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Inhibition of adenosine accumulation into guinea pig ventricle by benzodiazepines
European Journal of Pharmacology, 78 (1982) 241-24z~
241
Elsevier Biomedical Press
Short communication I N H I B I T I O N OF A D E N O S I N E A C C U M U L A T I O N I N T O G U I N E A P I G V E N T R I C L E BY BENZODIAZEPINES PHILIP H. BARKER and ALEXANDER S. CLANACHAN * Department of Pharmacology, University of Alberta, Edmonton, Alberta, T6G 2H7 Canada
Received 24 November 1981, accepted 28 December 1981
P.H. BARKER and A.S. CLANACHAN, Inhibition of adenosine accumulation into guineapig ventricle by benzodiazepines, European J. Pharmacol. 78 (1982) 2,~1-244. Several benzodiazepines were compared with dipyridamole for their ability to inhibit the nucleoside transporter specific accumulation of [3HJadenosine into guinea pig ventricle. Diazepam inhibited adenosine accumulation but was considerably less potent than dipyridamole. Lorazepam and flurazepam also inhibited accumulation, but were less potent than diazepam. Coronary vasodilation by diazepam or by dipyridamole probably is mediated via potentiation of adenosine following inhibition of the nucleoside transport system. Benzodiazepines
Coronary vasodilation
Dipyridamole Adenosineaccumulation
1. Introduction The central actions of benzodiazepines (BDZs) are considered to involve an interaction with a specific, high affinity binding site which results in a potentiation of the inhibitory effects of 3'aminobutyric acid (GABA). Interactions between benzodiazepines and adenosine have also been reported. Diazepam potentiates adenosine action in the CNS in vivo (Phillis, 1979) and in cardiac and smooth muscle preparations in vitro (Clanachan and Marshall, 1980a). Diazepam also increases coronary blood flow in man (Ikram et al., 1973) and potentiates adenosine-induced coronary vasodilation in anaesthetized dogs (Clanachan and Marshall, 1980b). The inability of diazepam to potentiate the negative inotropic effect of 2chloroadenosine (Clanachan and Marshall, 1980a), an analogue which appears not to be a substrate for the nucleoside transport system, suggests that diazepam may potentiate adenosine effects through inhibition of adenosine inactivation by the nucleoside transport system. Estimation of the affinities of several BDZs for the nucleoside transport system in human erythrocytes ( H a m m o n d et • To whom all correspondence should be addressed, 0014-2999/82/0000-0000/$02.75 © 1982 Elsevier BiomedicalPress
al., 1981) through inhibition of the site-specific binding of a potent nucleoside transport inhibitor, nitrobenzylthioinosine, revealed that diazepam has an apparent K i for the transporter of 5-10 ~M. This is also the range of concentrations of diazepam which has been shown previously to potentiate adenosine effects in vitro. In view of the importance of the role of adenosine in the regulation of coronary blood flow (Berne, 1963) and the observation that diazepam exerts significant effects in the heart (Clanachan and Marshall, 1980b), we have examined the effects of several BDZs on the nucleoside transporter mediated accumulation of adenosine in guinea pig left ventricle.
2. Materials and methods Female guinea pigs (350-400 g)were killed by a blow on the head followed by exsanguination. The hearts were quickly removed and placed in oxygenated Krebs solution (37°C). The left ventricle was dissected free from other heart tissue and cut into slices weighing about 10 mg each. Ventricle slices were first equilibrated for 30 rain at 37°C in oxygenated Krebs solution and then placed in 2'-deoxycorformycin (1/~M) Krebs solu-
242
tion for a further 30 rain. Each slice was then preincubated for 30 min in 2.0 ml of oxygenated Krebs solution containing 2'-deoxycorformycin (1/*M) and various concentrations of the potential inhibitors. The slices were finally incubated (2.0 ml final volume) in adenosine (1 #M) containing [3H]adenosine (2.5 #Ci), 2'-deoxycorformycin (1/*M) and various concentrations of the inhibitors. After 20 rain of incubation, the tissues were removed, blotted, weighed, dissolved (NCS 400 /*1), and neutralized with glacial acetic acid (10 #1). Tritium activity of the digests was measured by liquid scintillation spectrometry and expressed as adenosine equivalents (mean -+ S.E.). Significance levels for the difference between groups w e r e estimated using the Student's t-test and the difference was judged to be significant when P < 0.05. The Krebs solution had the following composition (mM): NaC1 116, KC1 5.4, CaC12 2.5, MgC12 1.2, NaHzPO 4 1.2, NaHCO 3 22, D-glucose 11.2. The solution (pH 7.4) was equilibrated with 95% O 2 and 5% CO z and maintained at 37°C. Drugs used were: [2-3H]adenosine (20 Ci/mmol; purchased from New England Nuclear); dipyridamole (donated by Boehringer-Ingelheim); diazepam and flurazepam (donated by HoffmannoLaRoche); lorazepam (donated by Wyeth Ltd.); morphine sulphate (BDH Chemicals); theophylline (Sigma Chemical Co.); hydroxynitrobenzylthioguanosine and 2'-deoxycorformycin (kindly donated by dr. A.R.P. Paterson, University of Alberta, Cancer Research Unit).
3. Results The total accumulation of adenosine by guinea pig ventricle was time-dependent and following incubation for 20 rain was equivalent to 4.1 ± 0.2 nmol adenosine/g wet weight (table 1), representing a tissue/medium ratio of about 4. Nucleoside transporter specific accumulation of adenosine was defined as the total accumulation minus the accumulation (presumably due to equilibration with the extracellular space and/or passive diffusion) which occurred in the presence of supramaximal nucleoside transport inhibiting concentrations of dipyridamole (10 #M) or hydroxynitroben-
TABLEI Accumulationof adenosine in guinea-pig ventricle. Tissues were continually exposed to 2'-deoxycorformycin (1/,M) to inhibit adenosine deaminase. They were incubated in the presence of [3H]adenosine (1 #M) for 20 rain, dissolved in NCS and prepared for liquid scintillation counting. Values of tritium accumulation, expressedas adenosineequivalents, represent the
mean-~S.E.,n=6. Accumulation (nmol/g
wet weight) Control
4.10±0.20
Hydroxynitrobenzylthioguanosine (10 ~M) Dipyridamole(10~M) Transporter specific
1.20~-0.20
1.05±0.18 2.90--+0.19
zylthioguanosine (10/~M). As nucleoside transport is a carrier-mediated facilitated diffusion process this apparent adenosine concentrating ability of cardiac muscle is presumably due to transport of the nucleoside followed by intracellular trapping by conversion to nucleotides by adenosine kinase. Dipyridamole (0.01-1 #M), a recognized inhibitor of nucleoside transport, produced a concentrationdependent inhibition of adenosine accumulation in guinea pig ventricle (table 2). Several BDZs also inhibited the nucleoside transporter specific accumulation of adenosine (table2), but these compounds were significantly weaker in this respect than dipyridamole. Diazepam was the most potent B D Z tested in the present study and had an IC50 of approximately 80 #M. Lorazepam, which is approximately five times more potent than diazepam as an anxiolytic agent, was significantly less potent than diazepam in reducing adenosine accumulation in cardiac muscle. Flurazepam was the weakest inhibitor of adenosine accumulation. It is unlikely that inhibition of adenosine accumulation was due to a non-specific effect of the high concentrations of BDZs tested as theophylline (300 #M), an adenosine receptor antagonist, or morphine (300 #M), failed to modify adenosine accumulation(table2).
243 TABLE 2 Inhibition of nucleoside transporter specific accumulation of adenosine by dipyridamole and several benzodiazepines. Tissues were continually exposed to 2'-deoxycorformycin (1 #M) to inhibit adenosine deaminase. They were incubated in the indicated concentrations of each potential transport inhibitor for 30 min before exposure to [3H]adenosine (1/~M) for 20 min. Tissues were dissolved in NCS and tritium activity (expressed as adenosine equivalents) was measured by liquid scintillation spectrometry. Values represent the mean---S.E., n~ 5. * Represents a significant difference (P<0.05, paired t-test) from the solvent control. NS, not significant. Drug
~M
Control
Nucleoside transporter specific accumulation (nmol/g)
% Inhibition
2.36 ±0.25
Solvent control
(DMSO)
2.53 ± 0.21
Dipyridamole
0.01 0.1 1.0 I0.0
1.99---0.32 1.31 ±0.43 0.34±0.25 0.00
21.3 * 48.2 * 86.6 * 100.0 *
Diazepam
10 100 300
2.18±0.33 0.99±0.25 0.20±0.30
13.8 * 60.9 * 92.1 *
Lorazepam
10 100 300
2.32 ± 0.44 2.50~0.27 0,76---0.14
NS NS 70.0 *
Flurazepam
10 100 300
1.42± 0.08 1.59---0.25 1.50±0.25
43.9 * 37.2 * 40.7 *
Theophylline
300
2.79 ± 0.35
NS
Morphine
300
2.78 ± 0.57
NS
4. Discussion -The accumulation of adenosine by guinea pig ventricle, which occurred by a nucleoside transporter specific mechanism, was inhibited by dipyridamole and b y several BDZs. The B D Z s were very weak inhibitors relative to dipyridamole and significant inhibition of accumulation was observed only with concentrations of the B D Z s in excess of 1/xM. The low apparent affinity of dia z e p a m for the nucleoside transport system (K i 9.8 # M ) was also observed in h u m a n erythrocytes ( H a m m o n d et al., 1981). It is unlikely, therefore, that significant inhibition of nucleoside transport would occur in the presence of concentrations of diazepam ( < I # M ) associated with its anxiolytic action. However, it is within the concentration range of 5 - 1 0 ptM that diazepam was shown previously to potentiate the negative inotropic action of
adenosine in guinea pig cardiac muscle (Clanachan and Marshall, 1980a). Further, this is the approximate peak plasma concentration of diazepam which can be associated with the decrease in c o r o n a r y vascular resistance in man. In smooth muscle in vitro, the ability to potentiate adenosine effects was not shared by all clinically active BDZs. L.orazepam, for example, failed to potentiate the presynaptic inhibitory action of adenosine (Clanachan and DeBoer, 1980). Furthermore, lorazepam has been shown to have approximately 7 - 1 0 times lower affinity than diazepam for the nucleoside transport system in h u m a n erythrocytes ( H a m m o n d et al., 198 I). These results are in agreement with the present demonstration that lorazepam was weaker than diazepam as an inhibitor of adenosine accumulation. Also, lorazepam, unlike diazepam, does not potentiate the negative inotropic action of adenosine on
244 g u i n e a pig atria (Barker a n d C l a n a c h a n , u n p u b lished). Similarly, f l u r a z e p a m showed low activity as an i n h i b i t o r of a d e n o s i n e a c c u m u l a t i o n in c a r d i a c muscle (ICs0 > 300 # M ) a n d as an inhibitor of n i t r o b e n z y l t h i o i n o s i n e b i n d i n g (ICso > 300 /~M) to the nucleoside t r a n s p o r t system in h u m a n e r y t h r o c y t e s ( H a m m o n d et al., 1981). C o r o n a r y v a s o d i l a t i o n b y d i p y r i d a m o l e is considered to be due to i n h i b i t i o n of the nucleoside t r a n s p o r t system resulting in a p o t e n t i a t i o n of the e x t r a c e l l u l a r effects of adenosine, m similar mecha n i s m m a y also e x p l a i n d i a z e p a m - i n d u c e d c o r o n a r y vasodilation. O n the o t h e r h a n d , the recent d e m o n s t r a t i o n of high affinity ' p e r i p h e r a l t y p e ' B D Z r e c o g n i t i o n sites for B D Z s ( K i for d i a z e p a m of 49 riM) in guinea pig c a r d i a c muscle (Davies a n d H u s t o n , 1981) has raised the possibility that c o r o n a r y v a s o d i l a t i o n m a y be m e d i a t e d via these sites. However, this is unlikely as d i a z e p a m a n d d i p y r i d a m o l e have c o m p a r a b l e affinities for the ' p e r i p h e r a l ' B D Z r e c o g n i t i o n site in c a r d i a c m u s c l e (Davies a n d H u s t o n , 1981) whereas dip y r i d a m o l e is c o n s i d e r a b l y m o r e p o t e n t t h a n dia z e p a m as a c o r o n a r y v a s o d i l a t o r ( C l a n a c h a n a n d M a r s h a l l , u n p u b l i s h e d ) a n d as an i n h i b i t o r of a d e n o s i n e a c c u m u l a t i o n . The site at which dia z e p a m acts to cause c o r o n a r y v a s o d i l a t i o n is p r o b a b l y the nucleoside t r a n s p o r t site at which d i a z e p a m d i s p l a y s low affinity ( K i a b o u t 5 - 1 0 # M ) , a n d which is distinct from the ' p e r i p h e r a l ' t y p e B D Z r e c o g n i t i o n site which has a high affinity for d i a z e p a m ( K i 49 nM).
Acknowledgements Supported by a grant from the Alberta Heart Foundation.
References Berne, R.M., 1963, Cardiac nucleotides in hypoxia: Possible role in regulation of coronary blood flow, Am. J. Physiol. 204, 317. Clanachan, A.S. and A. DeBoer, 1980, Effects of several benzodiazepines on adenosine mediated inhibition of neurotransmission in rat vas deferens in vitro, Proc. West. Pharmacol. Soc. 23, 231. Clanachan, A.S. and R.J. Marshall, 1980a, Potentiation of the effects of adenosine on isolated cardiac and smooth muscle by diazeparn, Br. J. Pharmacol. 71,459. Clanachan, A.S. and R.J. Marshall, 1980b, Diazepam potentiates the coronary vasodilator actions of adenosine in anaesthetized dogs, Br. J. Pharmacol. 70, 66p. Davies, L.P. and V. Huston, 1981, Peripheral benzodiazepine binding sites in heart and their interaction with dipyridamole, European J. Pharmacol. 73, 209. Hammond, J.R., A.R.P. Paterson and A.S. Clanachan, 1981, Benzodiazepine inhibition of the site-specific binding of nitrobenzylthioinosine, an inhibitor of nucleoside transport, Life Sci. 29, 2207. Ikram, H., A.P. Rubin and R.F. Jewkes, 1973, Effect of diazepam on myocardial blood flow of patients with and without coronary artery disease, Br. Heart J. 35, 626. Phillis, J.W., 1979, Diazepam potentiation of purinergic depression of central neurones, Can. J. Physiol. Pharmacol. 57, 432.
241
Elsevier Biomedical Press
Short communication I N H I B I T I O N OF A D E N O S I N E A C C U M U L A T I O N I N T O G U I N E A P I G V E N T R I C L E BY BENZODIAZEPINES PHILIP H. BARKER and ALEXANDER S. CLANACHAN * Department of Pharmacology, University of Alberta, Edmonton, Alberta, T6G 2H7 Canada
Received 24 November 1981, accepted 28 December 1981
P.H. BARKER and A.S. CLANACHAN, Inhibition of adenosine accumulation into guineapig ventricle by benzodiazepines, European J. Pharmacol. 78 (1982) 2,~1-244. Several benzodiazepines were compared with dipyridamole for their ability to inhibit the nucleoside transporter specific accumulation of [3HJadenosine into guinea pig ventricle. Diazepam inhibited adenosine accumulation but was considerably less potent than dipyridamole. Lorazepam and flurazepam also inhibited accumulation, but were less potent than diazepam. Coronary vasodilation by diazepam or by dipyridamole probably is mediated via potentiation of adenosine following inhibition of the nucleoside transport system. Benzodiazepines
Coronary vasodilation
Dipyridamole Adenosineaccumulation
1. Introduction The central actions of benzodiazepines (BDZs) are considered to involve an interaction with a specific, high affinity binding site which results in a potentiation of the inhibitory effects of 3'aminobutyric acid (GABA). Interactions between benzodiazepines and adenosine have also been reported. Diazepam potentiates adenosine action in the CNS in vivo (Phillis, 1979) and in cardiac and smooth muscle preparations in vitro (Clanachan and Marshall, 1980a). Diazepam also increases coronary blood flow in man (Ikram et al., 1973) and potentiates adenosine-induced coronary vasodilation in anaesthetized dogs (Clanachan and Marshall, 1980b). The inability of diazepam to potentiate the negative inotropic effect of 2chloroadenosine (Clanachan and Marshall, 1980a), an analogue which appears not to be a substrate for the nucleoside transport system, suggests that diazepam may potentiate adenosine effects through inhibition of adenosine inactivation by the nucleoside transport system. Estimation of the affinities of several BDZs for the nucleoside transport system in human erythrocytes ( H a m m o n d et • To whom all correspondence should be addressed, 0014-2999/82/0000-0000/$02.75 © 1982 Elsevier BiomedicalPress
al., 1981) through inhibition of the site-specific binding of a potent nucleoside transport inhibitor, nitrobenzylthioinosine, revealed that diazepam has an apparent K i for the transporter of 5-10 ~M. This is also the range of concentrations of diazepam which has been shown previously to potentiate adenosine effects in vitro. In view of the importance of the role of adenosine in the regulation of coronary blood flow (Berne, 1963) and the observation that diazepam exerts significant effects in the heart (Clanachan and Marshall, 1980b), we have examined the effects of several BDZs on the nucleoside transporter mediated accumulation of adenosine in guinea pig left ventricle.
2. Materials and methods Female guinea pigs (350-400 g)were killed by a blow on the head followed by exsanguination. The hearts were quickly removed and placed in oxygenated Krebs solution (37°C). The left ventricle was dissected free from other heart tissue and cut into slices weighing about 10 mg each. Ventricle slices were first equilibrated for 30 rain at 37°C in oxygenated Krebs solution and then placed in 2'-deoxycorformycin (1/~M) Krebs solu-
242
tion for a further 30 rain. Each slice was then preincubated for 30 min in 2.0 ml of oxygenated Krebs solution containing 2'-deoxycorformycin (1/*M) and various concentrations of the potential inhibitors. The slices were finally incubated (2.0 ml final volume) in adenosine (1 #M) containing [3H]adenosine (2.5 #Ci), 2'-deoxycorformycin (1/*M) and various concentrations of the inhibitors. After 20 rain of incubation, the tissues were removed, blotted, weighed, dissolved (NCS 400 /*1), and neutralized with glacial acetic acid (10 #1). Tritium activity of the digests was measured by liquid scintillation spectrometry and expressed as adenosine equivalents (mean -+ S.E.). Significance levels for the difference between groups w e r e estimated using the Student's t-test and the difference was judged to be significant when P < 0.05. The Krebs solution had the following composition (mM): NaC1 116, KC1 5.4, CaC12 2.5, MgC12 1.2, NaHzPO 4 1.2, NaHCO 3 22, D-glucose 11.2. The solution (pH 7.4) was equilibrated with 95% O 2 and 5% CO z and maintained at 37°C. Drugs used were: [2-3H]adenosine (20 Ci/mmol; purchased from New England Nuclear); dipyridamole (donated by Boehringer-Ingelheim); diazepam and flurazepam (donated by HoffmannoLaRoche); lorazepam (donated by Wyeth Ltd.); morphine sulphate (BDH Chemicals); theophylline (Sigma Chemical Co.); hydroxynitrobenzylthioguanosine and 2'-deoxycorformycin (kindly donated by dr. A.R.P. Paterson, University of Alberta, Cancer Research Unit).
3. Results The total accumulation of adenosine by guinea pig ventricle was time-dependent and following incubation for 20 rain was equivalent to 4.1 ± 0.2 nmol adenosine/g wet weight (table 1), representing a tissue/medium ratio of about 4. Nucleoside transporter specific accumulation of adenosine was defined as the total accumulation minus the accumulation (presumably due to equilibration with the extracellular space and/or passive diffusion) which occurred in the presence of supramaximal nucleoside transport inhibiting concentrations of dipyridamole (10 #M) or hydroxynitroben-
TABLEI Accumulationof adenosine in guinea-pig ventricle. Tissues were continually exposed to 2'-deoxycorformycin (1/,M) to inhibit adenosine deaminase. They were incubated in the presence of [3H]adenosine (1 #M) for 20 rain, dissolved in NCS and prepared for liquid scintillation counting. Values of tritium accumulation, expressedas adenosineequivalents, represent the
mean-~S.E.,n=6. Accumulation (nmol/g
wet weight) Control
4.10±0.20
Hydroxynitrobenzylthioguanosine (10 ~M) Dipyridamole(10~M) Transporter specific
1.20~-0.20
1.05±0.18 2.90--+0.19
zylthioguanosine (10/~M). As nucleoside transport is a carrier-mediated facilitated diffusion process this apparent adenosine concentrating ability of cardiac muscle is presumably due to transport of the nucleoside followed by intracellular trapping by conversion to nucleotides by adenosine kinase. Dipyridamole (0.01-1 #M), a recognized inhibitor of nucleoside transport, produced a concentrationdependent inhibition of adenosine accumulation in guinea pig ventricle (table 2). Several BDZs also inhibited the nucleoside transporter specific accumulation of adenosine (table2), but these compounds were significantly weaker in this respect than dipyridamole. Diazepam was the most potent B D Z tested in the present study and had an IC50 of approximately 80 #M. Lorazepam, which is approximately five times more potent than diazepam as an anxiolytic agent, was significantly less potent than diazepam in reducing adenosine accumulation in cardiac muscle. Flurazepam was the weakest inhibitor of adenosine accumulation. It is unlikely that inhibition of adenosine accumulation was due to a non-specific effect of the high concentrations of BDZs tested as theophylline (300 #M), an adenosine receptor antagonist, or morphine (300 #M), failed to modify adenosine accumulation(table2).
243 TABLE 2 Inhibition of nucleoside transporter specific accumulation of adenosine by dipyridamole and several benzodiazepines. Tissues were continually exposed to 2'-deoxycorformycin (1 #M) to inhibit adenosine deaminase. They were incubated in the indicated concentrations of each potential transport inhibitor for 30 min before exposure to [3H]adenosine (1/~M) for 20 min. Tissues were dissolved in NCS and tritium activity (expressed as adenosine equivalents) was measured by liquid scintillation spectrometry. Values represent the mean---S.E., n~ 5. * Represents a significant difference (P<0.05, paired t-test) from the solvent control. NS, not significant. Drug
~M
Control
Nucleoside transporter specific accumulation (nmol/g)
% Inhibition
2.36 ±0.25
Solvent control
(DMSO)
2.53 ± 0.21
Dipyridamole
0.01 0.1 1.0 I0.0
1.99---0.32 1.31 ±0.43 0.34±0.25 0.00
21.3 * 48.2 * 86.6 * 100.0 *
Diazepam
10 100 300
2.18±0.33 0.99±0.25 0.20±0.30
13.8 * 60.9 * 92.1 *
Lorazepam
10 100 300
2.32 ± 0.44 2.50~0.27 0,76---0.14
NS NS 70.0 *
Flurazepam
10 100 300
1.42± 0.08 1.59---0.25 1.50±0.25
43.9 * 37.2 * 40.7 *
Theophylline
300
2.79 ± 0.35
NS
Morphine
300
2.78 ± 0.57
NS
4. Discussion -The accumulation of adenosine by guinea pig ventricle, which occurred by a nucleoside transporter specific mechanism, was inhibited by dipyridamole and b y several BDZs. The B D Z s were very weak inhibitors relative to dipyridamole and significant inhibition of accumulation was observed only with concentrations of the B D Z s in excess of 1/xM. The low apparent affinity of dia z e p a m for the nucleoside transport system (K i 9.8 # M ) was also observed in h u m a n erythrocytes ( H a m m o n d et al., 1981). It is unlikely, therefore, that significant inhibition of nucleoside transport would occur in the presence of concentrations of diazepam ( < I # M ) associated with its anxiolytic action. However, it is within the concentration range of 5 - 1 0 ptM that diazepam was shown previously to potentiate the negative inotropic action of
adenosine in guinea pig cardiac muscle (Clanachan and Marshall, 1980a). Further, this is the approximate peak plasma concentration of diazepam which can be associated with the decrease in c o r o n a r y vascular resistance in man. In smooth muscle in vitro, the ability to potentiate adenosine effects was not shared by all clinically active BDZs. L.orazepam, for example, failed to potentiate the presynaptic inhibitory action of adenosine (Clanachan and DeBoer, 1980). Furthermore, lorazepam has been shown to have approximately 7 - 1 0 times lower affinity than diazepam for the nucleoside transport system in h u m a n erythrocytes ( H a m m o n d et al., 198 I). These results are in agreement with the present demonstration that lorazepam was weaker than diazepam as an inhibitor of adenosine accumulation. Also, lorazepam, unlike diazepam, does not potentiate the negative inotropic action of adenosine on
244 g u i n e a pig atria (Barker a n d C l a n a c h a n , u n p u b lished). Similarly, f l u r a z e p a m showed low activity as an i n h i b i t o r of a d e n o s i n e a c c u m u l a t i o n in c a r d i a c muscle (ICs0 > 300 # M ) a n d as an inhibitor of n i t r o b e n z y l t h i o i n o s i n e b i n d i n g (ICso > 300 /~M) to the nucleoside t r a n s p o r t system in h u m a n e r y t h r o c y t e s ( H a m m o n d et al., 1981). C o r o n a r y v a s o d i l a t i o n b y d i p y r i d a m o l e is considered to be due to i n h i b i t i o n of the nucleoside t r a n s p o r t system resulting in a p o t e n t i a t i o n of the e x t r a c e l l u l a r effects of adenosine, m similar mecha n i s m m a y also e x p l a i n d i a z e p a m - i n d u c e d c o r o n a r y vasodilation. O n the o t h e r h a n d , the recent d e m o n s t r a t i o n of high affinity ' p e r i p h e r a l t y p e ' B D Z r e c o g n i t i o n sites for B D Z s ( K i for d i a z e p a m of 49 riM) in guinea pig c a r d i a c muscle (Davies a n d H u s t o n , 1981) has raised the possibility that c o r o n a r y v a s o d i l a t i o n m a y be m e d i a t e d via these sites. However, this is unlikely as d i a z e p a m a n d d i p y r i d a m o l e have c o m p a r a b l e affinities for the ' p e r i p h e r a l ' B D Z r e c o g n i t i o n site in c a r d i a c m u s c l e (Davies a n d H u s t o n , 1981) whereas dip y r i d a m o l e is c o n s i d e r a b l y m o r e p o t e n t t h a n dia z e p a m as a c o r o n a r y v a s o d i l a t o r ( C l a n a c h a n a n d M a r s h a l l , u n p u b l i s h e d ) a n d as an i n h i b i t o r of a d e n o s i n e a c c u m u l a t i o n . The site at which dia z e p a m acts to cause c o r o n a r y v a s o d i l a t i o n is p r o b a b l y the nucleoside t r a n s p o r t site at which d i a z e p a m d i s p l a y s low affinity ( K i a b o u t 5 - 1 0 # M ) , a n d which is distinct from the ' p e r i p h e r a l ' t y p e B D Z r e c o g n i t i o n site which has a high affinity for d i a z e p a m ( K i 49 nM).
Acknowledgements Supported by a grant from the Alberta Heart Foundation.
References Berne, R.M., 1963, Cardiac nucleotides in hypoxia: Possible role in regulation of coronary blood flow, Am. J. Physiol. 204, 317. Clanachan, A.S. and A. DeBoer, 1980, Effects of several benzodiazepines on adenosine mediated inhibition of neurotransmission in rat vas deferens in vitro, Proc. West. Pharmacol. Soc. 23, 231. Clanachan, A.S. and R.J. Marshall, 1980a, Potentiation of the effects of adenosine on isolated cardiac and smooth muscle by diazeparn, Br. J. Pharmacol. 71,459. Clanachan, A.S. and R.J. Marshall, 1980b, Diazepam potentiates the coronary vasodilator actions of adenosine in anaesthetized dogs, Br. J. Pharmacol. 70, 66p. Davies, L.P. and V. Huston, 1981, Peripheral benzodiazepine binding sites in heart and their interaction with dipyridamole, European J. Pharmacol. 73, 209. Hammond, J.R., A.R.P. Paterson and A.S. Clanachan, 1981, Benzodiazepine inhibition of the site-specific binding of nitrobenzylthioinosine, an inhibitor of nucleoside transport, Life Sci. 29, 2207. Ikram, H., A.P. Rubin and R.F. Jewkes, 1973, Effect of diazepam on myocardial blood flow of patients with and without coronary artery disease, Br. Heart J. 35, 626. Phillis, J.W., 1979, Diazepam potentiation of purinergic depression of central neurones, Can. J. Physiol. Pharmacol. 57, 432.