Cholestasis, the loss of hepatic organic ion transport, is a prominent manifestation of inflammatory liver injury in sepsis, primary biliary cirrhosis, alcoholic hepatitis and viral hepatitis. Preliminary studies suggest that leukotrienes, lipid mediators of inflammation, activate chloride and organic ion channels in hepatocytes at the same time that transport of leukotrienes out of the cells is inhibited. The central hypothesis of this proposal is that leukotrienes are produced in hepatocytes and serve as messengers which activate ion channels and inhibit organic anion transport. Intracellular accumulation of leukotrienes leads to organic ion leak pathways which deplete the cells of metabolic intermediates and contribute to hepatocellular injury. The long range goal of this work is to understand how leukotrienes contribute to hepatocellular injury and to use this information as a basis for pharmacological therapy of inflammatory liver disease.
The specific aims are: 1. To determine whether intracellular leukotrienes activate CI channels, and to determine if the leukotriene transport protein, cMRP, mediates this effect. 2. To test whether cysteinyl leukotrienes are generated within hepatocytes and determine whether bacterial endotoxin increases expression of leukotriene synthesizing enzymes. 3. To determine whether tumor necrosis factor primes hepatocytes making them responsive to leukotrienes and ascertain the mechanism of this priming. 4. To learn whether intracellular leukotrienes down regulate secretion of themselves and other organic ions as a result of changes in CI channel activity. The role of leukotrienes in channel activation will be determined by whole-cell patch clamp and internal dialysis with leukotrienes, and single-channel analysis. The role of cMRP in binding leukotrienes and activating ion channels will be explored using mutant cells lacking cMRP expression. The capacity of hepatocytes to produce leukotrienes will be measured by HPLC and EIA, and expression of enzymes involved in leukotriene synthesis will be measured by northern blot, and RT-PCR. Effects of leukotrienes on hepatic secretory function will be determined using confocal florescence studies in internally dialyzed hepatocyte couplets, canalicular membrane vesicles and perfused liver. This work will identify new mechanisms by which inflammatory liver injury occurs and will lead to new therapeutic approaches to these diseases.
| Wozniak, Ann L; Griffin, Stephen; Rowlands, David et al. (2010) Intracellular proton conductance of the hepatitis C virus p7 protein and its contribution to infectious virus production. PLoS Pathog 6:e1001087 |
