Effect of oxidative stress and disruption of Ca2+ homeostasis on hepatocyte canalicular function in vitro.
Isolated rat hepatocyte couplets were used to study the effects of menadione and a rise in the intracellular concentration of calcium on biliary 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. Menadione induced a concentration-dependent inhibition of the canalicular vacuole accumulation (CVA) of CLF reaching 7.6 +/- 1.8% of control at 100 microM menadione. This disruption was not prevented by blocking receptor-operated calcium channels with Ni2+ (300 microM). The concentration range of menadione used did not deplete cellular ATP content. In contrast glutathione content was reduced to 52% of its control value by 100 microM menadione. A rise in cytosolic calcium induced by the calcium ionophore, A23187 (up to 30 microM) also disrupted CVA in a concentration-dependent manner. Release of endoplasmic reticulum calcium stores by thapsigargin (50 nM) affected the retention of canalicular contents to a much lesser extent, although it was able to stimulate a reduction in canalicular area to 40% of its original value, assumed to be due to canalicular contraction. Menadione (30 and 100 microM) reduced the fluorescence of phalloidin-FITC-labelled F-actin in both the total and pericanalicular cytoskeleton. Canalicular function was therefore disrupted by non-lethal concentrations of menadione via a mechanism which does not appear to involve ATP depletion or the entry of extracellular calcium, but is associated with a depletion of both cellular glutathione and F-actin. An increase in the concentration of intracellular calcium can stimulate canalicular contraction, and at relatively high concentrations calcium can also disrupt canalicular function.
Stone
V
V
School of Biochemistry, University of Birmingham, Edgbaston, U.K.
Johnson
G D
GD
Wilton
J C
JC
Coleman
R
R
Chipman
J K
JK
D000199:Actins / Q000032:analysis; D000255:Adenosine Triphosphate / Q000032:analysis; D000818:Animals; D001648:Bile Canaliculi / Q000187:drug effects* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D003599:Cytoskeleton / Q000187:drug effects; D005453:Fluorescence; D005978:Glutathione / Q000032:analysis; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D014812:Vitamin K / Q000494:pharmacology*
D000199:Actins; D013729:Terpenes; D014812:Vitamin K; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D005978:Glutathione; D002118:Calcium