Abstract
Nucleosides such as adenosine, as well as many nucleoside-based drugs, permeate cell membranes via a family of equilibrative nucleoside transporters (ENTs). We assessed the effects of (3-[1-(6,7-diethoxy-2-morpholino-quinazolin-4-yl)piperidin-4-yl]-1,6-dimethyl-2,4(1H,3H)-quinazolinedione hydrochloride (KF24345), a novel anti-inflammatory agent that potentiates the actions of adenosine, on the es (inhibitor-sensitive) and ei (inhibitor-resistant) subtypes of ENTs in human, mouse, and rat cells. KF24345 was similar to the prototypical high-affinity inhibitor nitrobenzylthioinosine (NBMPR) for blocking the human es transporter (KI of ∼0.4 nM), but was 50-fold more effective than NBMPR at blocking the human ei transporter (KI of ∼100 nM). KF24345 displayed significantly less species heterogeneity in its affinity for the es transporter than did dipyridamole, a widely used inhibitor of nucleoside transport; KF24345 may thus prove useful as an inhibitor for studies of nucleoside metabolism in a range of animal models. Furthermore, KF24345 seemed to act as a noncompetitive inhibitor of both [3H]NBMPR binding and [3H]nucleoside uptake by human es transporters, and these kinetics were consistent with an observed slow dissociation of KF24345 from the inhibitor binding site. KF24345 also exhibited unusual biphasic profiles for inhibition of [3H]NBMPR binding to membranes prepared from a recombinant human es transporter model (PK15-hENT1), suggesting the presence of multiple populations of NBMPR binding proteins in these membranes. The atypical tight binding interaction of KF24345 with the es transporter may prove useful for the molecular delineation of inhibitor binding domains and will facilitate its use as an in vivo inhibitor of nucleoside transport in studies focused on the biological effects of adenosine.
Footnotes
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This study was supported by grants to J.R.H. from the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Canada.
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DOI: 10.1124/jpet.103.060434.
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ABBREVIATIONS: ENT, equilibrative nucleoside transporter; es, equilibrative inhibitor-sensitive; NBMPR, nitrobenzymercaptopurine riboside (nitrobenzylthioinosine); ei, equilibrative inhibitor-insensitive; NBTGR, nitrobenzylthioguanosine; FBS, fetal bovine serum; ECGS, endothelial cell growth supplement; DMEM, Dulbecco's modified Eagle's medium; rMVEC, rat microvascular endothelial cell; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; MEM, minimum essential medium.
- Received September 25, 2003.
- Accepted November 17, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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