Hepatocytes contain many vesicular organelles, including lysosomes, Golgi, endocytic vesicles and protein secretory vesicles, the interior of which is acidified by an electrogenic proton pump (H+-ATPase). Such acidification appears essential for numerous cell functions including receptor-mediated endocytosis, degradation of exogenous and endogenous materials and efficient secretion of proteins. In turn, certain viruses and toxins exploit the acid pH of endocytic vesicles to gain entry to cell cytoplasm. The long-term goals of the proposed research are to characterize the function, distribution, and regulation of proton pumps in hepatic endocytic vesicles.
The specific aims of this proposal will test two new hypotheses. First, it is proposed that vesicles at distinct stages of the endocytic pathway are acidified to differing degrees, that this is due principally to changes in ion transport rather than to time-dependent acidification and that it reflects insertion of functional proton pumps into endocytic vesicles. If so, differences in the internal pH (pHi) of vesicles are likely to determine the endocytic stage(s) where pH-dependent processes differing in pH optimum occur. Second, it is proposed that endocytic vesicles take up and accumulate a number of small molecular weight solutes including calcium, cationic drugs, organic amines, and basic amino acids. This hypothesis further holds that the proton pump-generated electrochemical gradient drives vesicular uptake and storage of solutes via either H+/solute antiporters or protonation and sequestration of lipophilic weak bases. Stored solutes may be released to cytoplasm or transferred into bile as the vesicles in which they are contained fuse with the bile canalicular membrane. These hypotheses will be tested principally with purified endocytic vesicles using radiolabeled probes and fluorescent dyes. In complementary studies, proton pumps will be localized by means of electron microscopy employing specific antibodies. The relationship of vesicular uptake to overall hepatic extraction and biliary secretion of solutes will be defined in the perfused rat liver. The proposed studies are expected to provide new insight into poorly understood aspects of vesicle acidification and of solute transport across vesicular membranes in hepatocytes and in other cell types. They have important implications for a wide range of processes including receptor-mediated endocytosis, calcium homeostasis and signal transduction, biliary calcium secretion and gallstone formation, hepatic uptake and biliary secretion of drugs, amino acid metabolism, and drug-induced phospholipidoses.
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