Both B1R and B2R Act as Intermediate Signaling Molecules in High Glucose-Induced Stimulation of Glutamate Uptake in ARPE Cells

Bradykinin (BK) is a potent modulator of biological processes in the retina, and retinal pigment epithelial cells (RPE) and the regulation of glutamate are believed to be important in the pathogenesis of diabetic retinopathy. However, the mechanism by which BK regulates glutamate uptake in RPE cells in diabetic retinopathy is unknown. Here, we examined the involvement of BK receptors in high glucose-induced dysfunction of glutamate uptake in human ARPE cells. High glucose stimulated glutamate uptake and the expression of excitatory amino acid transporter-4 (EAAT4) mRNA, and these were blocked by treatment with small interfering RNA (siRNA) for BK1 receptor (B1R) and BK2 receptor (B2R), but not scrambled siRNA, supporting an involvement of B1R and B2R in this process. High glucose-stimulated glutamate uptake was also blocked by the B1R antagonist [des-Arg(10)]-HOE 140 and the B2R antagonist HOE 140. High glucose increased B1R and B2R mRNA and protein expression in a time-dependent manner, increased B1R and B2R translocation from the cytosol to the nucleus, and stimulated kininogen, kallikrein, and kininase 1 mRNA expression. We examined whether BK receptors were involved in high glucose-induced signaling pathways. High glucose stimulated arachidonic acid release, cytosolic phospholipase A(2) and cyclooxygenase-2 proteins, nuclear factor-kappa B activation, and inhibitor-kappa B activation; these events were blocked by treatment with B1R and B2R siRNAs, but not scrambled siRNA. In addition, high glucose-induced stimulation of glutamate uptake was blocked by the cyclooxygenase-2 inhibitors arachidonyl trifiuoromethyl ketone, mepacrine, 5-bromo-2-(4-fluorophenyl)-3-[4-(methyl-sulfonyl)phenyl]thiophene, and N-[2-cyclohexyloxy-4-nitrophenyl] methane-sulfonamide, and by the nuclear factor-kappa B inhibitors pyrrolidine dithiocarbamate and SN-50. J. Cell. Physiol. 221: 677-687, 2009. (C) 2009 Wiley-Liss, Inc.