ABSTRACT
EDHF-type relaxations are attenuated by the steroidal compounds ouabain and 18a-glycyrrhetinic acid and connexin-mimetic peptides homologous to the Gap 26 and 27 domains of connexin43 (Cx43). In the present study the effects of these agents on the function and integrity of gap junctions were investigated in cultured cells. HeLa cells were transfected with c D N A encoding a chimeric Cx43 protein in which the carboxyl terminus on Cx43 was fused to the amino terminus of Green Fluorescent Protein (GFP), thus establishing functional gap junctions that can be directly visualized by G F P autofluorescence. Functionality was monitored by microinjection of Lucifer yellow into individual cells. Incubation with either 4 3 G a p 26 or 4 3 G a p 27 (5 x 10~4 M ) resulted in about 50% reduction in dye transfer. Incubation with 18a-glycyrrhetinic acid (2.5 x 10~5 M ) reduced functionality by about 90% as did ouabain at concentrations of 1 x 1 0 ~ 6 M - 1 x 1 0 ~ 4 M , but ouabain was without effect at 1 x 10" 7 M . By contrast in A7r5 cells, ouabain concentrations of 3 x 10~4 M and above were necessary to attenuate the transfer of the dye. Visualization of Cx43-GFP by fluorescence microscopy showed that the peptides affected neither the number nor the distribution of gap junction plaques in the cell membrane. Time lapse microscopy of A7r5 cells expressing Cx43-GFP was used to analyse the effect of 18o;-glycyrrhetmic acid and ouabain on gap junction stability and integrity. Disassembly and internalization of plaques was observed around 30 minutes after the addition of 2.5 x 10~5 M 18a;-glycyrrhetinic acid to the cells. Gap junctions were not disrupted following treatment with 1 m M ouabain. The results suggest that connexin-mimetic peptides, 18a-glycyrrhetinic acid and ouabain block gap junctional communication by different mechanisms.
