Objective-In addition to their role in programmed cell death, cell survival, and cell growth, sphingolipid metabolites such as ceramide, sphingosine, and sphingosine-1-phosphate have vasoactive properties. Besides their occurrence in blood, they can also be formed locally in the vascular wall itself in response to external stimuli. This study was performed to investigate whether vasoactive compounds modulate sphingolipid metabolism in the vascular wall and how this might contribute to the vascular responses. Methods and Results-In isolated rat carotid arteries, the contractile responses to angiotensin II are enhanced by the sphingosine kinase inhibitor dimethylsphingosine. Endothelium removal or NO synthase inhibition by N -nitro-Larginine results in a similar enhancement. Angiotensin II concentration-dependently induces NO production in an endothelial cell line, which can be diminished by dimethylsphingosine. Using immunoblotting and intracellular calcium measurements, we demonstrate that this sphingosine kinase-dependent endothelial NO synthase activation is mediated via both phosphatidylinositol 3-kinase/Akt and calcium-dependent pathways. Sphingomyelinase catalyzes the hydrolysis of sphingomyelin to form ceramide. 1,2 The sequential action of ceramidase and sphingosine kinase converts ceramide to sphingosine and sphingosine-1-phosphate (S1P), and ceramide synthase and S1P phosphatase can reverse this process to form ceramide from S1P. 3,4 The sphingomyelin metabolites ceramide, sphingosine, and S1P are biologically active mediators that play important roles in cellular homeostasis. In this regard, ceramide and sphingosine on the one hand and S1P on the other hand frequently have opposite biological effects. For example, ceramide and sphingosine are generally involved in apoptotic responses to various stress stimuli and in growth arrest, 5,6 whereas S1P is implicated in mitogenesis, differentiation, and migration. 7,8 This homeostatic system is frequently referred to as the ceramide/S1P rheostat. 9 It can be hypothesized that this rheostat also plays a role in vascular contraction and relaxation because S1P, sphingosine, and ceramide are potentially counteracting, vasoactive compounds. 10,11 The molecular basis of ceramide effects has not been explored fully but is believed to involve stress-activated protein kinases, protein phosphatases such as protein phosphatases 1 and 2, guanylyl cyclase, and charybdotoxinsensitive K ϩ channels. 11,12 The molecular basis of S1P effects has been characterized in more detail. S1P can act on specific G protein-coupled receptors, of which 5 subtypes have been identified thus far, termed S1P 1-5 . These receptors couple to intracellular second messenger systems including intracellular Ca 2ϩ , adenylyl cyclase, phospholipase C, phosphatidylinositol 3 (PI3)-kinase, protein kinase Akt, mitogen-activated protein kinases, and Rho-and Ras-dependent pathways. 13 The cardiovascular system primarily expresses the receptor subtypes S1P 1-3 , and within the vasculature they are express...

We conclude that sphingomyelin metabolites are important endogenous modulators of vascular function, which may contribute to the pathophysiology of some diseases and be targets for therapeutic interventions.

Local formation of the sphingomyelin metabolite sphingosine-1-phosphate (S1P) within the vascular wall has been shown to modulate vascular reactivity. In this study we investigated whether sphingosine kinase, the enzyme responsible for S1P synthesis, plays a role in muscarinic receptor-mediated NO production and vascular relaxation in different blood vessel types. For this purpose, sphingosine kinase translocation and sphingolipid-dependent NO-production after muscarinic receptor stimulation were assessed in an endothelial cell line. Furthermore, we used the sphingosine kinase inhibitor N,N-dimethylsphingosine (DMS) to investigate the role of sphingosine kinase in the relaxant responses to the muscarinic agonist methacholine (MCh) in isolated rat aorta and mesenteric arteries. Activation of M(3)-receptors in an endothelial cell line induced a fast translocation of YFP-tagged sphingosine kinase-1 from the cytosol to the plasma membrane. Concomitant NO-production in this cell line was partially inhibited by DMS. Accordingly, in rat aorta the relaxant responses to MCh were attenuated in the presence of DMS, while the responses to the NO-donor sodium nitroprusside were unaltered. In contrast, DMS enhanced the relaxant responses to MCh in mesenteric artery preparations. This effect could also be observed in the presence of NO synthase and cyclooxygenase inhibitors, indicating that sphingosine kinase inhibition specifically enhanced endothelium-derived hyperpolarizing factor-mediated (i.e. non-NO and non-prostacyclin-dependent) relaxation. We conclude that sphingosine kinase differentially regulates vascular tone in different vessel types, enhancing NO-dependent vasorelaxation but counteracting EDHF-dependent vasorelaxation. This observation enhances our understanding of the complex mechanisms by which sphingolipids regulate vascular homeostasis. Moreover, a disturbed regulation of sphingolipid metabolism in the vascular wall may therefore play a role in the aetiology/pathology of disease states characterized by endothelial dysfunction.