Downregulation of Gap Junctions in Astrocytes by Monoclonal Antibodies against the Second Extracellular Loop of Connexin-43

Abstract

Downregulation of gap junctions by monoclonal antibodies against the second extracellular loop of connexin-43 (E2Cx43) was studied in a passaged culture of astrocytes. The results of confocal laser scanning microscopy demonstrated that, after two hours of coincubation of cells loaded with Calcein AM and Dil according to Goldberg et al. and unlabelled cells, the cytoplasmic dye Calcein AM was actively transferred to unlabelled cells through newly formed gap junctions. This transfer could be almost completely blocked by addition of 60 μg/ml of anti-E2Cx43 antibodies. Flow cytometric analysis showed that, in experiments carried out according to Goldberg et al., with approximately 2% of labeled cells added to unlabelled ones, about 2.5% of the total cell population took up Calcein AM through gap junctions, thus forming a cell pool characterized by low-intensity green fluorescence. In the presence of antibodies, the proportion of these cells was no more than 0.6%, which indicates an at least fourfold suppression of the gap junction function by E2Cx43 antibodies. The data obtained were reproduced in several independent series. Thus, we obtained monoclonal antibodies capable of modulating the gap junction function in cultures of Cx43-positive cells.