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GABAA ANTAGONIST AND NICOTINE-INDUCED ANTINOCICEPTION
pp. Copyright 01997 +
PII S0024-320S(97)00716-9
E
PHARMACOLOG”YLETTERS Accelerated Communication
GABAAANTAGONIST AND NICOTINE-INDUCED ANTINOCICEPTION Wui-Chiu Mui,,# Vlasta K. Zbuzek~+ and Wen-Hsien WU* Department of Anesthesiology, #Chia Yi Christian Hospital, 539 Chung Hsiao Road, Chia Yi, Taiwan, and *University of Medicine and Dentistry of New Jersey, New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103-2714, USA (SubmittedMay6, 1997; acceptedMay9, 1997; receivedin tinafformJune30,1997) Abstract Rats, pretreated with salineor GABAAantagonist bicuculline(BIC) at the doses of2, 4, 6, 10 and 20mg/kg, were injectedwith nicotine(NIC, lmg/kg ) 30 min later. Tail-flick(TF) latencies were measured before (baseline) and three times at 10 min interval after pretreated compounds, continued every 10 min up to 1 hr after NIC injection. In all groups, median TF latencies did not change from the baseline for the first 30 min after the pretreatment, Following NIC alone (control) and in the group pretreated with 2mg/kgBIC , 60°/0rats reached ceilingTF Iatencies(20 see) lasting for 10 min. In groups with higherBIC doses (4 to IOmg/kg), median TF latencies were in the range of 5-7 sec with 30°/0rats reaching the ceilingTF latencies. Following 20mg/kg BIC, one out of five rats reached 20 see; the median was in the range of 4-5 sec. Significantlylower responses were observed following4mg BIC and higher doses with no differenceamong the groups. In conclusion, our novel data show that BIC alone, injectedsystemically,does not possess any effect on the thermal nociceptive transmissionas measured by the tail flicktest. However, pretreatment with BIC partially prevents NIC-induced antinociception, in a non dose related manner. This suggests that GABAA receptors may, at least in part, contribute to the complex mechanisms involved in NIC-induced antinociception. Q 1997 nicotine,tail-fficktest,antinociception KqJ Wordr: bicucuffine, Introduction The mechanisms involved in nicotinic antinociception are both profound and perplexing. Accumulated evidence suggests that neuronal nicc)tinicacetylcholine receptors are involved in regulating neurotransmitter release at different synapses, such as dopaminergic, serotonergic, GABAergic, adrenergic, cholinergic and glutamatergic (l). Recent studies revealed that antinociception produced by nicotine is mediated via multiple spinal mediators including ctz adrenergic, serotonergic, and muscarinic cholinergic systems (2). In addition, subcorticalmicroinjection of a nicotinic agonist may activate descending pain inhibitory pathway, which, in tuw may activate the lumbar adrenergic, serotonergic, and cholinergiccircuits modulating pain transmission (3). Dopamine receptors D1 and D2 were shown not to be involved in antinociception induced by nicotine in mice, using the TF test (4).The Iigand-gated monotropic GABAAreceptors are one of the most significantreceptors in the CNS, regulating antinociception (5-7). However, the role of GABA* receptor in nicotine-induced antinociception has not yet been reported. + Author for correspondence
PL-222
NicotineAnalgesiaandBicuculline
Vol.61,No. 15,1997
In the present study, the TF test was employed to investigate the effect of different doses of the GABAAantagonist, bicuculline(BIC), on the antinociception induced by nicotine (NIC) in rats.
Material and Methods Experimental Animals: Males Sprague-Dawleyrats (n=54) weighting 300 to 350 gm were used in this study. They were kept two per cage at a 12:12 h light:dark cycle at the constant temperature of 22+.0.5°Cand food and water at libitum. The research protocol was approved by the Institutional Animal Care and Use Committee. Tail-Flick Test: Nociceptive responseswere assessedby the TF test, employingthe analgesiameter (model 33, Innovators in InstrumentationInc.) ~ describedin detail elsewhere(8,9). The nociceptive endpoint was set at 20 see, to prevent tissue damage. The light beam was always projected within a lcm in diameter, just 3cm above the tip of the tail as different tail areas may possess different sensitivity (10). The beam intensity dial was set to obtain basal TF latencies between 5-7 sec. Drugs and Dosage: Three drugs were used in this study: normal saline, injected S.C.in the volume of lmlkg; bicuculline(Sigma) 2,4,6,IO,and20 mg/kg, injected i.p. in the volume of 2mWg; nicotine free base (Sigma), lmg/ kg, injected S.C.in the volume of 2.5ml/kg, 30 minutes afler the injection of pretreated saline (control) and different doses of BIC. Experimental Protocol: All rats were acclimated in restrainers two hours a day for 2 days before the study. Experiments were carried out at the same time of day to avoid diurnal variation. Before each experiment,rats were rdlowedto rest in the restrainerfor 2 hours, then two consecutivebaseline TF latencywere measuredwith a 5 minuteinterval, before the injectionof the test compounds.Atler the baselineTF latencieswere recorded, saline or BIC at differentdoses were injected.Each rat was observed for any side effect following BIC injection, and TF latencies were measured every 10 minutesthree times. Then,NIC was injected to all rats. TF latencieswere recorded every 10 minutes for 60 minutes. Calculationand StatisticalAnalysis: The valuesof TF latencieswere calculatedas medianfor each group and expressed graphically. The area under the curve (AUC) was calculated for each rat, presented as mean =tSEfor each group. Statistical analysisof the AUC means was assessed by one way ANOVA. Tukey-Kramar HSD (honestly significant difference) test was used for multiple comparisons among the groups (P
Results No noticeable side effect was observed followingBIC i.p. injection. There was only, short period lasting, biting, scratching, and vocalization, occasionally noticed among all the rats. No seizure occurred throughout the whole course of the study. In control, saline pretreated group, 1 out of 10 rats experienced slight tremor within the first minute followingNIC injection. No tremor was observed in rats pretreated with BIC. The time course of the antinociceptive responses of all 6 groups are presented as median TF latencies in Fig. 1. The pretreatment with saline and all doses of BIC did not change the latencies from the baseline, ranging from 4 to 7 sec.
PL-223
NicotineAnalgesiaandBicucuIline
Vol.61,No. 15,1997
-40
-20
0
20
40
60
so
T I M E (m in)
Fig. L Median values of tail-flick. latency following the injection of nicotine (lmg/kg S.C.at time O) in rats pretreated at time -30 min with saline(o) and bicuculline;the doses in mg per kg body weight were: 2(=); 4 (~); 6 (V); 10 (+); and 20 (0).
TABLE I Mean values + SE of AUC and number (percentage) of rats responding to nicotine-inducedantinociceptionfollowingdifferentdoses of BIC, pretreated 30 min earlier. Groups
N
BIC(mg/kg) pretreated
No. responders
AUC Mean + SE
I
I
10
I
O
6 (60%)
I
817.35+ 41.48
II
I
10
I
2
6 (60%)
731.37 + 41.47
III
I
10
I
I
4
3 (30%)
I
539.38+ 41.48 *
IV
I
9
I
6
3 (30%)
I
519.49+ 43.’73*
3 (33.3%) I
466.14+ 40.48 *
1 (20%)
451.78+ 58.66 *
* significantlydifferent from control group; p <0.001
\
PL-224
NicotineAnalgesiaandBicuculline
Vol.61,No. 15,1997
Control rats and those pretreated with 2mg/kgBIC, exhibitedsimilarpattern atler NIC injection.The maximalantinociceptiveresponse occurred 10 min after MC, returningto the baselinewithin 30 min (Fig. 1). In both groups, 60% of rats reached maximalresponse (Table 1). Pretreatment with higher doses of BIC, from 4 to 20 mg/kg, reduced antinociceptive efl’ectof NIC, expressed as median TF latencies. The number of rats reachingthe ceilingTF Iatenciesdecreased by 50V0in groups pretreated with 4, 6 and 10 mg/kg BIC, compare to saline + NIC (control) and BIC 2mg/kg + NIC groups, Following 20mg/kg BIC, 1 out of5 rats (20%) responded to NIC (Table 1). One way ANOVA of the mean AUC values for six groups revealed significantdifference among the groups (p
Vol.61,
15,1997
NicotineAnalgesiaandBicuculline
PL-225
test) should be used. The other question is, whether intrathecally administrated BIC completely suppresses NIC-induced antinociception To answer this question, a preliminary study is in preparation. Our data are in the agreementwith the generalnotionthat, GABAAreceptors have an inhibitoryeffect in nociceptive transmission. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
L.W. ROLE and D.K. BERG, Neuron M 1077-1OS5(1996) D.T. ROGERS and E.T. IWAMOTO, J. Pharmacol. EXP.Ther. 2!5?341-349 (1993) E.T. IWAMOTO and L. MARION, J. Pharmacol. EXP.Ther. ~ 777-789 (1993) M.I. DAMAJ and B.R. MARTIN, Psychopharmacology~ 106-108 (1993) L.H. LIN, P. WHITING and R.A. HARRIS, J. Neurochem. @ 1548-1553 (1993) X.H. DING, X.Q. JI and K TSOU, Pain ~ 371-376 (1990) L.M. AANONSEN and G.L. WILCOX, J. Pharmacol. Exp. Ther.~ 1034-1038 (1989) C.Y. YANG, W.H. WU and V.K. ZBUZEK, psychopha~acology l!M 417-420 (1992) C.W.Y. CHIN, R.C. BLOCK, W.H. WU and V.K.ZBUZE~ Psychopharmacologym 497499 (1993) B.C. YOBURN,R. MORALES,D.D. KELLY and C.E. INTURRISI,Life Sci.M 1755-1762 (1984) T. ISHIKAWA and T.L. YAKSH, Japanese Journal of Anesthesiology.~ 439-444 (1996) T, YAMAMOTO and T.L. YAKSH, Pain. ~ 79-84 (1993) M.D. ACETO, R.S. BAGLEY, W.L.DEWEY, T.C. FU and B.R. MARTIN, Neurophannacol. ~ 1031-1036(1986)
PII S0024-320S(97)00716-9
E
PHARMACOLOG”YLETTERS Accelerated Communication
GABAAANTAGONIST AND NICOTINE-INDUCED ANTINOCICEPTION Wui-Chiu Mui,,# Vlasta K. Zbuzek~+ and Wen-Hsien WU* Department of Anesthesiology, #Chia Yi Christian Hospital, 539 Chung Hsiao Road, Chia Yi, Taiwan, and *University of Medicine and Dentistry of New Jersey, New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103-2714, USA (SubmittedMay6, 1997; acceptedMay9, 1997; receivedin tinafformJune30,1997) Abstract Rats, pretreated with salineor GABAAantagonist bicuculline(BIC) at the doses of2, 4, 6, 10 and 20mg/kg, were injectedwith nicotine(NIC, lmg/kg ) 30 min later. Tail-flick(TF) latencies were measured before (baseline) and three times at 10 min interval after pretreated compounds, continued every 10 min up to 1 hr after NIC injection. In all groups, median TF latencies did not change from the baseline for the first 30 min after the pretreatment, Following NIC alone (control) and in the group pretreated with 2mg/kgBIC , 60°/0rats reached ceilingTF Iatencies(20 see) lasting for 10 min. In groups with higherBIC doses (4 to IOmg/kg), median TF latencies were in the range of 5-7 sec with 30°/0rats reaching the ceilingTF latencies. Following 20mg/kg BIC, one out of five rats reached 20 see; the median was in the range of 4-5 sec. Significantlylower responses were observed following4mg BIC and higher doses with no differenceamong the groups. In conclusion, our novel data show that BIC alone, injectedsystemically,does not possess any effect on the thermal nociceptive transmissionas measured by the tail flicktest. However, pretreatment with BIC partially prevents NIC-induced antinociception, in a non dose related manner. This suggests that GABAA receptors may, at least in part, contribute to the complex mechanisms involved in NIC-induced antinociception. Q 1997 nicotine,tail-fficktest,antinociception KqJ Wordr: bicucuffine, Introduction The mechanisms involved in nicotinic antinociception are both profound and perplexing. Accumulated evidence suggests that neuronal nicc)tinicacetylcholine receptors are involved in regulating neurotransmitter release at different synapses, such as dopaminergic, serotonergic, GABAergic, adrenergic, cholinergic and glutamatergic (l). Recent studies revealed that antinociception produced by nicotine is mediated via multiple spinal mediators including ctz adrenergic, serotonergic, and muscarinic cholinergic systems (2). In addition, subcorticalmicroinjection of a nicotinic agonist may activate descending pain inhibitory pathway, which, in tuw may activate the lumbar adrenergic, serotonergic, and cholinergiccircuits modulating pain transmission (3). Dopamine receptors D1 and D2 were shown not to be involved in antinociception induced by nicotine in mice, using the TF test (4).The Iigand-gated monotropic GABAAreceptors are one of the most significantreceptors in the CNS, regulating antinociception (5-7). However, the role of GABA* receptor in nicotine-induced antinociception has not yet been reported. + Author for correspondence
PL-222
NicotineAnalgesiaandBicuculline
Vol.61,No. 15,1997
In the present study, the TF test was employed to investigate the effect of different doses of the GABAAantagonist, bicuculline(BIC), on the antinociception induced by nicotine (NIC) in rats.
Material and Methods Experimental Animals: Males Sprague-Dawleyrats (n=54) weighting 300 to 350 gm were used in this study. They were kept two per cage at a 12:12 h light:dark cycle at the constant temperature of 22+.0.5°Cand food and water at libitum. The research protocol was approved by the Institutional Animal Care and Use Committee. Tail-Flick Test: Nociceptive responseswere assessedby the TF test, employingthe analgesiameter (model 33, Innovators in InstrumentationInc.) ~ describedin detail elsewhere(8,9). The nociceptive endpoint was set at 20 see, to prevent tissue damage. The light beam was always projected within a lcm in diameter, just 3cm above the tip of the tail as different tail areas may possess different sensitivity (10). The beam intensity dial was set to obtain basal TF latencies between 5-7 sec. Drugs and Dosage: Three drugs were used in this study: normal saline, injected S.C.in the volume of lmlkg; bicuculline(Sigma) 2,4,6,IO,and20 mg/kg, injected i.p. in the volume of 2mWg; nicotine free base (Sigma), lmg/ kg, injected S.C.in the volume of 2.5ml/kg, 30 minutes afler the injection of pretreated saline (control) and different doses of BIC. Experimental Protocol: All rats were acclimated in restrainers two hours a day for 2 days before the study. Experiments were carried out at the same time of day to avoid diurnal variation. Before each experiment,rats were rdlowedto rest in the restrainerfor 2 hours, then two consecutivebaseline TF latencywere measuredwith a 5 minuteinterval, before the injectionof the test compounds.Atler the baselineTF latencieswere recorded, saline or BIC at differentdoses were injected.Each rat was observed for any side effect following BIC injection, and TF latencies were measured every 10 minutesthree times. Then,NIC was injected to all rats. TF latencieswere recorded every 10 minutes for 60 minutes. Calculationand StatisticalAnalysis: The valuesof TF latencieswere calculatedas medianfor each group and expressed graphically. The area under the curve (AUC) was calculated for each rat, presented as mean =tSEfor each group. Statistical analysisof the AUC means was assessed by one way ANOVA. Tukey-Kramar HSD (honestly significant difference) test was used for multiple comparisons among the groups (P
Results No noticeable side effect was observed followingBIC i.p. injection. There was only, short period lasting, biting, scratching, and vocalization, occasionally noticed among all the rats. No seizure occurred throughout the whole course of the study. In control, saline pretreated group, 1 out of 10 rats experienced slight tremor within the first minute followingNIC injection. No tremor was observed in rats pretreated with BIC. The time course of the antinociceptive responses of all 6 groups are presented as median TF latencies in Fig. 1. The pretreatment with saline and all doses of BIC did not change the latencies from the baseline, ranging from 4 to 7 sec.
PL-223
NicotineAnalgesiaandBicucuIline
Vol.61,No. 15,1997
-40
-20
0
20
40
60
so
T I M E (m in)
Fig. L Median values of tail-flick. latency following the injection of nicotine (lmg/kg S.C.at time O) in rats pretreated at time -30 min with saline(o) and bicuculline;the doses in mg per kg body weight were: 2(=); 4 (~); 6 (V); 10 (+); and 20 (0).
TABLE I Mean values + SE of AUC and number (percentage) of rats responding to nicotine-inducedantinociceptionfollowingdifferentdoses of BIC, pretreated 30 min earlier. Groups
N
BIC(mg/kg) pretreated
No. responders
AUC Mean + SE
I
I
10
I
O
6 (60%)
I
817.35+ 41.48
II
I
10
I
2
6 (60%)
731.37 + 41.47
III
I
10
I
I
4
3 (30%)
I
539.38+ 41.48 *
IV
I
9
I
6
3 (30%)
I
519.49+ 43.’73*
3 (33.3%) I
466.14+ 40.48 *
1 (20%)
451.78+ 58.66 *
* significantlydifferent from control group; p <0.001
\
PL-224
NicotineAnalgesiaandBicuculline
Vol.61,No. 15,1997
Control rats and those pretreated with 2mg/kgBIC, exhibitedsimilarpattern atler NIC injection.The maximalantinociceptiveresponse occurred 10 min after MC, returningto the baselinewithin 30 min (Fig. 1). In both groups, 60% of rats reached maximalresponse (Table 1). Pretreatment with higher doses of BIC, from 4 to 20 mg/kg, reduced antinociceptive efl’ectof NIC, expressed as median TF latencies. The number of rats reachingthe ceilingTF Iatenciesdecreased by 50V0in groups pretreated with 4, 6 and 10 mg/kg BIC, compare to saline + NIC (control) and BIC 2mg/kg + NIC groups, Following 20mg/kg BIC, 1 out of5 rats (20%) responded to NIC (Table 1). One way ANOVA of the mean AUC values for six groups revealed significantdifference among the groups (p
Vol.61,
15,1997
NicotineAnalgesiaandBicuculline
PL-225
test) should be used. The other question is, whether intrathecally administrated BIC completely suppresses NIC-induced antinociception To answer this question, a preliminary study is in preparation. Our data are in the agreementwith the generalnotionthat, GABAAreceptors have an inhibitoryeffect in nociceptive transmission. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
L.W. ROLE and D.K. BERG, Neuron M 1077-1OS5(1996) D.T. ROGERS and E.T. IWAMOTO, J. Pharmacol. EXP.Ther. 2!5?341-349 (1993) E.T. IWAMOTO and L. MARION, J. Pharmacol. EXP.Ther. ~ 777-789 (1993) M.I. DAMAJ and B.R. MARTIN, Psychopharmacology~ 106-108 (1993) L.H. LIN, P. WHITING and R.A. HARRIS, J. Neurochem. @ 1548-1553 (1993) X.H. DING, X.Q. JI and K TSOU, Pain ~ 371-376 (1990) L.M. AANONSEN and G.L. WILCOX, J. Pharmacol. Exp. Ther.~ 1034-1038 (1989) C.Y. YANG, W.H. WU and V.K. ZBUZEK, psychopha~acology l!M 417-420 (1992) C.W.Y. CHIN, R.C. BLOCK, W.H. WU and V.K.ZBUZE~ Psychopharmacologym 497499 (1993) B.C. YOBURN,R. MORALES,D.D. KELLY and C.E. INTURRISI,Life Sci.M 1755-1762 (1984) T. ISHIKAWA and T.L. YAKSH, Japanese Journal of Anesthesiology.~ 439-444 (1996) T, YAMAMOTO and T.L. YAKSH, Pain. ~ 79-84 (1993) M.D. ACETO, R.S. BAGLEY, W.L.DEWEY, T.C. FU and B.R. MARTIN, Neurophannacol. ~ 1031-1036(1986)