Characterization, expression, and functional analysis of proton-dependent oligopeptide and pharmaceutically relevant transporters

Stephanie Anne Mowery, Purdue University

Abstract

Solute Carrier (SLC) and ATP-Binding Cassette (ABC) transporters play an important role in nutrient uptake, and have been established as both pharmaceutically relevant transporters and potential transportophoric targets for increasing the bioavailability of pharmaceuticals. The pharmacokinetics of nutrients and therapeutic agents may be mediated via modifying their molecular structures to improve the role of SLC and/or ABC transporters in their absorption, distribution, metabolism, and excretion (ADME). Hence, it is of the utmost importance to characterize the expression patterns of these transporters in pharmaceutically relevant cell lines. The mRNA expression of 88 SLC and ABC family members where characterized under different cell culture conditions in Caco-2 cell lines stressing the variables of cell line homogeneity, media, and age. Results from these studies indicate that both the media and the age of the cell lines affect the expression levels of relevant transporters. In other transporter characterization studies using the HT-29 parental and HT-29 Cl.19A clonal cell line revealed a significant down regulation of P-glycoprotein in HT-29 Cl.19A cells. Functional and protein analysis was completed to confirm the observation of the mRNA down regulation. In addition to these studies, the localization and post translational modification (PTM) characterization of human Peptide Histidine Transporter 1 (hPHT1) was attempted. Understanding the localization and PTM characteristics of this transporter is essential to elucidating the function of hPHT1. The overall emphases of these studies is the understanding of the variability in in vitro cell based pharmaceutical modeling systems and how culturing conditions such as cell homogeneity, media selection, and age of the cell line affect SLC and ABC expression and function. Controlling the variables involved in a screening assay will lead to improved data comparisons within and across laboratories. ^

Degree

Ph.D.

Advisors

Gregory T. Knipp, Purdue University.

Subject Area

Health Sciences, Pharmacology|Health Sciences, Pharmacy

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