Intestinal cells have a poor capacity for de novo nucleoside biosynthesis and therefore have to rely heavily on salvage pathways for their nucleoside needs. This necessitates the intestinal epithelial cells to synthesis and express nucleoside transporters on their brush border membranes to allow for efficient salvage of nucleosides from the lumen. Since many antiviral and anti-cancer nucleoside drugs are substrates of nucleoside transporters, understanding their mechanism of uptake by the nucleoside transporters in the human intestine is important for prediction of potential beneficial or deleterious pharmacokinetic and pharmacodynamic drug-drug interactions and the design of nucleoside drugs with better oral absorption profiles. Therefore, the overall goal of this proposal is to elucidate the mechanisms of nucleoside transport in the human intestinal epithelium. In the current funding period, we have found that equilibrative (es and ei) and two concentrative (N1 and N2) nucleoside transporters are expressed in Xenopus-oocytes microinjected with intestinal mRNA. Because only the concentrative (N1 and N2) nucleoside transporters are present on the brush border membrane on the human intestinal epithelium, we have hypothesized that the equilibrative transporters are expressed on the basolateral membrane and perhaps on the intracellular membranes. Therefore, the specific aims of the present proposal are to: 1. Identify and map the critical structural determinants of the human es, N1 and the N2 transporters and of the nucleosides necessary for high affinity nucleoside-transporter interaction. We will accomplish this aim: a) by selecting PCR-generated mutant clones for null or reduced transporter activity in a high throughput expression system, Saccharomyces cerevisiae, and b) by binding a three dimensional model of the nucleoside binding sites of the transporters using Comparative Molecular Field Analysis (CoMFA). 2. Test the hypothesis that cellular differentiation in the crypt-to-villus axis results in a switch in the types and cellular localization of the expressed nucleoside transporters. We will accomplish this aim by characterizing the cellular localization and expression of the es and N2 nucleoside transporters and their mRNAs in the crypt-to-villus axis in both the adult and the fetal human intestine.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM054447-11
Application #
6490102
Study Section
Pharmacology A Study Section (PHRA)
Program Officer
Okita, Richard T
Project Start
1991-02-01
Project End
2003-12-31
Budget Start
2002-01-01
Budget End
2002-12-31
Support Year
11
Fiscal Year
2002
Total Cost
$316,026
Indirect Cost
Name
University of Washington
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
135646524
City
Seattle
State
WA
Country
United States
Zip Code
98195
Endres, Christopher J; Moss, Aaron M; Ishida, Kazuya et al. (2016) The role of the equilibrative nucleoside transporter 1 on tissue and fetal distribution of ribavirin in the mouse. Biopharm Drug Dispos 37:336-44
Moss, Aaron M; Endres, Christopher J; Ruiz-Garcia, Ana et al. (2012) Role of the equilibrative and concentrative nucleoside transporters in the intestinal absorption of the nucleoside drug, ribavirin, in wild-type and Ent1(-/-) mice. Mol Pharm 9:2442-9
Burnett, Lindsey A; Blais, Edik M; Unadkat, Jashvant D et al. (2010) Testicular expression of Adora3i2 in Adora3 knockout mice reveals a role of mouse A3Ri2 and human A3Ri3 adenosine receptors in sperm. J Biol Chem 285:33662-70
Eyal, Sara; Hsiao, Peng; Unadkat, Jashvant D (2009) Drug interactions at the blood-brain barrier: fact or fantasy? Pharmacol Ther 123:80-104
Endres, Christopher J; Endres, Michael G; Unadkat, Jashvant D (2009) Interplay of drug metabolism and transport: a real phenomenon or an artifact of the site of measurement? Mol Pharm 6:1756-65
Endres, Christopher J; Moss, Aaron M; Ke, Ban et al. (2009) The role of the equilibrative nucleoside transporter 1 (ENT1) in transport and metabolism of ribavirin by human and wild-type or Ent1-/- mouse erythrocytes. J Pharmacol Exp Ther 329:387-98
Endres, Christopher J; Moss, Aaron M; Govindarajan, Rajgopal et al. (2009) The role of nucleoside transporters in the erythrocyte disposition and oral absorption of ribavirin in the wild-type and equilibrative nucleoside transporter 1-/- mice. J Pharmacol Exp Ther 331:287-96
Govindarajan, Rajgopal; Leung, George P H; Zhou, Mingyan et al. (2009) Facilitated mitochondrial import of antiviral and anticancer nucleoside drugs by human equilibrative nucleoside transporter-3. Am J Physiol Gastrointest Liver Physiol 296:G910-22
Govindarajan, Rajgopal; Endres, Christopher J; Whittington, Dale et al. (2008) Expression and hepatobiliary transport characteristics of the concentrative and equilibrative nucleoside transporters in sandwich-cultured human hepatocytes. Am J Physiol Gastrointest Liver Physiol 295:G570-80
Govindarajan, Rajgopal; Bakken, Aimee H; Hudkins, Kelly L et al. (2007) In situ hybridization and immunolocalization of concentrative and equilibrative nucleoside transporters in the human intestine, liver, kidneys, and placenta. Am J Physiol Regul Integr Comp Physiol 293:R1809-22

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