Effect of culture time on the basal expression levels of drug transporters in sandwich-cultured primary rat hepatocytes.

Sandwich-cultured rat hepatocytes are used in drug discovery for pharmacological and toxicological assessment of drug candidates, yet their utility as a functional model for drug transporters has not been fully characterized. To evaluate the system as an in vitro model for drug transport, expression changes of hepatic transporters relative to whole liver and freshly isolated hepatocytes (day 0) were examined by real-time quantitative reverse transcription-polymerase chain reaction for 4 consecutive days of culture. No significant differences in transporter expression levels were observed between freshly isolated hepatocytes and whole liver. Two distinct mRNA profiles were detected over time showing 1) a more than 5-fold decline in levels of uptake transporters such as Na(+)-taurocholate cotransporting polypeptide (Ntcp), organic anion transporter (Oat) 2, organic anion-transporting polypeptide (Oatp) 1a1, Oatp1a4, and Oatp1b2 and 2) a greater than 5-fold increase of efflux transporters P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), and multidrug resistance-related proteins (Mrp) 1, 2, 3, and 4. In addition, protein levels and functional activities for selected transporters were also determined. Protein levels for Mrp2, Bcrp, P-gp, Ntcp, and Oatp1a4 corresponded to changes in mRNA. Functional activities of Oatps and Oct1 exhibited a 3- and 4-fold decrease on day 2 and day 4, respectively, relative to that on day 0, whereas a more than 10-fold reduction in Oat2 activity was observed. These results indicate that the cell culture conditions used herein did not provide an optimal environment for expression of all hepatic transporters. Significant time-dependent alterations in basal gene expression patterns of transporters were detected compared with those in liver or freshly isolated hepatocytes. Further work and new strategies are required to improve the validity of this model as an in vitro tool for in vivo drug transport or biliary clearance prediction.

Evaluation of thiol-based antioxidant therapeutics in cystic fibrosis sputum: Focus on myeloperoxidase.

Neutrophil-dependent reactions catalysed by myeloperoxidase (MPO) are thought to play important roles in the pulmonary pathobiology of cystic fibrosis (CF). Aerosolized thiol antioxidants such as glutathione (GSH) and N-acetylcysteine (NAC) are currently being utilized as therapeutics to modify CF respiratory tract oxidative processes. This study hypothesized that MPO in CF airway lining fluids may be a target of such therapeutics. MPO activity in sputum from 21 adult CF patients was found to be inversely associated with lung function (FEV(1)). In contrast, systemic inflammation (assessed by plasma C-reactive protein) was not correlated with lung function. Ex vivo studies revealed that GSH and NAC effectively scavenged N-chloramines in sputum and inhibited sputum MPO activity with potency exquisitely dependent upon MPO activity levels. Detailed kinetic analyses revealed that NAC and GSH inhibit MPO by distinct mechanisms. Activation of the key pro-inflammatory transcription factor NF-κB in cultured HBE1 cells was inhibited by GSH. The findings reveal that MPO activity and its reactive products represent useful predictors of the doses of inhaled thiol antioxidants required to ameliorate airway oxidative stress and inflammation in CF patients and provide mechanistic insight into the antioxidative/anti-inflammatory mechanisms of action of GSH and NAC when administered into the CF lung.

Myeloperoxidase-dependent oxidative metabolism of nitric oxide in the cystic fibrosis airway.

BACKGROUND: Decreased expired nitric oxide (eNO) is commonly observed in cystic fibrosis (CF) patients and is usually explained by dysregulation of NO synthase (NOS) isoforms in respiratory tract epithelium. Later stages of this disease are accompanied by intense airway infiltration of phagocytes with high NOS activity, abundant levels of the hemoprotein myeloperoxidase (MPO) and significant production of significant reactive oxygen species. METHODS: This study characterizes the contribution of the high airway levels of MPO to decreased eNO levels in adult CF patients. NO metabolites (NO(x)) and MPO levels in fresh sputum of control and adult CF patients were determined and related to measurements of eNO and to in vitro consumption of NO in CF sputum. RESULTS: Despite essentially equal levels of NO(x) in sputum, eNO was 2- to 3-fold lower in CF patients compared to healthy controls. In CF patients, eNO levels were negatively associated with sputum peroxidase activity. In vivo correlations were confirmed by ex vivo studies of NO consumption by MPO in CF sputum. Immunodepletion studies confirmed MPO as the major heme peroxidase in CF sputum contributing to the hydrogen peroxide (H(2)O(2))-dependent consumption of NO. CONCLUSIONS: In CF airways MPO acts as a phagocyte-derived NO oxidase that diminishes NO bioavailability at airway surfaces, possibly identifying this peroxidase as a potential target for therapeutic intervention.