title: Inflammatory cytokine signaling in insulin producing beta-cells enhances the colocalization correlation coefficient between L-type voltage-dependent calcium channel and calcium-sensing receptor.
abstract: The immunological processes in type 1 diabetes and metabolic/inflammatory disorder in type 2 diabetes converge on common signaling pathway(s) leading to beta-cell death in these two diseases. The cytokine-mediated beta-cell death seems to be dependent on voltage-dependent calcium channel (VDCC)-mediated Ca2+ entry. The Ca2+ handling molecular networks control the homeostasis of [Ca2+]i in the beta-cell. The activity and membrane density of VDCC are regulated by several mechanisms including G protein-coupled receptors (GPCRs). CaR is a 123-kDa seven transmembrane extracellular Ca2+ sensing protein that belongs to GPCR family C. Tumor necrosis factor-alpha (TNF-alpha), is a cytokine widely known to activate nuclear factor-kappaB (NF-kappaB) transcription in beta-cells. To obtain a better understanding of TNF-alpha-induced molecular interactions between CaR and VDCC, confocal fluorescence measurements were performed on insulin-producing beta-cells exposed to varying concentrations of TNF-alpha and the results are discussed in the light of increased colocalization correlation coefficient. The insulin producing beta-cells were exposed to 5, 10, 20, 30, and 50 ng/ml TNF-alpha for 24 h at 37 degrees . The cells were then immunolabelled with antibodies directed against CaR, VDCC, and NF-kappaB. The confocal fluorescence imaging data showed enhancement in the colocalization correlation coefficient between CaR and VDCC in beta-cells exposed to TNF-alpha thereby indicating increased membrane delimited spatial interactions between these two membrane proteins. TNF-alpha-induced colocalization of VDCC with CaR was inhibited by nimodipine, an inhibitor of L-type VDCC thereby suggesting that VDCC activity is required for spatial interactions with CaR. The 3-D confocal fluorescence imaging data also demonstrated that addition of TNF-alpha to RIN cells led to the translocation of NF-kappaB from the cytoplasm to the nucleus. Such molecular interactions between CaR and VDCC in tissues possibly provide control over Ca2+ channel activity via direct protein-protein contact.
authors.affiliation: Robert Stempel School of Public Health, Department of Environmental and Occupational Health, Florida International University, Miami, FL 33199, USA. parkashj@fiu.edu
authors.forename: Jai
authors.initials: J
authors.lastname: Parkash
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000818:Animals; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D007249:Inflammation / Q000378:metabolism*; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D051381:Rats; D044169:Receptors, Calcium-Sensing / Q000378:metabolism*; D015398:Signal Transduction; D013223:Statistics as Topic; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D020746:Calcium Channels, L-Type; D016328:NF-kappa B; D044169:Receptors, Calcium-Sensing; D014409:Tumor Necrosis Factor-alpha