Neurochemical Research, Vol. 25, No. 7, 2000, pp. 949–955
New Benzodiazepines Alter Acetylcholinesterase and ATPDase Activities Maria R. C. Schetinger,1,4 Neuza M. Porto,3 Maria B. Moretto,2 Vera M. Morsch,1 João Batista T. da Rocha,1 Vânia Vieira,1 Franciele Moro,1 Roseli Tatto Neis,1 Sandra Bittencourt,1 Hélio G. Bonacorso,1 and Nilo Zanatta1 (Accepted April 11, 2000)
This study examines the effect of new 1,5 benzodiazepines on acetylcholinesterase (AChE) and ATPDase (apyrase) activities from cerebral cortex of adult rats. Simultaneously, the effects of the classical 1,4-benzodiazepine on these enzymes were also studied for comparative purpose. The compounds 2-trichloromethyl-4-phenyl-3H-1,5-benzodiazepin and 2-trichloromethyl-4(p-methyl-phenyl)-3H-1,5-benzodiazepin significantly inhibited acetylcholinesterase activity (p < 0.01) when tested in the range of 0.18–0.35 mM. The inhibition caused by these two new benzodiazepines was noncompetitive in nature. Similarly, at concentrations ranging from 0.063 to 0.25 mM, the 1,5 benzodiazepines inhibited ATP and ADP hydrolysis by synaptosomes from cerebral cortex (p < 0.01). However, the inhibition of nucleotide hydrolysis was uncompetitive in nature. Our results suggest that, although diazepam and the new benzodiazepines have chemical differences, they both presented an inhibitory effect on acetylcholinesterase and ATPDase activities.
KEY WORDS: Benzodiazepines; acetylcholinesterase; ATPDase; cerebral cortex.
INTRODUCTION
there is also evidence showing that an ATPase and adenylate kinase can participate in ATP hydrolysis in the synaptic cleft (10,11). ATPDase is a general designation for enzymes that hydrolyze ATP and ADP (adenosine diphosphate) to the monophosphate ester plus inorganic phosphate (12–15). The neurotransmitter acetylcholine is hydrolyzed by acetylcholinesterase, an important regulatory enzyme that controls the transmission of nerve impulses across cholinergic synapses (16). Benzodiazepines are widely used clinically as muscle relaxants, anticonvulsants, sedatives, hypnotics and anxiolytics. The use of benzodiazepines is favored by the fact that they are relatively safe substances even when administered at high doses and normally do not produce hepatic toxicity (17,18). The mechanism underlying the therapeutic efficacy of 1,4 benzodiazepines is related to an increase in the activity of the inhibitory neurotransmitter γ-aminobutyric acid (GABA)
ATP (adenosine triphosphate) is a cotransmitter released together with various neurotransmitters including acetylcholine (1). ATP and acetylcholine are involved in the regulation of a variety of neurophysiological processes (2–9) and after exerting their effects they are hydrolyzed by ATPDase (E.C. 3.6.1.5) and acetylcholinesterase, respectively. With respect to ATP, 1 2
3
4
Departamento de Química, Centro de Ciências Naturais e Exatas. Departamento de Análises Clinicas, Centro de Ciências da Saúde, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brasil. Departamento de Ciências Biológicas, Centro de Educação, Ciências Biológicas e Artes, Universidade da Região da Campanha, 97300-000, São Gabriel, RS, Brasil. Address reprint request to: Maria Rosa Chitolina Schetinger, Departamento de Química, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, Santa Maria RS Brasil 97105-900. Fax: 0+55552208031 E-mail:
[email protected]
949 0364-3190/00/0700-0949$18.00/0 © 2000 Plenum Publishing Corporation
950 (19,20). However, considering the fact that acetylcholinesterase inhibitors are currently being used as therapeutic agents in the treatment of degenerative diseases (21,22), it is important to investigate the inhibitory effect of new compounds such as the 1,5 benzodiazepines on enzymes such as acetylcholines-terase and ATPDase. Previous research on Alzheimer Disease has focused on the identification of drugs aimed at limiting the deficient cognitive function associated with this disorder. For example, the synthetic cholinesterase inhibitor tetrahydroaminoacridine (THA) has been approved for patient treatment in several countries, and is effective in reducing cognitive impairment in approximately 30 % of Alzheimer Disease cases; however side effects such as nausea and liver damage are recognized as serious problems in the use of such drugs (23). Recently, it was demonstrated that THA, inhibited in vitro the ATPDase activity of synaptosomes from the cerebral cortex and hippocampus of adult rats (24). Furthermore, Barcellos et al. (25) reported that diazepam (a classical 1,4 benzodiazepine) inhibited acetylcholinesterase and ATPDase activities from the cerebral cortex when tested at relatively high concentrations. Thus, the aim of the present investigation was to study the effect of two new benzodiazepine compounds, 2-trichloromethyl-4-phenyl-3H-1,5-benzodiazepin and 2-trichloromethyl-4-(p-tolyl)-3H-1,5-benzodiazepin on acetylcholinesterase and ATPDase activities (acetylcholine, ATP and ADP hydrolysis, respectively) in the cerebral cortex of adult rats and to compare it to that of diazepam in order to determine whether these effects are specific for a given class of benzodiazepines or can also be observed with 1,5 benzodiazepines. EXPERIMENTAL PROCEDURE Animals. Adult male Wistar rats (280–300 g) from our breeding stock were maintained on a natural light cycle in an air-conditioned constant-temperature colony room with water and food ad libitum. Materials. Nucleotides, Trizma Base (Tris(hydroxymethyl) aminomethane), acetylthiocholine and DTNB (5.5′-dithiobis(2-nitrobenzoic acid) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Percoll was obtained from Pharmacia (Uppsala, Sweden) and wes routinely filtered through Millipore AP15 prefilters in order to remove aggregated, incompletely coated particles. All other reagents used in the experiments were of analytical grade of the highest purity. Synthesis of the New 1,5-Benzodiazepines. The new benzodiazepines were synthesized by the method of Bonacorso et al. (26). New series of 2-trichloromethyl-4-aryl-3H-1,5-benzodiazepines were synthetized from the reaction of β-methoxyvinyl trichloromethyl ketones derived from acetals and ophenylenediamine. Acetylcholinesterase Assay. Cerebral cortex was dissected out on ice and homogenized (15 strokes at 1500 rpm) in 10 volumes of
Schetinger et al. medium containing 0.32 M sucrose, 0.1 mM EDTA (ethylenediaminetetraacetic acid) and 5 mM HEPES (N-[2-hydroxyethyl]piperazine-N′[2-ethanesulfonic acid), pH 7.5, and protein was adjusted to 0.5–0.7 mg/ml. Acetylcholinesterase (AChE, EC.3.1.1.7) activity was determined by the method of Ellman et al. (27) as described by Villescas et al. (28). Hydrolysis rates v were measured at various substrate (s) concentrations (0.008–0.8 mM) in 2 mL assay solutions with 30 mM phosphate buffer, pH 7.0, and 0.454 mM DTNB (5,5′dithiobis(2-nitronenzoic acid) at 25°C. Fifty µl of the enzyme preparation (30–50 µg protein) was added to the reaction mixture and preincubated for 3 min. The hydrolysis was monitored by formation of the thiolate dianion of DTNB at 412 nM for 2–3 min (intervals of 30s) using a Hitachi 2001 spectrophotometer. All samples ware run in duplicate or triplicate. The new benzodiazepines used ware diluted in ethanol or dimethylsulfoxide (DMSO) to a final concentration of 1% in assay tubes (this final concentration of ethanol or dimethylsulfoxide did not affect the enzyme activity). Subcellular Fractionation. The synaptosomes were isolated essentially as described by Nagy and Delgado-Escueta (29) using a discontinuous Percoll gradient. Briefly, cerebral cortex was dissected on ice, washed and homogenized (15 strokes at 300 rpm) in 10 volumes of medium containing 0.32 M sucrose, 0.1 mM EDTA and 5 mM HEPES, pH 7.5 (medium I), and then centrifugated at 1,000 g for 10 minutes. The supernatant was removed and centrifuged again at 12,000 g for 20 min. An aliquot of 0.5 ml of the crude mitochondrial fraction was mixed with 4.0 ml 8.5% Percoll by gentle hand mixing in a small-volume Teflon-glass homogenizer and the suspension was layered onto an isoosmotic discontinuous Percoll/sucrose gradient (10/16%). The synaptosomes that banded at the 10/16% Percoll interface were collected with a wide-tip disposable plastic transfer pipette. The synaptosomal fraction wes washed twice with an isoosmotic solution (medium I) by centrifugation at 15,000 g for 20 min to remove the contaminating Percoll. The pellet of the second centrifugation was resuspended in an isoosmotic solution to a final protein concentration of 0.4–0.6 mg/ml. Synaptosomes were prepared fresh daily, maintained at 0–4°C throughout the procedure and used for ATPDase assay. ATPDase Assay. ATPDase activity was determined in a reaction medium containing 5 mM KCl, 1.5 mM CaCl2, 0.1 mM EDTA, 10 mM glucose, 225 mM sucrose and 45 mM Tris-HCl buffer, pH 8.0, in a final volume of 200 µl as described previously (14). Twenty µl of the enzyme preparation (8–12 µg protein) was added to the reaction mixture and preincubated for 10 min at 37°C. The enzyme incubation times were chosen to ensure linearity of the reactions with time and protein content. The reactions were stopped by the addition of 200 µl of 10% trichloroacetic acid (TCA) to provide a final concentration of 5%. After chilling on ice for 10 minutes, 100 µl samples were taken for assay of released inorganic phosphate by the method of Chan (30) using malachite green as the colorimetric reagent and KH2PO4 as standard. Controls were carried out to correct for nonenzymatic hydrolysis by adding the synaptosomal fraction after TCA. All samples were run in triplicate. Enzyme specific activities are reported as nmol Pi released per min per mg of protein unless otherwise stated. The new benzodiazepines used were diluted in ethanol to a final concentration of 5% in assay tubes (this final concentration of ethanol did not affect the enzyme activity). Protein Determination. Protein was assayed by the method of Bradford (31) using bovine serum albumin as standard. Kinetic Determinations Acetylcholinesterase. The kinetic of the interaction of diazepam and 1,5 benzodiazepines with acetylcholinesterase was determined
1,5 Benzodiazepines Alter AChE and Apyrase Activities using the Lineweaver-Burk double reciprocal plot, by plotting 1/v against 1/s analyzed over a range of acetylthiocholine concentrations (0.008–0.8 mM) in the absence and in the presence of diazepam (0.18–0.35 mM) and 1,5 benzodiazepines (0.18–0.35 mM). K(m) values were obtained by two different estimations, 1/v vs. 1/s and v vs. v/s. IC(50) was calculated according to the Dixon plot (32) using inhibitor concentrations ranging from 0.18 to 0.35 mM. ATPDase. The kinetics of the interaction of diazepam and 1,5 benzodiazepines with ATPDase was determined using the Lineweaver-Burk double reciprocal plot, by plotting 1/v against 1/s analyzed over a range of ATP (0.015–1.5 mM) or ADP (0.015– 1.5 mM) concentrations in the absence and in the presence of diazepam (0.06–0.25 mM) and 1,5 benzodiazepines (0.06–0.25 mM). K(m) values were obtained from two different estimations, 1/v vs. 1/s and v vs. v/s. IC(50) was calculated according to the Dixon plot (32) using inhibitor concentrations ranging from 0.18 to 0.35 mM. K(i) values were determined by the method of Cornish-Bowden (33), which also provides a simple way of determining the inhibition constant for uncompetitive inhibitors. K(m) values were obtained by plotting 1/v against 1/s, v against v/s or s/v against s. We decided to estimate K(m) using these two procedures because the Lineweaver-Burk method of plotting kinetic data yields estimaties of V(max) and of K(m) which are much less reliable than those yielded by the other two methods. In addition, Dowd & Riggs (34) showed that the plot of v against v/s exaggerates any departure of the points from the “true” line predicted by Michaelis-Menten formulation and consequently has the advantage of warning investigators when their observation does not obey the linear relationship of Michaelis-Menten kinetics. Statistical Analysis. Data were analyzed by one-way analysis of variance (ANOVA) followed by post-hoc Duncan’s multiple range
Fig. 1. Chemical structures of Diazepam (A), 2-trichloromethyl-4phenyl-3H-1,5-benzodiazepin (B) and 2-trichloromethyl-4-(ρ-tolyl)3H-1,5-benzodiazepin (C).
951 test when appropriate. Differences between groups were considered significant when P < 0.05. All analyses were performed using the Statistical Package for Social Sciences (SPSS) software.
RESULTS The effect of diazepam and 1,5 benzodiazepines on acetylcholinesterase activity from cerebral cortex are shown in Fig. 2. All benzodiazepines inhibited AChE activity when tested in the range of 0.18 to 0.35 mM. Analysis of kinetic data indicated that the inhibition caused by all the benzediazepines was noncompetitive in nature. Accordingly, the values of K(m) were not modified by benzodiazepines, which however, caused a concentration-dependent decrease in V(max). The K(m) values obtained under these conditions for cerebral cortex preparations in the presence or absence of diazepam (compound A), 2-trichloromethyl-4-phenyl-3H1,5-benzodiazepin (compound B) and 2-trichloromethyl-4-(p-tolyl)-3H-1,5-benzodiazepin (compound C) are shown in Table I. The concentration of benzodiazepines required to inhibit 50% of acetylcholinesterase activity was calculated according to the Dixon plot (32) using inhibitor concentrations ranging from 0.18 to 0.35 mM. The IC(50) values obtained are shown in Table II. The potency of benzodiazepines as inhibitors of AcSCh hydrolysis was similar; however, compound B (2-trichloromethyl-4-phenyl-3H-1,5-benzodiazepin) was a significantly more potent inhibitor than diazepam (compound A) and 2-trichloromethyl-4-(ρ-tolyl)-3H-1,5benzodiazepin (compound C). All benzodiazepines inhibited ATP and ADP hydrolysis when tested in the 0.06–0.25 mM range (Fig. 3 and 4). The analysis of kinetic data indicated that the
Fig. 2. Kinetic analysis of the inhibition of acetylcholinesterase by diazepam (A), 2-trichloromethyl-4-phenyl-3H-1,5-benzodiazepin (B) and 2-trichloromethyl-4-(ρ-tolyl)-3H-1,5-benzodiazepin (C) in cerebral cortex. The graphs show double-reciprocal plots of the acetylcholinesterase experiments in the absence and in the presence of 0.18 and 0.35 mM of the drugs. Hydrolysis rates v were measured at various substrate (S) concentrations (0.008–0.8 mM) in 2 ml assay solutions with 30 mM phosphate buffer (pH 7.0) and 0.454 mM DTNB (5,5′-dithiobis (2-nitronenzoic acid) with 30–50 µg of protein and preincubated for 3 min. All experiments were repeated at least three times and similar results were obtained. Data presented were from the media of the three individual experiments. SD values were within 10% of the mean values.
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inhibition caused by all these compounds on ATP and ADP hydrolysis of cerebral cortex synaptosomes was uncompetitive in nature. Accordingly, benzodiazepine caused a decrease in the estimaties K(m) and V(max) for ATP and ADP hydrolysis. K(m) values with two different estimations are shown in Table III. The K(i) and IC(50) values for inhibition of ATP and ADP hydrolysis are shown in Table II. K(i) for ATP hydrolysis was lower than that for ADP hydrolysis for diazepam and 2-trichlorometyl-4phenyl (Table II). In contrast, the K(i) values for ATP
Table I. K(m) Values for Acetylcholinesterase: Influence of Benzodiazepines.
A Control 0.018 0.35 B Control 0.18 0.35 C Control 0.18 0.35
1/V vs. 1/S
V vs. V/S
0.0485 0.0432 0.0456
0.0440 0.0460 0.0440
0.0485 0.0498 0.0549
0.0570 0.0600 0.0570
0.0490 0.0564 0.0543
0.0600 0.0630 0.0660
A: Diazepam, B: 2-trichloromethyl-4-phenyl-3H-1,5-benzodiazepin and C:2-trichloromethyl-4-(ρ-tolyl)-3H-1,5-benzodiazepin. K(m) is expressed as mM concentration of AcSCh obtained by plotting 1/V vs 1/S or V vs V/S. Hydrolysis rates v were measured at various substrate (S) concentrations (0.008–0.8 mM) in 2 ml assay solutions with 30 mM phosphate buffer, pH 7.0, and 0.454 mM DTNB (5,5′dithiobis(2-nitronenzoic acid) plus 30–50 µg of protein. Protein was preincubated for 3 min before substrate addition. Values correspond to the mean of three independent experiments done in triplicate.
and ADP were not significantly different for 2-tricholromethyl-4-(p-tolyl) (compound C). For IC(50) determination, the results were similar to those reported for K(i), i.e., diazepam (compound A) and 2-trichloromethyl-4-phenyl-3H-1,5-benzodiazepin (compound B) inhibited ATP hydrolysis with higher potency than ADP hydrolysis, while for 2-trichloro-methyl-4-(p-tolyl) (compound C) no difference was found in IC(50) for ATP and ADP hydrolysis. DISCUSSION Acetylcholine and ATP are important neurotransmitters (35,36) and the study of compounds that interfere with their metabolism is of potential clinical, pharmacological and toxicological interest. The three drugs tested exert an inhibitory effect on the degradation of two neurotransmitters that are co-released in the synaptic cleft and, consequently, the pharmacological use of these compounds may interfere with the functionality of the central nervous system. The structural formula of the three compounds used in this study is shown in Fig. 1. There are subtle chemical differences between these three compounds. Diazepam has a Cl at position C7 that is involved in the pharmacological activity of this molecule. The two new benzodiazepines also have Cl, but at the C2 position. Also, another difference is the position of the aryl group, which is C5 in diazepam and C4 in the new drugs. From the present results it is clear that ATPDase activity is more sensitive to the inhibitory effect of benzodiazepines than acetylcholinesterase. These may be related to the high hydrophobicity of these types of compound and to the fact that this enzyme contains various hydrophobic domains where the com-
Table II. K(i) and IC(50) Values for Acetylcholinesterase and ATPDase A AcSCh ATP ADP
IC(50) IC(50) K(i) IC(50) K(l)
(mM) (mM) (mM) (mM) (mM)
0.65 ± 0.17 ± 0.17 ± 0.34 ± 0.40 ±
B 0.05a 0.03b 0.04a 0.06c 0.08b
0.53 0.16 0.20 0.41 0.43
± ± ± ± ±
C 0.05a 0.02b 0.02a 0.04c 0.04b
0.67 0.21 0.25 0.29 0.30
± ± ± ± ±
0.06a 0.08b 0.01 0.04b 0.01
A: Diazepam, B: 2-trichloromethyl-4-phenyl-3H-1,5-benzodiazepin and C:2-trichloromethyl-4-(ρ-tolyl)-3H-1,5-benzodiazepin. IC(50) was calculated according to the Dixon plot (32) using concentrations of inhibitor ranging from 0.18 to 0.35 mM for acetylcholinesterase and from 0.06 to 0.25 mM for ATPDase. K(i) values were determined by the method of Cornish-Bowden plotting s/v against [l]. Values are expressed as mean ± SD of three independent experiments performed in triplicate. Means not sharing the same superscript letter were different at p < 0.05 (one-way ANOVA followed by Duncan’s multiple range test) for IC(50) or K(i).
1,5 Benzodiazepines Alter AChE and Apyrase Activities
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Fig. 3. Kinetic analysis of the inhibition of ATPDase by diazepam (A), 2-trichloromethyl-4-phenyl-3H-1,5-benzodiazepin (B) and 2-trichloromethyl-4-(ρ-tolyl)-3H-1,5-benzodiazepin (C) in synaptosomes from cerebral cortex. The graphs show double-reciprocal plots of ATPDase with ATP concentrations (0.015–1.5 mM) in the absence and in the presence of 0.06, 0.125 and 0.25 mM of the drugs. ATPDase activity was determined in a reaction medium containing 5 mM KCl, 1.5 mM CaCl2, 0.1 mM EDTA, 10 mM glucose, 225 mM sucrose and 45 mM TrisHCl buffer, pH 8.0, in a final volume of 200 µl, as described previously (14). Twenty µl of the enzyme preparation (10–20 µg protein) was added to the reaction mixture and preincubated for 10 min at 37°C. All experiments were repeated at least three times and similar results were obtained. Data presented were from the media of the three individual experiments. SD values were within 10% of the mean values.
Fig. 4. Kinetic analysis of the inhibition of ATPDase by diazepam (A), 2-trichloromethyl-4-phenyl-3H-1,5-benzodiazepin (B) and 2-trichloromethyl-4-(ρ-tolyl)-3H-1,5-benzodiazepin (C) in synaptosomes from cerebral cortex. The graphs show double-reciprocal plots of ATPDase with ADP concentrations (0.015–1.5 mM) in the absence and in the presence of 0.06, 0.125 and 0.25 mM of the drugs. ATPDase activity was determined in a reaction medium containing 5 mM KCl, 1.5 mM CaCl2, 0.1 mM EDTA, 10 mM glucose, 225 mM sucrose, and 45 mM TrisHCl buffer, pH 8.0, in a final volume of 200 µl. Twenty µl of the enzyme preparation (10–20 µg protein) was added to the reaction mixture and preincubated for 10 min at 37°C. All experiments were repeated at least three times and similar results were obtained. Data presented were from the media of the three individual experiments. SD values were within 10% of the mean values.
Table III. Kinetic Parameters for Synaptosomal ATPDase from Cerebral Cortex of Adult Rats A 1/V vs. 1/S
B V vs. V/S
1/V vs. 1/S
C V vs. V/S
1/V vs. 1/S
V vs. V/S
[mM]
ATP
ADP
ATP
ADP
ATP
ADP
ATP
ADP
ATP
ADP
ATP
ADP
0 0.060 0.125 0.250
39 30 24 17
27 23 19 15
45 38 35 22
31 28 26 22
43 22 19 16
32 28 26 24
39 33 30 26
40 35 33 30
44 28 21 19
37 30 26 24
43 39 34 29
37 34 31 28
A: Diazepam, B: 2-trichloromethyl-4-phenyl-3H-1,5-benzodiazepin and C:2-trichloromethyl-4-(ρ-tolyl)-3H-1,5-benzodiazepin. K(m) is expressed as µM concentration of ATP or ADP obtained by plotting 1/V vs 1/S or V vs V/S. ATPDase activity was determined in a reaction medium containing 5 mM KCl, 1.5 mM CaCl2, 0.1 mM EDTA, 10 mM glucose, 225 mM sucrose, and 45 mM Tris-HCl buffer, pH 8.0, in a final volume of 200 µl. Twenty µl of the enzyme preparation (10–20 µg protein) was added to the reaction mixture and preincubated for 10 min at 37°C. Values correspond to the mean of three different experiments. SD values were within 10% of the mean values.
954 pounds can bind (37). Furthermore, a direct effect on the membrane can not be ruled out. In the case of acetylcholinesterase it is probable that these compounds bind to a hydrophobic peripheral site located at the gorge of the enzyme. Binding of ligands at these site changes both the rate of substrate association and the rate of product dissociation, as suggested recently (38). The relatively low potency of benzodiazepines to interact with this peripheral anionic site may be related to the fact that these compounds do not contain a positive region that is known to facilitate the interaction of ligands with this site (39). Also, the IC(50) values for these drugs were similar, indicating the same sensitivity of acetylcholinesterase for these 1,4 and 1,5 benzodiazepines. The kinetic analysis of the effects of diazepam and 1,5 benzodiazepines on acetylcholinesterase activity indicated a noncompetitive inhibition for all compounds because they did not change the K(m) and caused a decrease in V(max). We propose the following scheme for the reactions: 1) AChE + Bz ⇔ AChE.Bz 2) AChE.Bz + S ⇔ AChE.Bz.S 3) AChE.Bz.S ⇔ AChE.S + Bz 4) AChE.S ⇔ AChE + P Both binary (AChE.Bz) and ternary (AChE.Bz.S) complexes could be formed, which are catalytically inactive. Consequently, the rate of Bz dissociation from these complexes determines the inhibitory effect of benzodiazepines. Although the experiments described in this study was carried out after pre-incubation, it should be emphasized that the inhibitory effect of benzodiazepines on AcSCh hydrolysis was not significantly modified when the assay was carried out without pre-incubation (data not shown). In contrast to acetylcholinesterase, the inhibition caused by 1,4 and 1,5 benzodiazepines on ATPDase was uncompetitive as evidenced by a decrease in apparent K(m) and V(max) when ATP or ADP was used as substrate. Consequently, the 1,4 and 1,5 benzodiazepines seem to promote a similar mechanism of ATPDase inhibition regardless of the substrate used. However, the K(i) and IC(50) for inhibition of ATP hydrolysis assessed in the presence of diazepam and 2-trichlorometyl-4-phenyl-3H-1,5-benzodiazepin were significantly lower than those for inhibition of ADP hydrolysis, indicating that the interaction of these drugs with the enzyme is different depending on the substrate that is forming the complex ES. Alternatively, benzodiazepines could be inhibiting with a high potency an ATPase co-expressed with ATPDase at the synaptic cleft (37).
Schetinger et al. Considering the kinetic data for ATPDase inhibition by benzodiazepines, for which a uncompetitive type of inhibition was found, and the fact that a uncompetitive inhibitor binds only to the ES form of the enzyme, we propose the following scheme of reaction for ATPDase in the presence of benzodiazepines: 1) ATPDase + S (ATP or ADP) ⇔ ATPDase.S 2) ATPDase.S + Bz ⇔ ATPDase.S.Bz 3) ATPDase.S.Bz ⇔ ATPDase.S + Bz 4) ATPDase.S ⇔ ATPDase + P In conclusion, it was shown that 1,4 and 1,5 benzodiazepines inhibited AcSCh, ATP and ADP hydrolysis in synaptosomes from cerebral cortex of adult rats. Nonetheless, the concentrations of benzodiazepines needed to inhibit acetylcholinesterase and ATP and ADP hydrolysis are relatively high and apparently are not related to the classical pharmacological action of these drugs. However, the possibility that inhibition of these enzymes may contribute to some of the pharmacological and/or toxicological properties of these compounds cannot be ruled out. In fact, preliminary results from our laboratory have shown that administration of high doses of these benzodiazepines inhibits brain acetylcholinesterase from rats.
ACKNOWLEDGMENTS The authors wish to thank Conselho Nacional de Desenvolvimento e Tecnológico (CNPq/PADCT III- Proj. 62.0228/97-0-QEQ) and FAPERGS 98/1711-0 for financial support. M. R. C. Schetinger (N° 524365/96.2) and J. B. T Rocha (N° 523761/95-3) are recipients of CNPq fellowships.
REFERENCES 1. Richardson, P. J., and Brown, S. J. 1987. ATP release from affinity-purified rat cholinergic nerve terminals. J. Neurochem. 48:622–630. 2. Wess, J. 1993. Molecular basis of muscarinic acetylcholine receptor function. Trends Pharmacol. Sci. 14:308–313. 3. Buccafusco, J. J., and Brezenoff, H. E. 1986. Brain neurotransmitters and the development and maintenance of hypertension. Prog. Drug. Res. 30:127–150. 4. Lundberg, J. M. 1996. Pharmacology of cotransmission in the autonomic nervous system: integrative aspects on amines, neuropeptides, adenosine triphosphate, amino acids and nitric oxide. Pharmacol. Rev. 48:113–178. 5. Phillis, J. W., and Wu, P. H. 1981. The role of adenosine and its nucleotide in central synaptic transmission. Prog. Neurobiol. 16:187–239. 6. Silinski, E. M., Gerzanich, V., and Vanner, S. M. 1992. ATP mediates excitatory synaptic transmission in mammalian neurones. Br. J. Pharmacology 106:762–763. 7. Edwards, F. A., Gibb, A. J., and Colquhoun, D. 1992. ATP receptor-mediated synaptic currents in the central nervous system. Nature 359:144–147.
1,5 Benzodiazepines Alter AChE and Apyrase Activities 8. Schetinger, M. R. C., Bonan, C. D., Frasetto, S., Wyse, A. T., Schierholt, R. Webber, A, Dias, R. D., Sarkis, J. J. F., and Netto, C. A. 1999. Pre-conditioning to global ischemia changes hippocampal acetylcholinesterase in the rat. Biochem. Mol. Biol. Int. 47:473–478. 9. Schetinger, M. R. C., Bonan, C. D., Schierholt, R. C., Webber, A, Arteni, N., Emanuelli, T., Dias, R. D., Sarkis, J. J. F., and Netto, C. A 1998. Nucleotide Hydrolysis in rats submitted to global cerebral ischemia: a possible link between preconditioning and adenosine production. J. Stroke Cerebrovasc. Dis. 7:281–286. 10. Nagy, A. K. 1996. Ecto-ATPases- Recent Progress on Structure and Function. Pages 1–13, in Plesner, L., Kirley. T., and Knowles, F. (eds.), Ecto-ATPases of the nervous system. Plenum Press, New York. 11. Zimmermann, H., Grondal, E. J. M., and Keller, F. 1986. Cellular Biology of Ecto-Enzymes. Pages 35–48, in Kreutzberg, G. W., Reddington, M. and Zimmermann, H. (eds), Hydrolysis of ATP and formation of adenosine at the surface of cholinergic nerve endings. Springer-Verlag, Heidelberg, Berlin, New York, Tokyo. 12. Zimmermann, H. 1996. Biochemistry, localization and functional roles of ectonucleotidases in the nervous system. Prog. Neurobiol. 49:589–618. 13. Schetinger, M. R. C., Falquembach, F., Michelot, F., Mezzomo, A., and Rocha, J. B. T. 1998. Heparin modulates adenine nucleotide hydrolysis by synaptosomes from cerebral cortex. Neurochem. Int. 33:243–249. 14. Battastini, A. M. O., Rocha, J. B. T., Barcellos, C. K., Dias, R. D., and Sarkis, J. J. F. 1991. Characterization of an ATPDase (EC 3.6.1.5) in synaptosomes from cerebral cortex of adult rats. Neurochem. Res. 16:1303–1310. 15. Meyerhof, O. 1945. The origin of the reaction of Harden and Young in cell-free alcoholic fermentation. J. Biol. Chem. 157: 105–119. 16. Milatovic, D., and Dettbarn, W-D. 1996. Modification of acetylcholinesterase during adaptation to chronic, subacute paraoxon application in rat. Toxicol. Appl. Pharmacol. 136:20–28. 17. Black, H. E., Szot, R. J., Arthaud L. E., Massa, T., Mylecraine, M., Klein, M., Lake, R., Fabry, A, Kaminska, G. Z., Sinha, D. P., and Schwartz, E. 1987. Preclinical safety evaluation of the benzodiazepine quazepam. Arzneim.-Forsch.(Drug Res). 37: 906–913. 18. Owen, G., Smith, T. H. F., and Agersborg Jr. H. P. K. 1970. Toxicity of some benzodiazepine compounds with CNS activity. Toxicol. Appl. Pharmacol. 16:556–570. 19. Braestrup, C., and Squires, R. F. 1977. Specific benzodiazepine receptor in the brain characterized by high affinity [3H]diazepam binding. Proc. Natl. Acad. Sci. USA 74:3805–3809. 20. Barnard, E. A., Skolnick, R., Olsen, R. W., Mohler, H., Sieghart, W., Biggio, G., Braestrup, C., Bateson, A. N., and Langer, S. Z. 1998. International union of pharmacology. XV. Subtypes of γ-aminobutyric acid receptors: Classification on the basis of subunit structure and receptor function. Pharmacol. Rev. 50: 291–313. 21. Davis, K. L., and Mohs, R. C. 1982. Enhancement of memory process in Alzheimer’s disease with multiple-dose intravenous physostigmine. Am. J. Psychiatry, 139:1421–1424. 22. Miyamoto, M., Takahashi, H., Kato, K., Hirai, K., Ishihara, Y., and Goto, G. 1996. Effects of 3-[1-(Phenylmethyl)-4-Piperidinyl]-1(2,3,4,5-Tetrahydro-1H-1-Benzazepin-8-yl)-1-Propa-
955
23. 24.
25.
26.
27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.
38.
39.
none Fumarate (TAK - 147), a novel acetylcholinesterase inhibitor, on impaired learning and memory in animal models. J. Pharmacol. Exp. Ther. 277:1292–1304. Burns A, Russel, E., and Page, S. 1999. New drugs for Alzheimer’s Disease. Br. J. Psychiatry, 174:476–479. Bonan, C. D., Battastini, A. M. O., Schetinger, M. R. C., Moreira, C. M., Frasetto, S., Dias, R. D., and J. J. F. Sarkis. 1997. Effects of 9-amino-1,2,3,4-tetrahydroacridine (THA) on ATPDase (EC 3.6.1.5) and 5′-nucleotidase (EC 3.1.3.5) from rat brain synaptosomes. Gen. Pharmacol. 26:761–766. Barcellos, C. K., Schetinger, M. R. C., Dias, R. D., and Sarkis, J. J. F. 1998. In vitro effect of central nervous system active drugs on the ATPase-ADPase activity and acetylcholinesterase activity from cerebral cortex of adult rats. Gen. Pharmacol. 31:563–567. Bonacorso, H. G., Bittencourt, S. T., Wastowski, A. D., Wentz, A. P., Zanatta, N., and Martins, M. A. 1996. A convenient method for the synthesis of 2-trichloromethyl-4-p-substitutedphenyl-3H1,5-benzodiazepines. Tetrahedron Letters 37:9155–9156. Ellman, G. L. Courtney, K. D., Andres Jr. V., and Featherstone, R. M. 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7:88–95. Villescas, R., Ostwald, R., Morimoto, H., and Bennet, E. 1981. Effects of neonatal undernutrition and cold stress on behavior and biochemical brain parameters in rats. J. Nutr., 11:1103– 1110. Nagy, A., and Delgado-Escueta, A. V. 1984. Rapid preparation of synaptosomes from mammalian brain using a nontoxic isosmotic gradient (Percoll). J. Neurochem., 43:1114–1123. Chan, K., Delfert, D., and Junger, K. D. 1986. A direct colorimetric assay for Ca2+ ATPase activity. Anal. Biochem., 157: 375–380. Bradford, M. M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72:248–254. Dixon, M., and Webb, E. C. 1964. Enzymes, 2nd ed. p.950. Longmans, London. Cornish-Bowden, A. 1974. A simple graphical method for determinating the inhibition constant of mixed, uncompetitive and non-competitive inhibitors. Biochem. J. 137:143–144. Dowd, J., E. and Riggs, D., S. 1965. A comparison of estimaties of Michaelis-Menten kinetic constants from various linear transformations. J. Biol. Chem. 240:863–869. Role, L. W., and Berg, D. K. 1996. Nicotinic receptors in the development and modulation of CNS synapses. Neuron, 16: 1077–1085. Evans, R. J., Derkach, V., and Surprenant, A. 1992. ATP mediates fast synaptic transmission in mammalian neurons. Nature, 359:144–147. Kegel, B., Braun, N., Heine, P., Maliszewski, C. R. and Zimmermann, H. 1997. An ecto- ATPase and nan ecto-ATP diphosphohydrolase are expressed in rat brain. Neuropharmacol. 36:1189–1200. Szegletes, T., Mallander, W. D., Thomas, P. J., and Rosenberry, T. L. 1999. Substrate binding to the peripheral site of acetylcholinesterase initiates enzymatic catalysis. Substrate Inhibition arises as a secondary effect. Biochemistry 38:122–133. Wilson, I. B. 1957. Specificity in cholinesterase reactions. Pages 175–185, in Pauling, L., and Itano, H. (eds.), Acetylcholines-terase: Molecular Structure and Biological Specificity, American Institute of Biological Sciences, New York.
