Lactic acid inhibition of gap junctional intercellular communication in in vitro astrocytes as measured by fluorescence recovery after laser photobleaching

Abstract

Lactic acid can permeate plasma membranes, causing intracellular acidosis. Gap junctions are sensitive to pHi and can be reversibly uncoupled by weak acids. In this study, dye coupling between in vitro astrocytes, presumably mediated by gap junctions, was measured in the absence and presence of lactic acid. Fluorescence recovery after laser photobleaching (gap-FRAP analysis) was used to measure dye coupling. Astrocytes bathed in Eagle's minimum essential medium (EMEM) with lactic acid, pHo 5.5-6, showed no difference in their dye coupling (mean recovery of fluorescence 30%) when compared to control astrocytes (mean recovery of fluorescence 26%). However, 24 mM lactic acid in EMEM, pHo 4.5, decreased dye coupling (mean recovery of fluorescence 2.0%). This effect occurred within 5 min of treatment. When lactic acid-EMEM, pH 4.5, was removed from astrocytes after 30 min and the cells were incubated in EMEM for 24 hr, decreased coupling was not reversed (mean recovery 4.0%). When lactic acid-treated astrocytes were incubated in EMEM for 48 hr, the mean recovery of fluorescence increased to 15% (i.e., 42% of the recovery seen in controls). These observations suggest that brief exposure to high concentrations of lactic acid can have immediate and long-lasting effects on glial gap junctional communication. Under pathological circumstances, such a sequence could be initiated, and this might impair astrocytic control of the central nervous system microenvironment mediated by spatial buffering.