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Gabaergic drugs, morphine and morphine tolerance: A study in relation to nociception and gastrointestinal transit in mice
Neuropharmarology Vol. 22, No. 6, pp. 767-774, Printed in Great Britain. All rights reserved
0028-3908/83/060767-08$03.00/O Copyright 0 1983 Pergamon Press Ltd
1983
GABAERGIC DRUGS, MORPHINE AND MORPHINE TOLERANCE: A STUDY IN RELATION TO NOCICEPTION AND GASTROINTESTINAL TRANSIT IN MICE S. P. SWAM and I. K. Ho* Department
of Pharmacology
and Toxicology, University of Mississippi Medical MS 39216, U.S.A.
Center,
Jackson.
(Accepted 16 November 1982)
Summary-Agonists and antagonists of y-aminobutyric acid, i.e. GABAergic drugs, such as muscimol, baclofen or bicuculhne, alone or in combination, exhibited analgesic effects per se and enhanced the analgesia induced by morphine. The analgesic effects of GABAergic drugs were unaffected by admini tration of naloxone in a dose which antagonized the analgesia induced by morphine. The ED,, for the antinociceptive effect of muscimol, bicuculline, picrotoxin, gabaculhne or aminooxyacetic acid (AOAA) was not affected in the morphine-tolerant group as compared to the control group, in contrast to the increase in the ED,, for morphine under similar conditions; this indicated that there was no development of cross-tolerance between morphine and GABAergic drugs. Muscimol suppressed the abrupt withdrawaljumping induced by morphine and enhanced the suppression of this phenomenon by morphine. The GABAergic drugs also shared with morphine the property of inhibiting gastrointestinal (GIT) motility. Naloxone reversed the inhibition of motility induced by morphine but failed to influence that induced by GABAergic drugs. In the morphine-tolerant state, the sensitivity of gastrointestinal motility to morphine decreased, whereas. the sensitivity to GABAergic drugs remained unaltered. The results indicate that GABAergic drugs share some of the classical properties of morphine, such as analgesia and inhibition of gastrointestinal motility, but they probably do so by different mechanisms. Key words:
morphine,
GABA,
GABAergic
drugs,
y-Aminobutyric acid (GABA) has been well established as an inhibitory neurotransmitter at spinal and supraspinal levels of the central nervous system (Roberts, Chase and Tower, 1976; Johnston, 1978; Lal, Fielding, Malick, Shah and Usdin, 1980) and has been implicated in a variety of neurological and behavioral disorders (Enna, 1981). Several studies have indicated a role for GABA in analgesia and tolerance/dependence to opiates (Ho, Loh and Way, 1973, 1976; DeBoer, Metsalaar and Bruinvels, 1977; Tzeng and Ho, 1978; Buckett, 1980; Ho and Gilliland, 1979: Mantegazza, Tammiso, Vincetini, Zambotti and Zonta, 1980; Sivam, Nabeshima and Ho, 198 la, b, 1982) and also in the antagonism of opiateinduced convulsions (Dingledine, Iversen and Breuker, 1978). However, the pharmacological manipulation of the GABA system using GABAergic drugs as tools, have revealed useful but conflicting results regarding the role of GABA in the effects of opiate drugs (Ho et al., 1976; Biggio, Della Bella, Frigeni and Guidotti, 1977; Mantegazza et al., 1980; Spaulding, Little, McCormick and Fielding, 1980;
*Send correspondence to: Dr I. K. Ho, Department of Pharmacology and Toxicology University of Mississippi Medical Center 2500, North State Street, Jackson, MS 39216, U.S.A.
gastrointestinal
transit,
analgesia,
opiate
tolerance.
Izumi, Munekata, Yamamoto, Nakanishi and Barbeau, 1980; Zonta, Zambotti, Vicentini, Tammiso and Mantegazza, 1981). Both agonists and antagonists of GABA have been reported to possess analgesic activity (Spaulding et al., 1980; DeFeudis, 1982). Contrary to this, Zonta el al. (1981) reported that bicuculline, a postsynaptic GABA receptor antagonist. reversed the effects of muscimol, a GABA agonist, which itself antagonized the analgesia induced by morphine or /I-endorphin in rats. Thus, there are conflicting reports with respect to the interaction between GABAergic drugs themselves and also in combination with opiates. In order to clarify this aspect, the present study was undertaken to assess the effects of GABAergic drugs alone, or in combination with morphine, on nociception and gastrointestinal transit with special reference to the development of tolerance to morphine.
METHODS
Animals
Male ICR mice (Charles River Breeding Labs, Wilmington, MA) weighing 25-30 g were maintained in a room at a temperature 24 + 1°C under artificial 12/12 hr lighting cycles were used; water was given ad libitum.
S. P. SWAM and I. K. Ho
768 Chemicals
used
Muscimol, bicuculline, picrotoxin, aminooxyacetic acid and gabaculline were purchased from Sigma Chemical Company, St Louis, MO; baclofen was generously supplied by Ciba-Geigy, Summit, NJ. Morphine sulfate was obtained from Mallinckrodt Chemical Works, St Louis, MO. Naloxone hydrochloride was a gift from the Endo Laboratories, Garden City, NY. All chemicals were dissolved in saline (0.9% w/v NaCl) and injected in a volume 0.1 ml/10 g body weight. Induction
qf tolerance to morphine
Tolerance to morphine was induced by the pellet (75 mg morphine base, s.c.) implantation method of Way, Loh and Shen (1969). The groups referred to as “tolerant” were those in which the pellets were removed for 8 hr after a 72 hr implantation period. Control animals received placebo pellets. Influence ofmuscimol on the abstinence jumping due to abrupt withdrawal of morphine The effect of muscimol on the spontaneous jumping behavior observed 10 hr after the removal of the pellets, which were implanted for 72 hr to induce dependence on morphine, was studied by injecting muscimol and morphine alone or in combination. Assessment
of analgesia by antinociceptive
tests
(A) Tail immersion antinociceptive test. The method is essentially that reported by Sewell and Spencer (1976). The mouse was restrained in an individual, ventilated rigid rectangular Plexiglass container and the nociceptive reaction time (in seconds) was determined when the tail was immersed in a constanttemperature water bath set at 50°C. The nociceptive endpoint was characterized by either a violent jerk of the tail or a rapid “flinch” of the whole body of the mouse. A 20 set cut-off time was imposed for all animals that failed to respond to the stimulus. This particular test as claimed by Sewell and Spencer (1976) possesses two distinct advantages: first, the use of wet heat transfer has provided more consistent responses than the radiant heat and it is possible to use a relatively low temperature as the nociceptive stimulus; secondly, this low temperature also minimizes local tissue damage in the tail, permitting repetitive measurements to be made on individual animals at frequent intervals. The response to the graded doses of muscimol, given subcutaneously, alone or in combination with a fixed dose of morphine, was observed both in morphine-tolerant and non-tolerant (control) animals, at 0, 30, 60 and 90 min. The 0 time represents the responses taken 15 min prior to injection of the test drugs. In another set of experiments using naive mice, the effect of naloxone and bicuculline on the response to muscimol was investigated.
(B) Acetic acid-induced abdominal writhing (AA W) test. Tests were carried out to determine the antinociceptive effects of test drugs in terms of their ability to inhibit abdominal writhing induced by 0.6% acetic acid, 0.1 ml/10 g body weight, (Koster, Anderson and DeBeer, 1959). A writhing was defined as a characteristic contraction of abdominal muscles accompanied by an extension of the hind limbs. The number of writhes occurring during a 10 min period after 10min of the injection of acetic acid was recorded. The ED,, value (Litchfield and Wilcoxon, 1949) was defined as the dose of the test drug capable of reducing by 50% the number of writhes occurring in control mice. The test drugs were given subcutaneously immediately prior to the injection of acetic acid. The ED,, values for muscimol, bicuculline, picrotoxin, gabaculline, aminooxyacetic acid and morphine were determined both in morphine-tolerant and non-tolerant animals. In addition, the effect of naloxone and bicuculline on muscimol or baclofeninduced antinociception was assessed in a separate set of experiments. Assessment qfgastrointestinal coal meal test
(GIT)
transit by char-
The gastrointestinal transit was measured essentially following the method of Wong, Roberts and Wai (1980) by a charcoal meal test. The animals were fasted for 16 hr before use during which period they had access only to tap water. The animals were given 0.25 ml of an aqueous suspension of 2.57/, charcoal and 1.25% gum acacia by stomach tube as a per oral bolus. Twenty minutes later the mice were sacrificed by cervical dislocation and the stomach and intestines were excised from the gastro-esophageal junction to the ileo-caecal junction. The distance the charcoal meal had travelled from the pylorus was measured. The drugs were injected subcutaneously, (aminooxyacetic acid, intraperitoneally) 15 min before giving the charcoal meal. Control animals received saline injections at the corresponding time schedule. At least 6 animals were used for each dose. Different sites of injection were chosen when two or more drugs were used. Statistical
analysis and the expression
of results
For the ED,, calculations, the Litchfield and Wilcoxon (1949) method was used. The data on analgesic tests were expressed as a percentage of control values. The statistical significance of the treatments was calculated on the original data, i.e. before normalization into percentages by Student’s t-test [time-effect relationship with respect to the control (Figs l-3) or control versus treated groups (Figs 4, 7)] and analysis of variance followed by Student-Neuman-Keuls test (comparison of the means of different treated groups; the significant results are indicated in the “Results” section). The Chi-square test was used on the original data of the withdrawal jumping (based on jumping versus non-
GABAergic
1. Antinociceptive effect Fio (pkebo) and morphine-tolerant
of
drugs
muscimol in control mice using the tail-
immersion test. The results are expressed as the percentage increase from the control level at the respective time points. *P < 0.05; **P < 0.1; ***P < 0.001 compared to the con-
769
and morphine
Fig. 3. Effect of bicuculline and naloxone on the muscimolinduced analgesic response in naive mice using the tailimmersion test. Seven mice were used in each group. Other
explanations are as for Fig. 1.
trol (Student’s t-test). Eight mice were used in each group except the placebo-saline group which consisted of 7 mice.
50
r
jumping) before normalization into percentages of animals (Figs 5 and 6). A P-value of at least ~0.05 between two means was considered significant. RESULTS
Effect of GABAergic nociceptive test
drugs on tail immersion
anti-
The tail-flick response time in control animals was 3.74 k 0.45 set (mean k SE, n = 7). In control animals muscimol produced a dose-related (0.25,0.5 and 1 mg/kg) increase in analgesic threshold (Fig. 1). At 30 min, there was a significant difference (P < 0.05) between the 0.25 and 1 mg/kg dose; at 60 min there were significant differences between the three doses except between 0.5 and 1 mg/kg. In tolerant animals, a paradoxical response to muscimol was observed in that at 30 and 60 min, the larger dose (1 mg/kg) was less effective (P < 0.05) than the smaller (0.25-0.5 mg/kg) doses (Fig. 1). At 60 min, there was a significant difference (P < 0.05) between the smaller doses. The combination of a dose (0.25, 0.5 or 1 mg/kg) of muscimol with a fixed dose of morphine (4 mg/kg)
Fig. 4. Effect of GABAergic drugs and combinations on the acetic acid-induced writhing test in naive mice. MUS = muscimol; BIC = bicuculline; NAL = naloxone; BAC = baclofen. *P < 0.05; **P < 0.01; ***P < 0.001 compared to the control (Student’s r-test). Six animals were used in each group.
elicited in general an enhanced response compared to either agent alone. In control animals at 30 min, there was a significant difference (P < 0.05) between the saline plus morphine group and groups given 0.5 and 1 mg/kg ofmuscimol. Groups given 0.25 and 1 mg/kg of muscimol were also significantly different from each other. In the tolerant group, 0.25 mg/kg of
100
r ________O________~________~
80
----____*_ --._
‘\ 6.
‘\\\\
-___
.%._
-o--_____~-.-________. -.%____---_-&
‘\
40 20
j__
0
'\ '0.. 9 *.._ _/_ -..O__r-C
*
I 0
Fig. 2. Antinociceptive effect of muscimolkmorphine combination in placebo and morphine-tolerant mice using the tail-immersion test. Seven mice were used per group except the saline + saline groups which had 8 mice per group. Other explanations are as for Fig. I.
30
60
Illl”l
. 0 0
YUICllM 0I5mI,kI OSn~,kg YUIClloL 1om1,11 WLCHloL PLICml -----mtlln
90
IIME(min) Fig. 5. Effect ofmuscimol on spontaneous jumping behavior induced by abrupt withdrawal of morphine. The number of
animals used for saline, muscimol 0.25, 0.5 and 1.0 mg/kg, respectively
are: placebo:
7, 7, 8, 8; tolerant:
9, 10, 9, 8.
S. P. SWAM and I. K. Ho
770
----__--
.!@ct of GABAergic writhing (AA W) test
c----____-~__ --__ SALINE
+ SALINE
SALINE
+ MORPHINE
MUSCIMOL
(O.Smg/kg]
-
Ptlctso
-----
TOLERANT
+ MORPHINE
.’
0
--‘O
(IOmg/kgi
30
60
.’
r*
c’
P
90
TIMEImid Fig. 6. Effect of muscimol and morphine alone, and in combination with morphine, on spontaneous jumping behavior induced by abrupt withdrawal of morphine. The number of animals used for saline + saline, saline + morphine, muscimol + morphine groups, respectively are: placebo: 7. 10, 10; tolerant: 10, 10, 12.
Muscimol, bicuculline or baclofen produced inhibition of acetic acid-induced writhing, thereby showing an analgesic effect (Fig. 4). A combination of muscimol and bicuculline or baclofen and bicuculline was more effective (P < 0.05) than either agent alone. Naloxone (15 mg/kg) did not significantly antagonize the effects of muscimol, bicuculline or baclofen, although it did significantly (P < 0.05) antagonize the effect of a combination of muscimol and bicuculline; smaller doses of naloxone failed to influence the analgesic effects of GABAergic drugs (data not shown). The ED,+ of these drugs for analgesia were not different in tolerant animals compared to the placebo group (Table 1); in contrast, the ED,, for morphine was increased in the tolerant group. Effect of muscimol behaaior
muscimol enhanced the response to morphine significantly at all time points tested whereas 0.5 and 1 mg/kg of muscimol inhibited the response significantly at 60min (Fig. 2). In order to elucidate the nature of the analgesic response to muscimol, naloxone and bicuculline (an opiate and a GABA antagonist, respectively) were employed. As shown in Fig. 3, in naive animals bicuculline per se produced significant analgesic effects. Muscimol significantly enhanced the analgesia induced by bicuculline at 30min. Naloxone in a large dose (15 mg/kg) significantly inhibited analgesia induced by muscimol at 60 min but failed to influence analgesia induced by bicuculline, although the values were still significantly different from control values. Naloxone, in doses of 2.5 or 10 mg/kg, failed to influence analgesia induced by either bicuculline and muscimol; in contrast, 2.5 mg/kg naloxone was sufficient to inhibit analgesia induced by morphine (4mg/kg) (data not shown).
Table
drugs on acetic acid induced-
on morphine-abstinence
jumping
Morphine-tolerant animals exhibited the characteristic spontaneous withdrawal (abstinence) jumping. Muscimol produced a dose-related inhibition of the withdrawal jumping; however, the values were statistically significant only with a 1 mg/kg dose level at 30, 60 and 90 min (Fig. 5). Morphine suppressed the withdrawal jumping (Fig. 6); this effect was significantly enhanced at 30 and 60min when 0.5 mg/kg muscimol was given with morphine. Eflect of muscimol on the gastrointestinal sit
(GIT) tran -
In the control group, morphine induced a decrease in the gastrointestinal transit as did GABAergic drugs such as muscimol, baclofen, bicuculline and aminooxyacetic acid. The combined effect of morphine and GABAergic drugs was greater than that of either agent alone (Fig. 7). Naloxone (5mg/kg) antagonized the effect of morphine, but even in large
1. Effect of morphine and GABAergic drugs on acetic induced writhing in control and morphine-tolerant mice
Compound
Placebo
Morphine Muscimol Bicuculline Picrotoxin Gabaculline AOAA
0.51 (0.33 - 0.65) 0.76 (0.38 - I .4) 0.83 (0.54 - 1.5) 0.32 (0.2 - 0.48) 5.2 (2.9 - 9.4) 8.4 (4.4 - 16.0)
acid-
Tolerant 3.54 (2.3 - 4.6) 0.45 (0.21 - 0.94) 0.63 (0.42 - 0.92) 0.15 (0.1 - 0.23) 3.80 (2.1 - 6.84) 6.51 (3.83 - 11.1)
Acetic acid (0.6x, 0.1 ml/log body weight) was given intraperitoneally. The number of writhes occurring during a lo-min period, after 10 min of the injection of acetic acid was recorded. Test drugs were given immediately prior to the acetic acid injection. Gabaculline and aminooxyacetic acid (AOAA) were injected 24 and 5 hr prior to testing, respectively. At least 4 doses of each drug were used for ED,, calculations; 9-12 mice were used for each dose. The values represent ED,, (95% confidence limits) calculated according to Litchfield and Wilcoxon (1949).
GABAergic
drugs
and morphine
Fig. 7. Effect of GABAergic drugs alone or in combination on gastrointestinal transit (GIT-T) of mice. MOR = morphine; NAL = naloxone; MUS = muscimol; BIC = bicuculline; AOAA = aminooxyacetic acid. Each group consisted of 6 mice except the control which consisted of I6 mice. *P < 0.05; **P < 0.01; ***P < 0.001 compared to the control (Student’s t-test).
doses (10 or 15 mg/kg) it failed to influence the effect of GABAergic drugs. The development of tolerance to morphme in this test was also observed as shown by the decrease in sensitivity to morphine. However, the morphine-tolerant animals did not develop tolerance to any of the GABAergic drugs tested (Table 2). DISCUSSION
Several reports have provided evidence to ascribe a possible involvement of the GABA system in the analgesic and addictive properties of opiates (Ho er al., 1973, 1976; Ho and Loh, 1974; Kuriyama and Yoneda, 1978; Buckett, 1980; Mantegazza et al., 1980; Hynes, Shearman and Lal, 1980; Sivam et al., 1981a, b, 1982). The interpretation of the results of the studies of opiate-GABAergic drug interactions is complicated by the fact that the GABAergic drugs, irrespective of the taxonomic classification as agonist or antagonist, appear to possess analgesic properties per se. In the following paragraphs, an appraisal of the literature on opiate-GABAergic drug interactions with reference to the present study is presented. Antinociceptive @ects
of GABAergic agents per se
The present results showed that muscimol, a GABA agonist on the postsynaptic GABA receptors, as well as bicuculline, a postsynaptic GABA antagonist, or baclofen, which enhances the release of GABA, elicited analgesia. The analgesia induced by muscimol was enhanced by bicuculline. Naloxone, an opiate antagonist in doses which fully antagonized the analgesia induced by morphine did not influence the analgesia induced by muscimol or bicuculline. These results were seen in two different models of the generally employed analgesic tests, namely thermally mediated (tail-flick) and chemically induced [acetic
acid-induced writhing (AAW)] tests. Though the tail-flick test is considered to be mainly at the spinal reflex level, the acetic acid-induced writhing test appears to be more supraspinal (Mayer and Price, 1977). It has also been shown that opiate drugs could completely inhibit acetic acid-induced writhing when they were injected into the cerebral ventricle in nanogram quantities (Sivam et al., 1982; Sivam and Ho, unpublished observations). The present results are consistent with the earlier reports that GABAergic agents elicit analgesia in the mouse and rat using tail-flick (Spaulding et al., 1980; Saelens, Bernard and Wilson, 1980) or hot-plate tests (Liebman and Pastor, 1978; Christensen, Arnt and ScheelKriiger, 1978; Buckett, 1980; Spaulding et al., 1980). However, Biggio et a/. (1977) did not observe an analgesic effect of muscimol in the rat and mouse tail-flick test. Further, the GABA-T inhibitors such as aminooxyacetic acid, y-acetylenic GABA, y-vinyl GABA were reported to possess analgesic properties in the mouse and rat tail-flick or the hot-plate method (Contreras, Tamayo and Quijada, 1979; Buckett, 1980; Spaulding et al., 1980; Liischer, 1980). The present experiments and those of other workers clearly show that the GABAergic drugs per se elicit analgesia (see review, DeFeudis, 1982). Since the analgesia was shared by an antagonist as well, it probably was not mediated by the GABA receptor itself. However, it should be noted that GABA receptors which are insensitive to bicuculline have been postulated (Andrews and Johnston, 1979; Bowery, Doble, Hill, David, Hudson, Shaw, Turnbull and Warrington, 1981). Further, the GABA antagonist, bicuculline induced an additive analgesic effect, indicating a possible common mode of action for both agonist and antagonist. Naloxone, an opiate antagonist, in high doses only, partially antagonized
112
S. P. SWAM and I. K. Ho Table
2. Effect of GABAergic
drugs transit
on the gastrointestinal GIT transit
(GIT)
(cm)
Dose Compound Saline Morphine
Muscimol
Baclofen
Bicuculline
AOAA
(mg/kg) 0.25 0.5 1.0 2.0 4.0 0.5 1.0 2.0 3.0 3.0 6.0 12.0 24.0 0.25 0.5 1.0 1.5 5.0 10.0 20.0 40.0
Control 32.8 + 24.0 + 17.3 * Il.3 + 9.1* 30.0 23.3 18.2 14.3 29.5 28.0 20.3 18.4 33.3 26.0 22.1 21.3 32.0 34.0 24.0 23.3
1.4 4.3* 1.8*** 0.3*** 1.2***
* 2.1 e 4.1* k 3.2** &-4.0*** + 4.6 * 3.5 k 2.3* k 1.9** 5 2.9 k 3.5 * 1.5* k 1.7** k 3.2 * 2.0 k 1.O* + 3.5*
Tolerant 37.3 _t 1.5
30.7 * 1.8* 28.7 k 2.0* 17.7 * 0.3** 27.3 k 5.0 22.0 k 4.6* 20.3 k 4.5** 31.0 + 1.0 29.Ok2.1’ 27.3 k 2.9* 32.0 + 0.6 27.6 k 1.9* 22.5 f 1.8** 32.7 + 2.9 33.3 + 2.4 28.7 + 0.7*
Gastrointestinal (GIT) transit was assessed by a charcoal meal test (aqueous suspension of 1.25% gum accacia + 2.5% charcoal). The drugs were given 15 min prior to the charcoal meal. The animals were sacrificed 20min after the charcoal meal. Aminooxyacetic acid (AOAA) was given 5 hr prior to the charcoal meal. Each group consists of 6 mice, except for the saline control groups which consisted of 16 mice. All drugs were given subcutaneously, except AOAA which was given by the intraperitoneal route. *P < 0.05;
**p < 0.01; ***P < 0.001 compared to control (Student’s t-test).
the analgesia induced by muscimol in the tail immersion test; however, in the acetic acid-induced writhing test, the antagonism was restricted to the combination of muscimol and bicuculline and was not evident on the individual analgesic response. The results indicate that the GABAergic-induced analgesia may have some naloxone-sensitive component depending on the analgesic test used and acting probably through opiate-receptor interaction. However, in receptor binding studies, GABAergic drugs do not seem to displace labelled opiates (Enna and Snyder, 1975; Sivam and Ho, unpublished observations). On the other hand, opiates in very large concentrations (10m3 M) displace GABA receptor binding (Dingledine et ul., 1978; Sivam et al., 1982). How the naloxone-insensitive analgesia induced by the GABAergic drugs is mediated, remains unknown. It may be speculated that they act on some naloxone-insensitive opiate receptors, if they exist. At this juncture, it is relevant to cite that levonantradol, a cannabinoid analogue, has been shown to be l&30 times more potent than morphine as an analgesic, yet the analgesic action was only partially antagonized by naloxone in large doses (Jacob, Ramabadran and Campos-Medeiros, 1981). It is worth noting in this context that the analgesia induced by GABAergic drugs does not induce toler-
ance or dependence (Buckett, 1980) a classical effect of opiate drugs. Moreover, the primary site of action of GABAergic drugs has been suggested to be at the spinal level (Spaulding et al., 1980) unlike the wellknown effect of morphine on spinal and supraspinal loci (Dewey, Snyder, Harris and Howes, 1969; Mayer and Price, 1977). The present results using the acetic acid-induced writhing test, show that GABAergic drugs might also act at the supraspinal level. Injuence qf GABAergic compounds on tolerunce and dependence to opiates In the acetic acid-induced writhing test, GABAergic drugs exhibited analgesia in tolerant animals but there was no classical development of crosstolerance between these drugs and morphine. Here too, both agonist and antagonist behaved similarly. This further supports the idea that they may have some common mode of action. In the tail immersion test, muscimol enhanced the analgesic effect of morphine, both in non-tolerant and tolerant animals. However, larger doses inhibited analgesia induced by morphine in tolerant but not in non-tolerant animals; the nature of the action of the larger doses of muscimol to produce this paradoxical effect is unclear. Muscimol inhibited the characteristic abrupt morphine-induced withdrawal jumping, and en-
GABAergic drugs
hanced the suppression of this phenomenon by morphine. It is not clear whether the naloxonesensitive and/or the sedative effect of muscimol contributed to the suppression. A number of reports in the past have dealt with the influence of GABAergic agents on analgesia induced by opiates. However, the results are disparate extending from lack of effect to potentiation or antagonism. For example, muscimol or GABA-T inhibitors, or sodium v,alproate enhanced analgesia induced by morphine in the mouse or rat (Knoll and Zsilla, 1974; Kllrianen and Vikberg, 1976; Biggio et al., 1977; Yoneda, Takashima and Kuriyama, 1976; Contreras et al., 1979; Ostrovskaya and Bulaev, 1979; Buckett, 1980). Others have reported an antagonism of opiateinduced analgesia (Izumi et al., 1980). Christensen et al. (1978) reported that muscimol failed to affect analgesia induced by morphine on paw stimulation. The GABA uptake inhibitors such as nipecotic acid, guvacine, or 2,4_diaminobutyric acid have been found to inhibit analgesia induced by morphine (Ho and Loh. 1974; Mantegazza et al., 1980); similar results were observed for the GABA synthesis inhibitor, semicarbazide and for the GABA antagonist bicuculline (Yoneda et al., 1976; Ostrovskaya and Bulaev, 1979). Many of the discrepancies seem to arise from one or more of the following considerations: (a) lack of investigation of the time course of the response or dose-response; (b) the diverse criteria used to measure analgesia and the choice of analgesic tests; (c) lack of investigation of the effect of the pretreatment per se; (d) routes of administration (e.g. systemic versus intracerebral routes); and (e) an a priori assumption that an antagonist should “antagonize” in any event. Further, the complexity of the GABA system might have also contributed. Biochemical studies have revealed that the GABA receptors appears to be a part of the protein complex containing receptor sites for GABA, benzodiazepines and picrotoxin/barbiturate/ chloride ionophore (Olsen, 1981). In addition to the classical postsynaptic GABA receptors, there also appear to be receptors located on GABA terminals (autoreceptors) which, when activated, inhibit release of GABA (Brennan, Cantril and Kiogsgaard-Larsen, 1981). Electrophysiological studies have shown that virtually all neurons and glia in the CNS are responsive to iontophoretically applied GABA, even those that do not receive a GABAergic input (Crawford and Curtis, 1964). Because of these characteristics, it is extremely difficult to draw definite conclusions about the physiological relevance of an action observed following the administration of GABAergic agents. While GABA receptors are found in virtually all areas of the brain, recent ligand binding studies indicate that the affinity and distribution of GABA receptors varies between different brain regions (Young, Enna, Zukin and Snyder, 1976; Sivam et al., 1982). It is possible that GABAergic drugs may possess divergent abilities to alter
713
and morphine
GABA function in different regions resulting in a net change in the differential inputs from various regions. This may explain the divergent results obtained in the in vivo studies. Efect
of GABAergic
intestinal
(GIT)
drugs
and morphine
on gastro-
transit
In order to determine whether GABAergic drugs elicited responses of a similar nature to those observed in analgesic tests, tests were carried out on gastrointestinal transit. It was found that muscimol, bicuculline, baclofen and aminooxyacetic acid behaved in a qualitatively similar fashion to morphine in inhibiting transit. The effect of GABAergic drugs on gastrointestinal transit was not antagonized by naloxone even in large doses. Here too, GABAergic drugs appeared to act by a different mechanism than interaction with opiate receptors. This was further demonstrated by the fact that development of tolerance to morphine did not alter the responses to GABAergic drugs, unlike the decreased sensitivity observed for morphine. These results clearly indicate that morphine and GABAergic drugs share common properties. However, the mechanism underlying this process appears to be different for morphine and GABAergic drugs as a class. Acknowledgements-This study was supported by Grant DA-01 3 10 from National Institute on Drug Abuse. Thanks are due to MS Kathy Gibson for her technical assistance and Mrs Annette McGrigg and MS Jewel Harper for their secretarial assistance. REFERENCES Andrews I’. R. and Johnston G. A. R. (1979) GABA agonists and antagonists. Biochem. Pharmac. 28: 2697-2702. Biggio G., Della Bella D., Frigeni V. and Guidotti A. (1977) Potentiation of morphine analgesia by muscimol. Neuropharmacology 16: 149-150. Bowery N. G., Doble, Hill A., David D. R., Hudson A. L., Shaw J. S., Turnbull M. J. and Warrington R. (1981) Bicuculline-insensitive GABA receptors on peripheral autonomic nerve terminals. Eur. J. Pharmac. 71: 53-70. Brennan M. J., Cantril R. C. and Krogsgaard-Larsen P. (1981) In: GABA and Benzodiazepine Receptors (Costa E., DiChiara G. and Gessa G.. Eds)./ v. . 157. Raven Press. New York. Buckett W. R. (1980) Irreversible inhibitors of GABA transaminase induce antinociceptive effects and potentiate morphine. Neuropharmacology 19: 715-722. Christensen A. V., Arnt J. and Scheel-Kruger J. (1978) Muscimol antagonizes morphine hypermotility without potentiation of analgesia. Eur. J. Pharmac. 48: 459462. Contreras E., Tamayo L. and Quijada L. (1979) Effects of the irreversible inhibition of GABA transaminase upon some morphine effects. Neuropharmacology 18: 309-3 13. Crawford J. M. and Curtis D. R. (1964) The excitation and depression of mammalian cortical neurons by amino acids. Br. J. Pharmac. 23: 313-329. DeBoer T., Metselaar H. J. and Bruinvels J. (1977) Suppression of GABA-induced abstinence behavior in naive rats by morphine and bicuculline. Life Sci. 20: 933-942. DeFeudis F. V. (1982) GABAergic analgesia-a naloxoneinsensitive system. Pharmac. Res. Commun. 14: 3833390.
774
S. P. SWAM and 1. K. Ho
Dewey W. L., Synder J. W., Harris L. S. and Howes J. T. (1969)The effect of narcotics and narcotic antagonists on the tail-flick response in spinal mice. J. Pharm. Pharmac. 21: 548-550. Dingledine R., Iversen L. C. and Breuker E. (1978) Naloxone as a GABA antagonist: Evidence from iontophoretic, receptor binding and convulsant studies. Eur. J. Pharmac. 47: 19-28. Enna S. J. (1981) GABA receptor pharmacology: Functional considerations. Biochem. Pharmac. 30: 907-913. Enna S. J. and Snyder S. H. (1975) Properties of ~-aminobutyric acid (GABA) receptor binding in rat brain synaptic membrane fractions. Brnin Res. 100: 81-97. Ho I. K.. Loh H. H. and Way E. L. (1973) Effect of y-aminobutyric acid (GABA) on morphine analgesia, tolerance and physical dependence. Fe& Proc. Fedn Am. Sots exp. Biol. 32: 158. Ho I. K. and Loh II. H. (1974) Effect of L-2,4diaminobutyric acid and /I-alanine on morphine analgesia, tolerance and physical dependence. Fedn Proc. Fedn Am. SOCJ exp. Biol. 33: 501. Ho 1. K., Loh H. H. and Way E. L. (1976) Pharmacological manipulation of y-aminobutyric acid (GABA) in morphine analgesia, tolerance and physical dependence. L$e Sci. 18: 1111-1124. Ho. 1. K. and Gilliland T. (1979) Effects of acute and continuous adminjstration of morphine on the affinity of glutamic acid decarboxylase for pyridoxal S-phosphate. Biochem. Pharmac. 28: 355-360. Hynes M. D., Shearman G. T. and La1 H. (1980) Effect of selected GABA agonists and antagonists on morphine withdrawal body shakes in the rat. Brain Res. BUN. 4: 710. Izumi K.. Munekata E., Yamamoto H., Nakanishi T. and Barbeau A. (1980) Effect of taurine and ~-aminobutyric acid on akinesia, and analgesia induced by DAla?-Met-enkephalinamide in rats. Peprides 1: 139-145. Jacob J. J., Ramabadran K. and Campos-Medeiros M. (I 98 I) A pharmacological analysis of levonantradol antinociception in mice. J. clin. Pharmac. 21: 3723-3338. Johnston G. A. R. (1978) Nemopharmacology of amino acid neurotransmitters. A. Rec. Pharmac. Toxic. 18: 2699289. Kalrignen I. and Vikberg P. (1976) Effects of aminooxyacetic acid and badofen on catalepsy, striatal homovanillic acid increase and antinociception caused by methadone in rats. Acta pharmac. tax. 39: 536-544. Koster R., Anderson M. and DeBeer E. J. (1959) Acetic acid for analgesic screening. Fedn Proc. Fedn Am. Sots exp. Biol. 18: 412. Knoll J. and Zsilla G. (1974) Possible mechanism of the low tolerance capacity of azidomorphine and azidocodeine. Biochem. Pharmac. 23: 745-750. Kuriyama K. and Yoneda Y. (1978) Morphine-induced alterations of y-aminobutyric acid and taurine contents and glutamate decarboxylase activity in rat spinal cord and thalamus: Possible correlates with analgesia action of morphine. Bruin Rex. 148: 163-179. La1 H., Fielding S., Malick J., Shah N. and Usdin E. (1980) GABA neurotransmission: Current developments in ohvsioloav and neurochemistrv. Bruin Res. Bull. Vol. 5. Suppl. 2TAnkho International Inc., New York. Litchfield J. T. and Wilcoxon F. (1949) A simplified method of evaluating dose effect experiments. J. Pharmac. exp. Ther. 96: 99- 113.
Liebman J. M. and Pastor G. (1978) Analgesia elicited by intracerebral injections of baclofen and muscimol. Neurosci. Abstr. 4: 461. Loseher, W. (1980). A comparative study of the pharmacology of inhibitors of GABA-metabolism. ~uunynSchmiedbergs Arch. Pharmac. 315: 119-128. Mantegazza P., Tammiso R., Vincentini L., Zambotti F. and Zonta N. (1980) The effect of GABAergic agents on opiate analgesia. Pharmac. Res. Commun. 12: 239-241. Mayer D. and Price D. D. (1977) Central nervous system mechanism of analgesia. Pain 2: 379-404. Olsen R. W. i 198t) GABA-~nzodiazeoine-barbiturates receptor interactions. J. ~e~rochem. 3% I-13. Ostrovskaya R. U. and Bulaev V. M. (1979) Influence of GABAergic compounds on morphine analgesia effect. Byul. Eksp. Biol. Med. 88: 35-31. Roberts E., Chase T. N. and Tower D. B. (1976) GABA in Nervous System Function. Raven Press, New York. Saelens J. K., Bernard P. S. and Wilson D. E. (1980) Baclofen as an analgesic. Bruin Res. Bzdl. 5 (Suppl. 2): 553-579. Sewell R. D. E. and Spencer P. S. J. (1976) Antinociceptive activity of narcotic agonist and partial agonist analgesics and other agents in the tail-immersion test in mice and rats. Neuropharmacology 15: 683-688. Sivam S. P., Nabeshima T. and Ho I. K. (1981a) Alterations of regional GABA receptors in morphine tolerant mice. Biochem. Pharmoc. 30: 2187-2 190. Sivam S. P., Nabeshima T. and Ho I. K. (1981b) Involvement of GABA in the acute and chronic effects of morphine: nociceptive, GABA release and receptor binding studies. In: Advances in Endogenous and Exogenous Upioids, Proceedings of the International Narcotic Research Conference, Kyoto, Japan, July 26-30, 1981. Kondansha Ltd, Tokyo, pp. 449-451. Sivam S. P., Nabeshima T. and Ho I. K. (1982) An analysis of GABA receptor changes in the discrete regions of mouse brain after acute and chronic treatment of morphine, J. Neurochetn. 39: 933-939. Spaulding T. C., Little J., McCormick K. and Fielding S. (1980) The antinociceptive effects of GABA agonists and antagonists in mice. Brain Res. Bull. 4: 71 l-716. Tzeng S. F. and Ho I. K. (1978) Acute and continuous morphine administration of the y-aminobutyric acid system in the mouse. Prog. Neuro-Psychopharmac. 2: 55-64. Way E. L., Loh H. H. and Shen F. H. (1969) Simultaneous quantitative assessment of morphine tolerance and physical dependence. J. Phurmac. exp. Ther. 167: l-8. Wong C. L., Roberts M. B. and Wai M. (1980) Effect of morphine and naloxone and intestinal transit in mice. EMT. J. Pharmac. 64~ 289-295. Yoneda Y., Takashima S. and Kuriyama K. (1976) Possible involvement of GABA in morphine analgesia. Biochem. Pharmac. 25: 2669-2670. Young A. B., Enna S. J., Zukin S. R. and Snyder S. H. (1976) Synaptic GABA receptor in mammalian CNS. In: GABA in Nervous System Function (Roberts E., Chase T. N. and Tower D. B., Eds), pp. 305-317. Raven Press, New York. Zonta N., Zambotti F., Vincentini L., Tammiso R. and Mantegazza P. (1981) Effects of some GABA-mimetic drugs on the antinociceptive activity of morphine and /I-endorphin in rats. Naunyn-Schmiedbergs Arch. Pharmat. 316: 213-234.
0028-3908/83/060767-08$03.00/O Copyright 0 1983 Pergamon Press Ltd
1983
GABAERGIC DRUGS, MORPHINE AND MORPHINE TOLERANCE: A STUDY IN RELATION TO NOCICEPTION AND GASTROINTESTINAL TRANSIT IN MICE S. P. SWAM and I. K. Ho* Department
of Pharmacology
and Toxicology, University of Mississippi Medical MS 39216, U.S.A.
Center,
Jackson.
(Accepted 16 November 1982)
Summary-Agonists and antagonists of y-aminobutyric acid, i.e. GABAergic drugs, such as muscimol, baclofen or bicuculhne, alone or in combination, exhibited analgesic effects per se and enhanced the analgesia induced by morphine. The analgesic effects of GABAergic drugs were unaffected by admini tration of naloxone in a dose which antagonized the analgesia induced by morphine. The ED,, for the antinociceptive effect of muscimol, bicuculline, picrotoxin, gabaculhne or aminooxyacetic acid (AOAA) was not affected in the morphine-tolerant group as compared to the control group, in contrast to the increase in the ED,, for morphine under similar conditions; this indicated that there was no development of cross-tolerance between morphine and GABAergic drugs. Muscimol suppressed the abrupt withdrawaljumping induced by morphine and enhanced the suppression of this phenomenon by morphine. The GABAergic drugs also shared with morphine the property of inhibiting gastrointestinal (GIT) motility. Naloxone reversed the inhibition of motility induced by morphine but failed to influence that induced by GABAergic drugs. In the morphine-tolerant state, the sensitivity of gastrointestinal motility to morphine decreased, whereas. the sensitivity to GABAergic drugs remained unaltered. The results indicate that GABAergic drugs share some of the classical properties of morphine, such as analgesia and inhibition of gastrointestinal motility, but they probably do so by different mechanisms. Key words:
morphine,
GABA,
GABAergic
drugs,
y-Aminobutyric acid (GABA) has been well established as an inhibitory neurotransmitter at spinal and supraspinal levels of the central nervous system (Roberts, Chase and Tower, 1976; Johnston, 1978; Lal, Fielding, Malick, Shah and Usdin, 1980) and has been implicated in a variety of neurological and behavioral disorders (Enna, 1981). Several studies have indicated a role for GABA in analgesia and tolerance/dependence to opiates (Ho, Loh and Way, 1973, 1976; DeBoer, Metsalaar and Bruinvels, 1977; Tzeng and Ho, 1978; Buckett, 1980; Ho and Gilliland, 1979: Mantegazza, Tammiso, Vincetini, Zambotti and Zonta, 1980; Sivam, Nabeshima and Ho, 198 la, b, 1982) and also in the antagonism of opiateinduced convulsions (Dingledine, Iversen and Breuker, 1978). However, the pharmacological manipulation of the GABA system using GABAergic drugs as tools, have revealed useful but conflicting results regarding the role of GABA in the effects of opiate drugs (Ho et al., 1976; Biggio, Della Bella, Frigeni and Guidotti, 1977; Mantegazza et al., 1980; Spaulding, Little, McCormick and Fielding, 1980;
*Send correspondence to: Dr I. K. Ho, Department of Pharmacology and Toxicology University of Mississippi Medical Center 2500, North State Street, Jackson, MS 39216, U.S.A.
gastrointestinal
transit,
analgesia,
opiate
tolerance.
Izumi, Munekata, Yamamoto, Nakanishi and Barbeau, 1980; Zonta, Zambotti, Vicentini, Tammiso and Mantegazza, 1981). Both agonists and antagonists of GABA have been reported to possess analgesic activity (Spaulding et al., 1980; DeFeudis, 1982). Contrary to this, Zonta el al. (1981) reported that bicuculline, a postsynaptic GABA receptor antagonist. reversed the effects of muscimol, a GABA agonist, which itself antagonized the analgesia induced by morphine or /I-endorphin in rats. Thus, there are conflicting reports with respect to the interaction between GABAergic drugs themselves and also in combination with opiates. In order to clarify this aspect, the present study was undertaken to assess the effects of GABAergic drugs alone, or in combination with morphine, on nociception and gastrointestinal transit with special reference to the development of tolerance to morphine.
METHODS
Animals
Male ICR mice (Charles River Breeding Labs, Wilmington, MA) weighing 25-30 g were maintained in a room at a temperature 24 + 1°C under artificial 12/12 hr lighting cycles were used; water was given ad libitum.
S. P. SWAM and I. K. Ho
768 Chemicals
used
Muscimol, bicuculline, picrotoxin, aminooxyacetic acid and gabaculline were purchased from Sigma Chemical Company, St Louis, MO; baclofen was generously supplied by Ciba-Geigy, Summit, NJ. Morphine sulfate was obtained from Mallinckrodt Chemical Works, St Louis, MO. Naloxone hydrochloride was a gift from the Endo Laboratories, Garden City, NY. All chemicals were dissolved in saline (0.9% w/v NaCl) and injected in a volume 0.1 ml/10 g body weight. Induction
qf tolerance to morphine
Tolerance to morphine was induced by the pellet (75 mg morphine base, s.c.) implantation method of Way, Loh and Shen (1969). The groups referred to as “tolerant” were those in which the pellets were removed for 8 hr after a 72 hr implantation period. Control animals received placebo pellets. Influence ofmuscimol on the abstinence jumping due to abrupt withdrawal of morphine The effect of muscimol on the spontaneous jumping behavior observed 10 hr after the removal of the pellets, which were implanted for 72 hr to induce dependence on morphine, was studied by injecting muscimol and morphine alone or in combination. Assessment
of analgesia by antinociceptive
tests
(A) Tail immersion antinociceptive test. The method is essentially that reported by Sewell and Spencer (1976). The mouse was restrained in an individual, ventilated rigid rectangular Plexiglass container and the nociceptive reaction time (in seconds) was determined when the tail was immersed in a constanttemperature water bath set at 50°C. The nociceptive endpoint was characterized by either a violent jerk of the tail or a rapid “flinch” of the whole body of the mouse. A 20 set cut-off time was imposed for all animals that failed to respond to the stimulus. This particular test as claimed by Sewell and Spencer (1976) possesses two distinct advantages: first, the use of wet heat transfer has provided more consistent responses than the radiant heat and it is possible to use a relatively low temperature as the nociceptive stimulus; secondly, this low temperature also minimizes local tissue damage in the tail, permitting repetitive measurements to be made on individual animals at frequent intervals. The response to the graded doses of muscimol, given subcutaneously, alone or in combination with a fixed dose of morphine, was observed both in morphine-tolerant and non-tolerant (control) animals, at 0, 30, 60 and 90 min. The 0 time represents the responses taken 15 min prior to injection of the test drugs. In another set of experiments using naive mice, the effect of naloxone and bicuculline on the response to muscimol was investigated.
(B) Acetic acid-induced abdominal writhing (AA W) test. Tests were carried out to determine the antinociceptive effects of test drugs in terms of their ability to inhibit abdominal writhing induced by 0.6% acetic acid, 0.1 ml/10 g body weight, (Koster, Anderson and DeBeer, 1959). A writhing was defined as a characteristic contraction of abdominal muscles accompanied by an extension of the hind limbs. The number of writhes occurring during a 10 min period after 10min of the injection of acetic acid was recorded. The ED,, value (Litchfield and Wilcoxon, 1949) was defined as the dose of the test drug capable of reducing by 50% the number of writhes occurring in control mice. The test drugs were given subcutaneously immediately prior to the injection of acetic acid. The ED,, values for muscimol, bicuculline, picrotoxin, gabaculline, aminooxyacetic acid and morphine were determined both in morphine-tolerant and non-tolerant animals. In addition, the effect of naloxone and bicuculline on muscimol or baclofeninduced antinociception was assessed in a separate set of experiments. Assessment qfgastrointestinal coal meal test
(GIT)
transit by char-
The gastrointestinal transit was measured essentially following the method of Wong, Roberts and Wai (1980) by a charcoal meal test. The animals were fasted for 16 hr before use during which period they had access only to tap water. The animals were given 0.25 ml of an aqueous suspension of 2.57/, charcoal and 1.25% gum acacia by stomach tube as a per oral bolus. Twenty minutes later the mice were sacrificed by cervical dislocation and the stomach and intestines were excised from the gastro-esophageal junction to the ileo-caecal junction. The distance the charcoal meal had travelled from the pylorus was measured. The drugs were injected subcutaneously, (aminooxyacetic acid, intraperitoneally) 15 min before giving the charcoal meal. Control animals received saline injections at the corresponding time schedule. At least 6 animals were used for each dose. Different sites of injection were chosen when two or more drugs were used. Statistical
analysis and the expression
of results
For the ED,, calculations, the Litchfield and Wilcoxon (1949) method was used. The data on analgesic tests were expressed as a percentage of control values. The statistical significance of the treatments was calculated on the original data, i.e. before normalization into percentages by Student’s t-test [time-effect relationship with respect to the control (Figs l-3) or control versus treated groups (Figs 4, 7)] and analysis of variance followed by Student-Neuman-Keuls test (comparison of the means of different treated groups; the significant results are indicated in the “Results” section). The Chi-square test was used on the original data of the withdrawal jumping (based on jumping versus non-
GABAergic
1. Antinociceptive effect Fio (pkebo) and morphine-tolerant
of
drugs
muscimol in control mice using the tail-
immersion test. The results are expressed as the percentage increase from the control level at the respective time points. *P < 0.05; **P < 0.1; ***P < 0.001 compared to the con-
769
and morphine
Fig. 3. Effect of bicuculline and naloxone on the muscimolinduced analgesic response in naive mice using the tailimmersion test. Seven mice were used in each group. Other
explanations are as for Fig. 1.
trol (Student’s t-test). Eight mice were used in each group except the placebo-saline group which consisted of 7 mice.
50
r
jumping) before normalization into percentages of animals (Figs 5 and 6). A P-value of at least ~0.05 between two means was considered significant. RESULTS
Effect of GABAergic nociceptive test
drugs on tail immersion
anti-
The tail-flick response time in control animals was 3.74 k 0.45 set (mean k SE, n = 7). In control animals muscimol produced a dose-related (0.25,0.5 and 1 mg/kg) increase in analgesic threshold (Fig. 1). At 30 min, there was a significant difference (P < 0.05) between the 0.25 and 1 mg/kg dose; at 60 min there were significant differences between the three doses except between 0.5 and 1 mg/kg. In tolerant animals, a paradoxical response to muscimol was observed in that at 30 and 60 min, the larger dose (1 mg/kg) was less effective (P < 0.05) than the smaller (0.25-0.5 mg/kg) doses (Fig. 1). At 60 min, there was a significant difference (P < 0.05) between the smaller doses. The combination of a dose (0.25, 0.5 or 1 mg/kg) of muscimol with a fixed dose of morphine (4 mg/kg)
Fig. 4. Effect of GABAergic drugs and combinations on the acetic acid-induced writhing test in naive mice. MUS = muscimol; BIC = bicuculline; NAL = naloxone; BAC = baclofen. *P < 0.05; **P < 0.01; ***P < 0.001 compared to the control (Student’s r-test). Six animals were used in each group.
elicited in general an enhanced response compared to either agent alone. In control animals at 30 min, there was a significant difference (P < 0.05) between the saline plus morphine group and groups given 0.5 and 1 mg/kg ofmuscimol. Groups given 0.25 and 1 mg/kg of muscimol were also significantly different from each other. In the tolerant group, 0.25 mg/kg of
100
r ________O________~________~
80
----____*_ --._
‘\ 6.
‘\\\\
-___
.%._
-o--_____~-.-________. -.%____---_-&
‘\
40 20
j__
0
'\ '0.. 9 *.._ _/_ -..O__r-C
*
I 0
Fig. 2. Antinociceptive effect of muscimolkmorphine combination in placebo and morphine-tolerant mice using the tail-immersion test. Seven mice were used per group except the saline + saline groups which had 8 mice per group. Other explanations are as for Fig. I.
30
60
Illl”l
. 0 0
YUICllM 0I5mI,kI OSn~,kg YUIClloL 1om1,11 WLCHloL PLICml -----mtlln
90
IIME(min) Fig. 5. Effect ofmuscimol on spontaneous jumping behavior induced by abrupt withdrawal of morphine. The number of
animals used for saline, muscimol 0.25, 0.5 and 1.0 mg/kg, respectively
are: placebo:
7, 7, 8, 8; tolerant:
9, 10, 9, 8.
S. P. SWAM and I. K. Ho
770
----__--
.!@ct of GABAergic writhing (AA W) test
c----____-~__ --__ SALINE
+ SALINE
SALINE
+ MORPHINE
MUSCIMOL
(O.Smg/kg]
-
Ptlctso
-----
TOLERANT
+ MORPHINE
.’
0
--‘O
(IOmg/kgi
30
60
.’
r*
c’
P
90
TIMEImid Fig. 6. Effect of muscimol and morphine alone, and in combination with morphine, on spontaneous jumping behavior induced by abrupt withdrawal of morphine. The number of animals used for saline + saline, saline + morphine, muscimol + morphine groups, respectively are: placebo: 7. 10, 10; tolerant: 10, 10, 12.
Muscimol, bicuculline or baclofen produced inhibition of acetic acid-induced writhing, thereby showing an analgesic effect (Fig. 4). A combination of muscimol and bicuculline or baclofen and bicuculline was more effective (P < 0.05) than either agent alone. Naloxone (15 mg/kg) did not significantly antagonize the effects of muscimol, bicuculline or baclofen, although it did significantly (P < 0.05) antagonize the effect of a combination of muscimol and bicuculline; smaller doses of naloxone failed to influence the analgesic effects of GABAergic drugs (data not shown). The ED,+ of these drugs for analgesia were not different in tolerant animals compared to the placebo group (Table 1); in contrast, the ED,, for morphine was increased in the tolerant group. Effect of muscimol behaaior
muscimol enhanced the response to morphine significantly at all time points tested whereas 0.5 and 1 mg/kg of muscimol inhibited the response significantly at 60min (Fig. 2). In order to elucidate the nature of the analgesic response to muscimol, naloxone and bicuculline (an opiate and a GABA antagonist, respectively) were employed. As shown in Fig. 3, in naive animals bicuculline per se produced significant analgesic effects. Muscimol significantly enhanced the analgesia induced by bicuculline at 30min. Naloxone in a large dose (15 mg/kg) significantly inhibited analgesia induced by muscimol at 60 min but failed to influence analgesia induced by bicuculline, although the values were still significantly different from control values. Naloxone, in doses of 2.5 or 10 mg/kg, failed to influence analgesia induced by either bicuculline and muscimol; in contrast, 2.5 mg/kg naloxone was sufficient to inhibit analgesia induced by morphine (4mg/kg) (data not shown).
Table
drugs on acetic acid induced-
on morphine-abstinence
jumping
Morphine-tolerant animals exhibited the characteristic spontaneous withdrawal (abstinence) jumping. Muscimol produced a dose-related inhibition of the withdrawal jumping; however, the values were statistically significant only with a 1 mg/kg dose level at 30, 60 and 90 min (Fig. 5). Morphine suppressed the withdrawal jumping (Fig. 6); this effect was significantly enhanced at 30 and 60min when 0.5 mg/kg muscimol was given with morphine. Eflect of muscimol on the gastrointestinal sit
(GIT) tran -
In the control group, morphine induced a decrease in the gastrointestinal transit as did GABAergic drugs such as muscimol, baclofen, bicuculline and aminooxyacetic acid. The combined effect of morphine and GABAergic drugs was greater than that of either agent alone (Fig. 7). Naloxone (5mg/kg) antagonized the effect of morphine, but even in large
1. Effect of morphine and GABAergic drugs on acetic induced writhing in control and morphine-tolerant mice
Compound
Placebo
Morphine Muscimol Bicuculline Picrotoxin Gabaculline AOAA
0.51 (0.33 - 0.65) 0.76 (0.38 - I .4) 0.83 (0.54 - 1.5) 0.32 (0.2 - 0.48) 5.2 (2.9 - 9.4) 8.4 (4.4 - 16.0)
acid-
Tolerant 3.54 (2.3 - 4.6) 0.45 (0.21 - 0.94) 0.63 (0.42 - 0.92) 0.15 (0.1 - 0.23) 3.80 (2.1 - 6.84) 6.51 (3.83 - 11.1)
Acetic acid (0.6x, 0.1 ml/log body weight) was given intraperitoneally. The number of writhes occurring during a lo-min period, after 10 min of the injection of acetic acid was recorded. Test drugs were given immediately prior to the acetic acid injection. Gabaculline and aminooxyacetic acid (AOAA) were injected 24 and 5 hr prior to testing, respectively. At least 4 doses of each drug were used for ED,, calculations; 9-12 mice were used for each dose. The values represent ED,, (95% confidence limits) calculated according to Litchfield and Wilcoxon (1949).
GABAergic
drugs
and morphine
Fig. 7. Effect of GABAergic drugs alone or in combination on gastrointestinal transit (GIT-T) of mice. MOR = morphine; NAL = naloxone; MUS = muscimol; BIC = bicuculline; AOAA = aminooxyacetic acid. Each group consisted of 6 mice except the control which consisted of I6 mice. *P < 0.05; **P < 0.01; ***P < 0.001 compared to the control (Student’s t-test).
doses (10 or 15 mg/kg) it failed to influence the effect of GABAergic drugs. The development of tolerance to morphme in this test was also observed as shown by the decrease in sensitivity to morphine. However, the morphine-tolerant animals did not develop tolerance to any of the GABAergic drugs tested (Table 2). DISCUSSION
Several reports have provided evidence to ascribe a possible involvement of the GABA system in the analgesic and addictive properties of opiates (Ho er al., 1973, 1976; Ho and Loh, 1974; Kuriyama and Yoneda, 1978; Buckett, 1980; Mantegazza et al., 1980; Hynes, Shearman and Lal, 1980; Sivam et al., 1981a, b, 1982). The interpretation of the results of the studies of opiate-GABAergic drug interactions is complicated by the fact that the GABAergic drugs, irrespective of the taxonomic classification as agonist or antagonist, appear to possess analgesic properties per se. In the following paragraphs, an appraisal of the literature on opiate-GABAergic drug interactions with reference to the present study is presented. Antinociceptive @ects
of GABAergic agents per se
The present results showed that muscimol, a GABA agonist on the postsynaptic GABA receptors, as well as bicuculline, a postsynaptic GABA antagonist, or baclofen, which enhances the release of GABA, elicited analgesia. The analgesia induced by muscimol was enhanced by bicuculline. Naloxone, an opiate antagonist in doses which fully antagonized the analgesia induced by morphine did not influence the analgesia induced by muscimol or bicuculline. These results were seen in two different models of the generally employed analgesic tests, namely thermally mediated (tail-flick) and chemically induced [acetic
acid-induced writhing (AAW)] tests. Though the tail-flick test is considered to be mainly at the spinal reflex level, the acetic acid-induced writhing test appears to be more supraspinal (Mayer and Price, 1977). It has also been shown that opiate drugs could completely inhibit acetic acid-induced writhing when they were injected into the cerebral ventricle in nanogram quantities (Sivam et al., 1982; Sivam and Ho, unpublished observations). The present results are consistent with the earlier reports that GABAergic agents elicit analgesia in the mouse and rat using tail-flick (Spaulding et al., 1980; Saelens, Bernard and Wilson, 1980) or hot-plate tests (Liebman and Pastor, 1978; Christensen, Arnt and ScheelKriiger, 1978; Buckett, 1980; Spaulding et al., 1980). However, Biggio et a/. (1977) did not observe an analgesic effect of muscimol in the rat and mouse tail-flick test. Further, the GABA-T inhibitors such as aminooxyacetic acid, y-acetylenic GABA, y-vinyl GABA were reported to possess analgesic properties in the mouse and rat tail-flick or the hot-plate method (Contreras, Tamayo and Quijada, 1979; Buckett, 1980; Spaulding et al., 1980; Liischer, 1980). The present experiments and those of other workers clearly show that the GABAergic drugs per se elicit analgesia (see review, DeFeudis, 1982). Since the analgesia was shared by an antagonist as well, it probably was not mediated by the GABA receptor itself. However, it should be noted that GABA receptors which are insensitive to bicuculline have been postulated (Andrews and Johnston, 1979; Bowery, Doble, Hill, David, Hudson, Shaw, Turnbull and Warrington, 1981). Further, the GABA antagonist, bicuculline induced an additive analgesic effect, indicating a possible common mode of action for both agonist and antagonist. Naloxone, an opiate antagonist, in high doses only, partially antagonized
112
S. P. SWAM and I. K. Ho Table
2. Effect of GABAergic
drugs transit
on the gastrointestinal GIT transit
(GIT)
(cm)
Dose Compound Saline Morphine
Muscimol
Baclofen
Bicuculline
AOAA
(mg/kg) 0.25 0.5 1.0 2.0 4.0 0.5 1.0 2.0 3.0 3.0 6.0 12.0 24.0 0.25 0.5 1.0 1.5 5.0 10.0 20.0 40.0
Control 32.8 + 24.0 + 17.3 * Il.3 + 9.1* 30.0 23.3 18.2 14.3 29.5 28.0 20.3 18.4 33.3 26.0 22.1 21.3 32.0 34.0 24.0 23.3
1.4 4.3* 1.8*** 0.3*** 1.2***
* 2.1 e 4.1* k 3.2** &-4.0*** + 4.6 * 3.5 k 2.3* k 1.9** 5 2.9 k 3.5 * 1.5* k 1.7** k 3.2 * 2.0 k 1.O* + 3.5*
Tolerant 37.3 _t 1.5
30.7 * 1.8* 28.7 k 2.0* 17.7 * 0.3** 27.3 k 5.0 22.0 k 4.6* 20.3 k 4.5** 31.0 + 1.0 29.Ok2.1’ 27.3 k 2.9* 32.0 + 0.6 27.6 k 1.9* 22.5 f 1.8** 32.7 + 2.9 33.3 + 2.4 28.7 + 0.7*
Gastrointestinal (GIT) transit was assessed by a charcoal meal test (aqueous suspension of 1.25% gum accacia + 2.5% charcoal). The drugs were given 15 min prior to the charcoal meal. The animals were sacrificed 20min after the charcoal meal. Aminooxyacetic acid (AOAA) was given 5 hr prior to the charcoal meal. Each group consists of 6 mice, except for the saline control groups which consisted of 16 mice. All drugs were given subcutaneously, except AOAA which was given by the intraperitoneal route. *P < 0.05;
**p < 0.01; ***P < 0.001 compared to control (Student’s t-test).
the analgesia induced by muscimol in the tail immersion test; however, in the acetic acid-induced writhing test, the antagonism was restricted to the combination of muscimol and bicuculline and was not evident on the individual analgesic response. The results indicate that the GABAergic-induced analgesia may have some naloxone-sensitive component depending on the analgesic test used and acting probably through opiate-receptor interaction. However, in receptor binding studies, GABAergic drugs do not seem to displace labelled opiates (Enna and Snyder, 1975; Sivam and Ho, unpublished observations). On the other hand, opiates in very large concentrations (10m3 M) displace GABA receptor binding (Dingledine et ul., 1978; Sivam et al., 1982). How the naloxone-insensitive analgesia induced by the GABAergic drugs is mediated, remains unknown. It may be speculated that they act on some naloxone-insensitive opiate receptors, if they exist. At this juncture, it is relevant to cite that levonantradol, a cannabinoid analogue, has been shown to be l&30 times more potent than morphine as an analgesic, yet the analgesic action was only partially antagonized by naloxone in large doses (Jacob, Ramabadran and Campos-Medeiros, 1981). It is worth noting in this context that the analgesia induced by GABAergic drugs does not induce toler-
ance or dependence (Buckett, 1980) a classical effect of opiate drugs. Moreover, the primary site of action of GABAergic drugs has been suggested to be at the spinal level (Spaulding et al., 1980) unlike the wellknown effect of morphine on spinal and supraspinal loci (Dewey, Snyder, Harris and Howes, 1969; Mayer and Price, 1977). The present results using the acetic acid-induced writhing test, show that GABAergic drugs might also act at the supraspinal level. Injuence qf GABAergic compounds on tolerunce and dependence to opiates In the acetic acid-induced writhing test, GABAergic drugs exhibited analgesia in tolerant animals but there was no classical development of crosstolerance between these drugs and morphine. Here too, both agonist and antagonist behaved similarly. This further supports the idea that they may have some common mode of action. In the tail immersion test, muscimol enhanced the analgesic effect of morphine, both in non-tolerant and tolerant animals. However, larger doses inhibited analgesia induced by morphine in tolerant but not in non-tolerant animals; the nature of the action of the larger doses of muscimol to produce this paradoxical effect is unclear. Muscimol inhibited the characteristic abrupt morphine-induced withdrawal jumping, and en-
GABAergic drugs
hanced the suppression of this phenomenon by morphine. It is not clear whether the naloxonesensitive and/or the sedative effect of muscimol contributed to the suppression. A number of reports in the past have dealt with the influence of GABAergic agents on analgesia induced by opiates. However, the results are disparate extending from lack of effect to potentiation or antagonism. For example, muscimol or GABA-T inhibitors, or sodium v,alproate enhanced analgesia induced by morphine in the mouse or rat (Knoll and Zsilla, 1974; Kllrianen and Vikberg, 1976; Biggio et al., 1977; Yoneda, Takashima and Kuriyama, 1976; Contreras et al., 1979; Ostrovskaya and Bulaev, 1979; Buckett, 1980). Others have reported an antagonism of opiateinduced analgesia (Izumi et al., 1980). Christensen et al. (1978) reported that muscimol failed to affect analgesia induced by morphine on paw stimulation. The GABA uptake inhibitors such as nipecotic acid, guvacine, or 2,4_diaminobutyric acid have been found to inhibit analgesia induced by morphine (Ho and Loh. 1974; Mantegazza et al., 1980); similar results were observed for the GABA synthesis inhibitor, semicarbazide and for the GABA antagonist bicuculline (Yoneda et al., 1976; Ostrovskaya and Bulaev, 1979). Many of the discrepancies seem to arise from one or more of the following considerations: (a) lack of investigation of the time course of the response or dose-response; (b) the diverse criteria used to measure analgesia and the choice of analgesic tests; (c) lack of investigation of the effect of the pretreatment per se; (d) routes of administration (e.g. systemic versus intracerebral routes); and (e) an a priori assumption that an antagonist should “antagonize” in any event. Further, the complexity of the GABA system might have also contributed. Biochemical studies have revealed that the GABA receptors appears to be a part of the protein complex containing receptor sites for GABA, benzodiazepines and picrotoxin/barbiturate/ chloride ionophore (Olsen, 1981). In addition to the classical postsynaptic GABA receptors, there also appear to be receptors located on GABA terminals (autoreceptors) which, when activated, inhibit release of GABA (Brennan, Cantril and Kiogsgaard-Larsen, 1981). Electrophysiological studies have shown that virtually all neurons and glia in the CNS are responsive to iontophoretically applied GABA, even those that do not receive a GABAergic input (Crawford and Curtis, 1964). Because of these characteristics, it is extremely difficult to draw definite conclusions about the physiological relevance of an action observed following the administration of GABAergic agents. While GABA receptors are found in virtually all areas of the brain, recent ligand binding studies indicate that the affinity and distribution of GABA receptors varies between different brain regions (Young, Enna, Zukin and Snyder, 1976; Sivam et al., 1982). It is possible that GABAergic drugs may possess divergent abilities to alter
713
and morphine
GABA function in different regions resulting in a net change in the differential inputs from various regions. This may explain the divergent results obtained in the in vivo studies. Efect
of GABAergic
intestinal
(GIT)
drugs
and morphine
on gastro-
transit
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