Diazepam and flurazepam inhibit adenosine uptake by rat brain synaptosomes

The suggestion has beenmade in earlier publications that benzodiazepines may exert some of their therapeutic effects by potentiating the pharmacological actions of endogenously released adenosine (Phillis 1979; Phillis et al 1979a). Adenosine and the adenine nucleotides are highly potent depressants of the firing of many central neurons. Diazepam potentiates the depressant actions of adenosine and 5’-adenosine monophosphate (5’AMP) on the spontaneous firing of cerebral cortical neurons and in largeramounts it evokes a similar depression of neuronal firing. The depressant actions of another benzodiazepine, flurazepam, are inhibited by the adenosine antagonist, theophylline. Both sets of observations are consistent with the postulate that benzodiazepines exert their actions on cortical neurons through a purinergic link. Mah & Daly (1976) have shown that diazepam inhibits the uptake of adenosine by rat brain slices but in their experiments high concentrations (5 x 10-5-2 x lo-‘ M) of diazepam were required to elicit such an inhibition. These concentrations are well above those which are likely to occur in the brain or in plasma (up to 1 0 6 ~ ) during the therapeutic administration of diazepam to man (Hillestad et al 1974; Skolnick et a1 1979). To validate our hypothesis that diazepam may elicit at least some of its therapeutic actions by inhibiting adenosine uptake, it is necessary to demonstrate that this agent, in therapeutically relevant concentrations, can in fact significantly affect such uptake. The results presented in this communication show that diazepam, at concentrations as low as 10-8-10-7 M, reduces adenosine uptake. A second benzodiazepine, flurazepam, which is less potent than diazepam as a therapeutic agent (Mohler & Okada 1978), also inhibited uptake, but somewhat less effectively. Synaptosomes were prepared by the method of Gray & Whittaker (1962) as modified by White (1975). Briefly, 4 male Wistar rats were killed by cervical dislocation. The cerebral cortices were removed, weighed and homogenized in 10 volumes (w/v) of ice-cold 0.32 M sucrose solution, p H 7.5. The homogenate was centrifuged at 1OOO g for 10 min. The debris was discarded and the remaining supernatant was centrifuged again a t 12 OOO g for 20 min at 4°C. The resultant pellet was suspended in 12 ml of ice-cold 0.32 M sucrose solution, p H 7.5, and transferred to a discontinuous sucrose gradient and centrifuged at 150 OOO g for 60 min. The synaptosomal fraction was collected and diluted to 40 ml with 0.32 M sucrose solution and then centrifuged at 20 000 g for 20 min at 4°C. The synaptosomal pellet