Figure 1.

Effects of suramin on hemichannel permeability. (A) Effects of suramin on cellular uptake of EtBr following removal of extracellular Ca²⁺. NRK cells were pretreated with the indicated concentrations of suramin for 20 min. After that, they were exposed to either normal or Ca²⁺-free culture medium that contained 10 μM EtBr in the presence of the same concentrations of suramin for an additional 15 min. The cellular uptake of EtBr was photographed (magnification, ×320). (B) Quantitation of the cellular fluorescent intensity in (A). Results are expressed as means ± SEM (n = 10, *P < 0.05, #P < 0.01 vs. zero control). (C) Blockade of EtBr uptake by suramin and hemichannel inhibitor. Cells were treated the same as earlier in the presence of 300 μM suramin or 100 μM lindane (magnification, ×160). (D) Effects of suramin on Ca²⁺ depletion triggers ATP release. NRK cells were exposed to Ca²⁺-free culture medium in the presence of the indicated concentrations of suramin for the indicated time intervals. Cell supernatants were collected and quantitated for ATP concentration. Results are expressed as relative light unit (RLU; mean ± SE, n = 3). #P < 0.01 versus zero point control. (E) Effects of suramin and hemichannel inhibitors on ATP release. Cells were exposed to Ca²⁺-free medium in the presence or absence of 300 μM suramin or 100 μM lindane. (F) Effects of suramin and down-regulation of Cx43 with specific siRNA on ATP release. NRK cells were treated with either control siRNA or siRNA against Cx43 for 48 h. Thereafter, cells were exposed to Ca²⁺-free medium in the presence or absence of 300 μM suramin for 5 min. Cell supernatants were collected and quantitated for ATP concentration. Results are expressed as RLU (mean ± SE, n = 3). #P < 0.01 versus control. To verify the effectiveness of Cx43 siRNA in down-regulation of Cx43, the cellular lysates extracted from siControl and siCx43 were subjected to Western blot analysis of Cx43 (Figure 1F, insert). Note the obvious reduced level of Cx43 in Cx43 siRNA-treated cells (right lane).