18-beta-Glycyrrhetinic acid (BGA) as an electrical uncoupler for intracellular recordings in confluent monolayer cultures.
In the study of epithelial cell biology, primary cell cultures or cell lines grown to confluency offer considerable advantages compared with isolated cells and cell clusters. This is due mainly to the development of appropriate cell-to-cell contacts that are a prerequisite for cell polarity and thus vectorial solute transport. On the other hand, electrical coupling via gap junctions in most instances significantly hinders the use of voltage-clamp techniques for electrophysiological analysis of transport processes in single cells. In the present study we employed the gap junctional blocker 18-beta-glycyrrhetinic acid (BGA) to reduce electrical cell-to-cell coupling in confluent primary cultures of rat hepatocytes. In current-clamp experiments, 40 micromol/l BGA reversibly increased apparent cell input resistance approximately tenfold. Due to this partial electrical isolation of cells, two-channel voltage-clamp experiments became feasible and, for the first time, the hypertonicity-induced Na+ conductance of rat hepatocytes could be analysed quantitatively. In ion substitution experiments, however, it became obvious that BGA, while leaving Na+ and K+ conductances virtually unchanged, completely blocked cell membrane Cl- conductance. This additional effect of BGA necessitates independent control experiments to ensure that the transport process under consideration is itself not changed by the compound. Nevertheless, BGA may serve as a powerful tool for the quantitative electrophysiological study of epithelial cells that are in quasi physiological contact with their neighbours.
Böhmer
C
C
Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
Kirschner
U
U
Wehner
F
F
D000818:Animals; D001692:Biological Transport; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism; D004553:Electric Conductivity; D017629:Gap Junctions / Q000502:physiology*; D006034:Glycyrrhetinic Acid / Q000031:analogs & derivatives / Q000494:pharmacology*; D022781:Hepatocytes / Q000187:drug effects* / Q000502:physiology*; D018408:Patch-Clamp Techniques; D051381:Rats; D012964:Sodium / Q000378:metabolism
D002712:Chlorides; D012964:Sodium; D006034:Glycyrrhetinic Acid