Proceedings of The Physiological Society

University of York (2002) J Physiol 539P, S017

Communications

The efflux of purine nucleobases and nucleosides from the rat brain

Aleksandra J. Isakovic*, Malcolm B. Segal† and Zoran B. Redzic*†

*Institute of Biochemistry, School of Medicine, Belgrade, Yugoslavia and †Division of Physiology, GKT School of Biomedical Sciences, King's College, London, UK


The efflux of purine nucleobases and their nucleosides from the rat brain was investigated using the brain efflux index (BEI) method (Kakee et al. 1996). Wistar rats (200-250 g; either sex) were anaesthetised with thiopenthone-Na (40 mg kg-1 I.P.) according to the Animals Scientific Procedures Act (1986), placed in a stereotaxic frame and a small hole drilled in the skull. A 0.4 mm needle was lowered to the point known as Par2 (0.2 mm anterior, 5.5 mm lateral to the Bregma and 4.5 mm deep) and artificial interstitial fluid (0.5 µl) was injected slowly over 2 s, containing 0.2 µCi of [14C] nucleobase or nucleoside as a test and 0.1 µCi of [3H] sucrose as a reference molecule. At different times after injection the animal was decapitated, left cerebrum collected, prepared for scintillation counting and the BEI calculated as the relative percentage of drug which had effluxed from the brain to the circulating blood across the BBB compared with the amount of drug injected into the brain. The efflux rate constant (Keff) of the test molecule was obtained from the slope of the semilogarithmic plot of (100 - BEI) values vs. time curve and can be considered as an index of the BBB permeability. The calculated BEI values gave a Keff for 0-3 min of (mean ± S.E.M., n = 6) 210.0 ± 60.0 X 10-3 min-1 for hypoxanthine and 133.3 ± 26.0 X 10-3 min-1 for adenine, while for adenosine and inosine these values were 57.15 ± 15.67 (n = 5) and 69.3 ± 32.0 X 10-3 min-1 (n = 6), which suggests that purine nucleobases have more rapid initial efflux then their nucleosides. After this time the velocity of efflux decreased for all test molecules, so Keff values for 3-15 min were 5.6 ± 0.8, 4.9 ± 5.7, 1.1 ± 1.0 and 13.7 ± 8.0 X 10-3 min-1 for hypoxanthine, adenine, adenosine and inosine, respectively, which was probably due to the intracellular trapping of labelled molecules and was confirmed by the capillary depletion technique (Triguero et al. 1990). The results of self- and cross-inhibition studies suggested that the efflux of test molecules was saturable and that nucleobases competed for the same transport systems with their nucleosides. The structure of the sugar moiety seemed to be important for the efflux of nucleosides from the brain, since a significant difference in the efflux velocity between ribo-and deoxyribonucleosides was observed.


Where applicable, experiments conform with Society ethical requirements