Nitric Oxide and Peroxynitrite Regulate Transporter Transcription in Rat Liver Slices
Biological and Pharmaceutical Bulletin
Lipopolysaccharide (LPS) administration to rats results in cholestasis associated with changes in the expression of several liver transporters. 1) The down-regulation of canalicular efflux transporters bile salt export pump (Bsep), multidrug resistance-associated protein 2 (Mrp2) and multidrug resistance protein 1a (Mdr1a), which causes cholestasis, is thought to be compensated by down-regulation of basolateral influx transporters, as well as up-regulation of basolateral and canalicular efflux
... canalicular efflux transporter, Mrp3 and Mdr1b, which protect the liver from the accumulation of endogenous compounds, such as bile acid and bilirubin, as well as exogenous toxic compounds. LPS activates the production of proinflammatory cytokines and nitric oxide (NO) in the liver (Fig. 1A ). The first LPS action is the induction of proinflammatory cytokine production, including tumor necrosis factor (TNF)-a and interleukin (IL)-1b, in Kupffer cells (KCs), resident macrophages in the sinusoid of the liver, via toll-like receptor signaling. Subsequently, proinflammatory cytokines released from KCs lead to the induction of inducible NO synthase (iNOS) to produce NO in hepatocytes and KCs. 2) Furthermore, NO reacts with superoxide anion (O 2 Ϫ ), derived from KCs and produced from mitochondria stimulated by NO, to form peroxynitrite (ONOO Ϫ ). 3) Proinflammatory cytokines are known as major mediators in transcriptional regulation of liver transporter genes, including Na ϩ -taurocholate cotransporting polypeptide (Ntcp), organic anion transporting polypeptides (Oatp1a1/Oatp1, Oatp1a4/Oatp2), Mrp2, Mrp3, Bsep, Mdr1a and Mdr1b. 4, 5) Although NO inhibits nuclear factor (NF)-kB activation 6) and transcriptional activities of nuclear receptors such as retinoid X receptor (RXR) and hepatocyte nuclear factor (HNF) 4a, 7,8) which regulate rat liver transporter transcription. 9) However, the role of NO has been demonstrated only in the regulation of organic anion transporter 2 (Oat2) transcription in liver. 10) In a previous report, we showed the possible regulation of rat liver transporter transcription by both proinflammatory cytokines and NO in vivo, the specific roles of which could not be separately determined. 11) Recently, precision-cut liver slices have shown potential in the study of biological phenomena under the control of hepatocytes and nonparenchymal cells due to the preservation of the original cellular architecture and cell interactions within the slice. 12) Here, we used precision-cut slices to investigate the role of NO and ONOO Ϫ in transcriptional regulation of basolateral influx transporters (Ntcp, Oatp1a1, Oatp1a4, Oatp1b2/Oatp4, Oat2, Oat3, organic cation transporter 1 (Oct1)), canalicular efflux transporters (Mrp2, Bsep, Mdr1a, Mdr1b, Mdr2), and basolateral efflux transporter Mrp3 (Fig. 1B) . To clarify the role of KCs in NO and ONOO Ϫ action, we also used gadolinium chloride-pretreated slices (Gdslices), in which gadolinium chloride hexahydrate (Gd) leads to a decrease of proinflammatory cytokine, O 2 Ϫ and NO production by KCs.    MATERIALS AND METHODS Reagents Lipopolysaccharide (LPS, Escherichia coli, serotype 0111:B4) and William's medium E (WME) were purchased from Sigma-Aldrich (MO, U.S.A.); spermine NONOate (N-[4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino] butyl]-1,3-propanediamin, SpNO) from Cayman Chemicals (MI, U.S.A.); SIN-1 (3-(4-morpholinyl) sydnonimine hydrochloride, SIN), Gd, kanamycin monosulfate and To determine the role of nitric oxide (NO) in rat liver transporter regulation, we investigated in precisioncut liver slices the effect of NO and peroxynitrite (ONOO ؊ ), a reaction product of NO with superoxide (O 2 ؊ ), on mRNA levels of 13 influx and efflux transporters. To inactivate Kupffer cells (KCs), liver slices were prepared from rats treated with gadolinium chloride (Gd). Transporter mRNA levels were determined after incubation of untreated (normal-slices) and Gd-pretreated slices (Gd-slices) for 18 h with Spermine NONOate (SpNO), an NO donor, and SIN-1 (3-(4-morpholinyl) sydnonimine hydrochloride, SIN), a ONOO ؊ donor. SpNO and SIN varied all transporter mRNA levels examined, except organic anion transporting polypeptide 1b2 (Oatp1b2/Oatp4). SpNO in normal-slices and SIN in Gd-and normal-slices generally decreased influx and increased efflux transporter transcription. In contrast, these effects were not observed in Gd-slices treated with SpNO. SpNO and SIN in normal-slices commonly decreased organic anion transporter 2 (Oat2) and increased multidrug resistance-associated protein 2 (Mrp2) transcription, but differentially regulated bile salt export pump (Bsep) and multidrug resistance protein 2 (Mdr2) transcription, the up-regulation by SpNO and the down-regulation by SIN. In addition, the induction of tumor necrosis factor (TNF)-a a and interleukin (IL)-1b b was not observed after incubation with SpNO or SIN. These findings suggest that NO and ONOO ؊ play a role in the regulation of rat transporter transcription in hepatocytes, which communicate with KCs, in a proinflammatory cytokine-independent manner.