Signaling modulation of bile salt-induced necrosis in isolated rat hepatocytes.
Hydrophobic bile salts induce either necrosis or apoptosis depending on the severity of the injury caused by them. Since bile salt-induced apoptosis is influenced by Ca2+- and protein kinase-signaling pathways, and both necrosis and apoptosis share common initiating mechanisms, we analyzed whether these signaling cascades also influence bile salt-induced necrosis in isolated rat hepatocytes. Taurochenodeoxycholate (TCDC, 0.25-1.50 mM, 2 h) reduced, in a dose-dependent manner, the percentage of viable hepatocytes, and increased the release of the cytosolic enzyme, lactate dehydrogenase (LDH) and alanine aminotransferase (ALAT), and that of the plasma membrane enzyme, alkaline phosphatase (AP). The PKC inhibitors, H7 (100 microM) and chelerythrine (2.5 microM), both prevented significantly TCDC-induced necrosis. On the contrary, the PKA activator, dibutyryl-cAMP, exacerbated TCDC-induced cell damage in a dose-dependent manner; this effect was more likely due to cAMP-mediated PKA activation, as the PKA inhibitor, KT5720 (1 microM), counteracted this effect. Instead, the intracellular Ca2+ chelator, BAPTA/AM (20 microM), was without effect. TCDC (1 mM) increased lipid peroxidation from 0.7 +/- 0.2 to 7.5 +/- 0.9 nmol of malondialdehyde per mg of protein, p < 0.001; the addition of the free radical scavenger, diphenyl-p-phenylendiamine, completely blocked this increase and prevented significantly TCDC-induced necrosis. PKC inhibition induced only a slight attenuation of TCDC-induced lipid peroxidation. Possible mechanisms accounting for the modulatory effect of signal transduction pathways on TCDC-induced necrosis, including signaling influence on TCDC transport events and TCDC-induced oxidative stress, are discussed.
Borgognone
Mariana
M
Institute of Experimental Physiology, CONICET-National University of Rosario, Rosario (S2002LRL), Argentina.
Pérez
Leonardo M
LM
Basiglio
Cecilia L
CL
Ochoa
Justina E
JE
Roma
Marcelo G
MG
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001647:Bile Acids and Salts / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D008054:Lipid Peroxides / Q000378:metabolism; D008297:Male; D009336:Necrosis / Q000201:enzymology; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000187:drug effects*; D013655:Taurochenodeoxycholic Acid / Q000494:pharmacology
D001647:Bile Acids and Salts; D004791:Enzyme Inhibitors; D008054:Lipid Peroxides; D013655:Taurochenodeoxycholic Acid; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D002118:Calcium