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The present study tested the hypothesis that endostatin stimulates superoxide (O2*-) production through a ceramide-mediating signaling pathway and thereby results in an uncoupling of bradykinin (BK)-induced increases in intracellular Ca2+ concentration ([Ca2+]i) from nitric oxide (NO) production in coronary endothelial cells. With the use of high-speed, wavelength-switching, fluorescence-imaging techniques, the [Ca2+]i and NO levels were simultaneously monitored in the intact endothelium of freshly isolated bovine coronary arteries. Under control conditions, BK was found to increase NO production and [Ca2+]i in parallel. When the arteries were pretreated with 100 nM human recombinant endostatin for 1 h, this BK-induced NO production was reduced by 89%, whereas [Ca2+]i was unchanged. With the conversion rate of L-[3H]arginine to L-[3H]citrulline measured, endostatin had no effect on endothelial NO synthase (NOS) activity, but it stimulated ceramide by activation of sphingomyelinase (SMase), whereby O2*-. production was enhanced in endothelial cells. O2*-. scavenging by tiron and inhibition of NAD(P)H oxidase by apocynin markedly reversed the effect of endostatin on the NO response to BK. These results indicate that endostatin increases intracellular ceramide levels, which enhances O2*-. production through activation of NAD(P)H oxidase. This ceramide-O2*-. signaling pathway may contribute importantly to endostatin-induced endothelial dysfunction.

. Cyclic ADP ribosemediated Ca 2ϩ signaling in mediating endothelial nitric oxide production in bovine coronary arteries. Am J Physiol Heart Circ Physiol 290: H1172-H1181, 2006. First published October 21, 2005 doi:10.1152/ajpheart.00441.2005.-The present study tested the hypothesis that cyclic ADP ribose (cADPR) serves as a novel second messenger to mediate intracellular Ca 2ϩ mobilization in coronary arterial endothelial cells (CAECs) and thereby contributes to endothelium-dependent vasodilation. In isolated and perfused small bovine coronary arteries, bradykinin (BK)-induced concentrationdependent vasodilation was significantly attenuated by 8-bromocADPR (a cell-permeable cADPR antagonist), ryanodine (an antagonist of ryanodine receptors), or nicotinamide (an ADP-ribosyl cyclase inhibitor). By in situ simultaneously fluorescent monitoring, Ca 2ϩ transient and nitric oxide (NO) levels in the intact coronary arterial endothelium preparation, 8-bromo-cADPR (30 M), ryanodine (50 M), and nicotinamide (6 mM) substantially attenuated BK (1 M)-induced increase in intracellular [Ca 2ϩ ] by 78%, 80%, and 74%, respectively, whereas these compounds significantly blocked BKinduced NO increase by about 80%, and inositol 1,4,5-trisphosphate receptor blockade with 2-aminethoxydiphenyl borate (50 M) only blunted BK-induced Ca 2ϩ -NO signaling by about 30%. With the use of cADPR-cycling assay, it was found that inhibition of ADP-ribosyl cyclase by nicotinamide substantially blocked BK-induced intracellular cADPR production. Furthermore, HPLC analysis showed that the conversion rate of ␤-nicotinamide guanine dinucleotide into cyclic GDP ribose dramatically increased by stimulation with BK, which was blockable by nicotinamide. However, U-73122, a phospholipase C inhibitor, had no effect on this BK-induced increase in ADP-ribosyl cyclase activity for cADPR production. In conclusion, these results suggest that cADPR importantly contributes to BK-and A-23187-induced NO production and vasodilator response in coronary arteries through its Ca 2ϩ signaling mechanism in CAECs.nucleotide; vasodilation; endothelium; endothelium-derived relaxing factor CYCLIC ADP RIBOSE (cADPR), endogenously generated from nicotinamide (Ni) adenine dinucleotide, has emerged as a novel intracellular Ca 2ϩ -mobilizing second messenger in a wide variety of mammalian cells. The actions of this signaling nucleotide are completely independent of the inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3 ]-signaling pathway, but it exerts its action by targeting ryanodine (Ry) receptors (RyRs) (8,15,16,(31)(32)(33)44). Elevation of intracellular cADPR levels followed by Ca 2ϩ mobilizition and activation of Ca 2ϩ -dependent effects within cells has been found to be essential for versatile physiological processes, such as cell cycle regulation, cell proliferation, gene expression, egg fertilization, neurotransmitter release, muscle contraction, and nitric oxide signaling (10,29,55). Recent studies (17,35,36,52,55,57) in our laboratory and by others have reported that in va...

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