Sodium-dependent nucleoside transport in mouse intestinal epithelial cells. Two transport systems with differing substrate specificities.

D Vijayalakshmi,J A Belt

doi:10.1016/s0021-9258(19)77649-2

D Vijayalakshmi, J A Belt

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https://doi.org/10.1016/s0021-9258(19)77649-2

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Abstract

Nucleoside transport was examined in freshly isolated mouse intestinal epithelial cells. The uptake of formycin B, the C nucleoside analog of inosine, was concentrative and required extracellular sodium. The initial rate of sodium-dependent formycin B transport was saturable with a Km of 45 +/- 3 microM. The purine nucleosides adenosine, inosine, guanosine, and deoxyadenosine were all good inhibitors of sodium-dependent formycin B transport with 50% inhibition (IC50) observed at concentrations less than 30 microM. Of the pyrimidine nucleosides examined, only uridine (IC50, 41 +/- 9 microM) was a good inhibitor. Thymidine and cytidine were poor inhibitors with IC50 values greater than 300 microM. Direct measurements of [3H]thymidine transport revealed, however, that the uptake of this nucleoside was also mediated by a sodium-dependent mechanism. Thymidine transport was inhibited by low concentrations of cytidine, uridine, adenosine, and deoxyadenosine (IC50 values less than 25 microM), but not by formycin B, inosine, or guanosine (IC50 values greater than 600 microM). These data indicate that there are two sodium-dependent mechanisms for nucleoside transport in mouse intestinal epithelial cells, and that formycin B and thymidine may serve as model substrates to distinguish between these transporters. Neither of these sodium-dependent transport mechanisms was inhibited by nitrobenzylmercaptopurine riboside (10 microM), a potent inhibitor of one of the equilibrative (facilitated diffusion) nucleoside transporters found in many cells.

Highlights

Nucleoside transport was examined in freshly iso- rine riboside (NBMPR)’ [1, 3,4,5]
The lates with inhibition of nucleoside transport [7]. This transinitial rate of sodium-dependentformycin B transport porter has a very broad substrate specificity accepting both was saturable with a K, of 45 f 3 p ~ T.he purine the ribosides and deoxyribosides of the physiological purine nucleosides adenosine, inosine, guanosine, and deoxy- and pyrimidine bases, as well as numerous cytotoxic nucleoadenosine were all good inhibitors of sodium-depend- side analogs [8].The NBMPR-insensitive transporter is siment formycin B transport with 50% inhibition (ICSO) ilar to the NBMPR-sensitivetransporter with respect to observed at concentrations less than 30 pM
Distinguishing substrates Formycin B Thymidine Tubercidin. These studies have demonstrated through direct measurements of 3H-labelednucleoside uptake that thepermeation of uridine, formycin B, and thymidine into mouse intestinal epithelial cells is dependent upon extracellular sodium

Summary

RESULTS

Equal to that in the extracellular medium at an uptake valueof 1 pmol/106 cells. The values shown are the means of duplicate assays. Uridine Uptake and Metabolism-As described previously and are representative of results obtained in three similar experifor guinea pig intestinal cells [11],the uptake of uridine by ments. Formycin B (10p ~ up)take was determined as described under “Experimental Procedures.”. The intracellular concentration of 3H was approximately equal to that in the extracellular medium at an uptake value of 10 pmol/106 cells. The values shown are the means of duplicate assays and are representative of results obtained in four similar experiments, Formycin B (PM). Inhibition of sodium-dependent formycinB and thymidine transport by other nucleosides. Sodium-dependent formycin B (1p ~an)d thymidine (1PM)transport were determined using a 10-suptake interval as described under “Experimental Procedures.”.

Pyrimidine nucleosides Uridine Cytidine Thymidine

Findings

DISCUSSION