Nitric oxide and peroxynitrite regulate transporter transcription in rat liver slices.

To determine the role of nitric oxide (NO) in rat liver transporter regulation, we investigated in precision-cut 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)-alpha and interleukin (IL)-1beta 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.

Effect of aminoguanidine on lipopolysaccharide-induced changes in rat liver transporters and transcription factors.

To determine the role of nitric oxide (NO) in rat liver transporter regulation, we investigated whether NO mediates lipopolysaccharide (LPS)-induced changes in transporters and their transcription factor expression using aminoguanidine (AG), an inhibitor of induced nitric oxide synthase (iNOS). We confirmed that LPS decreased mRNA levels for Ntcp, Oatp1, Oatp2, Oatp4, Oct1, Mrp2, Mdr1a and increased those for Mdr1b at 16 h after administration. AG attenuated these decreases for Ntcp, Oatp1 and Oatp4 (retinoid X receptor (RXR)alpha- and hepatocyte nuclear factor (HNF)4alpha-dependent genes) and increase for Mdr1b (nuclear factor (NF)-kappaB-dependent gene). Concomitantly, it suppressed LPS-induced NF-kappaB-dependent gene transcription, such as those for proinflammatory cytokines (cytokines; tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6) and iNOS, and also suppressed IL-1beta release from Kupffer cells (KCs) at post-translational levels, but had little effect on the LPS-induced decreases in RXRalpha and HNF4alpha transcriptional activities. These findings indicate that hepatocytes were stimulated directly by LPS, which lead to the activation of NF-kappaB and reduction of RXRalpha and HNF4alpha transcriptional activities as early responses, and indirectly by cytokines and NO released from KCs via activation of NF-kappaB by LPS as delayed responses. We conclude that AG, which suppresses LPS-induced NF-kappaB activation in both hepatocytes and KCs and then the release of cytokines and NO from KCs, attenuates LPS-induced changes of Ntcp, Oatp1, Oatp4 and Mdr1b transcription in hepatocytes. The roles of cytokines and NO could not be distinguished, however. Further in vitro study is needed to clarify the role of NO in transporter regulation.