CONCURRENT ANALYSIS OF INTRACELLULAR GLUTATHIONE CONTENT AND GAP JUNCTIONAL INTERCELLULAR COMMUNICATION

The potential for performing dual analysis of intracellular glutathion e levels and assessment of gap junctional intercellular communication with thiol-specific fluorescent probes in anchored cells was evaluated . Gap junction-mediated diffusion of monochlorobimane and 5-chlorometh ylfluorescein diacetate following intracellular loading and conjugatio n with glutathione was compared with 5-carboxyfluorescein diacetate (w hich is routinely used in laser cytometry to monitor intercellular com munication) by means of fluorescence recovery after photobleaching usi ng a variety of communication-competent and communicationin-competent cells. The rate of diffusion of fluorescence among communication-compe tent cells was inversely proportional to the size of the fluorescent p robe employed. The thiol-specific probes were also employed to monitor depletion and synthesis of glutathione following treatments to inhibi t glutathione synthesis or consume glutathione by adduct formation. An alysis of gap junctional intercellular communication following glutath ione depletion revealed a direct correlation between glutathione level s and intercellular communication. These studies support the utility o f the thiol-specific probes to monitor the respective role of cellular glutathione and intercellular communication in the mechanisms of cell ular injury. (C) 1993 Wiley-Liss, Inc.