Anti-NPPB monoclonal antibody, clone 24C5

Liu R, Juncos LA. GTPase-Rac enhances depolarization-induced superoxide production by the macula densa during tubuloglomerular feedback. Am J Physiol Regul Integr Comp Physiol 298: R453-R458, 2010. First published December 9, 2009; doi:10.1152/ajpregu.00622.2009.-Superoxide (O-2(-)) enhances tubuloglomerular feedback (TGF) by scavenging nitric oxide at the macula densa (MD). The primary source of O-2(-) in the MD during TGF is NADPH oxidase, which is activated by membrane depolarization. While Rac, a small GTP-binding protein, has been shown to enhance NADPH oxidase activity, its role in O-2(-) generation by the MD is unknown. We hypothesized that depolarization of the MD leads to translocation of Rac to the apical membrane, and its activation, in turn, augments O-2(-) generation during TGF. We tested this by measuring membrane potential and increased O-2(-) levels during TGF responses in isolated, perfused tubules containing the intact MD plaque. Switching tubular NaCl from 10 to 80 mM, which induces TGF, depolarized membrane potential by 28.4 +/- 4.5% from control (P < 0.05) and O-2(-) levels from 124 +/- 19 to 361 +/- 27 U/min. This NaCl-induced depolarization and O-2(-) generation were blocked by a Cl- channel blocker, 5-nitro-2(3-phenylpropylamino) benzoic acid (NPPB; 10(-6) M). Inhibition of Rac blunted NaCl-induced O-2(-) generation by 47%. When the NaCl content of the MD perfusate was increased from 10 to 80 mM, immunointensity of Rac on the apical side increased from 32 +/- 3.1 to 46 +/- 2.5% of the total immunofluorescence in the MD, indicating that high NaCl induces the translocation of Rac to the apical membrane. This NaCl-induced Rac translocation was blocked by a Cl- channel blocker, NPPB, indicating that depolarization of the MD induced Rac translocation. In conclusion, we found that depolarization of the MD during TGF leads to translocation of Rac to the apical membrane, which enhances O-2(-) generation by the MD.

The aim of this study was to investigate the proliferation, adhesion, and migration function of chloride ion channels in HLEC B-3, a human lens epithelial cell line, and provide experimental basis for studying the mechanism of human after-cataract as well as the effective methods for controlling and treating it. MTT and Scratch assay methods were applied to detect the proliferation, adhesion, and migration of HLEC B-3 after applying 5, 10, 20, 50, 100, and 200 mu mol/L of chloride ion channel blockers (NPPB). In the cell proliferation experiment, no apparent difference was found in the first 12 and 24 h after treatment with 10, 20, 50, 100 and 200 mu mol/L NPPB or 48 h after treatment with 20, 50, 100 and 200 mu mol/L NPPB. The cell proliferations were obviously inhibited compared with the control group(without NPPB treatment), and the difference was significant (P < 0.05 or P < 0.01). Cell adhesion experimental results showed that when the cells were treated with 10, 20, 50, 100, and 200 mu mol/L NPPB for 12 or 24 h, cell adhesions were obviously inhibited. The difference was very significant (P < 0.05 or P < 0.01) compared with the control group. The activity of chloride ion channels can inhibit the proliferation, adhesion, and migration of LECs; therefore, inhibiting its activity can control the occurrence of human after-cataracts.