Bicarbonate was determined in bile with a Beckman Synchron CX3 analyzer (Beckman, Albertville, MN). NO and NO in bile and cell supernatants were measured with a nitrate/nitrite colorimetric assay kit from Cayman Chemical (Ann Arbor, MI). The hepatic glutathione concentration was quantified with a commercial kit from Sigma, and the total NOS activity in liver tissue was measured with a radioactivity-based NOS activity assay kit from Cayman Chemical. The protein concentration in samples was determined according to Bradford’s method.20 Total SNOs and low-molecular-weight nitrosothiols (LMw-SNOs) were measured in bile with 4,5-diaminofluorescein MK1775 (Calbiochem).21

This compound (10 μL) was added to 1-mL diluted bile samples (200 μL in phosphate-buffered saline with or without 0.2% HgCl2). After 10 minutes of incubation at room temperature, fluorescence was measured at an excitation wavelength of 495 nm and an emission wavelength of 515 nm. The SNO content (FSNO) was estimated as the difference between the fluorescence measured in the presence of HgCl2 (F1) and the fluorescence measured in its absence (F2; i.e., FSNO = F1 − F2). In addition, 300-μL bile samples were filtered with Centricon devices with a molecular weight cutoff of 10 kDa (Millipore,

Billerica, MA), and the LMw-SNO content was measured in the filtrate as described previously. To characterize Lumacaftor biliary glutathione and GSNO, MS analysis was performed after protein precipitation via the mixing of a 500-μL sample with an equal volume of 98% acetonitrile and 0.2%

formic medchemexpress acid. After 30 minutes of incubation on ice, samples were centrifuged at 4000g. Supernatants were then infused with a 100-μL syringe connected to a Q-TOF Micro instrument (Waters, Milford, MA) through a PicoTip nanospray ionization source (Waters). The heated capillary temperature was 80°C, and the spray voltage was 1.8 to 2.2 kV. MS data were collected and processed with Masslynx 4.1 (Waters). Homogenates from liver samples, common bile ducts, and NRCs were subjected to western blot analysis as described22 with antibodies against iNOS (Santa Cruz Biotechnology, Santa Cruz, CA), Akt, or phosphorylated Akt (Ser473; Cell Signaling, Beverly, MA). For a loading control, a β-actin antibody (Sigma) was employed. Results are expressed as means and standard errors of the mean. Comparisons of quantitative variables among groups were made with analysis of variance or Kruskal-Wallis tests (followed by the Student t test or Mann-Whitney nonparametric test, respectively), as required. A P value < 0.05 was considered to be significant. UDCA administration through the femoral vein in anesthetized rats with a cannulated common bile duct (i.e., the isPRL model) induced a dose-related increase in both the biliary total amount (Fig. 1A) and the concentration (data not shown) of the NO-breakdown products NO and NO, and this reflected increased biliary NO secretion (Fig. 1A).