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benzodiazepine receptor complex of rat cerebellum
Life Sciences, Vol. 28, pp. 307-313 Printed in the U.S.A.
Pergamon Press
MODULATION BY PICROTOXIN AND IPTBO OF 3H-FLUNITRAZEPAM BINDING TO THE GABA/BENZODIAZEPINE RECEPTOR COMPLEX OF RAT CEREBELLUM Manfred Karobath, Gerhard Drexler and Porntip Supavilai Department of Biochemical Psychiatry Psychiatrische Universit~tsklinik Vienna, Austria A-1090 (Received in final form November 5, 1980)
su~mry Picrotoxin and IPTBO, two CNS convulsants which block chloride permeability of GABA receptor regulated chloride ion channels, have modulatory effects on 3H-flunitrazepambinding to the G A B A ~ n z o d i a zepine receptor complex of the cerebellum. Their actions on 3H-flunitrazepambinding to membranes from rat cerebellum include: (i) both drugs in high concentrations stimulate 3H-flunitrazepam binding malnly by increasing the apparent number of binding sites. This effect is chloride ion dependent and reversible; (ii) they inhibit 3H-flunitrazepam binding stimulated by the pyrazolopyridine etazolate (SQ 20009), and this effect is observed in bindingexperiments performed at low (0 "C) and at physiological (35 °C) temperature; (iii) they inhibit GABA stimulated binding in a chloride ion dependent mode. This action is much more potent when the binding experiments are performed at physiological temperatures. These results demonstrate that drugs which act on GABAreceptor associated chloride ion channels have potent modulatory effects on benzodiazepine receptor binding in cerebellum. Benzodiazepine receptor binding can be modulated by GABA (1-6) and in addition by a number of drugs structurally unrelated to GABA which appear to exert at least part of their CNS effects by altering the effects of GABA on neuronal membranes (7-14). Thus, the pyrazolopyridines (7,8) and pentobarbital (9) have been found to stimulate benzodiazepine receptor binding in the presence of halide ions, and this action is sensitive to inhibition by picrotoxin (7-9). In addition, benzodiazepine receptor binding is also modulated by avermectin Bla (10), a macrocyclic lactone disaccharide anthelmintic agent (ii). In invertebrates, avermectin B1 a increases chloride permeability of membranes presumably by a long lasting and picrotoxin sensitive opening of GABAreceptOr regulated chloride channels (ii), and in rats and mice it potentiates the muscle relaxant effects of diazepam in vivo (i0). Picrotoxin, picrotoxinin, and IPTBO (4-(isopropyl)-l-phospho-2,6,7-trioxabicyclo (2,2,2)octane-l-oxide) are powerful convulsants in the mammalian and invertebrate central nervous system which exert their CNS effects by decreasing chloride permeability of GABA receptor, regulated chloride channels (12,13). Thus, these drugs antagonize the effects of GABA in many tissues by acting at a site uhrelated to GABArecognition sites. We now report that these drugs have also modulatory effects on benzodiazepine receptor binding to membranes from rat cerebellum, which include potent inhibitory actions on binding stimulated by CgHBA and by the pyrazolopyridine etazolate.
0024-3205/81/030307-07502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
308
Picrotoxin and 3H-Flunitrazepam Binding
Vol. 28, No. 3, 1981
Materials and Methods 3H-flunitrazepam (79.3 Ci/mmol) was purchased from New England Nuclear, Boston, Mass., USA. Picrotoxin was obtained from Fluka, Buchs, Switzerland, and picrotoxinin from Sigma, Corp. St. Louis, MI, USA. IPTBO (4-(isopropyl)-l-phospho2,6,7-trioxabicyclo- (2,2,2)-octane-l-oxide) was kindly provided by J. S. Collins, City of London, Polytechnic, London, UK., and etazolate (SQ 20009) was donated by S. J. Lucania of E. R. Squibb & Sons, Inc., Princeton, N.J., USA. Male Sprague Dawley rats (200 - 250 g, Forschungsinstitut fttr Versuchstierzucht, Himberg, Austria) were decapitated, and their brains were removed immediately. The cerebellum was dissected and homogenized with an Ultra-Turrax for 30 sec. in 50 vol of ice-cold 50 mM Tris-citrate buffer, pH 7.1, and centrifuged for i0 min at 48,000 g. The pellets were washed five times by resuspension and recentrifugation in the same vol of buffer and stored at -20 °C for at least 18 hours. After thawing the membrane fractions were washed once in 50 mM Triscitrate buffer and finally resuspended in 50 vol of the same buffer for binding assays. In some experiments, portions of the membrane fractions were treated with 0.05 % Triton-X i00 as described (17). For binding assays performed at 0 °C, 0.1 ml membrane suspension derived from 2 mg tissue wet weight were usually incubated for 90 min with 3H-flunitrazepam (3H-FLU) in 1 ml of an incubation medium which contained 50 mM Tris-citrate buffer, pH 7.1, and the compounds to be tested. The samples were then filtered under vacuum through Whatman GF/B filters and immediately washed twice with 5 ml ice-cold buffer. In binding assays performed at physiological temperature (35 °C), incubations were performed for 30 min with membranes derived from 4 mg tissue wet weight, and the samples were then filtered as described above and washed with buffer which was kept at 35 °C. Radioactivity on the filter was then determined by liquid scintillation counting. Non-specific binding determined in the presence of 2 ~M unlabelled diazepam was subtracted from total binding to give specific binding (3). Non-specific binding was i0 % or approximately 50 % of total binding in experiments performed at 0 °C or at 35 °C respectively. Results In binding experiments performed at 0 °C, picrotoxin, picrotoxinin and IPTBO caused a concentration dependent increase of the binding of 0.25 nM 3H-FLU to membranes from rat cerebellum (Figure i). All three drugs increased binding mainly by enhancing the apparent number of binding sites (Figure I). The effects of picrotoxin on 3H-FLU binding were reversible since preincubation of the membranes and resuspension in drug free buffer did not lead to an alteration of 3H-FLU binding (Figure i). The direct stimulatory effects of picrotoxin and IPTBO on 3H-FLU binding at 0 °C were greatly diminished in the absence of chloride ions and abolished when the binding experiments were performed with membranes which had been partially denatured by pretreatment with 0.05 % Triton-X i00 (data not shown). The magnitude of the direct stimulatory effect of picrotoxin and IPTBO on 3H-FLU binding was variable in different membrane preparations. Although extensive washing of the membranes appeared to diminish the direct stimulatory effects of picrotoxin (data not shown), we were, despite considerable effords, unable to evaluate all factors responisble for this variability. Thus, in 14 membrane preparations, the apparent+number of binding,sites for 3H-FLU in the presence of i00 ~M picrotoxin was 121 - 5 percent (mean -~ s.e.m.) of the respective control groups. In a few membrane preparations picrotoxin slightly enhanced the apparent affinity of 3H-FLU binding sites to a greater extent than shown in Figure i.
Vol. 28, No. 3, 1981
Picrotoxin and 3H-Flunitrazepam Binding
309
A
Z
o
u
of o
.o
/
ee v
0
O
z
m o.
:E m
u.
# lOO 2O 4O 6O 3H-FLUNITRAZEPAM BOUND (ImoteSlmll)
DRUG CONC (M)
FIGURE 1 Effect of picrotoxin, picrotoxinin and IPTBO on the binding of 3H-FLU to membranes from rat cerebellum at 0 °C. 150 mM NaCI was present in all incubations. On the left concentrationresponse curves for picrotoxin (o), picrotoxinin (D), and IPTBO (A) on the binding of 0.25 nM 3H-FLU are shown. On the right, Scatchard plots of saturation of specific 3H-FLU birding (0.2 to 4.0 nM) are shown. Experiments were performed in 50 mM Tris-citrate buffer, pH 7.1, which contained 150 mM NaCI (e), or in the same buffer containing i00 ~M picrotoxin (o), 100 ~M picrotoxinin (~), or 30 ~M IPTBO (a). In one experiment (m) membrane suspensions werepreincubated with I00 ~M picrotoxin for 90 rain at 0 °C, subsequently washed by centrifugation and resuspended in drug free buffer before saturation analysis.
FIGIRE 2
2°° ""'a
o
|
! 110-.6
' IPTBO ( M )
;-.4
10-6
10-4
PICROTOXIN ( M )
Tne effects of picrotoxin and IPTBO on basal, GABA- or etazolate-stimulated 3H-FLU binding to membranes from rat cerebellum. Binding experiments were performed at 0 °C for 90 min in 50 ~M Tris-citrate buffer, pH 7.1, which contained 150 mM NaCl. Shown are the concentration-response curves for IPTBO or picrotoxin alone (e) or in the presence of i0 ~M etazolate (m), or of 2 ~M GABA (D). Tne results are mean values of duplicate determinations. The experiment was repeated twice with similar results. 3H-FLU concentration was 0.25 nM.
310
Picrotoxin and 3H-Flunitrazepam Binding
Vol. 28, No. 3, 1981
Consistent effects of picrotoxin and IPTBO on 3H-FLU binding included inhibition of binding stimulated by etazolate or by GABA (Figure 2). In binding experiments performed at 0 °C, both d~x~s potent] y interfered with 3H-FLU binding stimulated by i0 UM etazolate which confirms and extends our previous observations (7). It can be seen from Figure 2 that, in the presence of etazolate and of IPTBO or picrotoxin, binding can be less than that in the presence of either drug alone. These observations suggest mutual inhibitory effects on 3H-FLU binding of IPTBO or picrotoxin on one hand, and of etazolate on the other hand. Picrotoxin and IPTBO in high concentrations also interfered with binding stimulated by GABA (Figure 2). Since IPTBO appeared to be slightly more effective than picrotoxin in this action (Figure 2), we investigated its effects on concentration response curves for stimulation of 3H-FLU binding by GABA. In Figure 3 it is shown that IPTBO interfered with the actions of GABA by decreasing the apparent maximal response to GABA.
°/°~-°
250
O/~
200
~4taMIPTBO ,~20pMPTSO
,
FIGURE 3 +100 pM IPTBO
~s
~OOIilli 10-7
i GABA ( M )
i
$ 10-4
Effect of IPTBO on the stimulation of 3H-FLU binding by GABA. the binding of 0.25 nM 3H-FLU to membranes from rat cerebellum was determined at 0 °C in 50 mM Tris-citrate buffer, pH 7. i, to which 150 mM NaCI was added. The results are the mean of duplicate determinations. The experiment was repeated twice with similar results.
Recently we observed that GABA stimulation of benzodiazepine receptor binding exhibits strong temperature and chloride ion dependency (15). We therefore reinvestigated the actions of picrotoxin and IPTBO on 3H-FLU binding to membranes from rat cerebellum at physiological temperature (35 °C).
In agreement with previous.observations (15), but unlike to results obtained at 0 °C (4), we found that in binding experiments performed at physiological temperature with membranes from rat cerebellum, chloride ions slightly inhibited basal 3H-FLU binding. However, at this temperature picrotoxin and IPTBO potently antagonized 3H-FLU binding stimulated by GABA (Table i) with hal E maximal in~+ibition of the GABA stimulated component of 3H-~LU binding at 3.3 - 1.4 ~M (mean - S.E.M. of 3 membrane perparations) and at 1.6 -~0.7 ~M, respectively. These actions of picrotoxin and IPTBO, but not those of bicuculline-methiodide were observed only in the presence of chloride ions (Table i). Picrotoxin and IPTBO antagonized also etazolate (SQ 20009) stimulated 3H-FLU binding in experiments performed at physiological temperature, in the presence of 150 mM NaCI (Table 1 ).
Vol. 28, No. 3, 1981
Picrotoxin and 3H-Flunitrazepam Binding
311
TABLE 1 Effect of various drugs on 3H-FLU binding to membranes from rat cerebelltun in binding experiments performed at physiological temperature (35 °C).
A4~i tions (~tM)
specific 3H-FLU binding at 35 °C fm~les/ngwet tissue weight means - s.e.m. (n = 3) (% of controls) 50 mM Tris-citrate buffer pH 7.1
50 mM Tris-citrate buffer pH 7.1 plus 150 mM NaCI
none
1.17 t 0.09 (i00)
0.85 ~ 0.04 (i00)
3 1.0t G A ~
2.20 t 0.26 (188)
2.10 ~ 0.07 (247)
0.3 ~M IPTBO
2.28 ~ 0.37 (195) +
1.70 ~ 0. ii (200)
3.0 ~M IPTBO
2.19 ~ 0.17 (187) +
1.29 ~ 0.09 (152)
+ 30.0 ~M IPTBO
2.18 t 0.17 (186) +
0.95 t 0.05 (iii)
1.49 t 0.06 (127)
0.67 Z 0.03 (79)
none
1.14 ~ 0.06 (i00)
0.96 -+ 0.i0 (i00)
3 UM GABA
2.23 ~ 0.07 (196)
2.55 -+ 0.ii (266)
0.3 ~M picrotoxin
2.37 ~ 0.04 (208) +
2.39 +- 0.13 (249)
3.0 ~M picrotoxin
2.07 ~ 0.01 (182)
1.91 + 0.13 (199)
2.24 ~ 0.09 (196) +
1.58 -+ 0.09 (164)
1.37 ~ 0.08 (120)
1.13 + 0.07 (115)
none
1.28 -+ 0.04 (i00)
1.16 + 0.02 (i00)
3 ~M GABA " + i0 ~M bicucullinemethiodide
2.17 -+ 0.03 (170) 1.54 + 0.13 (121)
1.03 -+ 0.09 (89)
i0 ~M bicuculline-methiodide
1.17 -+ 0.06 (91)
0.71 + 0.06 (61)
i0 ~M etazolate
1.02 + 0.04 (79)
1.89 + 0.07 (164)
EXPERIMENT 1
|!
+ +
30 ~M IPTBO
EXPERIMENT 2
+
ii
+
+ 30.0 ~M picrotoxin 30 UM picrotoxin EXPERIMENT 3
2.21 -+ 0.01 (191) ,
,
"
+ i0 ~M picrotoxin
1.43 -+ 0.06 (112)*
1.24 + 0.05 (107)*
"
+ i0 ~g IPTBO
1.35 -+ 0.02 (106)*
0.86 + 0.04 ( 74)*
The binding of i. 0 nM 3H-FLU was determined as described in methods. p < 0.01 compared to binding in the presence of 3 ~M GABA or of 10 bum etazolate +
p > 0.i
compared to binding in the presence of 3 ~M GABA.
312
Picrotoxln and 3H-Flunitrazepam Binding
Vol. 28, No. 3, 1981
Discussion There is evidence that picrotoxin exerts its CNS convulsant effects by interacting with a specific drug receptor which is in close association with GABA receptor regulated chloride ion channels (9,16). This interaction apparently triggers allosteric effects which lead to a decrease of chloride permeability of GABA receptor associated chloride channels (9,12-14,16). The present observations demonstrate that picrotoxin has also potent allosteric effects on 3H-FLU binding to the GABA/benzodiazepine receptor complex of the cerebellum. At physiological temperature picrotoxin and IPTBO inhibit GABA stimulated but not unstimulated 3H-FLU bindinq in concentrations which are comparable to their respective affinities for JH-dihydropicrotoxinin binding sites (9,16). Thus the interaction of picrotoxin or IPTBO with their site of action leads to an apparent desensitization of benzodiazepine binding sites towards stlmulation by GABA suggesting that the benzodiazepine binding protein of the cerebellum is also modulated by the chloride ionophore. Since picrotoxin and IPTBO do not interfere with the binding of labeled ligands for GABA recognition sites (17), our results point to indirect actions of these drugs on benzodiazepine receptor binding. The effects of ~icrotoxin and IPTBO but not those of bicuculline-methiodide on GABA stimulated ~H-FLU binding are absolute chloride ion dependent which adds further support to the notion (18) that these drugs interfere with the function of GABA by different mechanisms. There are also interactions between the effects of pyrazolopyridines on one hand, and those of picrotoxin or IPTBO on the other hand as modulators of benzodiazepine receptor binding. It has been suggested that the possible site of action of pyrazolopyridines is closely related to the chloride conductance mechanism since the actions of pyrazolopyridines on 3H-FLU binding are chloride ion dependent and blocked by picrotoxin (7) and IPTBO. This notion has been supported by recent observations demonstrating that etazolate inhibits the binding of 3H-dihydropicrotoxinin (9). However, it cannot be excluded that the pyrazolopyridenes (or chloride ions ) exert their modulatory effects on GABA receptor binding (19,20) and on benzodiazepine receptor binding (7,8) by acting at a distinct modulatory site. The actions of picrotoxin and IPTBO on GABA stimulated 3H-FLU binding exhibit strong temperature dependency, and they are more than hundred fold weaker in binding experiments performed at 0 °C. This and the weak chloride ion dependent intrinsic stimulatory effects of high concentrations of these drugs on 3H-FLU binding investigated at 0 °C may be the reason why no effects of picrotoxin on benzediazepine receptor binding have previously been observed. In addition, the present observations of a temperature dependency of the actions of picrotoxin and IPTBO further support the notion (15) that the allosteric properties of the GABA/benzodiazepine receptor con~01ex of the cerebellum exhibit strong temperature dependency, and that the investigation of benzodiazepine receptor binding at physiological temperature may be a better in vitro model for the examination of GABA receptor properties than binding studies performed at low temperature.
Acknowledgements We thank J. Reisenhofer and T. Ab~cherli for secretarial help, J. S. Collins for the donation of IPTBO and S. J. Lucania for etazolate. Supported by "Fonds zur F~rderung der wissenschaftlichen Forschung in ~sterreich".
Vol. 28, No. 3, 1981
Picrotoxin and 3H-Flunitrazepam Binding
313
References i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15. 16.
17. 18. 19. 20.
M.S. BRILEY and S.Z. LANGER, Europ. J. Pharmacol. 5 2 124 (1978). Y. DUDAI, Brain Res. 167 422-425 (1979). M. KAROBATH and G. SPERK, Proc. natn. Acad. Sci. USA 76 1004-1006 (1979). I.L. MARTIN and J.M. CANDY, Neuropharmacology 1 7 993-998 (1978). J.F. TALLMAN, J.W. THOMAS and D.W. GAI/2%GER, Nature 274 111-113 (1979). G.W. WASTEK, R.C. SPETH, T.D. REISINE and H.I. YAMAMURA, Europ. J. Pharmacol. 50 445-447 (1978). P--?SUPAVILAI and M. KAROBATH, Europ. J. Pharmacol. 60 111-113 (1979). P. SUPAVILAI and M. KAROBATH, Europ. J. Pharmacol. 6 4 91-93 (1980). R.W. OLSEN and F. LEEB-I//NDBERG, in C ~ and Benzodiazepine Receptors, eds.: E. Costa, G. DiChiara and G.L. Gessa, Raven Press, New York, in press (1980). M. WILLIAMS and G.Y. YARBROUGH, Europ. J. Pharmacol. 5 6 273-276 (1979). L.C. FRITZ, C.C. WANG and A. GORIO, Proc. natl. Acad. Sci. USA 7 6 2063-2066 (1979). A. NISTRI and A. CONSTANTINI, Prog. Neurobiol. 1 3 117-235 (1979). J.S. ~.T.y, K. KRNJEVIC, M.E. MORRIS and G.K.W. YIM, Exptl. Brain Res. 7 11-31 (1968). R.A. NICOLL, J.C. E ~ S , T.C. OSHIMA and F. RUBIA, Nature 258 625-627 (1975) P. SUPAVILAI and M. KAROBATH, Neurosci. Lett. in press (1980). R.W. OLSON, M.K. TICKU, D. (~EENT.~ and P. VAN NESS, in GABA-Neurotrans,titters, eds. : P. Krogsgaard-Larsen, J. Scheel-Krt~er and H. Kofod, Munksgaard, Copenhagen, 165-178 (1979). S.J. ENNA, J.F. COLLINS and S.H. SNYDER, Brain Res. 124 185-190 (1977). M. A. SIMMONDS, Neuropharmacology 1 9 30-45 (1980). P. PLACHETA and M. KAROBATH, Europ. J. Pharmacol. 62 225-228 (1980). M. KAROBATH, P. S U P A ~ , P. PLACHETA and W. SIEGHART in Recent Advances in NeuropsychoFharmacology , eds. : B. Angrist et al., Pergamon Press, Oxford, in press (1980).
Pergamon Press
MODULATION BY PICROTOXIN AND IPTBO OF 3H-FLUNITRAZEPAM BINDING TO THE GABA/BENZODIAZEPINE RECEPTOR COMPLEX OF RAT CEREBELLUM Manfred Karobath, Gerhard Drexler and Porntip Supavilai Department of Biochemical Psychiatry Psychiatrische Universit~tsklinik Vienna, Austria A-1090 (Received in final form November 5, 1980)
su~mry Picrotoxin and IPTBO, two CNS convulsants which block chloride permeability of GABA receptor regulated chloride ion channels, have modulatory effects on 3H-flunitrazepambinding to the G A B A ~ n z o d i a zepine receptor complex of the cerebellum. Their actions on 3H-flunitrazepambinding to membranes from rat cerebellum include: (i) both drugs in high concentrations stimulate 3H-flunitrazepam binding malnly by increasing the apparent number of binding sites. This effect is chloride ion dependent and reversible; (ii) they inhibit 3H-flunitrazepam binding stimulated by the pyrazolopyridine etazolate (SQ 20009), and this effect is observed in bindingexperiments performed at low (0 "C) and at physiological (35 °C) temperature; (iii) they inhibit GABA stimulated binding in a chloride ion dependent mode. This action is much more potent when the binding experiments are performed at physiological temperatures. These results demonstrate that drugs which act on GABAreceptor associated chloride ion channels have potent modulatory effects on benzodiazepine receptor binding in cerebellum. Benzodiazepine receptor binding can be modulated by GABA (1-6) and in addition by a number of drugs structurally unrelated to GABA which appear to exert at least part of their CNS effects by altering the effects of GABA on neuronal membranes (7-14). Thus, the pyrazolopyridines (7,8) and pentobarbital (9) have been found to stimulate benzodiazepine receptor binding in the presence of halide ions, and this action is sensitive to inhibition by picrotoxin (7-9). In addition, benzodiazepine receptor binding is also modulated by avermectin Bla (10), a macrocyclic lactone disaccharide anthelmintic agent (ii). In invertebrates, avermectin B1 a increases chloride permeability of membranes presumably by a long lasting and picrotoxin sensitive opening of GABAreceptOr regulated chloride channels (ii), and in rats and mice it potentiates the muscle relaxant effects of diazepam in vivo (i0). Picrotoxin, picrotoxinin, and IPTBO (4-(isopropyl)-l-phospho-2,6,7-trioxabicyclo (2,2,2)octane-l-oxide) are powerful convulsants in the mammalian and invertebrate central nervous system which exert their CNS effects by decreasing chloride permeability of GABA receptor, regulated chloride channels (12,13). Thus, these drugs antagonize the effects of GABA in many tissues by acting at a site uhrelated to GABArecognition sites. We now report that these drugs have also modulatory effects on benzodiazepine receptor binding to membranes from rat cerebellum, which include potent inhibitory actions on binding stimulated by CgHBA and by the pyrazolopyridine etazolate.
0024-3205/81/030307-07502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
308
Picrotoxin and 3H-Flunitrazepam Binding
Vol. 28, No. 3, 1981
Materials and Methods 3H-flunitrazepam (79.3 Ci/mmol) was purchased from New England Nuclear, Boston, Mass., USA. Picrotoxin was obtained from Fluka, Buchs, Switzerland, and picrotoxinin from Sigma, Corp. St. Louis, MI, USA. IPTBO (4-(isopropyl)-l-phospho2,6,7-trioxabicyclo- (2,2,2)-octane-l-oxide) was kindly provided by J. S. Collins, City of London, Polytechnic, London, UK., and etazolate (SQ 20009) was donated by S. J. Lucania of E. R. Squibb & Sons, Inc., Princeton, N.J., USA. Male Sprague Dawley rats (200 - 250 g, Forschungsinstitut fttr Versuchstierzucht, Himberg, Austria) were decapitated, and their brains were removed immediately. The cerebellum was dissected and homogenized with an Ultra-Turrax for 30 sec. in 50 vol of ice-cold 50 mM Tris-citrate buffer, pH 7.1, and centrifuged for i0 min at 48,000 g. The pellets were washed five times by resuspension and recentrifugation in the same vol of buffer and stored at -20 °C for at least 18 hours. After thawing the membrane fractions were washed once in 50 mM Triscitrate buffer and finally resuspended in 50 vol of the same buffer for binding assays. In some experiments, portions of the membrane fractions were treated with 0.05 % Triton-X i00 as described (17). For binding assays performed at 0 °C, 0.1 ml membrane suspension derived from 2 mg tissue wet weight were usually incubated for 90 min with 3H-flunitrazepam (3H-FLU) in 1 ml of an incubation medium which contained 50 mM Tris-citrate buffer, pH 7.1, and the compounds to be tested. The samples were then filtered under vacuum through Whatman GF/B filters and immediately washed twice with 5 ml ice-cold buffer. In binding assays performed at physiological temperature (35 °C), incubations were performed for 30 min with membranes derived from 4 mg tissue wet weight, and the samples were then filtered as described above and washed with buffer which was kept at 35 °C. Radioactivity on the filter was then determined by liquid scintillation counting. Non-specific binding determined in the presence of 2 ~M unlabelled diazepam was subtracted from total binding to give specific binding (3). Non-specific binding was i0 % or approximately 50 % of total binding in experiments performed at 0 °C or at 35 °C respectively. Results In binding experiments performed at 0 °C, picrotoxin, picrotoxinin and IPTBO caused a concentration dependent increase of the binding of 0.25 nM 3H-FLU to membranes from rat cerebellum (Figure i). All three drugs increased binding mainly by enhancing the apparent number of binding sites (Figure I). The effects of picrotoxin on 3H-FLU binding were reversible since preincubation of the membranes and resuspension in drug free buffer did not lead to an alteration of 3H-FLU binding (Figure i). The direct stimulatory effects of picrotoxin and IPTBO on 3H-FLU binding at 0 °C were greatly diminished in the absence of chloride ions and abolished when the binding experiments were performed with membranes which had been partially denatured by pretreatment with 0.05 % Triton-X i00 (data not shown). The magnitude of the direct stimulatory effect of picrotoxin and IPTBO on 3H-FLU binding was variable in different membrane preparations. Although extensive washing of the membranes appeared to diminish the direct stimulatory effects of picrotoxin (data not shown), we were, despite considerable effords, unable to evaluate all factors responisble for this variability. Thus, in 14 membrane preparations, the apparent+number of binding,sites for 3H-FLU in the presence of i00 ~M picrotoxin was 121 - 5 percent (mean -~ s.e.m.) of the respective control groups. In a few membrane preparations picrotoxin slightly enhanced the apparent affinity of 3H-FLU binding sites to a greater extent than shown in Figure i.
Vol. 28, No. 3, 1981
Picrotoxin and 3H-Flunitrazepam Binding
309
A
Z
o
u
of o
.o
/
ee v
0
O
z
m o.
:E m
u.
# lOO 2O 4O 6O 3H-FLUNITRAZEPAM BOUND (ImoteSlmll)
DRUG CONC (M)
FIGURE 1 Effect of picrotoxin, picrotoxinin and IPTBO on the binding of 3H-FLU to membranes from rat cerebellum at 0 °C. 150 mM NaCI was present in all incubations. On the left concentrationresponse curves for picrotoxin (o), picrotoxinin (D), and IPTBO (A) on the binding of 0.25 nM 3H-FLU are shown. On the right, Scatchard plots of saturation of specific 3H-FLU birding (0.2 to 4.0 nM) are shown. Experiments were performed in 50 mM Tris-citrate buffer, pH 7.1, which contained 150 mM NaCI (e), or in the same buffer containing i00 ~M picrotoxin (o), 100 ~M picrotoxinin (~), or 30 ~M IPTBO (a). In one experiment (m) membrane suspensions werepreincubated with I00 ~M picrotoxin for 90 rain at 0 °C, subsequently washed by centrifugation and resuspended in drug free buffer before saturation analysis.
FIGIRE 2
2°° ""'a
o
|
! 110-.6
' IPTBO ( M )
;-.4
10-6
10-4
PICROTOXIN ( M )
Tne effects of picrotoxin and IPTBO on basal, GABA- or etazolate-stimulated 3H-FLU binding to membranes from rat cerebellum. Binding experiments were performed at 0 °C for 90 min in 50 ~M Tris-citrate buffer, pH 7.1, which contained 150 mM NaCl. Shown are the concentration-response curves for IPTBO or picrotoxin alone (e) or in the presence of i0 ~M etazolate (m), or of 2 ~M GABA (D). Tne results are mean values of duplicate determinations. The experiment was repeated twice with similar results. 3H-FLU concentration was 0.25 nM.
310
Picrotoxin and 3H-Flunitrazepam Binding
Vol. 28, No. 3, 1981
Consistent effects of picrotoxin and IPTBO on 3H-FLU binding included inhibition of binding stimulated by etazolate or by GABA (Figure 2). In binding experiments performed at 0 °C, both d~x~s potent] y interfered with 3H-FLU binding stimulated by i0 UM etazolate which confirms and extends our previous observations (7). It can be seen from Figure 2 that, in the presence of etazolate and of IPTBO or picrotoxin, binding can be less than that in the presence of either drug alone. These observations suggest mutual inhibitory effects on 3H-FLU binding of IPTBO or picrotoxin on one hand, and of etazolate on the other hand. Picrotoxin and IPTBO in high concentrations also interfered with binding stimulated by GABA (Figure 2). Since IPTBO appeared to be slightly more effective than picrotoxin in this action (Figure 2), we investigated its effects on concentration response curves for stimulation of 3H-FLU binding by GABA. In Figure 3 it is shown that IPTBO interfered with the actions of GABA by decreasing the apparent maximal response to GABA.
°/°~-°
250
O/~
200
~4taMIPTBO ,~20pMPTSO
,
FIGURE 3 +100 pM IPTBO
~s
~OOIilli 10-7
i GABA ( M )
i
$ 10-4
Effect of IPTBO on the stimulation of 3H-FLU binding by GABA. the binding of 0.25 nM 3H-FLU to membranes from rat cerebellum was determined at 0 °C in 50 mM Tris-citrate buffer, pH 7. i, to which 150 mM NaCI was added. The results are the mean of duplicate determinations. The experiment was repeated twice with similar results.
Recently we observed that GABA stimulation of benzodiazepine receptor binding exhibits strong temperature and chloride ion dependency (15). We therefore reinvestigated the actions of picrotoxin and IPTBO on 3H-FLU binding to membranes from rat cerebellum at physiological temperature (35 °C).
In agreement with previous.observations (15), but unlike to results obtained at 0 °C (4), we found that in binding experiments performed at physiological temperature with membranes from rat cerebellum, chloride ions slightly inhibited basal 3H-FLU binding. However, at this temperature picrotoxin and IPTBO potently antagonized 3H-FLU binding stimulated by GABA (Table i) with hal E maximal in~+ibition of the GABA stimulated component of 3H-~LU binding at 3.3 - 1.4 ~M (mean - S.E.M. of 3 membrane perparations) and at 1.6 -~0.7 ~M, respectively. These actions of picrotoxin and IPTBO, but not those of bicuculline-methiodide were observed only in the presence of chloride ions (Table i). Picrotoxin and IPTBO antagonized also etazolate (SQ 20009) stimulated 3H-FLU binding in experiments performed at physiological temperature, in the presence of 150 mM NaCI (Table 1 ).
Vol. 28, No. 3, 1981
Picrotoxin and 3H-Flunitrazepam Binding
311
TABLE 1 Effect of various drugs on 3H-FLU binding to membranes from rat cerebelltun in binding experiments performed at physiological temperature (35 °C).
A4~i tions (~tM)
specific 3H-FLU binding at 35 °C fm~les/ngwet tissue weight means - s.e.m. (n = 3) (% of controls) 50 mM Tris-citrate buffer pH 7.1
50 mM Tris-citrate buffer pH 7.1 plus 150 mM NaCI
none
1.17 t 0.09 (i00)
0.85 ~ 0.04 (i00)
3 1.0t G A ~
2.20 t 0.26 (188)
2.10 ~ 0.07 (247)
0.3 ~M IPTBO
2.28 ~ 0.37 (195) +
1.70 ~ 0. ii (200)
3.0 ~M IPTBO
2.19 ~ 0.17 (187) +
1.29 ~ 0.09 (152)
+ 30.0 ~M IPTBO
2.18 t 0.17 (186) +
0.95 t 0.05 (iii)
1.49 t 0.06 (127)
0.67 Z 0.03 (79)
none
1.14 ~ 0.06 (i00)
0.96 -+ 0.i0 (i00)
3 UM GABA
2.23 ~ 0.07 (196)
2.55 -+ 0.ii (266)
0.3 ~M picrotoxin
2.37 ~ 0.04 (208) +
2.39 +- 0.13 (249)
3.0 ~M picrotoxin
2.07 ~ 0.01 (182)
1.91 + 0.13 (199)
2.24 ~ 0.09 (196) +
1.58 -+ 0.09 (164)
1.37 ~ 0.08 (120)
1.13 + 0.07 (115)
none
1.28 -+ 0.04 (i00)
1.16 + 0.02 (i00)
3 ~M GABA " + i0 ~M bicucullinemethiodide
2.17 -+ 0.03 (170) 1.54 + 0.13 (121)
1.03 -+ 0.09 (89)
i0 ~M bicuculline-methiodide
1.17 -+ 0.06 (91)
0.71 + 0.06 (61)
i0 ~M etazolate
1.02 + 0.04 (79)
1.89 + 0.07 (164)
EXPERIMENT 1
|!
+ +
30 ~M IPTBO
EXPERIMENT 2
+
ii
+
+ 30.0 ~M picrotoxin 30 UM picrotoxin EXPERIMENT 3
2.21 -+ 0.01 (191) ,
,
"
+ i0 ~M picrotoxin
1.43 -+ 0.06 (112)*
1.24 + 0.05 (107)*
"
+ i0 ~g IPTBO
1.35 -+ 0.02 (106)*
0.86 + 0.04 ( 74)*
The binding of i. 0 nM 3H-FLU was determined as described in methods. p < 0.01 compared to binding in the presence of 3 ~M GABA or of 10 bum etazolate +
p > 0.i
compared to binding in the presence of 3 ~M GABA.
312
Picrotoxln and 3H-Flunitrazepam Binding
Vol. 28, No. 3, 1981
Discussion There is evidence that picrotoxin exerts its CNS convulsant effects by interacting with a specific drug receptor which is in close association with GABA receptor regulated chloride ion channels (9,16). This interaction apparently triggers allosteric effects which lead to a decrease of chloride permeability of GABA receptor associated chloride channels (9,12-14,16). The present observations demonstrate that picrotoxin has also potent allosteric effects on 3H-FLU binding to the GABA/benzodiazepine receptor complex of the cerebellum. At physiological temperature picrotoxin and IPTBO inhibit GABA stimulated but not unstimulated 3H-FLU bindinq in concentrations which are comparable to their respective affinities for JH-dihydropicrotoxinin binding sites (9,16). Thus the interaction of picrotoxin or IPTBO with their site of action leads to an apparent desensitization of benzodiazepine binding sites towards stlmulation by GABA suggesting that the benzodiazepine binding protein of the cerebellum is also modulated by the chloride ionophore. Since picrotoxin and IPTBO do not interfere with the binding of labeled ligands for GABA recognition sites (17), our results point to indirect actions of these drugs on benzodiazepine receptor binding. The effects of ~icrotoxin and IPTBO but not those of bicuculline-methiodide on GABA stimulated ~H-FLU binding are absolute chloride ion dependent which adds further support to the notion (18) that these drugs interfere with the function of GABA by different mechanisms. There are also interactions between the effects of pyrazolopyridines on one hand, and those of picrotoxin or IPTBO on the other hand as modulators of benzodiazepine receptor binding. It has been suggested that the possible site of action of pyrazolopyridines is closely related to the chloride conductance mechanism since the actions of pyrazolopyridines on 3H-FLU binding are chloride ion dependent and blocked by picrotoxin (7) and IPTBO. This notion has been supported by recent observations demonstrating that etazolate inhibits the binding of 3H-dihydropicrotoxinin (9). However, it cannot be excluded that the pyrazolopyridenes (or chloride ions ) exert their modulatory effects on GABA receptor binding (19,20) and on benzodiazepine receptor binding (7,8) by acting at a distinct modulatory site. The actions of picrotoxin and IPTBO on GABA stimulated 3H-FLU binding exhibit strong temperature dependency, and they are more than hundred fold weaker in binding experiments performed at 0 °C. This and the weak chloride ion dependent intrinsic stimulatory effects of high concentrations of these drugs on 3H-FLU binding investigated at 0 °C may be the reason why no effects of picrotoxin on benzediazepine receptor binding have previously been observed. In addition, the present observations of a temperature dependency of the actions of picrotoxin and IPTBO further support the notion (15) that the allosteric properties of the GABA/benzodiazepine receptor con~01ex of the cerebellum exhibit strong temperature dependency, and that the investigation of benzodiazepine receptor binding at physiological temperature may be a better in vitro model for the examination of GABA receptor properties than binding studies performed at low temperature.
Acknowledgements We thank J. Reisenhofer and T. Ab~cherli for secretarial help, J. S. Collins for the donation of IPTBO and S. J. Lucania for etazolate. Supported by "Fonds zur F~rderung der wissenschaftlichen Forschung in ~sterreich".
Vol. 28, No. 3, 1981
Picrotoxin and 3H-Flunitrazepam Binding
313
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17. 18. 19. 20.
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