Reactive oxygen species (ROS) as signaling molecules in hepatocytes: PKC mediates tert-buthyl-hydroperoxide (tBOOH)-induced actin cytoskeletal disorganization without affecting tBOOH-induced ROS formation.

PEREZ LM; OCHOA JE; BASIGLIO CL; SANCHEZ POZZI EJ; ROMA MG

San Salvador de Bahia

Congreso; Biennial Meeting of the International Association for the Study of the Liver (IASL); 2004

IASL

Background and aim: ROS can produce deleterious effects by modulating intracellular signaling pathways rather than by directly oxidizing the affected structures. Since ROS activates PKC, we analyze in rat hepatocytes the role of this signalling molecule in oxidative stress-induced actin disorganization, an early event in ROS-mediated cell damaging. A possible effect of PKC at a pre-oxidative level by modulating tBOOH-induced ROS formation was also investigated. Results: tBOOH (100 µM) increased ROS production, as evaluated by the intracellular formation of the fluorescent probe 2´,7´-dichlorofluorescein (+37 ± 4%, P<0.005). Instead, cell viability, LDH release, total GSH and GSSG to total GSH ratio remained unaffected, indicating moderate oxidative stress level. tBOOH increased the amount of cells displaying plasma membrane blebs, a preliminary marker of actin disorganization (68 ± 4% vs 18 ± 2%, P<0.001). This was further confirmed in isolated hepatocyte couplets, as F-actin (visualized by FITC-phalloidin fluorescent staining followed by confocal microscopy) suffered extensive redistribution from the pericanalicular area to the remaining cellular body (% of pericanalicular location: 73 ± 0.4% vs 30 ± 2%, P<0.001). Both events were prevented by the PKC inhibitors, H7 (100 µM), staurosporin (1 µM) and Gö6976 (2 µM), or by the PKA activator, dibutyryl-cyclic AMP (500 µM); this latter effect was counteracted by the PKA inhibitor KT5720 (5 µM). Conversely, the capacity of tBOOH to induce ROS production was not modified by any of the signalling modulators studied. Conclusions: ROS induces actin cytoskeletal disorganization by a PKC-mediated mechanism rather than by a direct oxidizing effect, an event which is counter-regulated by PKA. PKC seems therefore to act as an intermediate constituent in a transductional cascade initiated by ROS, thus confirming a role of these free radicals as signalling molecules in the hepatocyte.