Hepatocytes use intracellular vesicles to transport proteins and macromolecules into, out of and across cells as well as into or out of plasma membranes, processes termed endocytosis and exocytosis. Most of these vesicles are acidified by a unique electrogenic proton pump that usually functions in parallel with a chloride conductance to maximally acidify vesicle interior and limit membrane potential. Vesicle acidification is essential for efficient sorting and delivery of proteins, for degradation of macromolecules, for transport of small solutes into or out of acidified vesicles and for post-translational processing, delivery and function of lysosomal enzymes. Viruses and toxins also exploit the acidic environment of endocytic vesicles to trigger infection. Although vesicular transport and acidification have been identified in many cells, little is known regarding regulation of these important cell functions. My own work has shown that cholera and pertussis toxins, cAMP and protein kinase A all increase endosome acidification, findings that have implications for overall vesicular transport. In addition, work from others suggests that vesicle movement itself may be regulated, by a variety of agents, including cAMP, perhaps related, in part, to changes in vesicle acidification. Furthermore, endocytosis is of increasing clinical importance as a number of diseases affecting hepatic endocytosis have been identified, including familial hypercholesterolemia, alcoholic liver disease, diabetes, copper overload and lysosomal storage disorders. Finally, endocytosis is being employed for gene therapy. The long-term objectives of my research are to characterize, quantitate and compare mechanisms of vesicular acidification, to identify regulatory processes for acidification and for vesicular transport and to determine the role(s) of these regulatory processes in overall hepatic function. Based on work by myself and others, I have formulated a series of hypotheses within the context of these objectives that will be tested in the studies proposed in this application. I propose that receptor-coupled G proteins, located on intracellular vesicles, regulate endosome acidification and function with Gs proteins stimulating and Gi proteins, perhaps coupled to somatostatic, effecting tonic inhibition of vesicle acidification. I propose that these effects are mediated by cAMP, protein kinase A and, possibly, protein kinase C. I propose that these agents change vesicle acidification through effects on the proton pump, associated ion transporters, and/or changes in vesicle size, and that changes in acidification are associated with changes in rates of endocytosis and vesicular transport. I will test these hypotheses using isolated endocytic vesicles as well as the isolated perfused rat liver and cultured rat hepatocytes in conjunction with a variety of probes including toxins (cholera and pertussis toxins) and hormones known to affect G proteins as well as specific protein kinase inhibitors and agonists. The findings are expected to shed light on normal hepatic physiology and on mechanisms of those diseases affecting hepatic vesicular transport.
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