The goals of this project are to understand the mechanisms by which hepatocytes transport organic ions. These transport processes result in large concentration gradients, are impaired in cholestatic diseases, and are important considerations in the development of therapeutic and diagnostic agents.
The specific aims are: 1) to determine the electrical and chemical driving forces for uptake of organic ions. 2) To measure the contribution of electrogenic and ATP-dependent mechanisms to the secretion of organic ions into the canaliculus. 3) To determine the role of the intracellular binding capacity in organic solute uptake. The experimental design will use direct electrophysiological measurements in individual hepatocytes and secreting isolated hepatocyte couplets to measure uptake and secretion. Electrogenic transport currents will be measured with microelectrodes after blocking other membrane conductances and by tight-seal whole-cell patch clamp. Uptake of fluorescent organic ions such s bile salts and fluorescein derivatives into single cells will be directly determined in impaled cells. Secretion into the biliary space will be studied in couplets by determining the rate of fluorescence appearance in the canalicular space as a function of membrane voltage and cellular ATP. ATP-dependent organic ion efflux has previously been observed in liver and may be an important component of canalicular secretion. This hypothesis will be tested by maneuvers to deplete ATP in couplets and by observing secretion in whole-cell-path- clamped couplets in which the intracellular ATP concentration is varied directly. The role of intracellular binding in transport will be evaluated by observing the effect of varying the concentration of intracellular ligands and binding proteins on apparent unidirectional influx rate. These studies will provide much needed information on the driving forces for organic ion accumulation and secretion in the liver. Ultimately, this information could be used to limit hepatocellular damage from transported toxins, to improve targeting of therapeutic agents to hepatocytes and hepatocellular carcinomas, and to assist in the development of diagnostic studies that can be applied to extremely small samples of liver, such as that from a liver biopsy.
| 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 |
