Investigations into the molecular and functional characteristics of human peptide/histidine transporter 1
Abstract
One of the primary focuses of our laboratory has been to elucidate the pathways by which oligopeptides permeate biological barriers. During the course of these investigations, we have focused on the recently identified and cloned human isoform of a novel member of the Proton-Coupled Oligopeptide Transporter (POT) superfamily, termed Peptide/Histidine Transporter 1 (hPHT1). Initial investigations into the molecular and functional characteristics of hPHT1 were conducted by our laboratory and provided insights into its potential effects on the biopharmaceutical characteristics of perorally delivered drug products. However, these studies were far from comprehensive. This dissertation is divided into several distinct, but complementary sections. Chapter 1, entitled "Mammalian Oligopeptide Transporters", is a recently published book chapter (with some minor modifications) that provides a comprehensive literature review of the expression, function and regulation of members of the POT superfamily (solute carrier family 15). This paper also provides insights into the functional significance of these transporters with respect to the biopharmaceutical properties of drug products, as well as methodologies to model these aspects during the drug screening process. Given the conflicting nature of data generated in our laboratory at the onset of our evaluations, Chapter 2 focuses on our attempts to characterize hPHT1 function in the human adenocarcinoma cell line HT-29. Here, we utilized both transiently and stably transfected HT-29 cells to over-express hPHT1 to determine its functional activity from background kinetics. While these studies were ultimately unsuccessful in demonstrating differences in overall apical-basolateral, or basolateral-apical transport kinetics in hPHT1 transfected cells versus empty vector controls, they did provide insights into the functional activity of hPHT1 in the gastrointestinal epithelium. Chapters 3 and 4 further our overall goals of studying oligopeptide permeation through the utility of a novel blood brain barrier (BBB) cell line, hCMEC/D3. Chapter 3 focuses on our efforts to characterize and optimize the hCMEC/D3 cell line for drug screening purposes, specifically with respect to known POT substrates, while Chapter 4 expands on our attempts to elucidate hPHT1 function, utilizing stable hCMEC/D3/PHT1 transfection. In contrast to results obtained in Chapter 2, these studies clearly demonstrate changes in both apical-basolateral and basolateral-apical transport of potential substrates, indicative of apical influx, basolateral efflux, or concerted apical-basolateral transport function. Chapter 5 of this dissertation originated from the functional evaluations of Chapters 2 and 4. This Chapter focused on hPHT1 cellular localization in both HT-29 and hCMEC/D3 cells, via numerous techniques. Taken together, the studies compiled in this body of work provide additional insights into hPHT1's basic physiologic function in both gastrointestinal absorption and brain distribution of relevant oligopeptide substrates.
Degree
Ph.D.
Advisors
Knipp, Purdue University.
Subject Area
Pharmacology|Pharmacy sciences|Physiology
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