The protein kinase inhibitor 1-(5-isoquinolinylsulfonyl)-2-methyl-piperzine (H-7) prevents and reverses Ca(2+)-mediated injury in isolated rat hepatocyte couplets.
We have recently shown that protein kinase C (PKC) activation induces similar morphological and functional alterations in couplets to that caused by increments of intracellular Ca(2+). Since certain PKC isoforms are activated by Ca(2+), we tested whether the PKC inhibitor H-7 can counteract the alterations induced by this ion in couplets. The Ca(2+) ionophore A23187, which can mobilize Ca(2+) from extracellular and intracellular sources, decreased, in a dose-dependent manner, the percentage of couplets accumulating the fluorescent bile acid analogue cholyl-lysyl-fluorescein (CLF) in their canalicular vacuoles, i.e., in the canalicular vacuolar accumulation test (cVA of CLF), a measure of the overall capability of the couplets to secrete and retain CLF. To a similar extent, A23187 also decreased the percentage of couplets retaining CLF once secreted, i.e., in the canalicular vacuole retention test (cVR of CLF), a measure of tight junctional integrity. ATP (50 microM), another Ca(2+)-elevating compound, altered canalicular function in a similar extent to A23187. All these functional changes were prevented by H-7 in a dose-dependent manner. Canalicular dysfunction was accompanied by bleb formation and extensive redistribution of F-actin from the pericanalicular area to the cell body, which was also fully prevented by H-7; the intracellular Ca(2+) chelator, 1, 2-bis(o-aminophenoxy)-ethene-N,N,N',N'-tetraacetate tetrakis-(acetomethylester), (BAPTA/AM) (20 microM) had virtually the same preventive effects as H-7. Both H-7 and BAPTA/AM not only prevented but also reversed the decrease in cVA of CLF and blebbing induced by A23187. Thus, H-7 can both prevent and reverse Ca(2+)-mediated hepatocellular injury.
Roma
M G
MG
School of Biochemistry, The University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom.
Ahmed-Choudhury
J
J
Coleman
R
R
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000494:pharmacology*; D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D002793:Cholic Acids; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005452:Fluoresceins; D005453:Fluorescence; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D007476:Ionophores / Q000494:pharmacology; D008099:Liver / Q000187:drug effects* / Q000201:enzymology / Q000648:ultrastructure; D008297:Male; D047428:Protein Kinase Inhibitors; D051381:Rats; D017208:Rats, Wistar; D019108:Tight Junctions / Q000187:drug effects; D014617:Vacuoles / Q000378:metabolism
D000199:Actins; D002614:Chelating Agents; D002793:Cholic Acids; D004791:Enzyme Inhibitors; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007476:Ionophores; D047428:Protein Kinase Inhibitors; C072313:cholyl-lysylfluorescein; D000001:Calcimycin; D004533:Egtazic Acid; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000255:Adenosine Triphosphate; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium