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.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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General Medicine A Subcommittee 2 (GMA)
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University of Michigan Ann Arbor
Internal Medicine/Medicine
Schools of Medicine
Ann Arbor
United States
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Van Dyke, Rebecca W (2004) Heterotrimeric G protein subunits are located on rat liver endosomes. BMC Physiol 4:1
Van Dyke, R W (2000) Effect of cholera toxin and cyclic adenosine monophosphate on fluid-phase endocytosis, distribution, and trafficking of endosomes in rat liver. Hepatology 32:1357-69
Van Dyke, R W; Ervin, L L; Lewis, M R et al. (2000) Effect of cholera toxin on rat liver lysosome acidification. Biochem Biophys Res Commun 274:717-21
Van Dyke, R W (1997) Cholera and pertussis toxins increase acidification of endocytic vesicles without altering ion conductances. Am J Physiol 272:C1123-33
Van Dyke, R W; Root, K V; Hsi, R A (1996) cAMP and protein kinase A stimulate acidification of rat liver endosomes in the absence of chloride. Biochem Biophys Res Commun 222:312-6
Anbari, M; Root, K V; Van Dyke, R W (1994) Role of Na,K-ATPase in regulating acidification of early rat liver endocytic vesicles. Hepatology 19:1034-43
Van Dyke, R W; Belcher, J D (1994) Acidification of three types of liver endocytic vesicles: similarities and differences. Am J Physiol 266:C81-94
Root, K V; Engelhardt, J F; Post, M et al. (1994) CFTR does not alter acidification of L cell endosomes. Biochem Biophys Res Commun 205:396-401
Van Dyke, R W (1993) Acidification of rat liver lysosomes: quantitation and comparison with endosomes. Am J Physiol 265:C901-17
Van Dyke, R W; Root, K V (1993) Ethinyl estradiol decreases acidification of rat liver endocytic vesicles. Hepatology 18:604-13

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