Blood–Brain Barrier Efflux Transport of Pyrimidine Nucleosides and Nucleobases in the Rat

Abstract

The brain efflux index (BEI), a measurement of blood-brain barrier (BBB) efflux transport, was estimated at 15 s, 30 s, 1 min, 3 min and 10 min after intracerebral injection of [14C]pyrimidines. An initial steep increase of the BEI values over time was observed for [14]uracil and [14C]thymine, followed by a more moderate increase after 1 min. For the corresponding nucleosides, [14C]uridine and [14C]thymidine, the increase of BEI values over time was less steep and linear between 30 s and 3 min. The apparent BBB efflux clearances for [14C]uridine, [14C]thymidine, [14C]uracil and [14C]thymine were (microl/min/g): 95.2 +/- 12.1, 125.3 +/- 18.4, 290.4 +/- 28 and 358.5 +/- 32.5, respectively, which is at least several folds higher than the predicted BBB influx clearances of uridine, uracil and thymidine. Quick depletion of brain parenchyma from brain microvasculature has revealed that [14C] radioactivity accumulated in brain microvessels after injection of nucleosides [14C]thymidine and [14C]uridine, but that was not observed when nucleobases, [14C]thymine and [14C]uracil, were injected. Reverse transcriptase-PCR revealed that the rat brain and liver (positive control) express dihydropyrimidine dehydrogenase, a key enzyme in pyrimidine nucleobase catabolism. Two bands representing spliced variants have been detected with the relative density of the bands (expressed relative to the density of glyceraldehyde3-phosphate dehydrogenase bands, mean +/- SEM from 3 separate samples) 0.16 +/- 0.06 and 0.04 +/- 0.01 (brain) and 0.49 +/- 0.1 and 0.07 +/- 0.01 (liver). Overall, these results indicate that the net direction of pyrimidine BBB transport is the efflux transport; rapid BBB efflux transport and metabolic breakdown of pyrimidine nucleobases appear to be important for brain homeostasis.

Molecular Biology of Nucleoside Transporters and their Distributions and Functions in the Brain

“…Apical transport of adenosine by rCNT2/3 + rENT2 and basolateral transport by rENT1 + rENT2 is therefore indicated. There is similar evidence of sodium-independent transport of nucleosides and nucleobases across the basolateral face of the isolated in situ perfused sheep choroid plexus [100][101][102], and of mixed sodium-dependent and sodium-independent transport of hypoxanthine across the apical membrane of primary cultures of sheep choroid plexus epithelial cells [98]. Cross-inhibiton findings in rat and sheep suggest the presence of at least two basolateral nucleobase transport processes [97,101].…”

“…Abluminal transport of adenosine by rCNT2 + rENT2 and luminal transport by rENT1 + rENT2 is therefore indicated. Efflux studies across rat BBB following intracerebral injection of radiolabeled nucleosides or nucleobases have also been undertaken for adenosine [96], hypoxanthine [97], and pyrimidine nucleosides and nucleobases [98]. Self-and cross-inhibiton findings consistent with possible rENT2 involvement were reported.…”

“…Endothelial cells of the BBB and BCSFB epithelial cells of the choroid plexus interface with the peripheral blood supply and have key roles in CNS nucleoside and nucleobase homeostasis [1,95,98,107,110]. Reviewed in Section 3.7, these cells exhibit multiple NTs on both brain-and bloodfacing membrane surfaces, and bi-directional transcellular movement of nucleosides and nucleobases across the BBB and BCSFB has been demonstrated.…”

“…Within the CNS, nucleosides and their metabolites freely exchange between the interstitial fluid and CSF, such that both the BBB and BCSFB contribute to overall nucleoside homeostasis in the brain [1,110]. Notwithstanding that concentrative NTs are localized exclusively to membranes facing brain fluids, nucleosides and nucleobases enter as well as exit the CNS [1,97,98,107,110]. Nucleosides and nucleobases are present in plasma and the CSF at broadly similar concentrations, the CSF pool undergoing rapid turnover [1,110,111].…”

“…Nucleosides and nucleobases are present in plasma and the CSF at broadly similar concentrations, the CSF pool undergoing rapid turnover [1,110,111]. CNS entry provides access to salvageable nucleosides and nucleobases for metabolism, while exit may be especially important in removing adenosine and its metabolites from brain extracellular fluids ( [98,107] and Section 4.2). There is evidence of substantial nucleoside metabolism in BBB endothelial and BCSFB epithelial cells, such that molecules entering cells in one form may exit in another.…”