of protein per ml) was loaded into a 10-ml MonoS column equili-

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...

SummaryIn arterial myocytes the Ca 2+ mobilizing messenger NAADP evokes spatially restricted Ca 2+ bursts from a lysosome-related store that are subsequently amplified into global Ca 2+ waves by Ca 2+ -induced Ca 2+ -release from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs). Lysosomes facilitate this process by forming clusters that co-localize with a subpopulation of RyRs on the SR. We determine here whether RyR subtypes 1, 2 or 3 selectively co-localize with lysosomal clusters in pulmonary arterial myocytes using affinity purified specific antibodies. The density of: (1) αlgP120 labelling, a lysosome-specific protein, in the perinuclear region of the cell (within 1.5 μm of the nucleus) was ~4-fold greater than in the sub-plasmalemmal (within 1.5 μm of the plasma membrane) and ~2-fold greater than in the extra-perinuclear (remainder) regions; (2) RyR3 labelling within the perinuclear region was ~4-and ~14-fold greater than that in the extraperinuclear and sub-plasmalemmal regions, and ~2-fold greater than that for either RyR1 or RyR2; (3) despite there being no difference in the overall densities of fluorescent labelling of lysosomes and RyR subtypes between cells, co-localization with αlgp120 labelling within the perinuclear region was ~2-fold greater for RyR3 than for RyR2 or RyR1; (4) co-localization between αlgp120 and each RyR subtype declined markedly outside the perinuclear region. Furthermore, selective block of RyR3 and RyR1 with dantrolene (30μM) abolished global Ca 2+ waves but not Ca 2+ bursts in response to intracellular dialysis of NAADP (10nM). We conclude that a subpopulation of lysosomes cluster in the perinuclear region of the cell and form junctions with SR containing a high density of RyR3 to comprise a trigger zone for Ca 2+ signalling by NAADP.