Inhibition of adenosine uptake into rat brain synaptosomes by the benzodiazepines

(;en Pharmac Vol 12, pp. 67 to 70 Pergamon Press Lid 1981 Printed in Great Britain

0306-3623 81 0101-{Xh57S02{X}'0

I N H I B I T I O N O F A D E N O S I N E U P T A K E INTO RAT BRAIN S Y N A P T O S O M E S BY THE B E N Z O D I A Z E P I N E S J. W. PmLels, P. H. Wu and A. S. BI'NDrR Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, Canada, S7N OW0 (Received 22 April 1980)

Abstract--l. Thirteen benzodiazepines have been tested for their ability to inhibit the uptake of adenosine by rat brain synaptosomes. 2. All of these benzodiazepines were able to inhibit adenosine uptake with IC2o values ranging from 5 × 10 9 M (clonazepam) to 2.0 x 10 5 M (RO 11-6893). 3. Clonazepam, nitrazepam, lorazepam, RO I 1-6896, diazepam, flunitrazepam, medazepam, and flurazepam were the most potent compounds, having IC20 values of less than 10 -6 M. Adenosine uptake would therefore be appreciably reduced at the levels achieved with therapeutic doses (circa 1 l~M for diazepam). 4. The d-steroisomer RO 11-6896 was approximately 200 times more active than the 1-isomer, RO 11-6893. 5. The high IC5o values (10-5_ 10-3 M) indicate that dose response curves for the benzodiazepines arc rather shallow. 6. The results are consistent with the hypothesis that benzodiazepines exert some of their actions via an enhancement of the extracellular levels of adenosine.

INTRODUCTION

potentiates the depressant action of adenosine on the firing of cerebral cortical neurons and the depressant actions of flurazepam are inhibited by the adenosine antagonist, theophylline. Furthermore, diazepam enhances the release of adenosine and related purines from the in t,iro rat cerebral cortex (Phillis et al., 1980) and the depressant effects of diazepam on acetylcholine release from the rat cortex are abolished by theophylline, an adenosine antagonist. Diazepam and flurazepam have been shown to inhibit the uptake of adenosine by brain preparations (Mah & Daly, 1976; Traversa & Newman 1979; Hertz et al., 1979; Bender et al., 1980a) and should therefore enhance the levels of extracellular adenosine. Such an action would account for both the potentiation of adenosine's actions and the increased efflux of adenosine from the cerebral cortex described in the preceding paragraph. An enhancement of the levels of extracellular adenosine would also account for the theophylline- and adenosine-deaminase-sensitive stimulation of brain cyclic AMP formation by benzodiazepines (Schultz, 1974; Palmer, 1979; Traversa & Newman, 1979). The results presented in this paper show that a series of benzodiazepines, at concentrations within the therapeutic range, can inhibit adenosine uptake into rat brain cortical synaptosomes. Furthermore, the inhibitory potency of the substances tested has a clear relationship to their therapeutic efficacy.

Despite their widespread employment in the treatment of psychiatric and seizure disorders, the mechanisms of action of the benzodiazepines have not been elucidated. Some of the biochemical and electrophysiological evidence suggests that these compounds facilitate GABA-crgic synaptic effects but the results have sometimes been contradictory (Costa et al., 1975; Keller et al., 1976; Braestrup & Squires, 1978; Costa & Guidotti, 1979; Pole et al., 1974; G~ihwiler, 1976; Curtis et al., 1976; Geller et al., 1978; Assumpc~o et al., 1979). Part of the disagreement may relate to the doses of benzodiazepines used. MacDonald & Barker (1978) have found that in cell cultures of mammalian neurons, bcnzodiazepines potentiate GABA responses at very low doses but may antagonize them at higher doses. Another possible reason for the contradiction may be the existence of two or more biochemically and pharmacologically distinct benzodiazepine receptors IKlepner et al., 1979; Squires et al., 1979). Klepner et al. (1979) have proposec~ the existence of two biochemically distinct benzodiazepine receptors in rat brain which arc responsible for the mediation of different pharmacological activities. The type 1 receptors according to this scheme, which are not coupled to GABA receptors, mediate anxiolytic actions. Type II receptors are coupled to GABA receptors and mediate pharmacological effects other than anxiolytic activity such as sedation and ataxia. The suggestion has been made in earlier publications that bcnzodiazepines may exert some of their therapeutic actions by potentiating the pharmacological effects of endogenously released adenosine (Phillis, 1979; Phillis et al.. 1979a). Diazepam

MATERIALS A N D

METHODS

Preparation of sk,naptosome.s

Synaptosomes were prepared according to the method of Gray & Whittaker (1962), as modified by White (1975). 67

68

J . W . PHILLIS, P. H. W t and A. S. BENDI!R

Brielly. 4 male Wistar rats (weighing 150 250 g) were sacrificed. The cortices were removed and homogenized in 0.32 M sucrose solution, pH 7.5. The homogenate was centrifuged at 1000 O for 10 min. The pellet was discarded and the remaining supernatant was centrifuged again at 12.000 O for 20 min at 4 C. The resultant pellet was suspended in 12 ml of ice-cold 0.32 M sucrose solution pH 7.5 and transferred to a sucrose discontinuous gradient and centrifuged at 150,000 g for 60 min. The synaptosomal fraction was collected and diluted to 40 ml with 0.32 M sucrose solution. The synaptosomal suspension was centrifuged again at 20,000g for 2 0 m i n at 4~C. Finally, the pellet (synaptosomes) was suspended in 2 ml of ice-cold 0.32 M sucrose solution. 50,ul (0.4 ~ 0.5 mg protcin) of this preparation was used for the uptake studies. Protein was determined according to the method of Bio-Rad Binding Assay (Jan. 1979).

U p t a k e of 3 H - a d e n o s i n e (1 p M ) was linear with t i m e d u r i n g a 30 sec period a n d an i n c u b a t i o n time of 30 sec w a s therefore u s e d for m e a s u r e m e n t of uptake. U p t a k e of S H - a d e n o s i n e was s t u d i e d at c o n c e n t r a t i o n s b e t w e e n 0.25 p M a n d 2.0 ,uM. T h e r e a p p e a r e d to be o n e s a t u r a b l e t r a n s p o r t s y s t e m . T h e net u p t a k e (differences in the level of radioactivities b e t w e e n total u p t a k e a n d the non-specific b i n d i n g to) r u p t u r e d s y n a p t o s o m e s ) a p p e a r e d to be s a t u r a b l e at a d e n o s i n e c o n c e n t r a t i o n s a b o v e 1.4 I~M. Reciprocal plots cons t r u c t e d f r o m the d a t a revealed a h i g h affinity transport site with an a p p a r e n t K,,, of 0.9/.,M a n d a I,',.... of 5.26 p m o l / m g p r o t e i n / 3 0 sec.

Uptake of adenosine by s)'naptosonies

Metabolism of adenosine

50/.d (0.4 ~ 0.5 mg protein) was added to 940 ,ul of incubation solution containing (in final concentration); 1 2 0 m M NaCI, 4 . 7 5 m M KC1. 1.18mM MgSO4, 2 6 r a M N a H C O 3, 1.2 m M K H 2 P O , , 1.77 m M CaCI,, 5.5 m M glucose and 58.5 m M sucrose (White, 1975). The mixture was preincubated for a period of 2 rain and then the reaction was initiated by adding 10 ul of SH-adenosine (specific activity I uCi/nmole) so that the final concentration ranged from 0.25 to 2.0/aM in the incubation mixture. The reaction was allowed to continue for 30 seconds (or 0 - 60 sect at 37 C and was then stopped by adding 5.0 ml of washing solution (260 m M sucrose. 2 m M CaCI2, 2 m M MgCI2. and 20 m M Tris HCL pH 7.5) at room temperature to dilute the SH-adenosine concentration in the medium. The suspension was filtered through a W h a t m a n micro-glass fibre filter (GF/C) with suction. The particles retained on the micro-filter were subsequently washed twice with 5.0 ml of washing solution at room temperature. The filter was then transferred to a scintillation counting vial. To this 2 ml of PCS (Amersham) and 10ml Omnifluor-toluene solution were added. Ruptured synaptosomes were prepared by suspending synaptosomes in distilled H 2 0 (pH 7.5) and heating at 10(3 C for 3.5 min. The ruptured synaptosomes were incubated with SH-adenosine for 30 sec as described above for the intact synaptosomal preparation. This ruptured synaptosomal preparation was used for the determination of the nonspecific binding of SH-adenosine to synaptosomal membranes and the glass fibre filter and these values served as blanks for the adenosine uptake in our experiments. Radioactivity was determined in a Nuclear Chicago I S O C A P 300 spectrophotometer. The counting efficiency of the samples, as measured by the sample-channel ratio method, was approximately 42°0.

T h e d i s t r i b u t i o n of radioactivity in the p r o d u c t s of adenosine uptake and metabolism was determined after i n c u b a t i n g s y n a p t o s o m e s with I t i m S H - a d e n o sine (specific activity 1011Ci.,'nmol) for 3 0 s e e at 37 C. T h e s y n a p t o s o m a l c o n t e n t s were e x t r a c t e d a n d lyophilized a n d the m e t a b o l i t e s s e p a r a t e d by silica gel thin layer c h r o m a t r o g r a p h y a c c o r d i n g to the m e t h o d of S h i m i z u et al. (1969). 70", o f the a d e n o s i n e t a k e n u p r e m a i n e d as a d e n o s i n e a n d 12°. was c o n v e r t e d to nucleotides. I n o s i n e r e p r e s e n t e d the bulk of the degrad a t i v e m e t a b o l i t e s (14,o). o/ H y p o x a n t h i n e a n d cyclic A M P were f o u n d in trace a m o u n t s of 1.501, a n d 2.5°,i respectively.

Inhibition of adenosine uptake Bromazepam, chlordiazepoxide, clonazepam, diazepam, flunitrazepam, flurazepam, lorazepam, medazepam, nitrazepam, oxazepam, RO 11-6896, RO 11-6893 and RO 5-3636 were tested as inhibitors of adenosine uptake into synaptosomal preparations. 10~ul aliquots of various concentrations of the benzodiazepines were added to 980 td of the incubation mixture containing 50 ,ul (0.4 ~ 0.5 mg protein) of synaptosomal preparation and the mixture was preincubated for 2 min at 3 7 C . (The final concentrations of benzodiazepines in the incubation mixture were 1 0 4 10 -s, 10 '~', 10 -'7 and 10 -a M.) The limited solubility of many of the benzodiazepines restricted our use of higher concentrations in the incubation mixture). After preincubation, I0 ,ul of 3H-adenosine (1/aCi/nmole) was added to thc mixture for a final adenosine concentration of 2 x 10- 7 M. Incubation was allowed for 30 sec at 37~C and the samples were processed according to the procedures described in the previous section.

REStI.IS

Rapid uptake of adenosine

Inhibition ofadenosine uptake by tit(' henzodiazepine~ T h e results p r e s e n t e d in T a b l e I s h o w that the benzodiaz.epines i n h i b i t e d the u p t a k e of a d e n o s i n e into rat b r a i n cortical s y n a p t o s o m e s . T h e o r d e r of p o t e n c y as i n d i c a t e d by the IC2o values is c l o n a z e p a m , nitr a z e p a m , l o r a z e p a m , R O 11-6896, d i a z e p a m , flunitraTable

I. Inhibition

Compound Clonazepam Nitrazepam Lorazepam Ro 11-6896 Diazepam Flunitrazepam Medazepam Flurazepam Bromazepam Chlordiazepoxide RO5-3636 Oxazepam RO 11-6893

of adenosine uptake benzodiazepines+ IC2o value 5.0 3.0 6.0 8.4 9.0 1.6 2.6 2.7 1.1 3.7 6.0 9.1) 2.0

x × × x x x × x × × x x ×

I0 -') M 10-SM 10-SM 10 8 M 10 " M 10- v M 10-~M 10-~M 10 ('M 10-('M 10 -~'M 10 ~' M 10 s M

by

~arious

if's(, ~alucs 3.5 5.0 4.0 6.0 7.0 1.0 2.3 1.3 3.5 1.5 6.0 2.5

× x × x × x × x × x x x

10 s M 10 ~ M * 10SM 10-SM 10 s M 10 3 M* 10 "~M* 10-3M * 10-'* M* 10 3 M* 10 3 M . 10 -4 M*

* Data obtained by extrapolation of dose response curves. "1"IC2o and ICs0 values were obtained from semilogarithmic plots of dose response curves determined from bcnzodiazepine inhibition of adenosine uptake into rat brain cortical synaptosomes. The dose response curves were generated by linear regression analysis of the data obtained from six separate experiments, performed in triplicate. The coefficient of correlation value of the curve was in every instance greater than 0.988. The SEM in ever}' instance was less than seven percent.

Benzodiazepine inhibition of adenosine uptake zepam, medazepam, flurazepam, bromazepam, chlordiazepoxide, RO 5-3636, oxazepam and RO 11-6893. Some difficulty was experienced in the measurement of ICso values as the data for the less potent benzodiazepines had to be obtained by extrapolation of the dose-response curves. With a few exceptions, the general order of potency for adenosine uptake inhibition denoted by the ICs0 values was comparable with that indicated by the IC2o measurements. Bromazepam and oxazepam appeared to be slightly more potent at the higher concentration; flunitrazepam. flurazepam and nitrazepam were less potent. The nearly parallel nature of the dose response curves for the 13 benzodiazepines tested in this series gives a strong indication that the compounds inhibit adenosine uptake by similar mechanisms. The difference in uptake inhibitory potencies of the two steroisomers, RO 11-6896 and RO 11-6893 is of special significance as RO 11-6896 is known to be much more effective therapeutically than RO 11-6893. DISCUSSION These findings of potent inhibition of adenosine uptake by the benzodiazepines suggest that these compounds may evoke some of their therapeutic actions by enhancing extracellular adenosine levels. Adenosine is a powerful depressant of the spontaneous firing of neurons in many regions of the brain (Phillis et al., 1974, 1979a, b,c; Kuroda, 1978) and it appears to exert its action by depressing the release of transmitter from presynaptic nerve terminals (Harms et al., 1978, 1979). Adenosine antagonists (caffeine and theophylline) increase the rate of firing of cerebral cortical neurons, presumably by removing the inhibitory effects of endogenously released adenosine. Potentiators of adenosine (uptake inhibitors, adenosine deaminase inhibitors) depress the firing of cerebral cortical neurons (Phillis et al., 1979b,c). Agents, such as the benzodiazepines, which inhibit the uptake and removal of adenosine would thus elicit a suppression of the synaptic drive onto central neurones and a reduced level of excitability in the brain. Indeed diazepam has been shown to potentiate the depressant effects of adenosine on cortical neurons (Phillis, 1979) and theophylline antagonizes the depression of neuronal firing elicited by flurazepam (Phillis et al., 1979a). Moreover diazepam has been shown to increase the effiux of adenosine from the intact rat cerebral cortex, and the depressant effects of diazepam on acetylcholine release from the rat cortex are antagonized by theophylline (Phillis et al., 1980). Since acetylcholine has been implicated in central arousal, any reduction in its release might be expected to affect behavioural awareness (Karczmar, 1979). Reference was made in the introduction of this report of the possibility that there may be more than one type of benzodiazepine receptor (Klepner et al., 1979; Squires et al., 1979). Specifically, it has been suggested that the receptor which is coupled to GABA receptors may mediate such actions as sedation and ataxia whilst a second receptor, which is unrelated to the GABA receptor, may mediate anxiolytic actions. In the context it is interesting to consider the well known anxiety-provoking effects of the adenosine-antagonist, caffeine (Lutz, 1978; Greden,

69

1979). In the light of recent findings on adenosine's actions on the firing of neurons in the brain and the antagonism between adenosine and caffeine, it is reasonable to propose that adenosine may be critically involved in controlling levels of anxiety. According to this concept benzodiazepines may exert their anxiolytic effects by enhancing the levels of adenosine in the extracellular fluid around nerve cells and their processes. Adenosine removal is likely to be a function of both neuronal and glial elements in the brain (Shimizu et al., 1972; Hertz, 1978; Bender et al., 1980b), and the benzodiazepine effects are probably exerted on both types of cell (Hertz et al., 1979; Traversa & Newman, 1979; Bender et al., 1980a). The potency of the benzodiazepines as adenosine uptake inhibitors shows a good correlation with their clinical, pharmacological and receptor binding potencies (Cook & Sepinwall, 1975; M6hler & Okada, 1978; Braestrup & Squires, 1978; Duka et al., 19791. This correlation between the various pharmacological properties of the benzodiazepines and their ability to inhibit adenosine uptake clearly suggests that inhibition of adenosine uptake is an important factor in the central actions of these compounds. Acknowledgements--Supported by the Canadian Medical Research Council. We are grateful to Hoffman-La Roche Ltd., Canada for their gift of the benzodiazepines used in this study.

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