Nitrobenzylthioinosine (NBMPR) binding and nucleoside transporter ENT1 mRNA expression after prolonged wakefulness and recovery sleep in the cortex and basal forebrain of rat

We have previously shown that extracellular adenosine levels increase locally in the basal forebrain (BF) during prolonged wakefulness, yet the cellular mechanisms of this local accumulation have remained unknown. The extracellular adenosine levels are strictly regulated by adenosine metabolism and its transport through cell membrane by the nucleoside transporters. As we previously showed that the key adenosine metabolizing enzymes were not affected by prolonged wakefulness, we now focussed on potential changes in the nucleoside transporters. In the present study, we measured the binding of nitrobenzylthioinosine (NBMPR), an ENT1 transporter inhibitor, and the ENT1 transporter mRNA after prolonged wakefulness and recovery sleep. Rats were sleep-deprived for 3 or 6 h using gentle handling. After 6 h one group was allowed to sleep for 2 h. NBMPR binding was determined from BF and cortex by incubating tissue extracts with [3H] NBMPR. The in situ hybridization was carried out on 20 microm cryosections using [35S]dATP-labelled oligonucleotide probe for ENT1 mRNA. The NBMPR binding was significantly decreased in the BF, but not in the cortex, after 6 h sleep deprivation when compared with the time-matched controls, suggesting a decline in adenosine transport. The expression of ENT1 mRNA did not change during prolonged wakefulness or recovery sleep in either cortex or the BF, although circadian variations were measured in both areas. We conclude that the regional decrease in adenosine transport could contribute to the gradual accumulation of extracellular adenosine in the basal forebrain during prolonged wakefulness.