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Peripheral benzodiazepine binding sites in heart and their interaction with dipyridamole
European Journal of Pharmacology, 73 (1981) 209-211 Elsevier/North-Holland Biomedical Press
209
Short communication PERIPHERAL BENZODIAZEPINE BINDING SITES IN HEART AND THEIR INTERACTION WITH DIPYRIDAMOLE
LES P. DAVIES * and VICKI HUSTON Roche Research Institute of Marine Pharmacology, P.O. Box 255, Dee Why, N.S.W. 2099, Australia Received 26 May 1981, accepted 1 June 1981
L.P. DAVIES and V. HUSTON, Peripheral benzodiazepine binding sites in heart and their interaction with dipyridamole, European J. Pharmacol. 73 (1981) 209-211. Specific binding of [3 H]diazepam to rat and guinea-pig heart tissue has been found. These binding sites are similar to those in lung, liver, kidney and mast cells but differ from those in the brain. Dipyridamole interacts with this receptor and is several fold more potent as a displacer of [3 H]diazepam bound to heart preparations than to brain membranes. Specific binding per unit protein in the left ventricle, fight ventricle and interventricular septum is significantly higher than in the left and right atrium. Diazepam binding
Heart
Dipyridamole
1. Introduction
2. Materials and methods
We previously reported (Davies et al., 1980) that the coronary vasodilator drug dipyridamole was relatively potent as a displacer of [3H]diazepam bound to CNS benzodiazepine receptors. In view of current interest in peripheral benzodiazepine receptors which are pharmacologically distinct from the central receptors (Braestrup and Squires, 1977; Taniguchi et al., 1980) we have examined whether dipyridamole also interacts with peripheral receptors. Since heart tissue has not previously been examined for the presence of peripheral benzodiazepine receptors, we decided to investigate this tissue, particularly since diazepam has been reported to have a coronary vasodilator action in animals (Daniell, 1975) and man (Ikram et al., 1973). In the present study the regional distribution of binding with the heart was also examined in order to determine more accurately sites of diazepam binding.
Fi~llinsdorf rats (male) and Himalayan white guinea-pigs (either sex) were killed by decapitation and hearts removed into cold Krebs bicarbonate buffer. Hearts were dissected free of fat and aortic and vena caval stumps and homogenized or further dissected into the following regions: left atrium, right atrium, left ventricle, right ventricle, interventricular septum. Tissue pieces were homogenized in 20 vol of ice-cold 0.32 M sucrose u~ing a tissue homogenizer (Polytron), and a P2 p~llet prepared as described previously (Davies et al!, 1980). Pellets were resuspended in 50 mM T~is.HC1 buffer, pH 7.45 at 0-4°C and assays carried out in the same buffer according to established methods (M/Shler and Okada, 1977). Binding assays (final volume of 2 ml) were carried out on ice and were terminated after 15 min by addition of 4 rnl of ice-cold buffer and rapid filtration on Whatman G F / B glass-fibre filters. Tube and filters were washed by a further 8 ml of buffer. Non-specific binding to peripheral tissue was determined in the presence of 10 #M unlabelled diazepam. Protein was measured by the Lowry method. [Methyl-3H]diazepam (76.8- 87.6 Ci/mmol) was
* To whom reprint requests should be addressed at present address: Department of Pharmacology, Blackburn Building, University of Sydney, Sydney, N.S.W. 2006, Australia.
0014-2999/81/0000-0000/$02.50 © Elsevier/North-Holland Biomedical Press
210
supplied by New England Nuclear Boston; dipyridamole (Persantin), diazepam and clonazepam by Hoffmann-La Roche, Basel and the benzodiazepine Ro 5-4864 by Hoffmann-La Roche, Nutley, N.J.
3. Results
Incubation of heart extracts with labelled diazepam showed that saturable binding sites existed in both guinea-pig and rat heart tissue with Kos of 49 nM (mean of 4 determinations) and 80 nM (2 determinations) respectively. The amount of binding in heart was variable from experiment to experiment, with very low binding in tissue which was stored frozen prior t o assay. In 4 experiments with guinea-pig heart a Bma~ of 4.2 pmol/mg protein was obtained, which is just over twice that measured by us for rat brain diazepam binding (1.9 pmol/mg protein; Davies et al., 1980). The binding of [3H]diazepam to heart receptor sites was clearly differentiated from its binding to CNS sites by the use of clonazepam and Ro 5-4864 (table 1). Whereas clonazepam, specific for CNS receptors (Braestrup and Squires, 1977) did not displace heart diazepam binding, Ro 5-4864 which has been shown to bind only to peripheral benzodiazepine receptors (Braestrup and Squires, 1977) was very potent in displacing [3 H]diazepam bound to the heart preparations. Dipyridamole was quite TABLE 1 Competition by benzodiazepines and dipyridamole with specific [3H]diazepam binding to heart tissue. Each value (with 95% confidence limits) was calculated from log-probit plots of inhibition at four separate concentrations of inhibitor, with each point being determined in triplicate in 1-3 separate experiments. [3H]Diazepam concentration was 0.94 nM. Drug
IC50 (nM)
Guinea-pig
Diazepam Clonazepam Ro 5-4864 Dipyridamole
49 (27-86) 2 361 (1684-3 310} 2.2 (0.5-9.7) 24 (16-37)
Rat
Diazepam Clonazepam Ro 5-4864 Dipyridamole
73 (21-254) > 3 000 19 (I 1-34) 99 (93-105)
TABLE 2 The distribution of [3H]diazepam binding in five different regions of guinea-pig and rat hearts. Data are means-+ S.E.M. of triplicate binding determination carried out on pooled tissue from the hearts of two guinea-pigs and two rats. The concentration of diazepam used was approximately 0.55 nM. A similar pattern of binding was seen in 4 further experiments (3 using two guinea-pig hearts, 1 using two rat hearts). Binding in the left ventricle, right ventricle and interventricular septum was significantly higher (P<0.05) than in the left atrium and right atrium, both in guinea-pig heart (4 experiments) and rat heart (2 experiments). To check that the pharmacological specificity of the binding sites for dipyridamole was similar in each region, a concentration of drug close to its ICs0 for displacement of binding in whole heart extracts was used in 2 experiments on guinea-pig hearts. Dipyridamole (37.5 nM) produced the following displacements; LA 63.1%; RA 59.4%; LV 64.2%; RV 63.1%; I.V.S. 61.3%. Region
Left atrium Right atrium Left ventricle Right ventricle Interventricular septum
Specific [ 3H]diazepam bound (fmol/mg protein) Guinea-pig
Rat
41.1 _+2.0 43.0 -+ 5.3 189.1 _+2.4 114.4~+4.2 132.8+0.4
17.5 -4-4.5 10.7 -+ 5.5 101.1 -+4.8 93.5-+6.6 65.3-+6.0
potent as a diazepam displacer, both in rat and guinea-pig heart tissue (table 1 and 2). There are obvious differences in the number of binding sites per unit protein in different parts of the heart (table 2) such that the amount of binding in left ventricle, interventricular septum and fight ventricle is significantly greater than in right atrium or left atrium.
4. Discussion
In view of the reported coronary vasodilator action of diazepam it is interesting to note the presence of peripheral-type benzodiazepine receptors in heart tissue, and further, to note a strong interaction of these receptors with dipyridamole, a coronary vasodilator in clinical use. That the coronary vasodilator actions of these two drugs may be at a common site is further supported by the observed regional distribution of diazepam binding to heart which appears to parallel regional
211
coronary blood supply and hence the density of coronary blood vessels; in hearts the turnover rate and tissue uptake of radioactive tracer substances present in the blood is highest in the left ventricle and is progressively less in the right ventricle, left atrium and right atrium (Van Citters, 1965). Dipyridamole appears to act predominantly on small resistance vessels of the coronary bed by an inhibition of tissue uptake of adenosine which is a vasodilator (Drury and Szent-Gyt~rgyi, 1929). An alternative possibility is that binding sites are located on the Purkinje fibre system which is responsible for rapid transmission of the cardiac impulse throughout the ventricles. These fibres begin in the atrioventricular node (located on the lower part of the right atrium) and form into two major bundles which extend down the interventricular spectrum and separately spread along the walls of the right and left ventricles. The observed interaction of dipyridamole, a coronary vasodilator, with the diazepam-binding sites makes this possibility less likely. The potency of dipyridamole as a displacer of [3 H]diazepam was some 3-8 fold higher in rat and guinea-pig heart (IC50 99 and 24 nM respectively) than in rat and guinea-pig brain (IC50 200-300 nM; Davies et al., 1980 and unpublished results). The observed interaction of dipyridamole with diazepam binding sites may lead to further knowledge about the nature of the benzodiazepine receptor, although this interaction may be fairly nonselective. In addition to its potent inhibition of adenosine uptake, dipyridamole can also inhibit the transport of anions, purine bases, sugars (Plagemann and Richey, 1974) and amino acids, indicating an interaction with a component of the plasma membrane common to a number of transport systems/receptor sites. Although the majority of binding may be taking place on vascular tissue sites, the precise localization of these sites are unknown: in view of the presence of peripheral benzodiazepine receptors on mast cells (Taniguchi et al., 1980) it is possible that mast cells, which are distributed in many organs and located near walls of small blood vessels may account for the observed binding. However the number of mast cells observed in rat heart tissue is too low to account for more than a minor proportion of the binding seen in these experi-
ments (Yong et al., 1979, see fig. 1, and personal communication). Evidence that diazepam may be acting in the same manner as dipyridamole by inhibiting adenosine uptake has recently been published; Clanachan and Marshall (1980) have shown that diazepam potentiates the actions of adenosine on isolated cardiac and smooth muscle and the coronary vasodilator action of adenosine in anaesthetized dogs.
Acknowledgements The authors wish to thank Dr. J.F. Marwood for the dissection of heart tissue into separate regions and for helpful discussions.
References Braestrup, C. and R.F. Squires, 1977, Specific benzodiazepine receptors in rat brain characterized by high-affinity [3H]diazepam binding, Proc. Nat. Acad. Sci. U.S.A. 74, 3805. Clanachan, A.S. and R.J. Marshall, 1980, Diazepam potentiates the coronary vasodilator actions of adenosine in anaesthetized dogs, Br. J. Pharmacol. 70, 66P. Daniell, H.B., 1975, Cardiovascular effects of diazepam and chlordiazepoxide, European J. Pharmacol. 32, 58. Davies, L.P., A.F. Cook, M. Poonian and K.M. Taylor, 1980, Displacement of [3H]diazepam binding to rat brain by l-methylisoguanosine, a marine natural product with muscle-relaxant activity, Life Sci. 26, 1089. Drury, A.N. and A. Szent-Gy~rgyi, 1929, The physiological activity of adenosine compounds with especial reference to their action upon the mammalian heart, J. Physiol. 68, 213. 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. Mrhler, H. and T. Okada, 1977, Properties of [3H]diazepam binding to benzodiazepine receptors in rat cerebral cortex, Life Sci. 20, 2101. Plageman, P.G.W. and D.P. Richey, 1974, Transport of nucleosides, nucleic acid bases, choline and glucose by animal cells in culture, Biochim. Biophys. Acta 334, 263. Taniguchi, T., J.K.T. Wang and S. Spector, 1980, Properties [3H]diazepam binding to rat peritoneal mast cells, Life Sci. 27, .171. Van Citters, R.L., 1965, The coronary circulation: metabolism and nutrition of the heart; coronary disease, in: Physiology and Biophysics, 19th edn., eds. T.C. Ruch and H.D. Patton (W.B. Saunders, Philadelphia) p. 690. Yong, L.C., S.G. Watkins and J.E. Boland, 1979, The mast cell. III. Distribution and maturation in various organs of the young rat, Pathology 11,427.
209
Short communication PERIPHERAL BENZODIAZEPINE BINDING SITES IN HEART AND THEIR INTERACTION WITH DIPYRIDAMOLE
LES P. DAVIES * and VICKI HUSTON Roche Research Institute of Marine Pharmacology, P.O. Box 255, Dee Why, N.S.W. 2099, Australia Received 26 May 1981, accepted 1 June 1981
L.P. DAVIES and V. HUSTON, Peripheral benzodiazepine binding sites in heart and their interaction with dipyridamole, European J. Pharmacol. 73 (1981) 209-211. Specific binding of [3 H]diazepam to rat and guinea-pig heart tissue has been found. These binding sites are similar to those in lung, liver, kidney and mast cells but differ from those in the brain. Dipyridamole interacts with this receptor and is several fold more potent as a displacer of [3 H]diazepam bound to heart preparations than to brain membranes. Specific binding per unit protein in the left ventricle, fight ventricle and interventricular septum is significantly higher than in the left and right atrium. Diazepam binding
Heart
Dipyridamole
1. Introduction
2. Materials and methods
We previously reported (Davies et al., 1980) that the coronary vasodilator drug dipyridamole was relatively potent as a displacer of [3H]diazepam bound to CNS benzodiazepine receptors. In view of current interest in peripheral benzodiazepine receptors which are pharmacologically distinct from the central receptors (Braestrup and Squires, 1977; Taniguchi et al., 1980) we have examined whether dipyridamole also interacts with peripheral receptors. Since heart tissue has not previously been examined for the presence of peripheral benzodiazepine receptors, we decided to investigate this tissue, particularly since diazepam has been reported to have a coronary vasodilator action in animals (Daniell, 1975) and man (Ikram et al., 1973). In the present study the regional distribution of binding with the heart was also examined in order to determine more accurately sites of diazepam binding.
Fi~llinsdorf rats (male) and Himalayan white guinea-pigs (either sex) were killed by decapitation and hearts removed into cold Krebs bicarbonate buffer. Hearts were dissected free of fat and aortic and vena caval stumps and homogenized or further dissected into the following regions: left atrium, right atrium, left ventricle, right ventricle, interventricular septum. Tissue pieces were homogenized in 20 vol of ice-cold 0.32 M sucrose u~ing a tissue homogenizer (Polytron), and a P2 p~llet prepared as described previously (Davies et al!, 1980). Pellets were resuspended in 50 mM T~is.HC1 buffer, pH 7.45 at 0-4°C and assays carried out in the same buffer according to established methods (M/Shler and Okada, 1977). Binding assays (final volume of 2 ml) were carried out on ice and were terminated after 15 min by addition of 4 rnl of ice-cold buffer and rapid filtration on Whatman G F / B glass-fibre filters. Tube and filters were washed by a further 8 ml of buffer. Non-specific binding to peripheral tissue was determined in the presence of 10 #M unlabelled diazepam. Protein was measured by the Lowry method. [Methyl-3H]diazepam (76.8- 87.6 Ci/mmol) was
* To whom reprint requests should be addressed at present address: Department of Pharmacology, Blackburn Building, University of Sydney, Sydney, N.S.W. 2006, Australia.
0014-2999/81/0000-0000/$02.50 © Elsevier/North-Holland Biomedical Press
210
supplied by New England Nuclear Boston; dipyridamole (Persantin), diazepam and clonazepam by Hoffmann-La Roche, Basel and the benzodiazepine Ro 5-4864 by Hoffmann-La Roche, Nutley, N.J.
3. Results
Incubation of heart extracts with labelled diazepam showed that saturable binding sites existed in both guinea-pig and rat heart tissue with Kos of 49 nM (mean of 4 determinations) and 80 nM (2 determinations) respectively. The amount of binding in heart was variable from experiment to experiment, with very low binding in tissue which was stored frozen prior t o assay. In 4 experiments with guinea-pig heart a Bma~ of 4.2 pmol/mg protein was obtained, which is just over twice that measured by us for rat brain diazepam binding (1.9 pmol/mg protein; Davies et al., 1980). The binding of [3H]diazepam to heart receptor sites was clearly differentiated from its binding to CNS sites by the use of clonazepam and Ro 5-4864 (table 1). Whereas clonazepam, specific for CNS receptors (Braestrup and Squires, 1977) did not displace heart diazepam binding, Ro 5-4864 which has been shown to bind only to peripheral benzodiazepine receptors (Braestrup and Squires, 1977) was very potent in displacing [3 H]diazepam bound to the heart preparations. Dipyridamole was quite TABLE 1 Competition by benzodiazepines and dipyridamole with specific [3H]diazepam binding to heart tissue. Each value (with 95% confidence limits) was calculated from log-probit plots of inhibition at four separate concentrations of inhibitor, with each point being determined in triplicate in 1-3 separate experiments. [3H]Diazepam concentration was 0.94 nM. Drug
IC50 (nM)
Guinea-pig
Diazepam Clonazepam Ro 5-4864 Dipyridamole
49 (27-86) 2 361 (1684-3 310} 2.2 (0.5-9.7) 24 (16-37)
Rat
Diazepam Clonazepam Ro 5-4864 Dipyridamole
73 (21-254) > 3 000 19 (I 1-34) 99 (93-105)
TABLE 2 The distribution of [3H]diazepam binding in five different regions of guinea-pig and rat hearts. Data are means-+ S.E.M. of triplicate binding determination carried out on pooled tissue from the hearts of two guinea-pigs and two rats. The concentration of diazepam used was approximately 0.55 nM. A similar pattern of binding was seen in 4 further experiments (3 using two guinea-pig hearts, 1 using two rat hearts). Binding in the left ventricle, right ventricle and interventricular septum was significantly higher (P<0.05) than in the left atrium and right atrium, both in guinea-pig heart (4 experiments) and rat heart (2 experiments). To check that the pharmacological specificity of the binding sites for dipyridamole was similar in each region, a concentration of drug close to its ICs0 for displacement of binding in whole heart extracts was used in 2 experiments on guinea-pig hearts. Dipyridamole (37.5 nM) produced the following displacements; LA 63.1%; RA 59.4%; LV 64.2%; RV 63.1%; I.V.S. 61.3%. Region
Left atrium Right atrium Left ventricle Right ventricle Interventricular septum
Specific [ 3H]diazepam bound (fmol/mg protein) Guinea-pig
Rat
41.1 _+2.0 43.0 -+ 5.3 189.1 _+2.4 114.4~+4.2 132.8+0.4
17.5 -4-4.5 10.7 -+ 5.5 101.1 -+4.8 93.5-+6.6 65.3-+6.0
potent as a diazepam displacer, both in rat and guinea-pig heart tissue (table 1 and 2). There are obvious differences in the number of binding sites per unit protein in different parts of the heart (table 2) such that the amount of binding in left ventricle, interventricular septum and fight ventricle is significantly greater than in right atrium or left atrium.
4. Discussion
In view of the reported coronary vasodilator action of diazepam it is interesting to note the presence of peripheral-type benzodiazepine receptors in heart tissue, and further, to note a strong interaction of these receptors with dipyridamole, a coronary vasodilator in clinical use. That the coronary vasodilator actions of these two drugs may be at a common site is further supported by the observed regional distribution of diazepam binding to heart which appears to parallel regional
211
coronary blood supply and hence the density of coronary blood vessels; in hearts the turnover rate and tissue uptake of radioactive tracer substances present in the blood is highest in the left ventricle and is progressively less in the right ventricle, left atrium and right atrium (Van Citters, 1965). Dipyridamole appears to act predominantly on small resistance vessels of the coronary bed by an inhibition of tissue uptake of adenosine which is a vasodilator (Drury and Szent-Gyt~rgyi, 1929). An alternative possibility is that binding sites are located on the Purkinje fibre system which is responsible for rapid transmission of the cardiac impulse throughout the ventricles. These fibres begin in the atrioventricular node (located on the lower part of the right atrium) and form into two major bundles which extend down the interventricular spectrum and separately spread along the walls of the right and left ventricles. The observed interaction of dipyridamole, a coronary vasodilator, with the diazepam-binding sites makes this possibility less likely. The potency of dipyridamole as a displacer of [3 H]diazepam was some 3-8 fold higher in rat and guinea-pig heart (IC50 99 and 24 nM respectively) than in rat and guinea-pig brain (IC50 200-300 nM; Davies et al., 1980 and unpublished results). The observed interaction of dipyridamole with diazepam binding sites may lead to further knowledge about the nature of the benzodiazepine receptor, although this interaction may be fairly nonselective. In addition to its potent inhibition of adenosine uptake, dipyridamole can also inhibit the transport of anions, purine bases, sugars (Plagemann and Richey, 1974) and amino acids, indicating an interaction with a component of the plasma membrane common to a number of transport systems/receptor sites. Although the majority of binding may be taking place on vascular tissue sites, the precise localization of these sites are unknown: in view of the presence of peripheral benzodiazepine receptors on mast cells (Taniguchi et al., 1980) it is possible that mast cells, which are distributed in many organs and located near walls of small blood vessels may account for the observed binding. However the number of mast cells observed in rat heart tissue is too low to account for more than a minor proportion of the binding seen in these experi-
ments (Yong et al., 1979, see fig. 1, and personal communication). Evidence that diazepam may be acting in the same manner as dipyridamole by inhibiting adenosine uptake has recently been published; Clanachan and Marshall (1980) have shown that diazepam potentiates the actions of adenosine on isolated cardiac and smooth muscle and the coronary vasodilator action of adenosine in anaesthetized dogs.
Acknowledgements The authors wish to thank Dr. J.F. Marwood for the dissection of heart tissue into separate regions and for helpful discussions.
References Braestrup, C. and R.F. Squires, 1977, Specific benzodiazepine receptors in rat brain characterized by high-affinity [3H]diazepam binding, Proc. Nat. Acad. Sci. U.S.A. 74, 3805. Clanachan, A.S. and R.J. Marshall, 1980, Diazepam potentiates the coronary vasodilator actions of adenosine in anaesthetized dogs, Br. J. Pharmacol. 70, 66P. Daniell, H.B., 1975, Cardiovascular effects of diazepam and chlordiazepoxide, European J. Pharmacol. 32, 58. Davies, L.P., A.F. Cook, M. Poonian and K.M. Taylor, 1980, Displacement of [3H]diazepam binding to rat brain by l-methylisoguanosine, a marine natural product with muscle-relaxant activity, Life Sci. 26, 1089. Drury, A.N. and A. Szent-Gy~rgyi, 1929, The physiological activity of adenosine compounds with especial reference to their action upon the mammalian heart, J. Physiol. 68, 213. 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. Mrhler, H. and T. Okada, 1977, Properties of [3H]diazepam binding to benzodiazepine receptors in rat cerebral cortex, Life Sci. 20, 2101. Plageman, P.G.W. and D.P. Richey, 1974, Transport of nucleosides, nucleic acid bases, choline and glucose by animal cells in culture, Biochim. Biophys. Acta 334, 263. Taniguchi, T., J.K.T. Wang and S. Spector, 1980, Properties [3H]diazepam binding to rat peritoneal mast cells, Life Sci. 27, .171. Van Citters, R.L., 1965, The coronary circulation: metabolism and nutrition of the heart; coronary disease, in: Physiology and Biophysics, 19th edn., eds. T.C. Ruch and H.D. Patton (W.B. Saunders, Philadelphia) p. 690. Yong, L.C., S.G. Watkins and J.E. Boland, 1979, The mast cell. III. Distribution and maturation in various organs of the young rat, Pathology 11,427.