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Inhibition of GABA and benzodiazepine receptor binding by penicillins
Neuroscience Letters, 18 (1980) 309-312 © Elsevier/North-Holland Scientific Publishers Ltd.
309
INHIBITION OF G A B A A N D B E N Z O D I A Z E P I N E RECEPTOR BINDING
BY PENICILLINS
ADAM ANTONIADIS*, WALTER E. MULLER and UWE WOLLERT Pharmakologisches Institut der Universittit Mainz, Obere Zahlbacher Strasse 67, D-6500 Mainz (F.R.G.) (Received January 25th, 1980) (Revised version received April 8th, 1980) (Accepted April 14th, 1980)
SUMMARY
Penicillins are thought to be GABA receptor antagonists, In order to determine the affinities of various penicillin derivatives for the GABA receptor, their potencies as inhibitors of specific [3H]GABA binding to rat brain membranes were investigated. All investigated penicillins inhibit specific [3H]GABA binding, with IC50 values ranging from 2 to 60 mM. The results are consistent with the assumption that penicillins are weak GABA receptor antagonists.
Considerable evidence has been presented during the last years that penicillins reduce the synaptic activity of the inhibitory neurotransmitter 7-aminobutyric acid (GABA) [2, 4, 7, 9, 10]. This mechanism has been made responsible for the convulsive and epileptogenic properties of penicillin derivatives at high concentrations [2, 4, 7]. The question whether the GABA antagonistic activity is mediated directly at the GABA receptor is still controversial [2, 4, 7, 9, 10]. Specific, sodium-independent [3H]GABA binding was determined as described by Enna and Snyder [5]. A rat brain homogenate was pretreated with Triton X-100 0.05% for 30 min at 37°C, then centrifuged for 20 rain at 48,000 × g, and the pellet was resuspended in 0.1 M Tris.citrate buffer, pH 7.1. One ml aliquots of the membrane suspension (about 0.5 mg protein/ml) were incubated in triplicate for 10 min at 4°C together with 5 nM [3H]GABA and various concentrations of the displacers. The incubation was stopped by centrifugation (10 min at 48,000 × g) and the pellet was solubilized and radioactivity was measured by liquid scintillation * The results are part of a M.D. thesis of A.A.
310 spectrometry. Non-specific binding was obtained from experiments performed in the presence of 1 mM cold GABA. Under these conditions, specific [3H]GABA binding had all the characteristics of sodium-independent GABA binding [5, 8], as tested by the displacing activities of GABA, imidazoleacetic acid, and bicuculline (Table I). Benzodiazepine receptor binding studies were performed as previously described using a centrifugation technique and [3H]flunitrazepam as ligand [3]. One ml aliquots of a whole rat brain homogenate in 1/15 M phosphate buffer (about 1 mg protein/ml), pH 7.4, were incubated in triplicate together with about 0.1 mM [3H]flunitrazepam and different concentrations of the displacers for 30 rain at 4°C. Non-specific binding was obtained from experiments performed in the presence of cold diazepam 10 ~M. [3H]Flunitrazepam, specific activity 87.5 Ci/mmol and [3H]GABA, specific activity 36 Ci/mmol, were obtained from New England Nuclear, Dreieichenhain. The drugs used were gifts of the manufacturers. All other chemicals were obtained from commercial suppliers. Male Sprague-Dawley rats (150-200 g) were used. All investigated penicillin derivatives compete with [3H]GABA for sodiumindependent GABA binding in a concentration-dependent fashion. The 50% inhibitor concentrations (ICs0) are in the mmolar range. The highest affinity was found for the three cloxacillin derivatives (Table I). Oxacillin, pheneticillin, and benzylpenicillin have intermediate affinities for the GABA receptor, while that of TABLE l THE EFFECTS OF THE P E N I C I L L I N DERIVATIVE S ON G A B A A N D B E N Z O D I A Z E P I N E R E C E P T O R BINDING IN T H E RAT BRAIN. 1Cs0 values were determined by log-probit analysis, using 4 - 6 different concentrations of the displacing agents. All data are means of 4 - 6 individual determinations with S.E.M. values smaller than 20%. ICs0 values (raM) [3H]GABA Diazepam
-
Chlordiazepoxide GABA lmidazoleacetic acid Bicuculline Pentylenetetrazole Dicloxacillin Cloxacillin Flucloxacillin Oxacillin Pheneticillin Benzylpenicillin Ampicillin
0.000058 0.00055 0.0091 10 2.9 4.2 6.6 18.5 41 40 83
[3HlFlunitrazepam 0.000033 0.00050 1.0 5.0 5.3 9.1 15.8 26 4l 86
311 ampicillin is by far the lowest (Table I). The much lower ICs0 values of benzylpenicillin reported by Olsen et al. [12] for the inhibition of [3H]GABA binding to mouse brain membranes thoroughly washed but not treated with Triton X-100, could not be reproduced for rat brain membranes pretreated with Triton X100. The affinities of the penicillin derivatives for the GABA receptor are much lower than those of GABA itself and of the GABA agonist imidazoleacetic acid and of that of the GABA antagonist bicuculline (Table I). However, the affinity of pentylenetetrazole for the GABA receptor is of the same order of magnitude as those of oxacillin, pheneticillin, and benzylpenicillin, since pentylenetetrazole inhibits specific [3H]GABA binding by 40% at 10 mM, the highest concentration investigated (Table I). On the other hand, pentylenetetrazole, which is considered to have GABA antagonistic properties from electrophysiological measurements [9, 10], has a much higher affinity for the benzodiazepine receptor (Table I) than for the GABA receptor (Table I). Since the benzodiazepine receptor is thought to be part of a neuronal mechanism which facilitates GABAergic transmission [1], it has been suggested that pentylenetetrazole may reduce GABAergic transmission by an antagonistic activity at the benzodiazepine receptor [11]. A similar mechanism could also account for the GABA antagonistic properties of penicillin, since common synaptic mechanisms have been proposed for penicillin and pentylenetetrazole [9, 10, 13]. Accordingly, we have investigated the affinity of the penicillin derivatives for the benzodiazepine receptor, using [3H]flunitrazepam as ligand. Surprisingly, all investigated penicillin derivatives interact with the benzodiazepine receptor with affinities (ICs0 values) fairly similar to those reported for their interaction with the GABA receptor (Table I). The affinities of the penicillins for the benzodiazepine receptor are much lower than those of the benzodiazepines themselves, as demonstrated by the IC50 values of diazepam and chlordiazepoxide, two benzodiazepines with a high and a low affinity for the receptor (Table I). The interaction of the penicillin derivatives with both receptors shows a certain degree of specificity, since concentrations as high as 5 mM did not affect specific [3H]quinuclidinylbenzilate binding [14] to the muscarinic acetylcholine receptor. The concentrations of the penicillin derivatives needed to inhibit specific [3H]GABA binding or specific [3H]flunitrazepam binding are relatively high, but fit well in the concentration range used to demonstrate their convulsive or GABA antagonistic properties in vitro [6, 10]. Thus, the results may support the assumption that penicillins are weak GABA receptor antagonists. Furthermore, penicillins may interfere with GABAergic transmission not only at the GABA receptor, but also by blocking the benzodiazepine receptor, as has been suggested for the convulsant pentylenetetrazole. Whether the fairly similar affinities of the penicillin derivatives for the GABA as well as the benzodiazepine receptor indicate that penicillins are not bound to either receptor but to a penicillin binding site
312 (presumedly the chloride conductance mechanism), which can influence both receptors, will need further clarification. ACKNOWLEDGEMENT
This study was supported by a grant of the Deutsche Forschungsgemeinschaft. REFERENCES 1 2 3
4 5 6 7 8 9
10
11
12 13 14
Costa, E. and Guidotti, A., Molecular mechanisms in the receptor action of benzodiazepines, Ann. Rev. Pharmacol. Toxicol., 19 (1979) 531-545. Curtis, D.R., Game, C.J.A., Johnston, G.A.R. McCulloch, R.M. and MacLachlan, R.M., Convulsive action of penicillin, Brain Res., 43 (1972) 242-245. Damm, H.W., M~311er, W.E., Schl~fer, U. and Wollert, U., [3H]Flunitrazepam: its advantages as a ligand for the identification of benzodiazepine receptors in rat brain membranes, Res. Commun. Chem. Path. Pharmacol., 22 (1978) 597-600. Davidoff, R.A., Penicillin and inhibition in the cat spinal cord, Brain Res., 45 (1972) 638-642. Enna, S.J. and Snyder, S.H., Influences of ions, enzymes, and detergents on 7-aminobutyric acid-receptor binding in synaptic membranes of rat brain, Molec. Pharmacol., 13 (1977) 442-453. Gumick, M.J., Van Duijn, H. and Citri, N., Relative convulsant potencies of structural analogues of penicillin, Brain Res., 114 (1976) 139-143. Hochner, B., Spira, M.E. and Werman, R., Penicillin decreases chloride conductance in crustacean muscle: a model for the epileptic neuron, Brain Res., 107 (1976) 85-103. Horng, J.S. and Wong, D.T., 7-Aminobutyric acid receptors in cerebellar membranes of rat brain after a treatment with Triton X-100, J. Neurochem., 32 (1979) 1379-1386. MacDonald, R.L. and Barker, J.L., Pentylenetetrazole and penicillin are selective antagonists of GABA-mediated post-synaptic inhibition in cultured mammalian neurones, Nature (Lond.), 267 (1977) 720-721. MacDonald, R.L. and Barker, J.L., Specific antagonism of GABA-mediated postsynaptic inhibition in cultured mammalian spinal neurones: a common mode of convulsant action, Neurology (Minneap.), 28 (1978) 325-330. M~ller, W.E., Schl~fer, U. and Wollert, U., Benzodiazepine receptor binding: the interaction of some non-benzodiazepine drugs with specific [3H]diazepam binding to rat brain synaptosomal membranes, Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak., 305 (1978) 23 26. Olsen, R.W., Ticku, M.K., van Ness, P.C. and Greenlee, D., Effects of drugs on 7-aminobutyric acid receptors, uptake, release, and synthesis in vitro, Brain Res., 139 (1978) 277 294. Wilson, W.A. and Escueta, A.V., Common synaptic effects of pentylenetetrazole and penicillin, Brain Res., 72 (1974) 168-171. Yamamura, H.I. and Snyder, S.H., Muscarinic cholinergic binding in rat brain, Proc. nat. Acad. Sci. (Wash.), 71 (1974) 1725-1729.
309
INHIBITION OF G A B A A N D B E N Z O D I A Z E P I N E RECEPTOR BINDING
BY PENICILLINS
ADAM ANTONIADIS*, WALTER E. MULLER and UWE WOLLERT Pharmakologisches Institut der Universittit Mainz, Obere Zahlbacher Strasse 67, D-6500 Mainz (F.R.G.) (Received January 25th, 1980) (Revised version received April 8th, 1980) (Accepted April 14th, 1980)
SUMMARY
Penicillins are thought to be GABA receptor antagonists, In order to determine the affinities of various penicillin derivatives for the GABA receptor, their potencies as inhibitors of specific [3H]GABA binding to rat brain membranes were investigated. All investigated penicillins inhibit specific [3H]GABA binding, with IC50 values ranging from 2 to 60 mM. The results are consistent with the assumption that penicillins are weak GABA receptor antagonists.
Considerable evidence has been presented during the last years that penicillins reduce the synaptic activity of the inhibitory neurotransmitter 7-aminobutyric acid (GABA) [2, 4, 7, 9, 10]. This mechanism has been made responsible for the convulsive and epileptogenic properties of penicillin derivatives at high concentrations [2, 4, 7]. The question whether the GABA antagonistic activity is mediated directly at the GABA receptor is still controversial [2, 4, 7, 9, 10]. Specific, sodium-independent [3H]GABA binding was determined as described by Enna and Snyder [5]. A rat brain homogenate was pretreated with Triton X-100 0.05% for 30 min at 37°C, then centrifuged for 20 rain at 48,000 × g, and the pellet was resuspended in 0.1 M Tris.citrate buffer, pH 7.1. One ml aliquots of the membrane suspension (about 0.5 mg protein/ml) were incubated in triplicate for 10 min at 4°C together with 5 nM [3H]GABA and various concentrations of the displacers. The incubation was stopped by centrifugation (10 min at 48,000 × g) and the pellet was solubilized and radioactivity was measured by liquid scintillation * The results are part of a M.D. thesis of A.A.
310 spectrometry. Non-specific binding was obtained from experiments performed in the presence of 1 mM cold GABA. Under these conditions, specific [3H]GABA binding had all the characteristics of sodium-independent GABA binding [5, 8], as tested by the displacing activities of GABA, imidazoleacetic acid, and bicuculline (Table I). Benzodiazepine receptor binding studies were performed as previously described using a centrifugation technique and [3H]flunitrazepam as ligand [3]. One ml aliquots of a whole rat brain homogenate in 1/15 M phosphate buffer (about 1 mg protein/ml), pH 7.4, were incubated in triplicate together with about 0.1 mM [3H]flunitrazepam and different concentrations of the displacers for 30 rain at 4°C. Non-specific binding was obtained from experiments performed in the presence of cold diazepam 10 ~M. [3H]Flunitrazepam, specific activity 87.5 Ci/mmol and [3H]GABA, specific activity 36 Ci/mmol, were obtained from New England Nuclear, Dreieichenhain. The drugs used were gifts of the manufacturers. All other chemicals were obtained from commercial suppliers. Male Sprague-Dawley rats (150-200 g) were used. All investigated penicillin derivatives compete with [3H]GABA for sodiumindependent GABA binding in a concentration-dependent fashion. The 50% inhibitor concentrations (ICs0) are in the mmolar range. The highest affinity was found for the three cloxacillin derivatives (Table I). Oxacillin, pheneticillin, and benzylpenicillin have intermediate affinities for the GABA receptor, while that of TABLE l THE EFFECTS OF THE P E N I C I L L I N DERIVATIVE S ON G A B A A N D B E N Z O D I A Z E P I N E R E C E P T O R BINDING IN T H E RAT BRAIN. 1Cs0 values were determined by log-probit analysis, using 4 - 6 different concentrations of the displacing agents. All data are means of 4 - 6 individual determinations with S.E.M. values smaller than 20%. ICs0 values (raM) [3H]GABA Diazepam
-
Chlordiazepoxide GABA lmidazoleacetic acid Bicuculline Pentylenetetrazole Dicloxacillin Cloxacillin Flucloxacillin Oxacillin Pheneticillin Benzylpenicillin Ampicillin
0.000058 0.00055 0.0091 10 2.9 4.2 6.6 18.5 41 40 83
[3HlFlunitrazepam 0.000033 0.00050 1.0 5.0 5.3 9.1 15.8 26 4l 86
311 ampicillin is by far the lowest (Table I). The much lower ICs0 values of benzylpenicillin reported by Olsen et al. [12] for the inhibition of [3H]GABA binding to mouse brain membranes thoroughly washed but not treated with Triton X-100, could not be reproduced for rat brain membranes pretreated with Triton X100. The affinities of the penicillin derivatives for the GABA receptor are much lower than those of GABA itself and of the GABA agonist imidazoleacetic acid and of that of the GABA antagonist bicuculline (Table I). However, the affinity of pentylenetetrazole for the GABA receptor is of the same order of magnitude as those of oxacillin, pheneticillin, and benzylpenicillin, since pentylenetetrazole inhibits specific [3H]GABA binding by 40% at 10 mM, the highest concentration investigated (Table I). On the other hand, pentylenetetrazole, which is considered to have GABA antagonistic properties from electrophysiological measurements [9, 10], has a much higher affinity for the benzodiazepine receptor (Table I) than for the GABA receptor (Table I). Since the benzodiazepine receptor is thought to be part of a neuronal mechanism which facilitates GABAergic transmission [1], it has been suggested that pentylenetetrazole may reduce GABAergic transmission by an antagonistic activity at the benzodiazepine receptor [11]. A similar mechanism could also account for the GABA antagonistic properties of penicillin, since common synaptic mechanisms have been proposed for penicillin and pentylenetetrazole [9, 10, 13]. Accordingly, we have investigated the affinity of the penicillin derivatives for the benzodiazepine receptor, using [3H]flunitrazepam as ligand. Surprisingly, all investigated penicillin derivatives interact with the benzodiazepine receptor with affinities (ICs0 values) fairly similar to those reported for their interaction with the GABA receptor (Table I). The affinities of the penicillins for the benzodiazepine receptor are much lower than those of the benzodiazepines themselves, as demonstrated by the IC50 values of diazepam and chlordiazepoxide, two benzodiazepines with a high and a low affinity for the receptor (Table I). The interaction of the penicillin derivatives with both receptors shows a certain degree of specificity, since concentrations as high as 5 mM did not affect specific [3H]quinuclidinylbenzilate binding [14] to the muscarinic acetylcholine receptor. The concentrations of the penicillin derivatives needed to inhibit specific [3H]GABA binding or specific [3H]flunitrazepam binding are relatively high, but fit well in the concentration range used to demonstrate their convulsive or GABA antagonistic properties in vitro [6, 10]. Thus, the results may support the assumption that penicillins are weak GABA receptor antagonists. Furthermore, penicillins may interfere with GABAergic transmission not only at the GABA receptor, but also by blocking the benzodiazepine receptor, as has been suggested for the convulsant pentylenetetrazole. Whether the fairly similar affinities of the penicillin derivatives for the GABA as well as the benzodiazepine receptor indicate that penicillins are not bound to either receptor but to a penicillin binding site
312 (presumedly the chloride conductance mechanism), which can influence both receptors, will need further clarification. ACKNOWLEDGEMENT
This study was supported by a grant of the Deutsche Forschungsgemeinschaft. REFERENCES 1 2 3
4 5 6 7 8 9
10
11
12 13 14
Costa, E. and Guidotti, A., Molecular mechanisms in the receptor action of benzodiazepines, Ann. Rev. Pharmacol. Toxicol., 19 (1979) 531-545. Curtis, D.R., Game, C.J.A., Johnston, G.A.R. McCulloch, R.M. and MacLachlan, R.M., Convulsive action of penicillin, Brain Res., 43 (1972) 242-245. Damm, H.W., M~311er, W.E., Schl~fer, U. and Wollert, U., [3H]Flunitrazepam: its advantages as a ligand for the identification of benzodiazepine receptors in rat brain membranes, Res. Commun. Chem. Path. Pharmacol., 22 (1978) 597-600. Davidoff, R.A., Penicillin and inhibition in the cat spinal cord, Brain Res., 45 (1972) 638-642. Enna, S.J. and Snyder, S.H., Influences of ions, enzymes, and detergents on 7-aminobutyric acid-receptor binding in synaptic membranes of rat brain, Molec. Pharmacol., 13 (1977) 442-453. Gumick, M.J., Van Duijn, H. and Citri, N., Relative convulsant potencies of structural analogues of penicillin, Brain Res., 114 (1976) 139-143. Hochner, B., Spira, M.E. and Werman, R., Penicillin decreases chloride conductance in crustacean muscle: a model for the epileptic neuron, Brain Res., 107 (1976) 85-103. Horng, J.S. and Wong, D.T., 7-Aminobutyric acid receptors in cerebellar membranes of rat brain after a treatment with Triton X-100, J. Neurochem., 32 (1979) 1379-1386. MacDonald, R.L. and Barker, J.L., Pentylenetetrazole and penicillin are selective antagonists of GABA-mediated post-synaptic inhibition in cultured mammalian neurones, Nature (Lond.), 267 (1977) 720-721. MacDonald, R.L. and Barker, J.L., Specific antagonism of GABA-mediated postsynaptic inhibition in cultured mammalian spinal neurones: a common mode of convulsant action, Neurology (Minneap.), 28 (1978) 325-330. M~ller, W.E., Schl~fer, U. and Wollert, U., Benzodiazepine receptor binding: the interaction of some non-benzodiazepine drugs with specific [3H]diazepam binding to rat brain synaptosomal membranes, Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak., 305 (1978) 23 26. Olsen, R.W., Ticku, M.K., van Ness, P.C. and Greenlee, D., Effects of drugs on 7-aminobutyric acid receptors, uptake, release, and synthesis in vitro, Brain Res., 139 (1978) 277 294. Wilson, W.A. and Escueta, A.V., Common synaptic effects of pentylenetetrazole and penicillin, Brain Res., 72 (1974) 168-171. Yamamura, H.I. and Snyder, S.H., Muscarinic cholinergic binding in rat brain, Proc. nat. Acad. Sci. (Wash.), 71 (1974) 1725-1729.