- Email: [email protected]
benzodiazepine receptor complex
GABA Neurorrunsmission Brrrin Resrtrrch BuMin,
Vol. 5.
Suppl.
2, pp. 681-684.
Printed
in the U.S.A
Strychnine as a Potent Inhibitor of the Brain GABA/Benzodiazepine Receptor Complex CLAUS AIS Ferrosan,
BRAESTRUP
AND MOGENS
NIELSEN
5 Sydmarken, DK-2860 Soeborg and Set. Hans Mental Hospital, DK-4000 Roskilde, Denmark
BRAESTRUP, C. AND M. NIELSEN. Strychnine (IS LI potent inhibitor oj’the bruin GABAlhrnzodictzepinr rwrptor complex. BRAIN RES. BULL. 5: Suppl. 2,681-684, 1980.-The interaction between GABA receptors and benzodiazepine receptors at the molecular level was investigated on rat brain membranes in vitro, using RH-diazepam binding assays. Stereoselective interaction was confirmed using the two stereoisomers R- and S-trans4-methyl aminocrotonic acid. This finding further indicates that the GABA receptor recognition site involved in the GABA/benzodiazepine receptor complex is similar to electrophysiologically identified GABA receptors. Strychnine, bicuculline, bicuculline methiodide and THIP antagonized the increase in “H-diazepam binding induced by muscimol and GABA. Schild analysis indicated that strychnine was a potent competitive inhibitor having a K, value of I .9 PM, compared to K,=O.S PM for bicuculline, 4.4 PM for bicuculline methiodide, and 7.4 PM for THIP. Strychnine was a more potent inhibitor on hippocampal membranes than on cerebellar membranes. These results indicate that GABA-receptor recognition sites in rat forebrain exhibit some glycine receptor like properties. y-Aminobutyric Benzodiazepine
Strychnine acid CNS receptors
Stereoselective Rat brain
GABA agonists
BENZODIAZEPINES interact with the inhibitory neurotransmitter GABA (y-aminobutyric acid). The inhibitory effects of GABA is increased in most electrophysiological studies (for review, see [14]) but not in all [ 11, 13, 221. Synergism between GABA agonists and benzodiazepines has been observed in some behavioural studies, i.e. benzodiazepines potentiate muscimol effects on methylphenidate induced stereotypies [ 11. No potentiation, however, of “antianxiety” effects of benzodiazepines has yet been presented. The demonstration of benzodiazepine receptors in CNS [6, 18, 211 opened a new potential area of benzodiazepine/GABA interaction. Mutual interaction at the molecular level was proposed from the findings that GABA and muscimol increased the binding of “H-diazepam to brain membranes in vitro [23] and by the finding of increased in vitro “H-GABA binding after addition of several benzodiazepines [ 121. In the present investigation of potential antagonists for the GABAlbenzodiazepine receptor complex we observed surprisingly that the glycine antagonist strychnine strongly inhibits the effects of muscimol and GABA on SH-diazepam binding. METHOD
“H-diazepam
and cooled to 0-4°C on ice. Tissue was homogenized in 10 vol icecold 100 mM TRIS, citrate, pH 7.1 in a precooled Ultra Turrax homogenizer. The homogenate was centrifuged for 48.000 x g at O&C for 10 min and the pellet was rehomogenized for a few seconds in another 10 vol of the same buffer. The washing procedure was repeated five times and the final suspension was diluted to 160 ml/g original tissue and used directly for “H-diazepam assays. Assays
Membrane suspensions (2.5 ml corresponding to 15.6 mg original tissue) were incubated for 20 min at 0°C with 0.4 nM :‘H-diazepam (14.4 Ci/mmol, gift from Willy Haefely, Roche Basle). Ten ml ice cold buffer was added to the homogenate, which was immediately filtered through GF/C glass fiber filters. The filters were washed with an additional 10 ml of buffer, and tritium was measured by conventional scintillation counting. Non-specific binding, which is binding in the presence of 3 x 10mfiMdiazepam, was always subtracted from total binding to give specific binding. Presumed antagonists and agonists were added in that order to assays in duplicate just before addition of “H-diazepam. Results are based on at least two separate experiments.
Tissue Preparation
Materials
Male Wistar rats were killed and the whole forebrain, or specific brain areas when indicated, were rapidly excised
Muscimol HBr, R- and S-trans-4-methyl-aminocrotonic acid, THIP and piperidine-4-sulphonic acid (PSA) were syn-
Copyright
c 1980 ANKHO
International
Inc.-0361-9230/80/080681-04$00.90/O
BRA~STRUP
682
200
‘I‘ABLE I &VALUES (AFFINITY CONSTANTS) FOR SOME ANTA(;ONiS’I’S OF MUSCIMOL INDUCED ACTIVATION OF 3H-DIAZEPAM BINDING TO RAT FOREBRAIN MEMBRANES. K, VALUES WERE DETERMINED BY SCHILD ANALYSIS USING AT LEAST THREE CONCENTRATIONS OF ANTAGONIST
175 sf E :
150
l
I
0
AN5 NIELSEN
0
GABA
I,
TACA
Cl
g-t-1
4 -METHYL
TACA
m
R-i+)
4 -METHYL
TACA
Bicucuiline
0.51
(21
--
B~~uculline methiodide
4.4 I 1.3
(3)
-
Strychnine
1.9 t 0.7
(3)
-
1.2
(2)
>I000
THIP Muscimol GABA TACA S-( - )-4-methyt TACA
7.4 + 3 -
(6)
-
>lOOO 0.6 3.8 7.8 390
R-(+f-4-methyl TACA
-
>2ooo
Piperidine-4sulphonic acid
EC.,,,“. /&I. fol “H-diazepam activation
*Values from 141and Figure 1 (mean of two values).
I ”
10' F
’
Agent
K I%crM mean t SEM of (values)
10-o
I
I
10-3 c
10-4
/
10'"
1,M
FIG. 1. Dose-response curve for GABA, C-O; TACA (tram amino0-O; S-(-)4methyi TACA, a-U; and acid), ~-(+)-4-methyl TACA, 8-m on 3H-diazepam binding to rat forecrotonic
brain membranes.
thesized by P. Krogsgaard-Larsen, Copenhagen. Bicuculline (Sigma) was freshly prepared in dilute HCl; strychnineglycerophosphate (A/S Ferrosan, Copenhagen) and bicuculline methiodide (Pierce) were dissolved in water. RESULTS
The results in Fig. 1 and Table 1 confirm previous indications [4] that the activation of 3H-diazepam binding by GABA agonists is stereoselective. The GABA receptor active enantiomer S-(-)-4-methyl TACA (trans aminocrotonic acid) increased JH-diazepam binding while the GABA receptor inactive enantiomer R-(+)-4-methyl TACA was inactive at 2 x W3 M. The IC,-values for inhibition of 3H-GABA binding in rapidly frozen and thoroughly washed membranes are 4.1 PM and 148 PM, respectively [171. The proportion EC&JIG,,,=94 for S-(-)+methyl TACA is in good agreement with the reported value of 179 for nine full GABA agonists [43. Bicuculline and strychnine both decreased dose dependently the activation of 3H-diazepam binding induced by a submaximal concentration of the GABA agonist muscimol (3x 10-W) (Fig. 2). Schild analysis of the dose response for muscimol in the
presence of bicuculline (Fig. 3) indicates that bicuculline interacts competitively with muscimol in a 1. order interaction. Bicuculline is the most potent inhibitor yet tested. Also strychnine displaced the dose-response curves for muscimol almost parallelly to the right. Schild analysis exhibited a slope close to unity (Fig. 4) and indicates that strychnine inhibits muscimol competitively in a 1. order interaction. Strychnine was quite potent, having a low KI value agahW both muscimol (Table 1) and GABA (Ki=3.2 FM, Fig. 4). Table 1 shows Ki values of some other inhibitors for comparison. Regional susceptibility to strychnine was investigated in rat occipital cortex, frontal cortex, hippocampus, Corpus striatum, limbic forebrain, pons+medulla and cerebellum (dissection according to [6]). Strychnine (IO-“M) inhibited more than 60% of the effect of muscimol (3 x IO-@M) in all areas except cerebellum where only 25% inhibition was obtained. A detailed dose response for strychnine in hippocampus and cerebellum (experimental design as described in Fig. 2) confirmed that strychnine was a more potent inhibitor in hippocampus than in cerebellum (Table 2). DISCUSSION
The ph~macology of GABA receptors in the central nervous system can be investigated in several ways. Electrophysiological responses to GABA in intact neurons is generally accepted as the most conclusive investigational situation, despite difficulties in doing quantitative studies. High afkity binding sites on brain membranes for :sH-GABA and SH-muscimol exhibit several properties to be expected for physiologically relevant GABA receptors both with respect to drug (agent) specificity, organ specificity and cellular localization. High affinity binding sites for ‘Hmuscimol and 3H-GABA, however, exhibit some unexpected properties, such as the presence of two different af-
STRYCHNINE
683
AND CNS GABA RECEPTORS TABLE 2
200
INHIBITION OF MUSCIMOL AC~VATION OF :~H-DIAZEPAM BINDING BY STRYCHNINE IN RAT HIPPOCAMPUS AND CEREBELLUM Specific binding, cpmi15.6 mg tissue mean i- SEM of (values)
Brain area
Hippocampus Cerebellum
Control
+ Mus~imol
541 2 11 (4) 301 5 38 (4)
930 (2) 602 (2)
IC,,, for strychnine, I*M
2.5 37
Strychnine (four concentrations) or vehicle was added to “Hdiazepam assays just before addition of muscimol (3 x lo-” M) or vehicle. The B&value is the concentration of strychnine at which muscimol exhibits just half the percentual increase in “H-diazepam binding as without strychnine. The results are from a single experiment, which was repeated with very similar results.
125
SCHICD
50
,
.__
+_
..-. - ---
t
PLOT
v I
I i
25
t
/
. .
.;-
, -6
/
b
-6
1
i
-3
-4
BICUCULLINE
MUSCIMOL IOslC
0
I
[
13
FIG. 2. Inhibition of the effect of muscimol(3x IO-:‘M) on rat forebrain “H-diazepam binding by bieuculline (CiO) and strychnine (K!-Xl) in increasing contrations. Addition of strychnine without muscimol (cm). Control specific binding of “H-diazepam (no strychnine, bicuculline or muscimol added) was ca. 700 cpm /15.6 mg tissue; nonspecific binding (100 &ml clonazepam) was ca. 150 cpmi15.6 mg tissue.
l,M
FIG. 3. Left side: Dose response curves for the effect of muscimol on “H-diazepam binding to rat forebrain membranes. Various concentrations of bicuculline were added. 0, No addition; 0, lO_“M; +, IO-“M; A, 10e4M. Right side: Schild plots constructed from the EC,,, values for muscimol obtained in the left side. (The dose-response ratio DR, is the ratio of EC,,, values for muscimol with and without antagonist added). The EC,,, value is that concentration of GABA agonist which increases specific “H-diazepam binding to half the increase obtainable with optimal maximal concentrations (10~z-3x10-“) of muscimoi. The results shown in this figure are from a single experiment which was repeated with similar results. 200
c I
0
the one apparently below the actual concentration of agonists which affects neurons in vitro, and the unexpected low affinity of the potent and selective GABA antagonist bicuculline. A third way to investigate GABA receptors in CNS is to use the GABAlbenzodiazepine receptor complex in vitro and measure the increase in “H-diazepam binding induced by GABA agonists. The GABA receptor recognition sites involved in the increase in 3H-diazepam binding is probably very closely related, if not identical to the GABA receptor mediating electrophysiological changes on intact neurons. (1) The relevant concentrations of GABA are in the micromolar range for both systems. (2) The structura1 requirements for GABA agonists is consistent from one system to the other. Sixteen GABA mimetic compounds (Fig. 1, and ref. M) interact either as fuI1 or partial agonists with the finity constants,
180
SCHILD
I -7
Pt.01
I
-6
-5
-4
-3
MUSClMOL
ov STRYCHNINE
lonCl,M FIG. 4. See legend to Fig. 3. Strychnine was used instead of bicuculline; 0, 2x10GM; +, 2xIO~“M; & 2x W4M. 0, 2x10-“M strychnine and GABA as agonist; + 2 x IO-“M strychnine and GABA as agonist. The results shown in the figure are from a single experiment which was repeated with similar results.
684
GABAibenzodiazepine receptor complex (Fig. I, ref ]4,5]) and most of them interact with electrophysiologically identified systems in a very similar rank order [ 16,171. (3) The GABA/benzodiazepine receptor complex exhibit stereoselectivity for agonists. Stereoselectivity has not yet been demonstrated for electrophysiological responses to agonists. High affinity binding systems, however, exhibit the same stereoselectivity as the GABA/benzodiazepine receptor complex (Fig. 4, ref. [S]).(4) Bicucutline is a potent and competitive antagonist in both systems (Fig. 3, 191) and the affinity of bicuculline is similar to that of GABA in both (Table 1, ref. [3, 4, 191). Strychnine in reasonable doses is generally believed to block glycine receptors and not to interact with GABA receptors. This selectivity is clearly shown in the spinal cord [8] and is also described for Deiters nucleus [7] and for cuneo-thalamic relay cells [ 151at higher levels of CNS. The present finding, that strychnine is a very potent inhibitor, only 4 fold less potent than bicuculline, of the GABAibenzodiazepine receptor complex of rat forebrain membranes, is not fully unexpected. The differentiation between GABA like and glycine like (glycine, taurine, p-alanine) aminoacids, is very distinct in the spinal cord but is less clear in supraspinal CNS. This means that the inhibition of glycine like aminoacids by strychnine and of GABA like aminoacids by bicuculline is more specific in the spinal cord, where the selectivity is mainly established, than at higher levels of the CNS. For example in the cerebral cortex, the depressant
1. Arm,
J., A. V. Christensen and J. Scheel-Kruger. Benzodiazepines potentiate GABA-dopamine stereotyped dependent gnawing in mice. f. Pharm. Pharmac. 31: 5658, 1979. 2. Barker, J. L. and R. N. McBurney. GABA and glycine may share the same conductance channel on cultured mammalian neurones. Nature 277: 234-236, 1979. 3. Bowery, N. G. and D. A. Brown. Depolarizing actions of y-aminobutyric acid and related compounds on rat superior cervical ganglia in vitro. Br. J. Phnfmac~. 50: 205-218, 1974. 4. Braestrup, C., M. Nielsen, P. Krogsgaard-Larsen and E, Falch. Partial agonists for brain GAB~enz~i~epine receptor complex. Nature 28th 331-333, 1979. 5. Braestrup, C., M. Nielsen, P. Krogsgaard-Larsen and E, Falch. Two or more conformations of benzodiazepine receptors depending on GABA receptors and other variables. In: Int. Colloquium an Peptidps and Receptors, edited by G. Pepeu, M. Kuhar and S. Enna. (In press.) 6. Braestrup, C. and R. F. Squires. Specific benzodiazepine receptors in rat brain characterized by high-affinity [“Hldiazepam binding. Proc. n&n. Acnd. Sci. U.S.A. 74: 3805-3809, 1977. 7. Bruggencate, G. T. and I. Engberg. lontophoretic studies in deiters’ nucleus of the inhibitory actions of GABA and related amino acids and the interactions of strychnine and picrotoxin. Brain Res. 25: 431-448, 1971. 8. Curtis, D. R., A. W. Duggan and G. A. R. Johnston. The specificity of strychnine as a glycine antagonist in mammalian spinal cord. Expl Bruin Res. 12: 547-565, 1971. 9. Curtis, D. R., A. W. Duggan, D. Felix and G. A. R. Johnston. Bicuculline, an antagonist of GABA and synaptic inhibition of the spinal cord of the cat. Brain Res. 32: 69-96, 1971. 10. Curtis, D. R., A. W. Duggan, D. Felix, G. A. R. Johnston and H. McLennan. Antagonism between bicuculline and GABA in the cat brain. Bruin Res. 33: 57-73, 1971. 1I. Curtis, D. R., C. J. A. Game and D. Lodge. Benzodiazepines and central glycine receptors. Br. 1. Phnrmac. 56: 307-311, 1976. 12. Guidotti, A., G. Toffano and E. Costa. An endogenous protein modulates the affinity of GABA and benz~iazepine receptors in rat brain. Nnnrrc* 275: 553-555, 1978.
BRAESTRUP
AND NIELSEN
action of b-alanine (and taurine) is blocked by both strychnine and bicuculline [lo]. Furthermore, strychnine appears not to be selective towards glycine in the rat hippocampus, since both GABA, glycine and p-alanine can be antagonized by medium high currents (20-30 nA) of strychnine. In another brain area, the cerebellum, the action of glycine was selectively blocked by strychnine [to]. This apparent regional variation in receptor selectivity for strychnine was also found for the GABA/benzodi~~pine receptor complex (Table 2). The result thus indicate that the GABA receptors involved are less selective in hippocampus than in cerebellum. Other examples of inhibition of GABA effects by strychnine in vitro have been described in rat superior cervical ganglia [3], in rat olfactory cortex tract 1191, and in rat cuneate nucleus slices [20]. The concentrations of strychnine were S-10 fold higher than equivalent concentrations of bicuculline in these experiments. Glycine and GABA receptors share responsiveness to a zwitte~onic agonist and both receptors probably interact with a similar chloride channel 121. The present results indicate that also the receptor recognition sites in the CNS share structural elements. ACKNOWLEDGMENT
This study was aided by grants from Danish Medical Research Council Jr. No 512-8404. The generous supply of drugs by P. Krogsgaard-Larsen,
Copenhagen,
is highly appreciated.
13. Gahwiler, B. H. Diazepam and chlordiazepoxide: powerful GABA antagonists in explants of rat cerebellum. Bruin Res. 107: 176179, 1976. 14. Haefely, W., P. Pole,
R. SchatTner, H. H. Keller, L. Pieri and
H. Miihler. Facilitation of GABA-ergic transmission by drugs. In: GABA-Neurorrunsmitters, edited by P. Krogsgaard-Larsen, J. Scheel-Krtlger and H. Kofod. Copenhagen, Munksgaard and New York: Academic Press, 1979, pp. 357-375. 1s. Kelly, J. S. and L. P. Renaud. On the pharmacology of the glycine receptors on the cuneo-thalamic relay cells in the cat. Br. J. Pharmu~.
48: 387-395,
1973.
16. Krogsgaard-Larsen, P., H. Hjeds, D. R. Curtis, D. Lodge and G. A. R. Johnston. Dihvdromuscimol, t~omuscimol and related heterocyclic compounds as GABA analogues. .T. Nrurochem. 32: 1717-1724, 1979. 17. Krogsgaard-Larsen, P., T. Honort
and K. Thyssen. GABA reand structure-activity studies. In: GABA-Neurotrunsmitters, edited by P. Krogsgaard-Larsen, J. Scheel-Krtiger and H. Kofod. Copenhagen, Munksgaard and New York: Academic Press, 1979, pp. 201-216. 18. Mohler, H. and T. Okada. Properties of 3H-diazepam binding to benzodiazepine receptors in rat cerebral cortex. Lifp Sri. 20: 2101-2110. 1977. 19. Pickles, H: G. Presynaptic y-aminobu?y~c acid responses in the olfactorv Br. J. Phurmuc. 65: 223-228, 1979. --------, cortex. -20. Simmonds, M. A. Presynaptic actions of y-~~nobuty~c acid and some antagonists in a slice preparation of cuneate nucleus. ceptor
agonists:
design
Br. J. Pharmac. 63: 495-502, 1978. 21. Squires, R. F. and C. Braestrup. Benzodiazepine receptors in rat brain. Nature 266: 732-734, 1977. 22. Steiner, F. A. and D. Felix. Antagonistic effects of GABA and
benzodiazepines on vestibular and cerebellar neurones. Nature 260: 346-347, 1976. 23. Tallman, J. F., J. W. Thomas and D. W. Gallager. GABAergic modulation of benzodiazepine binding site sensitivity. Nature 274: 383-385, 1978.
Vol. 5.
Suppl.
2, pp. 681-684.
Printed
in the U.S.A
Strychnine as a Potent Inhibitor of the Brain GABA/Benzodiazepine Receptor Complex CLAUS AIS Ferrosan,
BRAESTRUP
AND MOGENS
NIELSEN
5 Sydmarken, DK-2860 Soeborg and Set. Hans Mental Hospital, DK-4000 Roskilde, Denmark
BRAESTRUP, C. AND M. NIELSEN. Strychnine (IS LI potent inhibitor oj’the bruin GABAlhrnzodictzepinr rwrptor complex. BRAIN RES. BULL. 5: Suppl. 2,681-684, 1980.-The interaction between GABA receptors and benzodiazepine receptors at the molecular level was investigated on rat brain membranes in vitro, using RH-diazepam binding assays. Stereoselective interaction was confirmed using the two stereoisomers R- and S-trans4-methyl aminocrotonic acid. This finding further indicates that the GABA receptor recognition site involved in the GABA/benzodiazepine receptor complex is similar to electrophysiologically identified GABA receptors. Strychnine, bicuculline, bicuculline methiodide and THIP antagonized the increase in “H-diazepam binding induced by muscimol and GABA. Schild analysis indicated that strychnine was a potent competitive inhibitor having a K, value of I .9 PM, compared to K,=O.S PM for bicuculline, 4.4 PM for bicuculline methiodide, and 7.4 PM for THIP. Strychnine was a more potent inhibitor on hippocampal membranes than on cerebellar membranes. These results indicate that GABA-receptor recognition sites in rat forebrain exhibit some glycine receptor like properties. y-Aminobutyric Benzodiazepine
Strychnine acid CNS receptors
Stereoselective Rat brain
GABA agonists
BENZODIAZEPINES interact with the inhibitory neurotransmitter GABA (y-aminobutyric acid). The inhibitory effects of GABA is increased in most electrophysiological studies (for review, see [14]) but not in all [ 11, 13, 221. Synergism between GABA agonists and benzodiazepines has been observed in some behavioural studies, i.e. benzodiazepines potentiate muscimol effects on methylphenidate induced stereotypies [ 11. No potentiation, however, of “antianxiety” effects of benzodiazepines has yet been presented. The demonstration of benzodiazepine receptors in CNS [6, 18, 211 opened a new potential area of benzodiazepine/GABA interaction. Mutual interaction at the molecular level was proposed from the findings that GABA and muscimol increased the binding of “H-diazepam to brain membranes in vitro [23] and by the finding of increased in vitro “H-GABA binding after addition of several benzodiazepines [ 121. In the present investigation of potential antagonists for the GABAlbenzodiazepine receptor complex we observed surprisingly that the glycine antagonist strychnine strongly inhibits the effects of muscimol and GABA on SH-diazepam binding. METHOD
“H-diazepam
and cooled to 0-4°C on ice. Tissue was homogenized in 10 vol icecold 100 mM TRIS, citrate, pH 7.1 in a precooled Ultra Turrax homogenizer. The homogenate was centrifuged for 48.000 x g at O&C for 10 min and the pellet was rehomogenized for a few seconds in another 10 vol of the same buffer. The washing procedure was repeated five times and the final suspension was diluted to 160 ml/g original tissue and used directly for “H-diazepam assays. Assays
Membrane suspensions (2.5 ml corresponding to 15.6 mg original tissue) were incubated for 20 min at 0°C with 0.4 nM :‘H-diazepam (14.4 Ci/mmol, gift from Willy Haefely, Roche Basle). Ten ml ice cold buffer was added to the homogenate, which was immediately filtered through GF/C glass fiber filters. The filters were washed with an additional 10 ml of buffer, and tritium was measured by conventional scintillation counting. Non-specific binding, which is binding in the presence of 3 x 10mfiMdiazepam, was always subtracted from total binding to give specific binding. Presumed antagonists and agonists were added in that order to assays in duplicate just before addition of “H-diazepam. Results are based on at least two separate experiments.
Tissue Preparation
Materials
Male Wistar rats were killed and the whole forebrain, or specific brain areas when indicated, were rapidly excised
Muscimol HBr, R- and S-trans-4-methyl-aminocrotonic acid, THIP and piperidine-4-sulphonic acid (PSA) were syn-
Copyright
c 1980 ANKHO
International
Inc.-0361-9230/80/080681-04$00.90/O
BRA~STRUP
682
200
‘I‘ABLE I &VALUES (AFFINITY CONSTANTS) FOR SOME ANTA(;ONiS’I’S OF MUSCIMOL INDUCED ACTIVATION OF 3H-DIAZEPAM BINDING TO RAT FOREBRAIN MEMBRANES. K, VALUES WERE DETERMINED BY SCHILD ANALYSIS USING AT LEAST THREE CONCENTRATIONS OF ANTAGONIST
175 sf E :
150
l
I
0
AN5 NIELSEN
0
GABA
I,
TACA
Cl
g-t-1
4 -METHYL
TACA
m
R-i+)
4 -METHYL
TACA
Bicucuiline
0.51
(21
--
B~~uculline methiodide
4.4 I 1.3
(3)
-
Strychnine
1.9 t 0.7
(3)
-
1.2
(2)
>I000
THIP Muscimol GABA TACA S-( - )-4-methyt TACA
7.4 + 3 -
(6)
-
>lOOO 0.6 3.8 7.8 390
R-(+f-4-methyl TACA
-
>2ooo
Piperidine-4sulphonic acid
EC.,,,“. /&I. fol “H-diazepam activation
*Values from 141and Figure 1 (mean of two values).
I ”
10' F
’
Agent
K I%crM mean t SEM of (values)
10-o
I
I
10-3 c
10-4
/
10'"
1,M
FIG. 1. Dose-response curve for GABA, C-O; TACA (tram amino0-O; S-(-)4methyi TACA, a-U; and acid), ~-(+)-4-methyl TACA, 8-m on 3H-diazepam binding to rat forecrotonic
brain membranes.
thesized by P. Krogsgaard-Larsen, Copenhagen. Bicuculline (Sigma) was freshly prepared in dilute HCl; strychnineglycerophosphate (A/S Ferrosan, Copenhagen) and bicuculline methiodide (Pierce) were dissolved in water. RESULTS
The results in Fig. 1 and Table 1 confirm previous indications [4] that the activation of 3H-diazepam binding by GABA agonists is stereoselective. The GABA receptor active enantiomer S-(-)-4-methyl TACA (trans aminocrotonic acid) increased JH-diazepam binding while the GABA receptor inactive enantiomer R-(+)-4-methyl TACA was inactive at 2 x W3 M. The IC,-values for inhibition of 3H-GABA binding in rapidly frozen and thoroughly washed membranes are 4.1 PM and 148 PM, respectively [171. The proportion EC&JIG,,,=94 for S-(-)+methyl TACA is in good agreement with the reported value of 179 for nine full GABA agonists [43. Bicuculline and strychnine both decreased dose dependently the activation of 3H-diazepam binding induced by a submaximal concentration of the GABA agonist muscimol (3x 10-W) (Fig. 2). Schild analysis of the dose response for muscimol in the
presence of bicuculline (Fig. 3) indicates that bicuculline interacts competitively with muscimol in a 1. order interaction. Bicuculline is the most potent inhibitor yet tested. Also strychnine displaced the dose-response curves for muscimol almost parallelly to the right. Schild analysis exhibited a slope close to unity (Fig. 4) and indicates that strychnine inhibits muscimol competitively in a 1. order interaction. Strychnine was quite potent, having a low KI value agahW both muscimol (Table 1) and GABA (Ki=3.2 FM, Fig. 4). Table 1 shows Ki values of some other inhibitors for comparison. Regional susceptibility to strychnine was investigated in rat occipital cortex, frontal cortex, hippocampus, Corpus striatum, limbic forebrain, pons+medulla and cerebellum (dissection according to [6]). Strychnine (IO-“M) inhibited more than 60% of the effect of muscimol (3 x IO-@M) in all areas except cerebellum where only 25% inhibition was obtained. A detailed dose response for strychnine in hippocampus and cerebellum (experimental design as described in Fig. 2) confirmed that strychnine was a more potent inhibitor in hippocampus than in cerebellum (Table 2). DISCUSSION
The ph~macology of GABA receptors in the central nervous system can be investigated in several ways. Electrophysiological responses to GABA in intact neurons is generally accepted as the most conclusive investigational situation, despite difficulties in doing quantitative studies. High afkity binding sites on brain membranes for :sH-GABA and SH-muscimol exhibit several properties to be expected for physiologically relevant GABA receptors both with respect to drug (agent) specificity, organ specificity and cellular localization. High affinity binding sites for ‘Hmuscimol and 3H-GABA, however, exhibit some unexpected properties, such as the presence of two different af-
STRYCHNINE
683
AND CNS GABA RECEPTORS TABLE 2
200
INHIBITION OF MUSCIMOL AC~VATION OF :~H-DIAZEPAM BINDING BY STRYCHNINE IN RAT HIPPOCAMPUS AND CEREBELLUM Specific binding, cpmi15.6 mg tissue mean i- SEM of (values)
Brain area
Hippocampus Cerebellum
Control
+ Mus~imol
541 2 11 (4) 301 5 38 (4)
930 (2) 602 (2)
IC,,, for strychnine, I*M
2.5 37
Strychnine (four concentrations) or vehicle was added to “Hdiazepam assays just before addition of muscimol (3 x lo-” M) or vehicle. The B&value is the concentration of strychnine at which muscimol exhibits just half the percentual increase in “H-diazepam binding as without strychnine. The results are from a single experiment, which was repeated with very similar results.
125
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FIG. 2. Inhibition of the effect of muscimol(3x IO-:‘M) on rat forebrain “H-diazepam binding by bieuculline (CiO) and strychnine (K!-Xl) in increasing contrations. Addition of strychnine without muscimol (cm). Control specific binding of “H-diazepam (no strychnine, bicuculline or muscimol added) was ca. 700 cpm /15.6 mg tissue; nonspecific binding (100 &ml clonazepam) was ca. 150 cpmi15.6 mg tissue.
l,M
FIG. 3. Left side: Dose response curves for the effect of muscimol on “H-diazepam binding to rat forebrain membranes. Various concentrations of bicuculline were added. 0, No addition; 0, lO_“M; +, IO-“M; A, 10e4M. Right side: Schild plots constructed from the EC,,, values for muscimol obtained in the left side. (The dose-response ratio DR, is the ratio of EC,,, values for muscimol with and without antagonist added). The EC,,, value is that concentration of GABA agonist which increases specific “H-diazepam binding to half the increase obtainable with optimal maximal concentrations (10~z-3x10-“) of muscimoi. The results shown in this figure are from a single experiment which was repeated with similar results. 200
c I
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the one apparently below the actual concentration of agonists which affects neurons in vitro, and the unexpected low affinity of the potent and selective GABA antagonist bicuculline. A third way to investigate GABA receptors in CNS is to use the GABAlbenzodiazepine receptor complex in vitro and measure the increase in “H-diazepam binding induced by GABA agonists. The GABA receptor recognition sites involved in the increase in 3H-diazepam binding is probably very closely related, if not identical to the GABA receptor mediating electrophysiological changes on intact neurons. (1) The relevant concentrations of GABA are in the micromolar range for both systems. (2) The structura1 requirements for GABA agonists is consistent from one system to the other. Sixteen GABA mimetic compounds (Fig. 1, and ref. M) interact either as fuI1 or partial agonists with the finity constants,
180
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-4
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ov STRYCHNINE
lonCl,M FIG. 4. See legend to Fig. 3. Strychnine was used instead of bicuculline; 0, 2x10GM; +, 2xIO~“M; & 2x W4M. 0, 2x10-“M strychnine and GABA as agonist; + 2 x IO-“M strychnine and GABA as agonist. The results shown in the figure are from a single experiment which was repeated with similar results.
684
GABAibenzodiazepine receptor complex (Fig. I, ref ]4,5]) and most of them interact with electrophysiologically identified systems in a very similar rank order [ 16,171. (3) The GABA/benzodiazepine receptor complex exhibit stereoselectivity for agonists. Stereoselectivity has not yet been demonstrated for electrophysiological responses to agonists. High affinity binding systems, however, exhibit the same stereoselectivity as the GABA/benzodiazepine receptor complex (Fig. 4, ref. [S]).(4) Bicucutline is a potent and competitive antagonist in both systems (Fig. 3, 191) and the affinity of bicuculline is similar to that of GABA in both (Table 1, ref. [3, 4, 191). Strychnine in reasonable doses is generally believed to block glycine receptors and not to interact with GABA receptors. This selectivity is clearly shown in the spinal cord [8] and is also described for Deiters nucleus [7] and for cuneo-thalamic relay cells [ 151at higher levels of CNS. The present finding, that strychnine is a very potent inhibitor, only 4 fold less potent than bicuculline, of the GABAibenzodiazepine receptor complex of rat forebrain membranes, is not fully unexpected. The differentiation between GABA like and glycine like (glycine, taurine, p-alanine) aminoacids, is very distinct in the spinal cord but is less clear in supraspinal CNS. This means that the inhibition of glycine like aminoacids by strychnine and of GABA like aminoacids by bicuculline is more specific in the spinal cord, where the selectivity is mainly established, than at higher levels of the CNS. For example in the cerebral cortex, the depressant
1. Arm,
J., A. V. Christensen and J. Scheel-Kruger. Benzodiazepines potentiate GABA-dopamine stereotyped dependent gnawing in mice. f. Pharm. Pharmac. 31: 5658, 1979. 2. Barker, J. L. and R. N. McBurney. GABA and glycine may share the same conductance channel on cultured mammalian neurones. Nature 277: 234-236, 1979. 3. Bowery, N. G. and D. A. Brown. Depolarizing actions of y-aminobutyric acid and related compounds on rat superior cervical ganglia in vitro. Br. J. Phnfmac~. 50: 205-218, 1974. 4. Braestrup, C., M. Nielsen, P. Krogsgaard-Larsen and E, Falch. Partial agonists for brain GAB~enz~i~epine receptor complex. Nature 28th 331-333, 1979. 5. Braestrup, C., M. Nielsen, P. Krogsgaard-Larsen and E, Falch. Two or more conformations of benzodiazepine receptors depending on GABA receptors and other variables. In: Int. Colloquium an Peptidps and Receptors, edited by G. Pepeu, M. Kuhar and S. Enna. (In press.) 6. Braestrup, C. and R. F. Squires. Specific benzodiazepine receptors in rat brain characterized by high-affinity [“Hldiazepam binding. Proc. n&n. Acnd. Sci. U.S.A. 74: 3805-3809, 1977. 7. Bruggencate, G. T. and I. Engberg. lontophoretic studies in deiters’ nucleus of the inhibitory actions of GABA and related amino acids and the interactions of strychnine and picrotoxin. Brain Res. 25: 431-448, 1971. 8. Curtis, D. R., A. W. Duggan and G. A. R. Johnston. The specificity of strychnine as a glycine antagonist in mammalian spinal cord. Expl Bruin Res. 12: 547-565, 1971. 9. Curtis, D. R., A. W. Duggan, D. Felix and G. A. R. Johnston. Bicuculline, an antagonist of GABA and synaptic inhibition of the spinal cord of the cat. Brain Res. 32: 69-96, 1971. 10. Curtis, D. R., A. W. Duggan, D. Felix, G. A. R. Johnston and H. McLennan. Antagonism between bicuculline and GABA in the cat brain. Bruin Res. 33: 57-73, 1971. 1I. Curtis, D. R., C. J. A. Game and D. Lodge. Benzodiazepines and central glycine receptors. Br. 1. Phnrmac. 56: 307-311, 1976. 12. Guidotti, A., G. Toffano and E. Costa. An endogenous protein modulates the affinity of GABA and benz~iazepine receptors in rat brain. Nnnrrc* 275: 553-555, 1978.
BRAESTRUP
AND NIELSEN
action of b-alanine (and taurine) is blocked by both strychnine and bicuculline [lo]. Furthermore, strychnine appears not to be selective towards glycine in the rat hippocampus, since both GABA, glycine and p-alanine can be antagonized by medium high currents (20-30 nA) of strychnine. In another brain area, the cerebellum, the action of glycine was selectively blocked by strychnine [to]. This apparent regional variation in receptor selectivity for strychnine was also found for the GABA/benzodi~~pine receptor complex (Table 2). The result thus indicate that the GABA receptors involved are less selective in hippocampus than in cerebellum. Other examples of inhibition of GABA effects by strychnine in vitro have been described in rat superior cervical ganglia [3], in rat olfactory cortex tract 1191, and in rat cuneate nucleus slices [20]. The concentrations of strychnine were S-10 fold higher than equivalent concentrations of bicuculline in these experiments. Glycine and GABA receptors share responsiveness to a zwitte~onic agonist and both receptors probably interact with a similar chloride channel 121. The present results indicate that also the receptor recognition sites in the CNS share structural elements. ACKNOWLEDGMENT
This study was aided by grants from Danish Medical Research Council Jr. No 512-8404. The generous supply of drugs by P. Krogsgaard-Larsen,
Copenhagen,
is highly appreciated.
13. Gahwiler, B. H. Diazepam and chlordiazepoxide: powerful GABA antagonists in explants of rat cerebellum. Bruin Res. 107: 176179, 1976. 14. Haefely, W., P. Pole,
R. SchatTner, H. H. Keller, L. Pieri and
H. Miihler. Facilitation of GABA-ergic transmission by drugs. In: GABA-Neurorrunsmitters, edited by P. Krogsgaard-Larsen, J. Scheel-Krtlger and H. Kofod. Copenhagen, Munksgaard and New York: Academic Press, 1979, pp. 357-375. 1s. Kelly, J. S. and L. P. Renaud. On the pharmacology of the glycine receptors on the cuneo-thalamic relay cells in the cat. Br. J. Pharmu~.
48: 387-395,
1973.
16. Krogsgaard-Larsen, P., H. Hjeds, D. R. Curtis, D. Lodge and G. A. R. Johnston. Dihvdromuscimol, t~omuscimol and related heterocyclic compounds as GABA analogues. .T. Nrurochem. 32: 1717-1724, 1979. 17. Krogsgaard-Larsen, P., T. Honort
and K. Thyssen. GABA reand structure-activity studies. In: GABA-Neurotrunsmitters, edited by P. Krogsgaard-Larsen, J. Scheel-Krtiger and H. Kofod. Copenhagen, Munksgaard and New York: Academic Press, 1979, pp. 201-216. 18. Mohler, H. and T. Okada. Properties of 3H-diazepam binding to benzodiazepine receptors in rat cerebral cortex. Lifp Sri. 20: 2101-2110. 1977. 19. Pickles, H: G. Presynaptic y-aminobu?y~c acid responses in the olfactorv Br. J. Phurmuc. 65: 223-228, 1979. --------, cortex. -20. Simmonds, M. A. Presynaptic actions of y-~~nobuty~c acid and some antagonists in a slice preparation of cuneate nucleus. ceptor
agonists:
design
Br. J. Pharmac. 63: 495-502, 1978. 21. Squires, R. F. and C. Braestrup. Benzodiazepine receptors in rat brain. Nature 266: 732-734, 1977. 22. Steiner, F. A. and D. Felix. Antagonistic effects of GABA and
benzodiazepines on vestibular and cerebellar neurones. Nature 260: 346-347, 1976. 23. Tallman, J. F., J. W. Thomas and D. W. Gallager. GABAergic modulation of benzodiazepine binding site sensitivity. Nature 274: 383-385, 1978.