Figure 4.

Vesicular ATP release by white adipocytes in response to Ca²⁺-free medium application. Effects of secretion inhibitors, knockdown of Cx43 and Ca²⁺ chelators on the vesicular secretion. (A,D) Images of the near-membrane localization of ATP-containing vesicles stained with quinacrine, obtained using TIRF microscopy before and after application of the Ca²⁺-free medium (+Ca²⁺-free), to control white adipocytes (A) and cells with Cx43 knockdown (Cx43-KD) (D). A single white adipocyte is presented. (B) Dynamics of ATP-containing vesicle secretion obtained using TIRF microscopy, reflecting increased secretion (decrease in quinacrine fluorescence) upon application of the Ca²⁺-free medium in the control (black curves) and with 50 ng/mL TeNT (red curves), an inhibitor of Ca²⁺-dependent vesicular fusion. (C) Effect of pre-incubation of white adipocytes for 40 min with 50 µM of the Ca²⁺ chelator, BAPTA-AM, on the Ca²⁺-free medium-induced Ca²⁺ oscillations of white adipocytes. Shown is the typical Ca²⁺ responses of the white adipocytes. Shown is the typical Ca²⁺ responses of 27 ± 11% (red line) and 32 ± 8% (black line) white adipocytes. (E) Summary data illustrating the peak frequency of the Ca²⁺-free medium-induced fusion of the ATP-containing vesicles recorded in white adipocytes without stimuli (Control), with Ca²⁺-free medium application and the Ca²⁺-free medium with 50 ng/mL tetanus toxin (TeNT), an inhibitor of Ca²⁺-dependent vesicular fusion, 1 µM Bafilomycin A1 (BafA), a vacuolar ATPase inhibitor, 50 µM BAPTA-AM (BAPTA), a Ca²⁺ chelator, and Cx43-KD—Cx43 gene knockdown using Gja1 siRNA. Statistical analyses were performed by paired t-test. Significance between groups means: ** p < 0.01 and *** p < 0.001.