Characterization of cationic amino acid transporter and its gene expression in rat hepatic stellate cells in relation to nitric oxide production

Abstract

Background/Aims: Nitric oxide is a potent mediator of hepatic sinusoidal hemodynamics and affects hepatic stellate cells (Ito cells, fat-storing cells). Although nitric oxide production may depend on the induction of inducible nitric oxide synthase and on transport of extracellular L-arginine, the precise mechanisms controlling nitric oxide production in stellate cells have not been well characterized. Methods: Using stellate cells prepared from the male Wistar rat, kinetic analysis of L-arginine transport and reverse transcription-polymerase chain reaction for cationic amino acid transporter were carried out. The effect of tumor necrosis factor-α and interferon-γ on L-arginine transport, nRNA expression of cationic amino acid transporter and inducible nitric oxide synthase, and nitric oxide production of stellate cells was assessed. Results: The L-arginine transport system functioning in the transformed hepatic stellate cells was system y +, possibly mediated by cationic amino acid transporter-1 and cationic amino acid transporter-2B (Km ∼50 μM). Tumor necrosis factor-α enhanced cationic amino acid transporter-2B mRNA expression and L-arginine transport, whereas cationic amino acid transporter-1 mRNA expression remained unchanged. Interferon-γ induced the expression of inducible nitric oxide synthase mRNA without obvious changes in L-arginine transport. Interferon-γ in combination with tumor necrosis factor-α induced nitric oxide production with an enhancement in cationic amino acid transporter-2B mRNA expression, inducible nitric oxide synthase mRNA expression, and L-arginine transport, while extracellular L-lysine competitively inhibited this nitric oxide production. Conclusions: In transformed hepatic stellate cells, tumor necrosis factor-α and interferon-γ have a crucial role in nitric oxide production, and extracellular L-arginine transport and inducible nitric oxide synthase expression are regulated in a differential cytokine-specific manner. As the estimated Km of L-arginine transporter in transformed hepatic stellate cells is very similar to the physiological L-arginine concentration in portel vein, we assume that increased portal L-arginine concentration may easily affect sinusoidal blood flow through enhancement of autocrine nitric oxide production in transformed hepatic stellate cells of diseased liver.