Brain Research, 542 (1991) 225-232 Elsevier
225
BRES 16356
Neurochemical action of the general anaesthetic propofol on the chloride ion channel coupled with GABAA receptors A. Concas, G. Santoro, M. Serra, E. Sanna and G. Biggio Department of Experimental Biology, Chair of Pharmacology, University of Cagliari, Cagliari (Italy) (Accepted 11 September 1990)
Key words: ~,-Aminobutyricaeid-A receptor; Chioride channel; [3H]~,-Aminobutyricacid binding; [35S]t-Butylbicyelophosphorothionate binding; ~CI- uptake; Propofol; General anesthetic; Barbiturate; Steroid; Rat brain
The effect of propofol, a novel short acting anaesthetic, on the function of the GABAA/ionophore receptor complex was studied in vitro in cortical membrane preparations from rat cerebral cortex and was compared with the action of pentobarbital and alphaxalone, two general anaesthetics known to enhance GABAerglc transmission. Propefol, mimicking the action of pentobarbital and alphaxalone, increased [3H]GABA binding, reduced [35S]TBPS binding and enhanced museimol-stimulated~CI- uptake in a concentration-dependent manner. While the efficacy of the drugs in affecting these biochemical parameters was similar, they differed markedly in potency being alphaxalone > propofol > pentobarbital. However, separate sites of action or different mechanisms for these drugs can be suggested by the result that the concomitant addition of propofol either with alphaxalone or pentobarbital or diazepam produced a simple additive inhibition of [35S]TBPS binding as well as an addictive enhancement of [3H]GABA binding and museimol-stimulated 36C1- uptake. The effect of propefol at the level of the GABA/ionophore receptor complex seems to be strictly dependent on the interaction of GABA with its recognition site. In fact, the specific GABA^ receptor antagonist bicueulline antagonized the decrease of [35S]TBPS binding as well as the enhancement of [3H]GABA binding and museimol-stimulated~CI- uptake induced by propefol. On the other hand, propofol was able to enhance [3I-I]GABAbinding in membranes previously incubated with the specific chloride channel blocker pierotoxin. Finally, the finding that propofol fails to affect [3H]flunitrasepam binding together with the failure of Ro 15-1788 and PK 11195 to antagonize its effect on [35S]TBPS binding excludes a direct interaction at the level of benzodiazepine recognition sites. Taken together these results strongly suggest that an enhancement in the function of the GABA^lionophore receptor complex may have a relevant role in mediating the anaesthetic effect of propofol as well as those of other general anaesthetics. INTRODUCTION One of the most important discoveries in the physiology and pharmacology of the central GABAergic transmission has been the finding that the G A B A A receptor complex is a site of action for several drugs with different chemical profile and diverse or opposite pharmacological effects. Thus, it has been well established that the GABAA/benzodiazepine ionophore receptor complex plays a major role in the pharmacology of several anxiolytic and anxiogenic, anticonvulsant and convulsant, hypnotic and somnolytic drugs as well as in the physiopathology of stress, anxiety, sleep disorders and epilepsy3-5.22,31.39. More recently, biochemical, pharmacological and electrophysiologicai studies have shown that the chloride ion channel coupled with G A B A A receptor is also a sensitive target for the action of different general anaesthetics 1' 10,14,15,17,27,33,38. In fact, a good correlation has been found between the pharmacological potency of chemically different non-volatile and inhalation anaesthetics
and their capability to specifically alter different biochemical ([3H]muscimol binding, [35S]TBPS binding, 36C1- uptake) and electrophysiological (chloride ion conductance) parameters currently used to evaluate 'in vitro' the action of positive and negative modulators of GABAA/ionophore receptor complex. Taken together the above experimental data have strongly suggested that the enhancement in the function of the GABA-coupled chloride channel rather than an alteration in the membrane fluidity might be important for the anaesthetic action of these drugs 26. Recently, the 2,6-diisopropylphenol (propofol) a novel short acting general anaesthetic chemically unrelated to other compounds with anaesthetic properties 19 has been shown to enhance, at clinically relevant concentrations, GABA-mediated transmission probably by an interaction with the G A B A A receptor complex in the rat olfactory cortex slice 6. Since no data are still available on the effect of propofol on biochemical parameters directly related to the function of the GABAA/ionophore receptor complex we studied the effect of this drug on [3H]GABA,
Correspondence: A. Concas, Department of Experimental Biology, Chair of Pharmacology, University of Cagliari, 09123 Cagliari, Italy. 0006-8993/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
226 [3H]flunitrazepam and [3sS]TBPS binding and on muscitool-stimulated 36C1- uptake. In fact, these biochemical parameters have been extensively used to characterize in vitro, the action of other general anaesthetics on the function of the GABAA-benzodiazepine receptor complex~3'l 5,25,27,35.
In the present study we have compared the capability of propofol to alter the function of the GABA-coupled chloride channel in homogenates from the rat cerebral cortex, with the action of pentobarbital and alphaxalone two non-volatile anaesthetics known to enhance the GABAergic transmission via the activation of allosteric recognition sites located at the level of the chloride ion channel-coupled with the GABAergic synapses 1°'18'38.
MATERIALS AND METHODS
Animals Male Sprague-Dawley CD R rats (Charles River, Como, Italy) weighing 180-200 g were used. The animals were kept under a 12 h light-dark cycle at a temperature of 23 + 2 °C with water and standard laboratory food ad libitum. The animals were killed by decapitation in the middle of the light phase. The brains were rapidly removed, the cerebral cortex was dissected out and was used for measurement of [3H]GABA binding, [3H]fiunitrazepam binding, [3sS]TBPS binding and 36C1- uptake.
[3H]GABA binding Fresh cerebral cortices were homogenized with a Polytron PT 10 (setting 5, for 30 s) in 10 vols. of ice-cold water and centrifuged for 10 min at 48,000 g at 0 °C. The pellet was washed once by resuspension and recentrifugation in 10 vols. of 20 mM K2HPO4/ KH2PO 4 buffer (pH 7.4) containing 50 mM KCI. The membranes were stored at -20 °C until used 1-15 days later. On the day of the assay, the membranes were thawed and centrifuged. The pellet was washed 3 additional times by resuspension and recentrifugation in ice-cold buffer. The tissue was resuspended in 50 vols. of the same buffer and 350/zl of membrane suspension (250-300/zg of protein) was added to plastic minivials. The drugs were added in 50 /~l aliquots. The total incubation volume was 500/~l. The incubation (10 rain at 4 °C) was started by the addition of 10 nM (final concentration) [3H]GABA and was stopped by centrifugation of the incubation mixture at 48,000 g for 10 rain. The supernatant was discarded and the pellet was gently washed twice with 4 ml of ice-cold distilled water and was then dissolved in 3 ml of scintillation fluid (Atomlight, New England Nuclear).
[3H]Flunitrazepam binding Cerebral cortices were homogenized in 50 vols. of ice-cold 50 mM Tris-citrate buffer (pH 7.4) + 100 mM NaCI and centrifuged at 20,000 g for 20 rain. The pellet was reconstituted in 50 vols. of Tris-citrate buffer without salts and used for the binding assay. Aliquots of 100/~1 of tissue homogenate (0.1-0.15 mg of protein) were incubated in the presence of [3H]flunitrazepam at a final concentration of 0.5 nM, in a total incubation volume of 500/A. The drugs were added in 50/~1 aliquots. After 60 min incubation at 0-4 °C, the assay was terminated by rapid filtration through glass fiber filter strips (Whatman GF/B). The filters were rinsed with two 4-ml portions of ice-cold Tris-citrate buffer with a Cell Harvester filtration manifold (model M-24, Brandel). Radioactivity bound to the filters was quantitated by liquid scintillation spectrometry. Non-specific binding was determined in the presence of 5 /~M diazepam.
FsS]TBPS binding Fresh cerebral cortices were homogenized with a Polytron PT 10 (setting 5, for 20 s) in 50 vols. of ice-cold 50 mM Tris-citrate buffer (pH 7.4 at 25 °C) containing 100 mM NaCI. The homogenate was centrifuged at 20,000 g for 20 min and reconstituted in 50 vols. of Tris-citrate buffer without salts for the binding assay. [35S]TBPS binding was determined in a final volume of 500/~1, consisting of: 200/~1 tissue homogenate (0.2-0.25 mg protein), 50/A [35S]TBPS at a final concentration of 2 nM, 50/~1 2 M NaC1, 50/tl drugs or solvent, and buffer to volume. Incubations (25 °C) were initiated by the addition of tissue homogenate and were termined 90 rain later by rapid filtration through glass fibre filter strips (Whatman GF/B) which were rinsed with two 4 ml portions of ice-cold Tris-citrate buffer as previously described. Non-specific binding was defined as binding in the presence of 100/~M picrotoxin. Scatchard plots were based on 8 different concentrations of ligand (2.5-500 nM). The specific activity of [35S]TBPS was kept constant at 2.5 nM and then diluted with unlabelled TBPS dissolved in dimethyl suphoxide. Values for BmaX (pmol/mg protein) and Ka (nM) were obtained by the linear regression of the binding isotherms.
36Cl uptake Membrane vesicles were prepared from the cerebral cortex which was dissected free from white matter. Approximately 1 g of tissue was homogenized in 7 ml of buffer (0 °C) containing 20 mM HEPES-Tris, 118 mM NaCI, 4.7 mM KCI, 1.18 mM MgSO4, 2.5 mM CaCI 2 and 10 mM o-glucose (adjusted to pH 7.5 with Tris base) using a glass-glass homogenizer (12 strokes). The homogenate was brought up to 30 ml with ice-cold buffer and centrifuged at 1000 g for 15 min at 4 °C. After discarding the supernatant the pellet was resuspended in the same volume of buffer and centrifuged at 1000 g for 15 min. The final pellet was gently resuspended in buffer to a final protein concentration of 16 mg/ml. Aliquots of tissue (100 ~1) were preincubated for 10 min at 30 °C prior to the addition of 100 /~1 of 360- (2.0/~Ci/ml) or a solution of 36C1- and muscimol (5/~M). Final incubation volume was 500 bd. Drugs were preincubated with tissue at 30 °C for 10 min. Uptake of 36C1- was terminated 5 s later by the addition of 3.5 ml ice-cold buffer followed by filtration through 2.5 cm Whatman GF/C filters (presoaked with 0.05% polyethyleneimine to reduce non-specific binding), using a Hoefer manifold (Hoefer Scientific, San Francisco, CA, U.S.A.). The filters were washed twice with 3.5 ml of ice-cold buffer and the 36CI~content of the filters was determined by liquid scintillation spectrometry. The amount of 36C1- bound to the filters in the absence of membranes was subtracted from all values. Protein contents were determined by the method of Lowry et al. 24, using bovine serum albumin as standard.
Chemicals Propofol (2,6-diisopropylphenol) and alphaxalone were kindly provided by ICI-Pharma (Milan, Italy) and by Glaxo Group Research Ltd. (Middlesex, England), respectively. Stock solutions of propofol (56 mM) and alphaxalone, diazepam, Ro 15-1788 and PK 11195 (10 mM) were dissolved in dimethyl sulfoxide and serial dilutions were made with the incubation buffer. For [3H]GABA binding same concentrations of drugs were prepared using ethanol instead of dimethyl sulfoxide. Control groups received the same amount of solvent. Pentobarbital (Sigma Chemical Co.) and bicuculline methiodide (Pierce Chemical Co.) were dissolved in buffer. All statistical analyses were performed utilizing the Student's t-test.
RESULTS
Propofol enhances [3H]GABA binding A s s h o w n in Fig. 1, p r o p o f o l e n h a n c e d in a c o n c e n tration
dependent
manner
the
specific
binding
of
227 90
z ! qt ~
70 60.
t
was completely antagonized by the specific G A B A A receptor antagonist bicuculline (100/~M). On the contrary, the specific chloride channel blocker picrotoxin (30 /zM), which per se decreased [3H]GABA binding by 78% failed to reverse the effect of propofol on this parameter as indicated by the greater enhancement (+118%) of [3H]GABA binding induced by the latter in the presence of picrotoxin.
..
40 30
2o 10 0
- L~ [ORUG].U Fig. 1. Increase of [3H]GABA binding by propofol (0), alphaxalone (&) and pentobarbital (1). [3H]GABA binding was measured in frozen-thawed membrane preparations from rat cerebral cortex with 10 nM [3H]GABA. Data are expressed as the percentage increase in binding above the control values and are the means _+ S.E.M. from 3-7 separate experiments, each run in quadruplicate. Specific binding of [3H]GABA in the control group was 0.221 ± 0.018 pmoi/mg protein (mean ± S.E.M., n = 10). *P < 0.05 and **P < 0.01 compared with the control value. [3H]GABA to washed, frozen and thawed membrane preparations from the rat cerebral cortex. The maximal increase (+ 59 + 10% above the control value) was observed in the presence of 300/~M propofol, whereas concentrations lower than 10/zM failed to significantly increase [3H]GABA binding. In agreement with previous reports 13'30"35'38, alphaxalone and pentobarbital produced a large and concentration-dependent enhancement of [3H]GABA binding (maximal enhancement alphaxalone + 70 + 12% at 100/~M; pentobarbital + 78 + 6.5% at 1 raM). Moreover, when propofol was added in combination with alphaxalone or pentobarbital the action of these drugs on [3H]GABA binding was additive (Table I). Finally, the effect of propofol on [3H]GABA binding
Failure of propofol to enhance l~H]flunitrazepam binding Table II shows that propofol even at very high concentrations (30-300/zM) fails to significantly modify [3H]flunitrazepam binding in membrane preparations from rat cerebral cortex. On the contrary, according to other authors 13'2°'3a alphaxalone and pentobarbital significantly enhanced [3H]flunitrazepam binding (results not shown). Propofol reduces f s S ] T B P S binding Fig. 2 shows the effect of propofol, alphaxalone and pentobarbital on [35S]TBPS binding to unwashed membrane preparations from the rat cerebral cortex. As previously reported alphaxalone and pentobarbital 1°' 15,32,37,3s inhibited in a concentration-dependent manner [35S]TBPS binding to this membrane preparation. Although these drugs show the same efficacy (maximal inhibition 100%), they have a different potency (alphaxalone ICs0 0.3/zM; pentobarbital ICso 40/~M). Propofol (300 n M - 3 0 / z M ) , like alphaxalone and pentobarbital, induced a marked and complete concentration-dependent inhibition of [35S]TBPS binding to the same membrane preparation. However, the rank order of potency was different being alphaxalone > propofol (ICso 4/~M) > pentobarbital. Saturation analysis of [3SS]TBPS bind-
TABLE II
TABLE I
Effect of alphaxalone, pentobarbital, bicuculline and picrotoxin on the propofol-induced increase of ffH]GABA binding in the rat cerebralcortex
Failure of propofol to modify f H]flunitrazepam binding to membranepreparationsfrom rat cerebralcortex
[aH]GABA binding was measured in frozen-thawed and washed membranes using 10 nM [aH]GABA. Each value is the mean + S.E.M. of 3-5 experiments.
Incubations of 0.5 nM [3H]flunitrazepamwere maintained at 0 °C for 60 min. Each value represents the mean + S.E.M. from 6 separate experiments, each run in triplicate. Specific binding of [3H]flunitrazepamin the control group was 339 + 31 fmol/mgprotein (mean + S.E.M., n = 6).
Specific [3H]GABA binding (fmol/mgproO
Concentration (#M)
ff H]Flunitrazepam binding (% of solvenO
0.01 0.1 0.3 1 3 10 30 100 300
105.8 ± 2.2 104.3 ± 6.7 105.0 + 2.9 106.8 + 2.4 108.8 + 3.4 110.8 ± 3.6 116.8 ± 5.9 121.0 + 6.6 120.0 + 9.7
Drugs Solvent Alphaxalone Pentobarbital Bicuculline Picrotoxin
Concentration Solvent (~M) 1 100 100 30
236 + 14 351 + 16" 283 + 9* 125 + 10" 54 ± 9*
+ Propofol (IO~M) 304 + 13" 415 + 18"* 349 + 11"* 131 ± 12"* 118 + 13"*
*P < 0.05 compared with solvent; **P < 0.05 compared with propofol.
228 I
r
PROPOFOL
100
0 _z
0.0 0.3
1
3
0.0 0.3
o.o o.5
0.0 0.3
uM
-10
7° ,;, --20
g
i i -30
v
pe~
~ '6
\ 0
tn
,
,
,
,
,
8
7
6
5
4
-40
"-" - 5 0
-f
-60
3
SOLVENT
-,-g [DRUa].u
+ PEICT~ARaTAL
+ ALPtMXM.O~
-70
Fig. 2. Decrease of [35S]TBPS binding by propofol (O), alphaxalone
I ÷ ~ M I I
(&) and pentobarbital (ll). [3sS]TBPS binding was measured in fresh, unwashed membrane preparations from rat cerebral cortex with 2 nM [3sS]TBPS. Data are expressed as the percentage decrease in binding from control values and are the means + S.E.M. from 4 separate experiments, each run in triplicate. Specific binding of [35S]TBPS in the control group was 51 + 4.6 fmol/mg protein (mean + S.E.M., n = 16).
Fig. 4. Additive effect of pentobarbital (10/~M), alphaxalone (0.1 /~M) and diazepam (0.3/zM) on the propofol (0.3-3/~M)-induced decrease of [3sS]TBPS in the rat cerebral cortex. [3sS]TBPS binding was assayed as described in Materials and Methods using 2 nM [35S]TBPS in the absence or presence of the indicated drugs. Data are expressed as the percentage decrease in binding from control values (without drugs) and are the means + S.E.M. from 3 separate experiments, each run in triplicate.
ing indicated that propofol, like alphaxalone 13 inhibited [35S]TBPS binding in an allosteric rather than in a competitive manner. In fact, both the apparent K d and Bma x of [35S]TBPS binding were altered by propofol (Fig. 3). Moreover, the same degree of [35S]TBPS binding inhibition by propofol was obtained using two different concentrations (2; 40 nM) of [35S]TBPS (data not shown). This finding further indicates that this anaesthetic does not interact with TBPS recognition sites in a competitive manner. To clarify whether the effect of propofol at the level of the GABA A receptor complex was mediated by the direct interaction of this drug with the recognition sites used by steroids and barbiturates we studied the effect of alphaxalone and pentobarbital in combination with pro-
pofol on [35S]TBPS binding. As shown in Fig. 4 the concomitant in vitro addition of propofol (0.3-3 /~M) either with alphaxalone (0.1 #M) or pentobarbital (10 /~M) (concentrations which per se decrease [35S]TBPS binding by 24% and 16%, respectively) produced a simple additive inhibitory effect of [35S]TBPS binding suggesting separate sites of action for these drugs. This effect is similar to that recently shown for alphaxalone and pentobarbital which added together to the membranes produced a mutual synergistic inhibition of [35S]TBPS binding aS. A similar additive effect was also obtained in the presence of propofol and diazepam (0.3 /~M) which per se decreased [35S]TBPS binding by 28%. The inhibitory effect of propofol on [35S]TBPS binding seems to be strictly dependent by the capability of the
30150
B/r 20-
O
•
e~
r'~
SOLVlmT
,°°
•
10-
0
0
,
,
,
500
1000
1500
2000
BOUND (fmol/mg protein)
Fig. 3. Scatchard analysis of [35S]TBPS binding to membrane preparation from rat cerebral cortex in the absence or presence of propofol (5/~M). [3sS]TBPS binding was measured as described in Materials and Methods using 8 concentrations (2,5-500 nM) of [35S]TBPS. Results are typical of one experiment that was replicated 5 times. Solvent (O): Bma~ = 1836 fmol/mg protein; K d = 78 nM. Propofol (©): Bmax = 1360 fmol/mg protein; K a = 115 nM.
0
$01.V(NT
~-'~'~
÷ ~ I UM
÷ _~.~J~-~_UN( IOuM
÷ RO 15--1788
+ PK 11195
Fig. 5. Effect of bicuculline (1-10/~M), Ro 15-1788 (1 ~M) and PK 11195 (1/~M) on propofoi (3 ~tM)-induced decrease of [3SS]TBPS binding in the rat cerebral cortex. [3SS]TBPS binding was assayed in the absence or presence of the indicated drugs. Data are the means + S.E.M. from 4 separate experiments each run in triplicate. *P < 0.01 compared with solvent; **P < 0.05 compared with propofol alone.
229 7O
'° t
o
50 -
40. 30. 20.
o -Loe[ORUa] .u
Fig. 6. Enhancement of muscimol-stimulated 360- uptake into membrane vesicles from rat cerebral cortex by propofol (O), alphaxalone (&) and pentobarbital (11). Membrane vesicles were preincubated for 10 min at 30 *(2 in the presence of increasing concentrations of drugs or solvent. Data are expressed as the percentage increase in uptake above the muscimol (5 /~M)stimulated ~CI- uptake. Data are the means + S.E.M. from 4-7 experiments, each run in quadruplicate. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the control value. drug to enhance the interaction of G A B A with its recognition site. In fact, as shown in Fig. 5, the specific G A B A A receptor antagonist bicuculline (1-10 /zM) completely reversed the effect of propofol (3/zM) on [35S]TBPS binding. Moreover, it is worth noting that in the presence of bicuculline (10/~M), propofol was able to enhance the action of the latter on [35S]TBPS binding. On the contrary, Ro 15-1788 and PK 11195, a central and a peripheral benzodiazepine receptor ligand 16,21, failed to antagonize the propofol-induced inhibition of [35S]TBPS binding (Fig. 5) indicating, in agreement with the results of Table II, that this anaesthetic does not interact with both central and peripheral benzodiazepine recognition sites.
Propofol enhances muscimol-stimulated 36C1- uptake To further investigate the molecular mechanism by which propofol potentiates the function of the GABAAcoupled chloride channel, we studied the ability of this drug to enhance muscimol-stimulated 36C1- uptake in membrane vesicles from rat cerebral cortex. The uptake of 36C1- into membrane vesicles was stimulated by 5 g M muscimol (90 + 4.9% above the basal value). As expected 1'15"28, muscimol-stimulated 36Cluptake was increased in a concentration-dependent manner by the presence of alphaxalone (1-30 /zM) and pentobarbital (10-100 /zM). The maximal effect of alphaxalone (+43 + 4%) and pentobarbital (+45 + 4%) was obtained at concentrations of 10 and 100 /~M, respectively (Fig. 6). Consistent with the action on [3H]GABA and [35S]TBPS binding, propofol mimicked the effect of alphaxalone and pentobarbital markedly potentiating muscimol-induced increase of 36C1- in membrane vesicles fom rat cerebral cortex. The maximal effect of propofol (+40 + 3%) was obtained at 10/zM, while a higher concentration (30/zM) of this compound failed to further increase the muscimol-stimulated uptake of 36C1-. Moreover, similarly to the effect on [35S]TBPS binding, the concomitant addition of propofol (1 #M) either with alphaxalone (0.3/zM), pentobarbital (30/zM) or diazepam (0.3 ~M) produced an additive increase of muscimol-stimulated 36C1- uptake (Fig. 7). Finally, in agreement with the results reported in Fig. 5, Table III shows that propofol-induced increase of 36C1uptake was completely antagonized by the specific G A B A A receptor antagonist bicuculline. This finding is
TABLE III 50' ~
Olll
40. 30"
Propofol enhances muscimol stimulated 36C1-uptake: antagonism by bicuculline Membrane vesicles were preincubated for 10 min at 30 °C in the presence of propofol, bieuculline or solvent before the addition of ~C1- and muscimol. Data are the mean + S.E.M. of 4 experiments performed in quadruplicate.
!= ~ If(
36CI-uptake (nmol/mgproO
20.
o
_I
Fig. 7. Additive effect of aiphaxalone (0.3/~M), diazepam (0.3/zM) and pentobarbital (30gM) on the propofol (1/zM)-induced increase of muscimol-stimulated 36CI- uptake into membrane vesicles from rat cerebral cortex. Membrane vesicles were preincubated for 10 rain at 30 *C in the presence or absence of the indicated drug. Data are the means + S.E.M. from 5 experiments each run in quadruplicate. *P < 0.05 compared with propofol and alphaxalone, **P < 0.05 compared with propofol and diazepam, ***P < 0.05 compared with propofol and pentobarbital.
Basal
+ Muscimol (SgM)
Solvent
12.4 + 0.8
23.8 + 1.1'
Propofoi 10/~M
11.5 + 1.3
34.5 + 1.9"*
Bicuculline 50/zM
10.6 + 1.7
10.8 + 1.4"*
Bicuculline 50/~M + Propofo110/~M
10.9 + 1.6
13.1 + 1.7"*
*P < 0.01 compared with basal. **P < 0.05 compared with muscimol.
230 consistent with the evidence that propofol, unlike GABA, muscimol and pentobarbital but similarly to alphaxalone and benzodiazepines 12'28'29'34 fails to enhance the basal value of 36C1- uptake. DISCUSSION It is well established that the pharmacological profile and efficacy of several sedative-hypnotic drugs is mainly related to their ability to enhance the function of the GABAA/ionophore receptor complex. Accordingly, biochemical and electrophysiological data have demonstrated that benzodiazepines and barbiturates enhance the GABAergic transmission by facilitating the interaction of G A B A with its specific recognition site thus potentiating the stimulatory action of this inhibitory amino acid on its coupled chloride channel (see for refs. 5, 31). More recently, a similar effect has been also described for the general anaesthetics including the steroid derivative alphaxalone14'17'27'33. In fact this and other non-volatile anaesthetics, like benzodiazepines and barbiturates, enhance the specific binding of [3H]GABA to washed, frozen and thawed membrane preparations from the tissue brain2'3°'35'36, increase the GABA dependent chloride flux in brain membrane vesicles1'15'29 and reduce the binding of [35S]TBPS to the chloride channel coupled to the GABA A receptor complex in rat brain membrane preparations7,9A°'lS'32,37,as. Taken together these findings strongly suggest that the GABA-dependent chloride channel is an excellent target site for anaesthetic drugs. Our present results show that propofol, a novel short acting general anaesthetic, mimicking the actions of alphaxalone and pentobarbital, increases [3H]GABA binding, enhances the muscimol-stimulated chloride uptake and inhibits [35S]TBPS binding to unwashed membrane preparations from the rat brain. All these effects are antagonized by bicuculline. These data suggest that propofol like barbiturates, alphaxalone and other general anaesthetics enhances the function of the chloride channel coupled to central GABAergic synapses. This biochemical finding is consistent with previous electrophysioiogical evidences showing that propofol potentiates GABA-mediated synaptic inhibition in cat spinal cord, in the rat olfactory cortex slice and in bovine chromaffin cells in culture6A1'23. The results show that the potency of propofol to enhance in vitro the function of GABAergic synapses is different from that of pentobarbital and alphaxalone. In fact, propofol increases [3H]GABA binding and muscimol-stimulated chloride uptake and decreases [35S]TBPS binding with a lower potency than alphaxalone. On the contrary it enhances the GABAergic transmission by a
greater potency respect to pentobarbital. On the basis of these results one of the major problems to be clarified is to identify the putative site of action or the molecular mechanism by which this drug enhances the function of the GABA-coupled chloride channel. The potent and selective enhancement exerted by propofol on the GABAergic transmission suggests that the effect of this compound might be mediated through a direct interaction with a specific molecular component of the GABA A supramolecular receptor complex, a mechanism already described for barbiturates 2° and also more recently suggested for alphaxalone1°'18"38. The failure of propofol to alter [3H]flunitrazepam binding together with the lack of Ro 15-1788 and PK 11195 to antagonize the action of the drug on [35SiTBPS and [3H]GABA binding and on muscimol-stimulated 36C1- uptake strongly suggests that the site of action of propofol is different from that of benzodiazepines. On the other hand, the finding that bicuculline, a selective GABA A receptor antagonist8, antagonized the enhancement of both [3H]GABA binding and muscimol-stimulated chloride uptake induced by propofol as well as the inhibitory effect of this compound on [35S]TBPS binding, suggests that propofol might have on GABAergic synapses a GABA like action. This conclusion might imply a possible direct interaction of propofol with the GABAA recognition site. However, the evidence that propofol enhances rather than reduces [3H]GABA binding should ruled out this hypothesis. The results that propofol affects both Bmax and Kd of [35S]TBPS binding and produces the same degree of inhibition using a low and a relatively high concentration of [35S]TBPS, allow to exclude a competitive interaction of this compound on picrotoxin/TBPS recognition site. This conclusion is further supported by the failure of picrotoxin to antagonize the effect of propofol on [3H]GABA binding. The similarities in the qualitative effects of propofol, pentobarbital and alphaxalone on GABAergic synapses suggest that recognition sites for steroids and/or barbiturates might be also involved in the action of propofol on GABA-coupled chloride channel. However, the results obtained using low concentrations of propofol in combination with a low concentration of pentobarbital or alphaxalone demonstrated that the effect of propofol on [3H]GABA binding, [35S]TBPS binding and muscimolstimulated 36C1- uptake was clearly additive to the effect elicited by the other drugs alone. A similar synergistic effect was also obtained using the combination of propofol and diazepam. The above results are consistent with the idea that different sites or mechanisms of action for these drugs are present at the level of the GABAA receptor complex. This conclusion is in agreement with
231 the finding that p r o p o f o l unlike p e n t o b a r b i t a l and a l p h a x a l o n e 13'2°'3s failed to increase [3H]flunitrazepam
protein is different from those of p e n t o b a r b i t a l and alphaxalone. Since p r o p o f o l like those c o m p o u n d s is able
binding and unlike p e n t o b a r b i t a l is not sensitive to picrotoxin 3°. In conclusion our results have shown that the general anaesthetic p r o p o f o l shares with pentobarbital, alphaxalone and o t h e r general anaesthetics some similarities in the capability to enhance the function of central G A B A ergic synapse. M o r e o v e r , the present d a t a also suggest that the target site of p r o p o f o l to the r e c e p t o r channel-
to induce an o p e n i n g o f the chloride channel b o t h in the presence and in the absence o f G A B A 6, the present finding further supports the i d e a of a crucial role for the central G A B A e r g i c synapses in the mechanism of action of some general anaesthetic drugs. Finally, the action of propofol on G A B A e r g i c transmission does not exclude the possible interaction of the drug with the lipid c o m p o n e n t of the m e m b r a n e .
REFERENCES
Cumin, R., Schaffner, R. and Haefely, W., Selective antagonists of benzodiazepines, Nature, 290 (1981) 514-516. 17 Keane, EE. and Biziere, K., The effects of general anaesthetics on GABAergic synaptic transmission, Life Sci., 41 (1987) 1437-1448. 18 Lambert, J.J., Peters, J.A. and Cottrell, G.A., Actions of synthetic and endogenous steroids on the GABAA receptor, Trends Pharmacol. Sci., 8 (1987) 224-227. 19 Langley, M.S. and Heel, R.C., Propofol a review of its pharmacodynamic and pharmacokinetic properties and use as an intravenous anaesthetic, Drugs, 35 (1988) 334-372. 20 Leeb-Lundberg, E, Snowman, A. and Olsen, R.W., Barbiturate receptors are coupled to benzodiazepine receptors, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 7468-7472. 21 Le Fur, G., Vander, N., Perrier, M.L., Flamier, A., Benavides, J., Renault, C., Dubroeneq, M.C., Gueremy, C. and Uzan, A.D., Differentiation between two ligands for peripheral benzodiazepine binding sites [3H]Ro 5-4864 and [3H]PK 11195, by thermodynamic studies, Life Sci., 33 (1983) 449-457. 22 Levy, R.H., Dreifuss, EE., Mattson, R.H., Meldrum, B.S. and Kiffin Penry, J. (Eds.), Antiepileptic Drugs, Raven, New York, 1989. 23 Lodge, D. and Anis, N.A., Effects of ketamine and three other anaesthetics on spinal reflexes and inhibitions in the cat, Br. J. Anaesthesiol., 56 (1984) 1143-1151. 24 Lowry, O.M., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 25 Majewska, M.D., Harrison, N.L., Schwartz, R.D., Barker, J.L. and Paul, S.M., Steroid hormone metabolites are barbituratelike modulators of the GABA receptor, Science, 232 (1986) 1004-1007. 26 Miller, K.W., Are lipids or proteins the targets of general anesthetic action?, Trends Neurosci., 9 (1986) 49-51. 27 Moody, E.J., Suzdak, ED., Paul, S.M. and Skolnick, P., Modulation of the benzodiazepine/y-aminobutyricacid receptor chloride-channel complex by inhalation anaesthetics, J. Neurochem., 51 (1988) 1386-1393. 28 Morrow, A.L. and Paul, S.M., Benzodiazepine enhancement of 7-aminobutyric acid-mediated chloride ion flux in rat brain synaptoneurosomes, J. Neurochem., 50 (1988) 302-307. 29 Obata, T. and Yamamura, H.I., The effect of benzodiazepines and fl-carbolines on GABA-stimulated chloride influx by membrane vesicles from the rat cerebral cortex, Biochem. Biophys. Res. Commun., 141 (1986) 1-6. 30 Olsen, R.W. and Snowman, A.M., Chloride-dependent barbiturate enhancement of GABA receptor binding, J. Neurosci., 2 (1982) 1812-1823. 31 Olsen, R.W. and Venter, J.S. (Eds.), Benzodiazepine/GABA Receptors and Chloride Channels: Structural and Functional Properties, Receptor Biochemistry and Methodology, Vol. 5, Alan R. Liss, New York, 1986. 32 Ramanjaneyulu, R.R. and Ticku, M.K., Binding characteristics and interactions of depressant drugs with [35S]t-butylbicyclophosphorotionate, a ligand that binds to the picrotoxin sites, J.
1 Allan, A.M. and Harris, R.A., Anesthetic and convulsant barbiturates alter ?-aminobutyric acid-stimulated chloride flux across brain membranes, Z Pharmacol. Exp. Ther., 238 (1986) 763-768. 2 Biggio, G., The action of stress, fl-carbolines, diazepam and Ro 15-1788 on GABA receptors in the rat brain. In G. Biggio and E. Costa (Eds.), Benzodiazepine Recognition Site Ligands: Biochemistry and Pharmacology, Raven, New York, 1983, pp. 105-117. 3 Biggio, G. and Costa, E. (Eds.), Benzodiazepine Recognition Site Ligands: Biochemistry and Pharmacology, Adv. Biochem. Psychopharmacol., Vol. 38, Raven, New York, 1983. 4 Biggio, G. and Costa, E. (Eds.), GABAergic Transmission and Anxiety, Adv. Biochem. Psychopharmacol., Vol. 41, Raven, New York, 1986. 5 Biggio, G. and Costa, E. (Eds.), Chloride Channels and their Modulation by Neurotransmitters and Drugs, Adv. Biochem. Psychopharmacol., Vol. 45, Raven, New York, 1988. 6 Collins, G.G.S., Effects of the anaesthetic 2,6-diisopropylphenol on synaptic transmission in the rat olfactory cortex slice, Br. J. Pharmacol., 95 (1988) 939-949. 7 Concas, A., Serra, M., Atsoggiu, T. and Biggio, G., Foot-shock stress and anxiogenic fl-carbolines increase t-[3ss]bu tylbicyclophosphorothionate binding in the rat cerebral cortex, an effect opposite to anxiolytics and 7-aminobutyric acid mimetics, J. Neurochem., 51 (1988) 1868-1876. 8 Curtis, D.R., Duggan, A.W., Felix, D. and Johnston, G.A.R., Bicuculline, an antagonist of GABA and synaptic inhibition in the spinal cord of the cat, Brain Research, 32 (1971) 69-96. 9 Gee, K.W., Lawrence, L.J. and Yamamura, H.I., Modulation of the chloride ionophore by benzodiazepine receptor ligands: influence of F-aminobutyric acid and ligand efficacy, Mol. Pharmacol., 30 (1986) 218-225. 10 Gee, K.W., Chang, W., Brinton, R.E. and McEwen, B., GABA-dependent modulation of the CI- ionophore by steroids in rat brain, Fur. J. Pharmacol., 136 (1987) 419-423. 11 Hales, T.G. and Lambert, J.J., Modulation of the GABA A receptor by propofol, Br. J. Pharmacol., 93 (1988) 84P. 12 Harris, R.A. and Allan, A.M., Functional coupling of GABA receptors to chloride channels in brain membranes, Science, 228 (1985) 1108-1110. 13 Harrison, N.L., Majewska, M.D., Harrington, J.W. and Barker, J.L., Structure-activity relationships for steriod interaction with the ~,-aminobutDic acid^ receptor complex, J. Pharmacol. Exp. Ther., 241 (1987) 346-353. 14 Harrison, N.L., Vicini, S. and Barker, J.L., A steroid anesthetic prolongs inhibitory postsynaptic currents in cultured rat hippocampal neurons, J. Neurosci., 7 (1987) 604-609. 15 Huidobro-Toro, J.P., Bleck, V., Allan, A.M. and Harris, R.A., Neurochemical actions of anesthetic drugs on the ~,-aminobutyric acid receptor-chloride channel complex, J. Pharmacol. Exp. Ther., 242 (1987) 963-969. 16 Hunkeler, W., Mohler, M., Pieri, L., Polc, P., Bonetti, E.P.,
232
Neurochem., 42 (1984) 221-229. 33 Robertson, B., Actions of anaesthetics and avermectin on GABAA chloride channels in mammalian dorsal root ganglion neurones, Br. J. Pharrnacol., 98 (1989) 167-176. 34 Schwartz, R.D., Skolnick, P., Seale, T.W. and Paul, S.M., Demonstration of GABA/barbiturate-receptor-mediated chloride transport in rat brain synaptoneurosomes: a functional assay of GABA receptor-effector coupling. In G. Biggio and E. Costa (Eds.), GABAergic Transmission and Anxiety, Raven, New York, pp. 33-49. 35 Simmonds, M.A., Turner, J.P. and Harrison, N.L., Interactions of steroids with the GABAA receptor complex, Neuropharmacology, 23 (1984) 877-878. 36 Skerritt, J.H. and Johnston, G.A.R., Enhancement of GABA
binding by benzodiazepines and related anxiolytics, Eur. J. Pharrnacol., 89 (1983) 193-198. 37 Squires, R.E, Casida, J.E., Richardson, M. and Saederup, E., [35S]t-butylbicyclophosphorothionatebinds with high affinity to brain-specific sites coupled to ~,-aminobutyric acid-A and ion recognition sites, Mol. Pharmacol., 23 (1983) 326-336. 38 Turner, D.M., Ransom, R.W., Yang, J.S.-J. and Olsen, R.W., Steroid anaesthetics and naturally occurring analogs modulate the 7-aminobutyric acid receptor complex at the site distinct from barbiturates, J. Pharrnacol. Exp. Ther., 248 (1989) 960966. 39 Wauquier, A., Gaillard, J.M., Monti, J.M. and Radulovacki, M. (Eds.), Sleep Neurotransmitters and Neuromodulators, Raven, New York, 1985.
225
BRES 16356
Neurochemical action of the general anaesthetic propofol on the chloride ion channel coupled with GABAA receptors A. Concas, G. Santoro, M. Serra, E. Sanna and G. Biggio Department of Experimental Biology, Chair of Pharmacology, University of Cagliari, Cagliari (Italy) (Accepted 11 September 1990)
Key words: ~,-Aminobutyricaeid-A receptor; Chioride channel; [3H]~,-Aminobutyricacid binding; [35S]t-Butylbicyelophosphorothionate binding; ~CI- uptake; Propofol; General anesthetic; Barbiturate; Steroid; Rat brain
The effect of propofol, a novel short acting anaesthetic, on the function of the GABAA/ionophore receptor complex was studied in vitro in cortical membrane preparations from rat cerebral cortex and was compared with the action of pentobarbital and alphaxalone, two general anaesthetics known to enhance GABAerglc transmission. Propefol, mimicking the action of pentobarbital and alphaxalone, increased [3H]GABA binding, reduced [35S]TBPS binding and enhanced museimol-stimulated~CI- uptake in a concentration-dependent manner. While the efficacy of the drugs in affecting these biochemical parameters was similar, they differed markedly in potency being alphaxalone > propofol > pentobarbital. However, separate sites of action or different mechanisms for these drugs can be suggested by the result that the concomitant addition of propofol either with alphaxalone or pentobarbital or diazepam produced a simple additive inhibition of [35S]TBPS binding as well as an addictive enhancement of [3H]GABA binding and museimol-stimulated 36C1- uptake. The effect of propefol at the level of the GABA/ionophore receptor complex seems to be strictly dependent on the interaction of GABA with its recognition site. In fact, the specific GABA^ receptor antagonist bicueulline antagonized the decrease of [35S]TBPS binding as well as the enhancement of [3H]GABA binding and museimol-stimulated~CI- uptake induced by propefol. On the other hand, propofol was able to enhance [3I-I]GABAbinding in membranes previously incubated with the specific chloride channel blocker pierotoxin. Finally, the finding that propofol fails to affect [3H]flunitrasepam binding together with the failure of Ro 15-1788 and PK 11195 to antagonize its effect on [35S]TBPS binding excludes a direct interaction at the level of benzodiazepine recognition sites. Taken together these results strongly suggest that an enhancement in the function of the GABA^lionophore receptor complex may have a relevant role in mediating the anaesthetic effect of propofol as well as those of other general anaesthetics. INTRODUCTION One of the most important discoveries in the physiology and pharmacology of the central GABAergic transmission has been the finding that the G A B A A receptor complex is a site of action for several drugs with different chemical profile and diverse or opposite pharmacological effects. Thus, it has been well established that the GABAA/benzodiazepine ionophore receptor complex plays a major role in the pharmacology of several anxiolytic and anxiogenic, anticonvulsant and convulsant, hypnotic and somnolytic drugs as well as in the physiopathology of stress, anxiety, sleep disorders and epilepsy3-5.22,31.39. More recently, biochemical, pharmacological and electrophysiologicai studies have shown that the chloride ion channel coupled with G A B A A receptor is also a sensitive target for the action of different general anaesthetics 1' 10,14,15,17,27,33,38. In fact, a good correlation has been found between the pharmacological potency of chemically different non-volatile and inhalation anaesthetics
and their capability to specifically alter different biochemical ([3H]muscimol binding, [35S]TBPS binding, 36C1- uptake) and electrophysiological (chloride ion conductance) parameters currently used to evaluate 'in vitro' the action of positive and negative modulators of GABAA/ionophore receptor complex. Taken together the above experimental data have strongly suggested that the enhancement in the function of the GABA-coupled chloride channel rather than an alteration in the membrane fluidity might be important for the anaesthetic action of these drugs 26. Recently, the 2,6-diisopropylphenol (propofol) a novel short acting general anaesthetic chemically unrelated to other compounds with anaesthetic properties 19 has been shown to enhance, at clinically relevant concentrations, GABA-mediated transmission probably by an interaction with the G A B A A receptor complex in the rat olfactory cortex slice 6. Since no data are still available on the effect of propofol on biochemical parameters directly related to the function of the GABAA/ionophore receptor complex we studied the effect of this drug on [3H]GABA,
Correspondence: A. Concas, Department of Experimental Biology, Chair of Pharmacology, University of Cagliari, 09123 Cagliari, Italy. 0006-8993/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
226 [3H]flunitrazepam and [3sS]TBPS binding and on muscitool-stimulated 36C1- uptake. In fact, these biochemical parameters have been extensively used to characterize in vitro, the action of other general anaesthetics on the function of the GABAA-benzodiazepine receptor complex~3'l 5,25,27,35.
In the present study we have compared the capability of propofol to alter the function of the GABA-coupled chloride channel in homogenates from the rat cerebral cortex, with the action of pentobarbital and alphaxalone two non-volatile anaesthetics known to enhance the GABAergic transmission via the activation of allosteric recognition sites located at the level of the chloride ion channel-coupled with the GABAergic synapses 1°'18'38.
MATERIALS AND METHODS
Animals Male Sprague-Dawley CD R rats (Charles River, Como, Italy) weighing 180-200 g were used. The animals were kept under a 12 h light-dark cycle at a temperature of 23 + 2 °C with water and standard laboratory food ad libitum. The animals were killed by decapitation in the middle of the light phase. The brains were rapidly removed, the cerebral cortex was dissected out and was used for measurement of [3H]GABA binding, [3H]fiunitrazepam binding, [3sS]TBPS binding and 36C1- uptake.
[3H]GABA binding Fresh cerebral cortices were homogenized with a Polytron PT 10 (setting 5, for 30 s) in 10 vols. of ice-cold water and centrifuged for 10 min at 48,000 g at 0 °C. The pellet was washed once by resuspension and recentrifugation in 10 vols. of 20 mM K2HPO4/ KH2PO 4 buffer (pH 7.4) containing 50 mM KCI. The membranes were stored at -20 °C until used 1-15 days later. On the day of the assay, the membranes were thawed and centrifuged. The pellet was washed 3 additional times by resuspension and recentrifugation in ice-cold buffer. The tissue was resuspended in 50 vols. of the same buffer and 350/zl of membrane suspension (250-300/zg of protein) was added to plastic minivials. The drugs were added in 50 /~l aliquots. The total incubation volume was 500/~l. The incubation (10 rain at 4 °C) was started by the addition of 10 nM (final concentration) [3H]GABA and was stopped by centrifugation of the incubation mixture at 48,000 g for 10 rain. The supernatant was discarded and the pellet was gently washed twice with 4 ml of ice-cold distilled water and was then dissolved in 3 ml of scintillation fluid (Atomlight, New England Nuclear).
[3H]Flunitrazepam binding Cerebral cortices were homogenized in 50 vols. of ice-cold 50 mM Tris-citrate buffer (pH 7.4) + 100 mM NaCI and centrifuged at 20,000 g for 20 rain. The pellet was reconstituted in 50 vols. of Tris-citrate buffer without salts and used for the binding assay. Aliquots of 100/~1 of tissue homogenate (0.1-0.15 mg of protein) were incubated in the presence of [3H]flunitrazepam at a final concentration of 0.5 nM, in a total incubation volume of 500/A. The drugs were added in 50/~1 aliquots. After 60 min incubation at 0-4 °C, the assay was terminated by rapid filtration through glass fiber filter strips (Whatman GF/B). The filters were rinsed with two 4-ml portions of ice-cold Tris-citrate buffer with a Cell Harvester filtration manifold (model M-24, Brandel). Radioactivity bound to the filters was quantitated by liquid scintillation spectrometry. Non-specific binding was determined in the presence of 5 /~M diazepam.
FsS]TBPS binding Fresh cerebral cortices were homogenized with a Polytron PT 10 (setting 5, for 20 s) in 50 vols. of ice-cold 50 mM Tris-citrate buffer (pH 7.4 at 25 °C) containing 100 mM NaCI. The homogenate was centrifuged at 20,000 g for 20 min and reconstituted in 50 vols. of Tris-citrate buffer without salts for the binding assay. [35S]TBPS binding was determined in a final volume of 500/~1, consisting of: 200/~1 tissue homogenate (0.2-0.25 mg protein), 50/A [35S]TBPS at a final concentration of 2 nM, 50/~1 2 M NaC1, 50/tl drugs or solvent, and buffer to volume. Incubations (25 °C) were initiated by the addition of tissue homogenate and were termined 90 rain later by rapid filtration through glass fibre filter strips (Whatman GF/B) which were rinsed with two 4 ml portions of ice-cold Tris-citrate buffer as previously described. Non-specific binding was defined as binding in the presence of 100/~M picrotoxin. Scatchard plots were based on 8 different concentrations of ligand (2.5-500 nM). The specific activity of [35S]TBPS was kept constant at 2.5 nM and then diluted with unlabelled TBPS dissolved in dimethyl suphoxide. Values for BmaX (pmol/mg protein) and Ka (nM) were obtained by the linear regression of the binding isotherms.
36Cl uptake Membrane vesicles were prepared from the cerebral cortex which was dissected free from white matter. Approximately 1 g of tissue was homogenized in 7 ml of buffer (0 °C) containing 20 mM HEPES-Tris, 118 mM NaCI, 4.7 mM KCI, 1.18 mM MgSO4, 2.5 mM CaCI 2 and 10 mM o-glucose (adjusted to pH 7.5 with Tris base) using a glass-glass homogenizer (12 strokes). The homogenate was brought up to 30 ml with ice-cold buffer and centrifuged at 1000 g for 15 min at 4 °C. After discarding the supernatant the pellet was resuspended in the same volume of buffer and centrifuged at 1000 g for 15 min. The final pellet was gently resuspended in buffer to a final protein concentration of 16 mg/ml. Aliquots of tissue (100 ~1) were preincubated for 10 min at 30 °C prior to the addition of 100 /~1 of 360- (2.0/~Ci/ml) or a solution of 36C1- and muscimol (5/~M). Final incubation volume was 500 bd. Drugs were preincubated with tissue at 30 °C for 10 min. Uptake of 36C1- was terminated 5 s later by the addition of 3.5 ml ice-cold buffer followed by filtration through 2.5 cm Whatman GF/C filters (presoaked with 0.05% polyethyleneimine to reduce non-specific binding), using a Hoefer manifold (Hoefer Scientific, San Francisco, CA, U.S.A.). The filters were washed twice with 3.5 ml of ice-cold buffer and the 36CI~content of the filters was determined by liquid scintillation spectrometry. The amount of 36C1- bound to the filters in the absence of membranes was subtracted from all values. Protein contents were determined by the method of Lowry et al. 24, using bovine serum albumin as standard.
Chemicals Propofol (2,6-diisopropylphenol) and alphaxalone were kindly provided by ICI-Pharma (Milan, Italy) and by Glaxo Group Research Ltd. (Middlesex, England), respectively. Stock solutions of propofol (56 mM) and alphaxalone, diazepam, Ro 15-1788 and PK 11195 (10 mM) were dissolved in dimethyl sulfoxide and serial dilutions were made with the incubation buffer. For [3H]GABA binding same concentrations of drugs were prepared using ethanol instead of dimethyl sulfoxide. Control groups received the same amount of solvent. Pentobarbital (Sigma Chemical Co.) and bicuculline methiodide (Pierce Chemical Co.) were dissolved in buffer. All statistical analyses were performed utilizing the Student's t-test.
RESULTS
Propofol enhances [3H]GABA binding A s s h o w n in Fig. 1, p r o p o f o l e n h a n c e d in a c o n c e n tration
dependent
manner
the
specific
binding
of
227 90
z ! qt ~
70 60.
t
was completely antagonized by the specific G A B A A receptor antagonist bicuculline (100/~M). On the contrary, the specific chloride channel blocker picrotoxin (30 /zM), which per se decreased [3H]GABA binding by 78% failed to reverse the effect of propofol on this parameter as indicated by the greater enhancement (+118%) of [3H]GABA binding induced by the latter in the presence of picrotoxin.
..
40 30
2o 10 0
- L~ [ORUG].U Fig. 1. Increase of [3H]GABA binding by propofol (0), alphaxalone (&) and pentobarbital (1). [3H]GABA binding was measured in frozen-thawed membrane preparations from rat cerebral cortex with 10 nM [3H]GABA. Data are expressed as the percentage increase in binding above the control values and are the means _+ S.E.M. from 3-7 separate experiments, each run in quadruplicate. Specific binding of [3H]GABA in the control group was 0.221 ± 0.018 pmoi/mg protein (mean ± S.E.M., n = 10). *P < 0.05 and **P < 0.01 compared with the control value. [3H]GABA to washed, frozen and thawed membrane preparations from the rat cerebral cortex. The maximal increase (+ 59 + 10% above the control value) was observed in the presence of 300/~M propofol, whereas concentrations lower than 10/zM failed to significantly increase [3H]GABA binding. In agreement with previous reports 13'30"35'38, alphaxalone and pentobarbital produced a large and concentration-dependent enhancement of [3H]GABA binding (maximal enhancement alphaxalone + 70 + 12% at 100/~M; pentobarbital + 78 + 6.5% at 1 raM). Moreover, when propofol was added in combination with alphaxalone or pentobarbital the action of these drugs on [3H]GABA binding was additive (Table I). Finally, the effect of propofol on [3H]GABA binding
Failure of propofol to enhance l~H]flunitrazepam binding Table II shows that propofol even at very high concentrations (30-300/zM) fails to significantly modify [3H]flunitrazepam binding in membrane preparations from rat cerebral cortex. On the contrary, according to other authors 13'2°'3a alphaxalone and pentobarbital significantly enhanced [3H]flunitrazepam binding (results not shown). Propofol reduces f s S ] T B P S binding Fig. 2 shows the effect of propofol, alphaxalone and pentobarbital on [35S]TBPS binding to unwashed membrane preparations from the rat cerebral cortex. As previously reported alphaxalone and pentobarbital 1°' 15,32,37,3s inhibited in a concentration-dependent manner [35S]TBPS binding to this membrane preparation. Although these drugs show the same efficacy (maximal inhibition 100%), they have a different potency (alphaxalone ICs0 0.3/zM; pentobarbital ICso 40/~M). Propofol (300 n M - 3 0 / z M ) , like alphaxalone and pentobarbital, induced a marked and complete concentration-dependent inhibition of [35S]TBPS binding to the same membrane preparation. However, the rank order of potency was different being alphaxalone > propofol (ICso 4/~M) > pentobarbital. Saturation analysis of [3SS]TBPS bind-
TABLE II
TABLE I
Effect of alphaxalone, pentobarbital, bicuculline and picrotoxin on the propofol-induced increase of ffH]GABA binding in the rat cerebralcortex
Failure of propofol to modify f H]flunitrazepam binding to membranepreparationsfrom rat cerebralcortex
[aH]GABA binding was measured in frozen-thawed and washed membranes using 10 nM [aH]GABA. Each value is the mean + S.E.M. of 3-5 experiments.
Incubations of 0.5 nM [3H]flunitrazepamwere maintained at 0 °C for 60 min. Each value represents the mean + S.E.M. from 6 separate experiments, each run in triplicate. Specific binding of [3H]flunitrazepamin the control group was 339 + 31 fmol/mgprotein (mean + S.E.M., n = 6).
Specific [3H]GABA binding (fmol/mgproO
Concentration (#M)
ff H]Flunitrazepam binding (% of solvenO
0.01 0.1 0.3 1 3 10 30 100 300
105.8 ± 2.2 104.3 ± 6.7 105.0 + 2.9 106.8 + 2.4 108.8 + 3.4 110.8 ± 3.6 116.8 ± 5.9 121.0 + 6.6 120.0 + 9.7
Drugs Solvent Alphaxalone Pentobarbital Bicuculline Picrotoxin
Concentration Solvent (~M) 1 100 100 30
236 + 14 351 + 16" 283 + 9* 125 + 10" 54 ± 9*
+ Propofol (IO~M) 304 + 13" 415 + 18"* 349 + 11"* 131 ± 12"* 118 + 13"*
*P < 0.05 compared with solvent; **P < 0.05 compared with propofol.
228 I
r
PROPOFOL
100
0 _z
0.0 0.3
1
3
0.0 0.3
o.o o.5
0.0 0.3
uM
-10
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i i -30
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pe~
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,
,
,
,
,
8
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-40
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-60
3
SOLVENT
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-70
Fig. 2. Decrease of [35S]TBPS binding by propofol (O), alphaxalone
I ÷ ~ M I I
(&) and pentobarbital (ll). [3sS]TBPS binding was measured in fresh, unwashed membrane preparations from rat cerebral cortex with 2 nM [3sS]TBPS. Data are expressed as the percentage decrease in binding from control values and are the means + S.E.M. from 4 separate experiments, each run in triplicate. Specific binding of [35S]TBPS in the control group was 51 + 4.6 fmol/mg protein (mean + S.E.M., n = 16).
Fig. 4. Additive effect of pentobarbital (10/~M), alphaxalone (0.1 /~M) and diazepam (0.3/zM) on the propofol (0.3-3/~M)-induced decrease of [3sS]TBPS in the rat cerebral cortex. [3sS]TBPS binding was assayed as described in Materials and Methods using 2 nM [35S]TBPS in the absence or presence of the indicated drugs. Data are expressed as the percentage decrease in binding from control values (without drugs) and are the means + S.E.M. from 3 separate experiments, each run in triplicate.
ing indicated that propofol, like alphaxalone 13 inhibited [35S]TBPS binding in an allosteric rather than in a competitive manner. In fact, both the apparent K d and Bma x of [35S]TBPS binding were altered by propofol (Fig. 3). Moreover, the same degree of [35S]TBPS binding inhibition by propofol was obtained using two different concentrations (2; 40 nM) of [35S]TBPS (data not shown). This finding further indicates that this anaesthetic does not interact with TBPS recognition sites in a competitive manner. To clarify whether the effect of propofol at the level of the GABA A receptor complex was mediated by the direct interaction of this drug with the recognition sites used by steroids and barbiturates we studied the effect of alphaxalone and pentobarbital in combination with pro-
pofol on [35S]TBPS binding. As shown in Fig. 4 the concomitant in vitro addition of propofol (0.3-3 /~M) either with alphaxalone (0.1 #M) or pentobarbital (10 /~M) (concentrations which per se decrease [35S]TBPS binding by 24% and 16%, respectively) produced a simple additive inhibitory effect of [35S]TBPS binding suggesting separate sites of action for these drugs. This effect is similar to that recently shown for alphaxalone and pentobarbital which added together to the membranes produced a mutual synergistic inhibition of [35S]TBPS binding aS. A similar additive effect was also obtained in the presence of propofol and diazepam (0.3 /~M) which per se decreased [35S]TBPS binding by 28%. The inhibitory effect of propofol on [35S]TBPS binding seems to be strictly dependent by the capability of the
30150
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10-
0
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,
,
,
500
1000
1500
2000
BOUND (fmol/mg protein)
Fig. 3. Scatchard analysis of [35S]TBPS binding to membrane preparation from rat cerebral cortex in the absence or presence of propofol (5/~M). [3sS]TBPS binding was measured as described in Materials and Methods using 8 concentrations (2,5-500 nM) of [35S]TBPS. Results are typical of one experiment that was replicated 5 times. Solvent (O): Bma~ = 1836 fmol/mg protein; K d = 78 nM. Propofol (©): Bmax = 1360 fmol/mg protein; K a = 115 nM.
0
$01.V(NT
~-'~'~
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÷ _~.~J~-~_UN( IOuM
÷ RO 15--1788
+ PK 11195
Fig. 5. Effect of bicuculline (1-10/~M), Ro 15-1788 (1 ~M) and PK 11195 (1/~M) on propofoi (3 ~tM)-induced decrease of [3SS]TBPS binding in the rat cerebral cortex. [3SS]TBPS binding was assayed in the absence or presence of the indicated drugs. Data are the means + S.E.M. from 4 separate experiments each run in triplicate. *P < 0.01 compared with solvent; **P < 0.05 compared with propofol alone.
229 7O
'° t
o
50 -
40. 30. 20.
o -Loe[ORUa] .u
Fig. 6. Enhancement of muscimol-stimulated 360- uptake into membrane vesicles from rat cerebral cortex by propofol (O), alphaxalone (&) and pentobarbital (11). Membrane vesicles were preincubated for 10 min at 30 *(2 in the presence of increasing concentrations of drugs or solvent. Data are expressed as the percentage increase in uptake above the muscimol (5 /~M)stimulated ~CI- uptake. Data are the means + S.E.M. from 4-7 experiments, each run in quadruplicate. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the control value. drug to enhance the interaction of G A B A with its recognition site. In fact, as shown in Fig. 5, the specific G A B A A receptor antagonist bicuculline (1-10 /zM) completely reversed the effect of propofol (3/zM) on [35S]TBPS binding. Moreover, it is worth noting that in the presence of bicuculline (10/~M), propofol was able to enhance the action of the latter on [35S]TBPS binding. On the contrary, Ro 15-1788 and PK 11195, a central and a peripheral benzodiazepine receptor ligand 16,21, failed to antagonize the propofol-induced inhibition of [35S]TBPS binding (Fig. 5) indicating, in agreement with the results of Table II, that this anaesthetic does not interact with both central and peripheral benzodiazepine recognition sites.
Propofol enhances muscimol-stimulated 36C1- uptake To further investigate the molecular mechanism by which propofol potentiates the function of the GABAAcoupled chloride channel, we studied the ability of this drug to enhance muscimol-stimulated 36C1- uptake in membrane vesicles from rat cerebral cortex. The uptake of 36C1- into membrane vesicles was stimulated by 5 g M muscimol (90 + 4.9% above the basal value). As expected 1'15"28, muscimol-stimulated 36Cluptake was increased in a concentration-dependent manner by the presence of alphaxalone (1-30 /zM) and pentobarbital (10-100 /zM). The maximal effect of alphaxalone (+43 + 4%) and pentobarbital (+45 + 4%) was obtained at concentrations of 10 and 100 /~M, respectively (Fig. 6). Consistent with the action on [3H]GABA and [35S]TBPS binding, propofol mimicked the effect of alphaxalone and pentobarbital markedly potentiating muscimol-induced increase of 36C1- in membrane vesicles fom rat cerebral cortex. The maximal effect of propofol (+40 + 3%) was obtained at 10/zM, while a higher concentration (30/zM) of this compound failed to further increase the muscimol-stimulated uptake of 36C1-. Moreover, similarly to the effect on [35S]TBPS binding, the concomitant addition of propofol (1 #M) either with alphaxalone (0.3/zM), pentobarbital (30/zM) or diazepam (0.3 ~M) produced an additive increase of muscimol-stimulated 36C1- uptake (Fig. 7). Finally, in agreement with the results reported in Fig. 5, Table III shows that propofol-induced increase of 36C1uptake was completely antagonized by the specific G A B A A receptor antagonist bicuculline. This finding is
TABLE III 50' ~
Olll
40. 30"
Propofol enhances muscimol stimulated 36C1-uptake: antagonism by bicuculline Membrane vesicles were preincubated for 10 min at 30 °C in the presence of propofol, bieuculline or solvent before the addition of ~C1- and muscimol. Data are the mean + S.E.M. of 4 experiments performed in quadruplicate.
!= ~ If(
36CI-uptake (nmol/mgproO
20.
o
_I
Fig. 7. Additive effect of aiphaxalone (0.3/~M), diazepam (0.3/zM) and pentobarbital (30gM) on the propofol (1/zM)-induced increase of muscimol-stimulated 36CI- uptake into membrane vesicles from rat cerebral cortex. Membrane vesicles were preincubated for 10 rain at 30 *C in the presence or absence of the indicated drug. Data are the means + S.E.M. from 5 experiments each run in quadruplicate. *P < 0.05 compared with propofol and alphaxalone, **P < 0.05 compared with propofol and diazepam, ***P < 0.05 compared with propofol and pentobarbital.
Basal
+ Muscimol (SgM)
Solvent
12.4 + 0.8
23.8 + 1.1'
Propofoi 10/~M
11.5 + 1.3
34.5 + 1.9"*
Bicuculline 50/zM
10.6 + 1.7
10.8 + 1.4"*
Bicuculline 50/~M + Propofo110/~M
10.9 + 1.6
13.1 + 1.7"*
*P < 0.01 compared with basal. **P < 0.05 compared with muscimol.
230 consistent with the evidence that propofol, unlike GABA, muscimol and pentobarbital but similarly to alphaxalone and benzodiazepines 12'28'29'34 fails to enhance the basal value of 36C1- uptake. DISCUSSION It is well established that the pharmacological profile and efficacy of several sedative-hypnotic drugs is mainly related to their ability to enhance the function of the GABAA/ionophore receptor complex. Accordingly, biochemical and electrophysiological data have demonstrated that benzodiazepines and barbiturates enhance the GABAergic transmission by facilitating the interaction of G A B A with its specific recognition site thus potentiating the stimulatory action of this inhibitory amino acid on its coupled chloride channel (see for refs. 5, 31). More recently, a similar effect has been also described for the general anaesthetics including the steroid derivative alphaxalone14'17'27'33. In fact this and other non-volatile anaesthetics, like benzodiazepines and barbiturates, enhance the specific binding of [3H]GABA to washed, frozen and thawed membrane preparations from the tissue brain2'3°'35'36, increase the GABA dependent chloride flux in brain membrane vesicles1'15'29 and reduce the binding of [35S]TBPS to the chloride channel coupled to the GABA A receptor complex in rat brain membrane preparations7,9A°'lS'32,37,as. Taken together these findings strongly suggest that the GABA-dependent chloride channel is an excellent target site for anaesthetic drugs. Our present results show that propofol, a novel short acting general anaesthetic, mimicking the actions of alphaxalone and pentobarbital, increases [3H]GABA binding, enhances the muscimol-stimulated chloride uptake and inhibits [35S]TBPS binding to unwashed membrane preparations from the rat brain. All these effects are antagonized by bicuculline. These data suggest that propofol like barbiturates, alphaxalone and other general anaesthetics enhances the function of the chloride channel coupled to central GABAergic synapses. This biochemical finding is consistent with previous electrophysioiogical evidences showing that propofol potentiates GABA-mediated synaptic inhibition in cat spinal cord, in the rat olfactory cortex slice and in bovine chromaffin cells in culture6A1'23. The results show that the potency of propofol to enhance in vitro the function of GABAergic synapses is different from that of pentobarbital and alphaxalone. In fact, propofol increases [3H]GABA binding and muscimol-stimulated chloride uptake and decreases [35S]TBPS binding with a lower potency than alphaxalone. On the contrary it enhances the GABAergic transmission by a
greater potency respect to pentobarbital. On the basis of these results one of the major problems to be clarified is to identify the putative site of action or the molecular mechanism by which this drug enhances the function of the GABA-coupled chloride channel. The potent and selective enhancement exerted by propofol on the GABAergic transmission suggests that the effect of this compound might be mediated through a direct interaction with a specific molecular component of the GABA A supramolecular receptor complex, a mechanism already described for barbiturates 2° and also more recently suggested for alphaxalone1°'18"38. The failure of propofol to alter [3H]flunitrazepam binding together with the lack of Ro 15-1788 and PK 11195 to antagonize the action of the drug on [35SiTBPS and [3H]GABA binding and on muscimol-stimulated 36C1- uptake strongly suggests that the site of action of propofol is different from that of benzodiazepines. On the other hand, the finding that bicuculline, a selective GABA A receptor antagonist8, antagonized the enhancement of both [3H]GABA binding and muscimol-stimulated chloride uptake induced by propofol as well as the inhibitory effect of this compound on [35S]TBPS binding, suggests that propofol might have on GABAergic synapses a GABA like action. This conclusion might imply a possible direct interaction of propofol with the GABAA recognition site. However, the evidence that propofol enhances rather than reduces [3H]GABA binding should ruled out this hypothesis. The results that propofol affects both Bmax and Kd of [35S]TBPS binding and produces the same degree of inhibition using a low and a relatively high concentration of [35S]TBPS, allow to exclude a competitive interaction of this compound on picrotoxin/TBPS recognition site. This conclusion is further supported by the failure of picrotoxin to antagonize the effect of propofol on [3H]GABA binding. The similarities in the qualitative effects of propofol, pentobarbital and alphaxalone on GABAergic synapses suggest that recognition sites for steroids and/or barbiturates might be also involved in the action of propofol on GABA-coupled chloride channel. However, the results obtained using low concentrations of propofol in combination with a low concentration of pentobarbital or alphaxalone demonstrated that the effect of propofol on [3H]GABA binding, [35S]TBPS binding and muscimolstimulated 36C1- uptake was clearly additive to the effect elicited by the other drugs alone. A similar synergistic effect was also obtained using the combination of propofol and diazepam. The above results are consistent with the idea that different sites or mechanisms of action for these drugs are present at the level of the GABAA receptor complex. This conclusion is in agreement with
231 the finding that p r o p o f o l unlike p e n t o b a r b i t a l and a l p h a x a l o n e 13'2°'3s failed to increase [3H]flunitrazepam
protein is different from those of p e n t o b a r b i t a l and alphaxalone. Since p r o p o f o l like those c o m p o u n d s is able
binding and unlike p e n t o b a r b i t a l is not sensitive to picrotoxin 3°. In conclusion our results have shown that the general anaesthetic p r o p o f o l shares with pentobarbital, alphaxalone and o t h e r general anaesthetics some similarities in the capability to enhance the function of central G A B A ergic synapse. M o r e o v e r , the present d a t a also suggest that the target site of p r o p o f o l to the r e c e p t o r channel-
to induce an o p e n i n g o f the chloride channel b o t h in the presence and in the absence o f G A B A 6, the present finding further supports the i d e a of a crucial role for the central G A B A e r g i c synapses in the mechanism of action of some general anaesthetic drugs. Finally, the action of propofol on G A B A e r g i c transmission does not exclude the possible interaction of the drug with the lipid c o m p o n e n t of the m e m b r a n e .
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