General anesthetics attenuate gap junction coupling in P19 cell line

Abstract

Gap junction communication is widespread throughout the mammalian nervous system among neurons as well as glia. We addressed the hypothesis that general anesthetics attenuate gap junction mediated coupling in P19 cell line that can differentiate into neuronal-like cells and astrocytes and oligodendrocytes. We characterized the extent of dye coupling over time in the P19 cell line using colocalization of chlormethylbenzamido-1,1 dioctadecyl-3,3,3',3'-tetramethylindocarbocyamine (CM-DiI) and calcein-AM in donor and recipient cells in cocultures. After seeding, the gap junction permeant dye calcein spreads from donor to recipient cells. CM-DiI and calcein fluorescence identified donor and recipient cells, respectively. The extent of intercellular connections was evaluated using cell counting and flow cytometry up to 2 hr after treatment. Clinically relevant concentrations of the intravenous anesthetics propofol (15 microM) and thiopental (10 microM) attenuated gap junction permeability in P19 cell cultures. In contrast, halothane, a volatile anesthetic in a concentration (0.64 mM) relevant to its free aqueous EC50 had no effect on gap junction coupling; however, very high halothane concentrations (2.8 mM) blocked dye transfer by approximately 90%. The results indicate that halothane concentrations pertinent to clinical anesthesia were unable to attenuate gap junction communication in a cell line that can express neuronal and glial gap junction proteins; however, clinically relevant concentrations of propofol and thiopental depressed gap junction coupling.