Azathioprine acts upon rat hepatocyte mitochondria and stress-activated protein kinases leading to necrosis: protective role of N-acetyl-L-cysteine.

Azathioprine is an immunosuppressant drug widely used. Our purpose was to 1) determine whether its associated hepatotoxicity could be attributable to the induction of a necrotic or apoptotic effect in hepatocytes, and 2) elucidate the mechanism involved. To evaluate cellular responses to azathioprine, we used primary culture of isolated rat hepatocytes. Cell metabolic activity, reduced glutathione, cell proliferation, and lactate dehydrogenase release were assessed. Mitochondria were isolated from rat livers, and swelling and oxygen consumption were measured. Mitogen-activated protein kinase pathways and proteins implicated in cell death were analyzed. Azathioprine decreased the viability of hepatocytes and induced the following events: intracellular reduced glutathione (GSH) depletion, metabolic activity reduction, and lactate dehydrogenase release. However, the cell death was not accompanied by DNA laddering, procaspase-3 cleavage, and cytochrome c release. The negative effects of azathioprine on the viability of hepatocytes were prevented by cotreatment with N-acetyl-L-cysteine. In contrast, 6-mercaptopurine showed no effects on GSH content and metabolic activity. Azathioprine effect on hepatocytes was associated with swelling and increased oxygen consumption of intact isolated rat liver mitochondria. Both effects were cyclosporine A-sensitive, suggesting an involvement of the mitochondrial permeability transition pore in the response to azathioprine. In addition, the drug's effects on hepatocyte viability were partially abrogated by c-Jun N-terminal kinase and p38 kinase inhibitors. In conclusion, our findings suggest that azathioprine effects correlate to mitochondrial dysfunction and activation of stress-activated protein kinase pathways leading to necrotic cell death. These negative effects of the drug could be prevented by coincubation with N-acetyl-L-cysteine.

Insulin receptor substrate-4 signaling in quiescent rat hepatocytes and in regenerating rat liver.

This study was designed to characterize insulin receptor substrate-4 (IRS-4) in isolated rat hepatocytes and to examine its role in liver regeneration. Subcellular fractionation revealed that 85% of IRS-4 is located at isolated hepatocyte plasma membranes. The distribution of IRS-4 among intracellular compartments remained unchanged in insulin-stimulated cells. Two bands corresponding to 145 and 138 kd were observed in immunoblotting experiments. Immunoprecipitation of hepatocyte lysates with a highly specific antibody against IRS-4 led to an insulin and insulin-like growth factor 1 (IGF-1)-dependent increase in phosphotyrosine residues of the 145-kd band. IRS-4 was found to be associated with Src homology 2 (SH2) domain-containing proteins (phosphatidylinositol 3-kinase [PI 3-kinase] and Src homology phosphatase [SHP-2]) and with protein kinase C zeta (PKC zeta). Insulin and IGF-1 elicited a rapid and dose-dependent binding of these 3 proteins to IRS-4. These data suggest that IRS-4 is insulin-/IGF-1-activated by phosphorylation and not by translocation, inducing the recruitment of SH2 domain-containing proteins and PKC zeta to the membrane. To evaluate the possible role of IRS-4 in liver regeneration, we also examined this system after partial hepatectomy (PH). One day after PH, IRS-1 expression increased, consistent with a stimulatory role in the regenerative process, whereas it decreased 7 days after liver resection. This drastic IRS-1 depletion occurred at the expense of increased IRS-2 and IRS-4 expression 7 days after PH. In addition, at this period of time after surgery, the in vivo insulin stimulation of remnant rat livers showed an increase in IRS-4/PI 3-kinase association. Given that 1 and 7 days after PH isolated hepatocytes responded similarly to insulin in terms of induced cell proliferation, a compensatory role is proposed for IRS-2/4 induction. In conclusion, IRS-4 is activated by insulin and IGF-1-like IRS-1 in rat hepatocytes, and the induced expression of IRS-4 is a compensatory mechanism that plays a role in conditions of liver regeneration.

Cyclosporin A induced internalization of the bile salt export pump in isolated rat hepatocyte couplets.

Isolated rat hepatocyte couplets were used to perform the comparative study of two widely used immunosuppressors, cyclosporin A (CsA) and tacrolimus (FK506) on hepatocanalicular function. We assessed canalicular function by counting the percentage of couplets that were able to accumulate the fluorescent cholephile, cholyl-lysyl-fluorescein (CLF), into the canalicular vacuole between the two cells, i.e., canalicular vacuole accumulation (CVA) of CLF. Compared to controls (DMSO-treated cells), CsA, in the approximate range of concentrations used therapeutically, caused inhibition of CVA of CLF, disorganization of the bile salt export pump (Bsep) localization at canalicular level resulting in its relocation into the cell, and disruption of the pericanalicular F-actin cytoskeleton. In contrast, FK506, at both approximately therapeutic and supratherapeutic concentrations, had no deleterious effect upon CVA of CLF, upon the localization of the bile salt transporter at the canalicular membrane, or on the organization of the pericanalicular F-actin cytoskeleton. These results point to transporter and cytoskeletal disorganization as contributors or determinants of CsA-induced cholestasis at canalicular level, whereas FK506 does not appear to produce these cholestasis-determining responses even at supratherapeutic concentrations.

Pretreatment with FK506 up-regulates insulin receptors in regenerating rat liver.

This report examines the effect of FK506 pretreatment on liver insulin receptor expression in partially (70%) hepatectomized rats. FK506 pretreatment led to an increased insulin receptor number 24 hours after hepatectomy, detected by means of insulin binding and cross-linking procedures. This increase was related to enhanced insulin receptor expression determined by in vitro mRNA translation and Western blot techniques. We also tested the functionality of the expressed insulin receptors by [(3)H] thymidine incorporation into DNA in insulin-stimulated hepatocytes. The results show that FK506 pretreatment elicits an increase in the amount of insulin receptor alpha-subunits as measured by Western blot. Maximum alpha-subunit expression recorded 24 hours after surgery was preceded by increased insulin receptor mRNA levels, which were detected 6 hours after hepatectomy. Moreover, in FK506-pretreated rat hepatocytes, obtained from remnant livers 24 hours after partial hepatectomy (PH), the increase in insulin receptor number was associated with improved sensitivity to the hormone. However, in both experimental groups (FK506-pretreated and nonpretreated rats), the sensitivity of hepatocytes toward epidermal growth factor (EGF) showed no significant change, which suggests a specific effect of FK506 on insulin receptor expression. In conclusion, our findings suggest that FK506 pretreatment induces insulin receptor expression in regenerating rat liver and promotes liver regeneration in hepatectomized rats.