Vasopressin-induced disruption of actin cytoskeletal organization and canalicular function in isolated rat hepatocyte couplets: possible involvement of protein kinase C.
The effect of vasopressin (VP) on canalicular function and hepatocellular morphology, with particular regard to actin cytoskeletal organization and the concomitant plasma membrane bleb formation, was studied in isolated rat hepatocyte couplets. VP induced the concentration-dependent formation of multiple plasma membrane blebs as well as simultaneous impairment in both canalicular vacuolar accumulation (cVA) and retention (cVR) of the fluorescent bile acid, cholyl-lysyl-fluorescein (CLF), which evaluate couplet secretory function and tight-junction integrity, respectively. These effects were mimicked by the protein kinase C (PKC) activator, phorbol dibutyrate (PDB), but not by the protein kinase A (PKA) activator, dibutyryl-cAMP. VP-induced bleb formation and canalicular dysfunction were fully prevented by the protein kinase inhibitor, H-7, but not by the PKA inhibitor, KT5720, further suggesting a specific role of PKC. VP-induced alterations were also prevented by pretreatment with the Ca2+-buffering agent, BAPTA/AM, but not with the calmodulin-dependent protein kinase II antagonist, calmidazolium. Neither the Ca2+-activated neutral protease inhibitor, leupeptin, nor the antioxidants, alpha-tocopherol or deferoxamine, were able to prevent either VP-induced plasma membrane blebbing or canalicular dysfunction. The Ca2+-ionophore, A23187, mimicked the VP-induced alterations, but its harmful effects were completely prevented by H-7. Bleb formation induced by VP and PDB was accompanied by an extensive redistribution of filamentous actin from the pericanalicular area to the cell body, and this effect was fully prevented by H-7. These results suggest that VP-induced canalicular and cytoskeletal dysfunction is mediated by PKC and that classical (Ca2+-dependent) PKC appear to be involved because intracellular Ca2+ is required for VP to induce its harmful effects.
Roma
M G
MG
Institute of Experimental Physiology (IFISE), School of Biochemical and Pharmacological Science, The University of Rosario-CONICET, Rosario, Argentina.
Stone
V
V
Shaw
R
R
Coleman
R
R
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000494:pharmacology; D000199:Actins / Q000378:metabolism*; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001648:Bile Canaliculi / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D003994:Bucladesine / Q000494:pharmacology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D002227:Carbazoles; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003599:Cytoskeleton / Q000187:drug effects / Q000648:ultrastructure*; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D008099:Liver / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D008297:Male; D008855:Microscopy, Electron, Scanning; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D011758:Pyrroles / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D019108:Tight Junctions / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D014617:Vacuoles / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D014667:Vasopressins / Q000494:pharmacology*
D000199:Actins; D000975:Antioxidants; D002227:Carbazoles; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D007093:Imidazoles; D007211:Indoles; D007976:Leupeptins; D011758:Pyrroles; D014667:Vasopressins; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D015240:Phorbol 12,13-Dibutyrate; C031938:calmidazolium; D004533:Egtazic Acid; C057416:KT 5720; D003994:Bucladesine; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C032854:leupeptin