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Our recent studies have indicated that there is an autocrine/paracrine pattern of superoxide production through NAD(P)H oxidase in coronary arterial myocytes (CAMs). The present study determined whether extracellular O2*- could serve as an autocrine factor to activate intracellular Ca2+ signaling pathway and explored related underlying mechanisms. Using a newly established dual fluorescence microscopic imaging technique, We found that M1-receptor agonist oxotremorine (Oxo) simultaneously increased the extracellular O2*- production and [Ca2+]i,, which were significantly attenuated by addition of superoxide dismutase (SOD) and catalase into the bath solution. NAD(P)H oxidase inhibitor, apocynin also inhibited this response of CAMs to Oxo. When ADP-ribosyl cyclase inhibitor -- nicotinamide, RyR/ Ca2+release channel antagonists -- ryanodine or cADPR antagonist -- 8-Br-cADPR was added into the bath solution, Oxo-induced increase in [Ca2+]i was abolished, while extracellular O2*- production remained. Furthermore, introduction of cADPR into CAMs by ultrasound microbubbles increased [Ca2+]i,,but it had no effect on extracellular O2*- production. These results provide direct evidence that extracellular O2*- derived from NAD(P)H oxidase contributes to Ca2+activation of CAMs through its autocrine action on cADPR signaling pathway (Supported by NIH grants HL057244 and HL075316). [ABSTRACT FROM AUTHOR]

The formation of lipid raft (LR)-redox signaling platforms on coronary arterial endothelial cell (CAEC) membrane is an important mechanism mediating transmembrane signaling of different death receptors and thereby resulting in endothelial dysfunction. This platform formation was associated with activation of acid sphingomyelinase (ASM). Given that lysosomal ASM is a major type of ASM, the present study tested whether lysosome function contributes to this process. By confocal microscopic analysis, we found that Fas ligand (Fas L) stimulated the clustering of individual LRs on the plasma membrane of CAECs and induced the aggregation of NAD(P)H oxidase complex subunits, gp91phox and p47phox to LR clusters. When the cells were pretreated with the lysosome function inhibitors, bafilomycin and GPN, Fas L-induced LR redox signaling platform formation was markedly abolished. Correspondingly, superoxide (02) production induced by Fas L was significantly decreased by lysosomal function inhibition. In isolated perfused small coronary arteries, Fas L-induced inhibition of endothelium-dependent vasorelaxation was also reduced by inhibition of lysosome function. These results demonstrate that lysosome function plays an important role in the formation of LR-redox signaling platforms and that inhibition of lysosome function may protect coronary artery from death receptor-mediated endothelial dysfunction. [ABSTRACT FROM AUTHOR]

Interaction of von Willebrand factor (VWF) with circulating blood platelets is the initial trigger for thrombosis in an arterial stenosis. Polymorphisms in the gene encoding the VWF receptor, platelet glycoprotein Ib (GpIb), may thus alter the risk of cardiovascular disease. One such polymorphism is a C/T dimorphism at nucleotide 482 in the GpIbα gene; it alters the codon for amino acid 161 from threonine to methionine within the VWF binding domain. We have devised a method based on the real-time polymerase chain reaction to assay this dimorphism. Another polymorphism involves a variable number of tandem repeats (VNTR) of a 39-nucleotide sequence encoding part of the extracellular portion of GpIbα this sequence may occur once (VNTR-D allele), twice (VNTR-C) or three times (VNTR-B). We have genotyped individuals from three racial groups for both polymorphisms. Our data indicate that the rarer 482T and VNTR-B alleles are much more prevalent in African Americans than in Caucasians or Asians. Moreover, the pattern of linkage disequilibrium among these polymorphisms is weaker in African Americans and Asians than in Caucasians. These observations suggest that studies in African Americans will be more powerful at elucidating the mechanism whereby either polymorphism alters the propensity for cardiovascular disease. [ABSTRACT FROM AUTHOR]

We have demonstrated that the formation of lipid raft (LR)-redox signaling platforms on the cell membrane serves as an important transmembrane signaling mechanism in endothelial cells. We wondered whether this LR signaling platforms participate in redox signaling induced by hyperhomocysteinemia in rat glomerular endothelial cells (GECs). The present study tested the hypothesis that homocysteine (Hcys) induces LRs clustering on the membrane of GECs and form a redox signaling platform by aggregation and activation of NAD(P)H oxidase subunits. By confocal microscopic analysis, we found that incubation of GECs with Hcys for 30 min not only stimulated the clustering and trafficking of individual LRs on the plasma membrane of GECs, but also induced NAD(P)H oxidase membrane-associated subunit gp91phox(Nox-2) aggregation. In addition, p47phox, a crucial cytosolic subunit and Rac GTPase were found translocated into LR clusters. This aggregation or translocation was substantially blocked by nystatin and methyl-β-cyclodextrin (MCD), two compounds for disruption of LRs. Correspondingly, superoxide (O2) production induced by Hcys was decreased by 52% in the presence of MCD, as measured by ESR spectrometry. These results suggest that Hcys can induce the formation of LR redox signaling platform in GECs even at an early stage of its action, which may represent an early event of Hcys-induced glomerular injury. [ABSTRACT FROM AUTHOR]

In our previous studies, homocysteine (Hcys) has been reported to stimulate de novo ceramide synthesis and thereby induce NAD(P)H oxidase activation by increase in Rac GTPase activity in rat mesangial (RMG) cells. However, the mechanism by which Hcys-enhances Rac GTPase activity is still unknown. Given Vav1, a member of guanine exchange factors (GEFs), plays an important role in the activation of Rho/Rac GTPase activity, the present study tested the hypothesis that Rac1/NAD(P)H oxidase signaling induced by Hcys is associated with Vav1 in RMG cells. Real-time RT PCR and Western blot analysis confirmed that Vav1 was present in RMG cells. By Rac GTPase pull down assay, it was found that NSC23766, an inhibitor by interfering Rac1 interaction with the Rac-specific GEF, blocked the Hcys-induced Rac GTPase activity by 42%. In Vav1-siRNA transfected RMG cells, Hcys-induced Rac GTPase activity decreased by 52%, which was accompanied by 55% reduction of Hcys-induced superoxide (O2) production, as measured by ESR spectrometry. By immunofluorescence assay, it was found that Hcys stimulated Vav1 phosphorylated and then translocated to cell membrane. Similar results were obtained by C16-ceramide treatments of RMG cells. Based on these results, we conclude that Rac1/NADPH oxidase signaling induced by Hcys is Vav1-depentent in RMG cells, which involves its phosphorylation and subsequently translocation, an essential step for the activation of this GEF. [ABSTRACT FROM AUTHOR]

Recently, we characterized lysosomal nicotinic acid adenine dinucleotide phosphate (NAADP)-sensitive Ca2+ release channels in coronary arterial smooth muscle cells (CASMCs). The present study was designed to further confirm the identity of these lysosomal Ca2+ release channels that possibly relate to transient receptor potential-mucolipin-1 (TRP-ML1) protein, a non-selective Ca2+ permeable cation channel. Western blot analysis demonstrated the existence of TRP-ML1 protein in purified lysosomes from CASMCs. By lipid bilayer reconstitution of these purified CASMCs lysosomes, this NAADP-activated channel activity in TRP-ML1-deprived lysosomal preparations was significantly reduced. In intact CASMCs, endothelin-1 (a lysosomal Ca2+ release agonist) induced a large lysosome-associated Ca2+ release, which was significantly attenuated by transfection of TRP-ML1 siRNA in the cells. However, oxotremorine-induced Ca2+ release in CASMCs was not altered by TRP-ML1 siRNA. In addition, ultrasound microbubble introduction of NAADP into TRP-ML4-human fibroblastcells did not produce any Ca2+ release response. However, when these TRP-ML-/- cells were transfected with a full-length TRP-ML1 gene, NAADP-induced Ca2+ release response was restored. We conclude that lysosomal TRP-ML1 is functioning as NAADP-sensitive Ca2+release channels. [ABSTRACT FROM AUTHOR]