Abstract-In a screen of potential lipid regulators of transient receptor potential (TRP) channels, we identified sphingosine-1-phosphate (S1P) as an activator of TRPC5. We explored the relevance to vascular biology because S1P is a key cardiovascular signaling molecule. TRPC5 is expressed in smooth muscle cells of human vein along with TRPC1, which forms a complex with TRPC5. Importantly, S1P also activates the TRPC5-TRPC1 heteromultimeric channel. Because TRPC channels are linked to neuronal growth cone extension, we considered a related concept for smooth muscle. We find S1P stimulates smooth muscle cell motility, and that this is inhibited by E3-targeted anti-TRPC5 antibody. Ion permeation involving TRPC5 is crucial because S1P-evoked motility is also suppressed by the channel blocker 2-aminoethoxydiphenyl borate or a TRPC5 ion-pore mutant. S1P acts on TRPC5 via two mechanisms, one extracellular and one intracellular, consistent with its bipolar signaling functions. The extracellular effect appears to have a primary role in S1P-evoked cell motility. The data suggest S1P sensing by TRPC5 calcium channel is a mechanism contributing to vascular smooth muscle adaptation. Key Words: vascular smooth muscle Ⅲ vein Ⅲ sphingosine-1-phosphate Ⅲ transient receptor potential Ⅲ calcium channel S phingosine-1-phosphate (S1P) has emerged as a major endogenous signaling phospholipid with diverse roles in yeast, plants, and mammals. 1 Proposed functions include the regulation of cell proliferation, migration, programmed death, and pathological processes including cancer, asthma, inflammation, and trauma. There has been particular interest in the role of S1P in the cardiovascular system, where it accumulates in atherosclerotic lesions and plays a role in ischemic preconditioning of the heart. 2-5 S1P is derived from the phosphorylation of sphingosine catalyzed by sphingosine kinase, sphingosine being from ceramide and ceramide from sphingomyelin, a constituent lipid of signaling microdomains of plasma membrane lipid rafts and caveolae. 3 S1P is detected in serum at almost 1 mol/L, although protein binding impacts on the available concentration and local concentrations may vary substantially. 6 S1P is quite unusual among signaling molecules in having separate intracellular and extracellular effects. 1,4,7,8 It affects vascular smooth muscle cell migration, 9,10 evokes contraction of rat mesenteric artery, 11 and slows pacemaker activity of the sino-atrial node of the heart. 12 The underlying mechanisms are only partially worked out, but vascular smooth muscle cells respond to S1P with transient followed by sustained elevation of the cytosolic Ca 2ϩ concentration, 10,11,13,14 whereas cardiac myocytes show activation of potassium current and S1P-evoked "Ca 2ϩ deregulation," depending on extracellular Ca 2ϩ . 12,15 Despite positive effects on Ca 2ϩ signaling, the molecular basis of a Ca 2ϩ channel stimulated by S1P is unknown. L-type voltage-gated Ca 2ϩ channels are inhibited by S1P. 12 The Drosophila transient receptor potential ...

Occlusive vascular disease is a widespread abnormality leading to lethal or debilitating outcomes such as myocardial infarction and stroke. It is part of atherosclerosis and is evoked by clinical procedures including angioplasty and grafting of saphenous vein in bypass surgery. A causative factor is the switch in smooth muscle cells to an invasive and proliferative mode, leading to neointimal hyperplasia. Here we reveal the importance to this process of TRPC1, a homolog of Drosophila transient receptor potential. Using 2 different in vivo models of vascular injury in rodents we show hyperplasic smooth muscle cells have upregulated TRPC1 associated with enhanced calcium entry and cell cycle activity. Neointimal smooth muscle cells after balloon angioplasty of pig coronary artery also express TRPC1. Furthermore, human vein samples obtained during coronary artery bypass graft surgery commonly exhibit an intimal structure containing smooth muscle cells that expressed more TRPC1 than the medial layer cells. Veins were organ cultured to allow growth of neointimal smooth muscle cells over a 2-week period. To explore the functional relevance of TRPC1, we used a specific E3-targeted antibody to TRPC1 and chemical blocker 2-aminoethoxydiphenyl borate. Both agents significantly reduced neointimal growth in human vein, as well as calcium entry and proliferation of smooth muscle cells in culture. The data suggest upregulated TRPC1 is a general feature of smooth muscle cells in occlusive vascular disease and that TRPC1 inhibitors have potential as protective agents against human vascular failure.