Disruption of canalicular function in isolated rat hepatocyte couplets caused by cyclosporin A

Isolated rat hepatocyte couplets were used to study the effects of different concentrations of cyclosporin A in relation to canalicular function. Canalicular function was assessed by counting the percentage of couplets which were able to accumulate the fluorescent cholephile cholyl lysyl fluorescein (CLF) into the canalicular vacuole between the two cells, i.e. canalicular vacuole accumulation (CVA). At lower doses, the immunosuppressor increased the CVA, reaching 121 +/- 3.86% of control at 25 nM cyclosporin A. However, higher doses of cyclosporin A induced a concentration-dependent inhibition of CVA to 64.0 +/- 3.51% of control at 100 nM. Modifications in canalicular area (as % couplet area) were also observed. Image analysis of the fluorescent image showed that cyclosporin A (25 nM) increased canalicular area by 25% (of control); however, this parameter decreased to 36% of control at 100 nM cyclosporin A. In addition, at 100 nM, cyclosporin A reduced the proportion of couplets retaining CLF within the canaliculus to 75.0 +/- 6.59% of control. Treatment of couplets with cyclosporin A (0-2 microM) for 15 min revealed that reduced glutathione (GSH) intracellular content does not change significantly at these doses. However, alteration in pericanalicular F-actin at 100 nM cyclosporin A may be an important factor in the disruption of the canalicular function induced by higher doses of the immunosuppressor.