Nitric Oxide Ameliorates Hydrophobic Bile Acid-induced Apoptosis in Isolated Rat Hepatocytes by Non-mitochondrial Pathways

Eric Gumpricht,Rolf Dahl,Baruch Yerushalmi,Michael W Devereaux,Ronald J Sokol

doi:10.1074/jbc.m112305200

Abstract

Hydrophobic bile acids are toxic to isolated rat hepatocytes by mechanisms involving mitochondrial dysfunction and oxidative stress. In the current study we examined the role of nitric oxide (NO), a potential mediator of apoptosis, during bile acid-induced apoptosis. Freshly isolated rat hepatocytes and hepatic mitochondria generated NO and peroxynitrite (ONOO(-)) in a concentration- and time-dependent manner when exposed to the toxic bile salt glycochenodeoxycholate (GCDC) (25-500 microm), which was prevented by the nitric-oxide synthase (NOS) inhibitors N(G)-monomethyl-N-arginine monoacetate (l-NMMA) and 1400W. Relationships between hepatocyte NO production and apoptosis were examined by comparing the effects of NOS inhibitors with other inhibitors of GCDC-induced apoptosis. Inhibitors of caspases 8 and 9, the mitochondrial permeability transition blocker cyclosporin A, and the antioxidant idebenone reduced NO generation and apoptosis in GCDC-treated hepatocytes. In contrast, NOS inhibitors had no effect on GCDC-induced apoptosis despite marked reduction of NO and ONOO(-). However, treatment with the NO donors S-nitroso-N-acetylpenicillamine and spermine NONOate [N-(-aminoethyl)N-(2-hydroxy-2-nitrohydrazino)-1,2-ethylenediamine) inhibited apoptosis and caspase 3 activity while significantly elevating NO levels above GCDC-stimulated levels. Neither NO donors nor NOS inhibitors affected GCDC-induced mitochondrial permeability transition or cytochrome c release from liver mitochondria or GCDC-induced mitochondrial depolarization from isolated hepatocytes, suggesting that NO inhibits bile acid-induced hepatocyte apoptosis by a non-mitochondrial-dependent pathway. In conclusion, whereas NO produced from GCDC-treated hepatocytes neither mediates nor protects against bile acid-induced apoptosis, higher levels of NO inhibit GCDC-induced hepatocyte apoptosis by caspase-dependent pathways.

Highlights

The accumulation and toxicity of hydrophobic bile acids within the liver play an important role in the pathogenesis of cholestatic liver disorders and congenital defects in bile acid synthesis and transport [1]
The objectives of this study were to determine 1) if nitric oxide (NO) or its reaction product with superoxide, ONOOϪ, was generated from isolated rat hepatocytes or liver mitochondria exposed to concentrations of GCDC previously observed to promote hepatotoxicity and the mitochondrial permeability transition (MPT), 2) if modulation of NO status by employing either NO synthases (NOS) inhibitors or NO donors prevented or promoted hepatocyte apoptosis induced by GCDC, and 3) the effect of NOS inhibitors and NO donors on mitochondrial pathways involved in GCDC-induced apoptosis
We have demonstrated that neither NO nor ONOOϪ play a role in mediating hepatocellular apoptosis induced by hydrophobic bile acids and that elevated NO levels may have an anti-apoptotic effect that does not involve mitochondrial pathways of apoptosis

Summary

Introduction

The accumulation and toxicity of hydrophobic bile acids within the liver play an important role in the pathogenesis of cholestatic liver disorders and congenital defects in bile acid synthesis and transport [1]. With little evidence assessing the importance of NO as a primary mediator of bile acid-induced hepatocyte apoptosis, the current study was performed to elucidate if NO promoted or protected against this injury In these studies, freshly isolated rat hepatocytes were used rather than cultured or hepatoma cell lines so that homeostasis of physiologic bile acid uptake [25] and endogenous antioxidant pathways [26] were maintained to better reflect the in vivo conditions observed during cholestatic liver diseases and so that physiologic apoptotic pathways were intact. The objectives of this study were to determine 1) if NO or its reaction product with superoxide, ONOOϪ, was generated from isolated rat hepatocytes or liver mitochondria exposed to concentrations of GCDC previously observed to promote hepatotoxicity and the MPT, 2) if modulation of NO status by employing either NOS inhibitors or NO donors prevented or promoted hepatocyte apoptosis induced by GCDC, and 3) the effect of NOS inhibitors and NO donors on mitochondrial pathways involved in GCDC-induced apoptosis

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Conclusion