Moreover, experiments carried out with freshly isolated rat hepatocytes HSP inhibitor indicated that these cells can release NO species when they are incubated with UDCA. UDCA-induced

release of NO from isolated hepatocytes was not observed in the presence of the protein synthesis inhibitor cycloheximide, and this supports the involvement of increased production of iNOS in these liver cells. It has been shown that SNOs prolong the NO half-life and allow this molecule to be transported in biological fluids to fulfill biological functions at places distant from the point at which it is produced.15 The considerable abundance of glutathione in bile makes this compound an excellent carrier for the transport of functional NO along the biliary tree. In our study, we observed a great rise in SNOs upon UDCA infusion, mainly at the expense of SNOs with a molecular weight less than 10 kDa, and this strongly suggests that GSNO is the most likely NO carrier in bile. MS analysis confirmed that, upon UDCA infusion, there was a rise in GSNO and putative GSNO derivatives in bile. Moreover, when the rat liver was depleted of glutathione with BSO, the infusion of UDCA failed to induce any increase in biliary NO, and this

further reinforces the view that GSNO has an essential role as the NO carrier in bile. ABCC2/Mrp2 is a canalicular protein involved in the transport of glutathione and glutathione conjugates to bile (reviewed by Ballatori et al.33). In TR− rats, which have defective ABCC2 function,27 there is a marked MCE impairment in biliary secretion of glutathione, the concentration of which falls from the normal Atezolizumab millimolar range to a micromolar range.28 This impairment is associated with decreased biliary NO secretion in response to UDCA. The level of total biliary NO (which is normally excreted at concentrations in the micromolar range) falls to less than half of normal

values, and the level of biliary SNOs falls to about one-third of normal. This observation reveals a role of ABCC2 in mediating, at least in part, the canalicular efflux of NO species from hepatocytes to bile. Furthermore, this finding is consistent with the existence of a link between biliary NO secretion and biliary transport of glutathione. Although GSNO might be the prevalent NO species in canalicular bile, its catabolism or degradation to nitrites/nitrates within the bile ducts may create a gradient of decreasing concentration along the biliary tree. Also, a portion of the GSNO that remains intact in the bile duct lumen may enter the bile duct epithelial cells. It thus seems probable that the actual concentration of GSNO that is secreted at the canaliculi and drains into the intrahepatic bile ducts is substantially higher than that detected at the end of the common bile duct. Our data indicate that the secretion of GSNO to bile is critical for the hypercholeretic activity of UDCA.