Kinetics of nucleoside uptake by the basolateral side of the sheep choroid plexus epithelium perfused in situ

Abstract

Sheep choroid plexus epithelium expresses equilibrative nucleoside transporters (ENT) 1 and 2 and concentrative nucleoside transporter 2 at the transcript level. This study aimed to explore the kinetics and functional role of these transporters at the basolateral side of the sheep choroid plexus epithelium perfused in situ. The cellular uptake of [3H]adenosine and [3H]uridine was insensitive to 1 μm nitrobenzylthioinosine (NBTI), and the uptake of [3H]adenosine was reduced significantly when 10 μm NBTI was present in low-Na+ Ringer solution. This might suggest that ENT2, a transporter sensitive to micromolar NBTI, is functionally active at the basolateral side of the choroid plexus epithelium while ENT1, a transporter sensitive to nanomolar NBTI, is not active. When low-Na+ Ringer solution was used for the in situ perfusion, the Na+ concentration in the venous effluent decreased to 14 mm; under these conditions the maximal uptake (Umax) of [3H]adenosine and [ 3H]uridine did not change significantly when compared with the U max obtained when Ringer solution that contained 145 mm Na + was used. Kinetic analysis revealed apparent Michaelis-Menten constants (Km,app) for cellular uptake of [3H]adenosine, [3H]inosine and [3H]thymidine of 1.2 ± 0.2, 15.7 ± 2.6 and 3.8 ± 0.9 μm, respectively. The HPLC and HPLC-fluorometric analysis of the sheep plasma and cerebrospinal fluid revealed nanomolar concentrations of adenosine and thymidine and micromolar levels of inosine and nucleobases. Considering the estimated Km,app values, it appears that under normal conditions inosine is the more important nucleoside substrate for uptake by the basolateral membrane of the choroid plexus epithelium than other nucleosides. © 2008 The Authors.