Nucleoside transporter subtype expression and function in rat skeletal muscle microvascular endothelial cells.

1. Microvascular endothelial cells (MVECs) form a barrier between circulating metabolites, such as adenosine, and the surrounding tissue. We hypothesize that MVECs have a high capacity for the accumulation of nucleosides, such that inhibition of the endothelial nucleoside transporters (NT) would profoundly affect the actions of adenosine in the microvasculature. 2. We assessed the binding of [(3)H]nitrobenzylmercaptopurine riboside (NBMPR), a specific probe for the inhibitor-sensitive subtype of equilibrative NT (es), and the uptake of [(3)H]formycin B (FB), by MVECs isolated from rat skeletal muscle. The cellular expression of equilibrative (ENT1, ENT2, ENT3) and concentrative (CNT1, CNT2, CNT3) NT subtypes was also determined using both qualitative and quantitative polymerase chain reaction techniques. 3. In the absence of Na(+), MVECs accumulated [(3)H]FB with a V(max) of 21+/-1 pmol microl(-1) s(-1). This uptake was mediated equally by es (K(m) 260+/-70 microm) and ei (equilibrative inhibitor-insensitive; K(m) 130+/-20 microm) NTs. 4. A minor component of Na(+)-dependent cif (concentrative inhibitor-insensitive FB transporter)/CNT2-mediated [(3)H]FB uptake (V(i) 0.008+/-0.005 pmol microl(-1) s(-1) at 10 microm) was also observed at room temperature upon inhibition of ENTs with dipyridamole (2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido-[5,4-d]pyrimidine)/NBMPR. 5. MVECs had 122,000 high-affinity (K(d) 0.10 nm) [(3)H]NBMPR binding sites (representing es transporters) per cell. A lower-affinity [(3)H]NBMPR binding component (K(d) 4.8 nm) was also observed that may be related to intracellular es-like proteins. 6. Rat skeletal muscle MVECs express es/ENT1, ei/ENT2, and cif/CNT2 transporters with characteristics typical of rat tissues. This primary cell culture model will enable future studies on factors influencing NT subtype expression, and the consequent effect on adenosine bioactivity, in the microvasculature.

Pharmacological analysis and molecular cloning of the canine equilibrative nucleoside transporter 1.

We studied the binding of [3H]nitrobenzylthioinosine (NBMPR) and the uptake of [3H]formycin B by the es (equilibrative inhibitor-sensitive) nucleoside transporter of Madin Darby Canine Kidney (MDCK) cells. NBMPR inhibited [3H]formycin B uptake with a Ki of 2.7+/-0.6 nM, and [3H]NBMPR had a KD of 1.3+/-0.3 nM for binding to these cells; these values are significantly higher than those obtained in human and mouse cell models. In contrast, other recognized es inhibitors, such as dipyridamole, were significantly more effective as inhibitors of [3H]NBMPR binding and [3H]formycin B uptake by MDCK cells relative to that seen for human cells. We isolated a cDNA encoding the canine es nucleoside transporter (designated cENT1), and assessed its function by stable expression in nucleoside transport deficient PK15NTD cells. The PK15-cENT1 cells displayed inhibitor sensitivities that were comparable to those obtained for the endogenous es nucleoside transporter in MDCK cells. These data indicate that the dog es/ENT1 transporter has distinctive inhibitor binding characteristics, and that these characteristics are a function of the protein structure as opposed to the environment in which it is expressed.

Interaction of the novel adenosine uptake inhibitor 3-[1-(6,7-diethoxy-2-morpholinoquinazolin-4-yl)piperidin-4-yl]-1,6-dimethyl-2,4(1H,3H)-quinazolinedione hydrochloride (KF24345) with the es and ei subtypes of equilibrative nucleoside transporters.

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 (K(I) of approximately 0.4 nM), but was 50-fold more effective than NBMPR at blocking the human ei transporter (K(I) of approximately 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 [(3)H]NBMPR binding and [(3)H]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 [(3)H]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.