Connexins regulate calcium signaling by controlling ATP release

Abstract

Forced expression of gap junction proteins, connexins, enables gap junction-deficient cell lines to propagate intercellular calcium waves. Here, we show that ATP secretion from the poorly coupled cell lines, C6 glioma, HeLa, and U373 glioblastoma, is potentiated 5- to 15-fold by connexin expression. ATP release required purinergic receptor-activated intracellular Ca2+ mobilization and was inhibited by Cl- channel blockers. Calcium wave propagation also was reduced by purinergic receptor antagonists and by Cl- channel blockers but insensitive to gap junction inhibitors. These observations suggest that cell-to-cell signaling associated with connexin expression results from enhanced ATP release and not, as previously believed, from an increase in intercellular coupling.

Figure 1

Uncoupling agents fail to reduce astrocytic calcium signaling despite a marked reduction in gap junction coupling. (A) Representative experiment of the propagation of a calcium wave in an astrocytic culture exposed to the gap junction inhibitor, αGA (10 μM) for 10 min. Culture was loaded with fluo-3 and imaged by confocal microscopy. Sequence of images was collected 1, 8, and 16 sec after focal mechanical stimulation (arrow). The color scale is the same as in Fig. 2 and indicates relative changes in fluo-3 signal (ΔF/F) (B) FRAP in matched sister cultures treated with αGA (Right) or vehicle (Left). Both cultures were loaded with the gap junction permeant fluorescence tracer CDCF (2 μM). Red rectangles outline area of photobleach (Top), Middle panels display culture immediately after bleach, and red arrows indicate refill of fluorescence 2 min later (Bottom). αGA reduced refill to 2%, whereas fluorescence recovered to 38% of prebleach level within 2 min in the control culture. (C) Data summarizing effects of αGA and octanol (0.5 mM) upon gap junction coupling (FRAP) and calcium signaling. The extent of astrocytic coupling and calcium signaling was examined before (control) and after addition of inhibitors. Gap junction coupling was calculated as percentage refill of fluorescence signal 2 min after photobleach. Radius of calcium waves was in the range of 200–300 μm during control conditions. ∗, statistically significant difference from control at P < 0.05 by t test; n = 5–15.

Figure 2

Desensitization of purinergic receptors inhibits the extent of calcium signaling in cultured astrocytes. First row shows mechanical-induced astrocytic calcium wave (white arrow). Sequence of images (1, 7, 15 sec after stimulation) depicts a typical calcium wave under control conditions. Second row shows the same field of astrocytes mobilize Ca²⁺i transiently in response to 2MeSATP (10 μM). Third row shows mechanical stimulation of a cell neighboring the cell stimulated in the first block (white arrow) failed to induce a propagating Ca²⁺ wave after exposure to 2MeSATP. Ca²⁺i increases are limited to a few cells surrounding the stimulated cell. Stimulation was performed when Ca²⁺i had returned to resting level after the exposure to 2MeSATP (5–10 min). Wave radius was on an average reduced to 11 ± 8 (n = 8) after exposure to 2MeSATP. Fourth row shows subsequent challenge with bradykinin (25 μM) demonstrating that intracellular calcium stores were not depleted by 2MeSATP exposure and that the cells remained viable.

Figure 3

Increased ATP release as a consequence of connexin expression in C6 glioma cells, HeLa cells, and the human-derived glioblastoma cell line, U373-MG. ATP release was evoked by exposure to 100 μM UTP and supernatant collected 10 min later. ATP release was quantified by using the luciferase–luciferin assay. Null-1 and null-2 are null-transfected C6 control cells, Wt represents wild-type C6 cells; Cx43, Cx32, and Cx26 are stable transfected clones overexpressing connexins. Results from C6 expressing Cx43 and Cx32 represent the mean of three independent clones. Wt/g and Wt/u are HeLa wild-type cells from Germany and U.S. U373 MG cells were transiently transfected with either cDNA for either Cx32 or β-galactosidase (β-gal). Cx expression enhanced ATP release 5- to 15-fold in all cell lines studied.