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Mecamylamine blockade of nicotine enhanced noradrenaline turnover in rat brain
Pergamon Press
Life Sciences, Vol . 24, pp . 417-420 Printed in the U .S .A .
MECAMYLAMINE BLOCKADE OF NICOTINE ENHANCED NORADRENALINE TURNOVER IN RAT BRAIN William W . Morgan and Karla A . Pfeil Department of Anatomy The University of Texas Health Science Center at San Antonio 7703 Floyd Curl Drive San Antonio, Texas 78284 U .S .A . (Received in final form December 13, 1978) Summa~ The administration of nicotine significantly enhanced the depletion in noradrenaline (NA) observed in the brains of male Sprague-Dawley rats following alpha methyl-pare-tyrosine (aMPT) administration . These data indicate that nicotine enhances the turnover of NA in the rat brain . This effect of nicotine was completely blocked by mecamylamine administration while mecamylamine alone had no observed effect on NA content or turnover . These data are consistent with the action of mecamylamine as an effective antagonist of the action of nicotine in the rat brain . There has been recent conflicting evidence as to whether mecamylamine is effective as a blocker of the action of nicotine in the central nervous system (1,2) . Since we had recently observed that the administration of nicotine increases the turnover of noradrenaline (NA) in the rat brain, the present study was undertaken to determine if mecamylamine could block this action of nicotine . Methods Adult male Sprague-Dawley rats (150-180 g ; Simonsen, California) were housed in our animal quarters for one week before experimentation . The animals were housed 4 per cage in an animal room equipped with a regulated 14 :10 light-dark lighting regimen with the lights on from 0600 to 2000 local time . Food and water were available ad libitum . Rats were assigned to experimental subgroups utilizing a completely randomized schedule which was also employed to determine the time of drug treatment and subsequent sacrifice of each animal . Drug treatments were not initiated before 1200 and all sacrifices were completed by 1800 . In the initial study nicotine was administered intraperitoneally (i .p .) at dosages of 0 .5 mg or 1 mg/kg at hourly intervals for 2 hours . In the second experiment mecamylamine HC1 was administered at a dosage of 5 mg/kg intravenously (via the tail vein) 30 minutes before each of the 2 hourly i .p . administrations of nicotine (1 mg/kg) . In all the experiments, control animals received a saline injection via the same route for each drug injection into the experimental animals . In a third study rats received i .v . 0300-9653/79/0129-041702 .00/0 Copyright (c) 1979 Pergamon Press
41 8
Nicotine Enhanced NA Turnover
Vol . 24, No . 5, 1979
administrations of mecamylamine HC1 (5 mg/kg) 30 minutes before each of 2 hourly administrations of saline . In all studies some of the animals in each control or experimental subgroup received a single injection of alpha-methyl-p-tyrosine (aMPT, 250 mg/kg, i .p ., methyl ester, Regis), a potent inhibitor of tyrosine hydroxylase, 2 hours before sacrifice . As with all other drugs, each animal received «MPT for the exact same time interval . The decline in NA content observed in the brain following aMPT administration served as an indirect measure of the turnover of this amine . Since we have previously observed that the decline in NA produced by aMPT is linear in the brain area of interest for at least 4 hours (submitted for publication), in this study NA levels were determined only 2 hours following uMPT . The rats were sacrificed by decapitation, and the brain was removed in less than 1 minute . A coronal section was made at the anterior margin of the pons, and the brain tissue rostral to this cut was immediately frozen on dry ice and stored . The dissected brain samples were homogenized in 5000 pL of 0 .1 N perchloric acid, and the levels of NA were determined in duplicate 25 uL aliquots of each homogenate following the procedure of Palkovits et al . (3) . The statistical significance of differences in the same parameter among the experimental groups was determined first with the analysis of variance (4) . If significant differences were demonstrated, the data were subjected to further statistical evaluation with the Student Newman Keuls test (5) . Results Two dosages of either 0 .5 or 1 mg/kg nicotine given 1 hour apart did not significantly affect the concentration of NA in the rat brain . On the other hand, the decline in NA levels in the brain following the administration of aMPT was significantly greater (p<0 .01) in those animals that received either dosage of nicotine when compared to the same parameter in rats which received saline (Table I) . These data are consistent with an increase in NA turnover in the brains of the nicotine-treated rats . TABLE I Effect of Nicotine on Noradrenaline in the Rat Brain . Treatment
ng/gm Noradrenaline ± S .E .M .b No aMPT
aMPT
Saline
201111(8)
1601 8(8)
Nicotine (0 .5 mg/kg)
1861 9(8)
13118(7) a
Nicotine (1 mg/kg)
193112(8)
12715(8) a
a -_-s~n
sal
from iné, rea e p< . significance of differences among nMPT-treated groups p<0 .01 b - standard error of the mean
Vol . 24, No . 5, 1979
Nicotine Enhanced NA 1~rnover
419
When mecamylamine HC1 was administered 30 minutes before each of 2 dosages of nicotine (1 mg/kg), the mecamylamine was effective in completely blocking the effect of nicotine on the a-MPT-induced decline in NA in the brain (Table II) . When mecamylamine was administered alone but by the same time schedule, this drug had no effect on either NA concentration or the decline in NA observed following aMPT administration (Table III) . TABLE II Ability of Meca~ylamine in Two Dosages to Block the Effect of Nicotine on Noradrenaline in the Rat Brain . Treatment
ng/gm Noradrenaline t S .E .M . No aMPT
aMPT
Saline
244± 9(8)
196± 8(8)
Nicotine
223±10(8)
151±10(8)a
Mecamylamine + Nicotine
226±15(8)
184± 8(8)
a -s~n3~an~rom sâlinë, aM~<~ Ö~ statistical significance among aMPT groups p<0.001 TABLE III Effect of Mecamylamine in Two Dosages on Noradrenaline in the Rat Brain . Treatment
ng/gm Noradrenaline t S .E .M . No aMPT
aMPT
Saline
211112(6)
146±8(6)
Mecar~lamine
195±13(6)
156±8(6)
Discussion Mecamylamine has been shown to possess the properties of a ganglionic blocking drug with no important qualitative differences from the quaternary ammonium ganglionic blocking agents (6) . Further, mecamylamine has a potent blocking action on the golgi recurrent collateral-Renshaw cell synapse in the spinal cord (7) . This particular action illustrates the nicotinic blocking action of mecamylamine and also the ability of this drug to penetrate the blood-brain barrier at least in small amounts . Since mecamylamine is structurally a secondary amine, the ability of this drug to cross the bloodbrain barrier would be predicted . Meca~ylamine is also a potent antagonist
42 0
Nicotine Enhanced NA Turnover
Vol, 24, No . 5, 1979
of nicotine induced convulsions which are presumably initiated through a Recently, Ahtee and Kaakkola (1) concluded central action of nicotine (8) . that mecargylamine decreased the turnover of striatal dopamine probably by a mechanism opposite to that of nicotine . Contrary to the evidence that suggests that mecamylamine is an effective nicotinic antagonist in the central nervous system, Morley and others (2) found that mecamylamine was ineffective in reducing the binding of radioactively labeled a-bungarotoxin, a specific nicotinic antagonist, in a number of brain regions of the rat . Considering the conflicting evidence as to the mode of action of mecamylamine in the central nervous system, it was decided to test the effect of this compound on the nicotine induced increase in NA turnover in the rat brain . The data presented show that nicotine administration increased the depletion of NA produced in the brain of rats following aMPT administration . These data are consistent with a nicotine-induced increase in NA turnover in the rat brain . A similar effect of nicotine has been observed previously (9, 10) . Mecamylamine pretreatment completely antagonized the action of nicotine on NA turnover while mecamylamine alone had no demonstrable effect on this same parameter . These data provide positive evidence that mecamylamine may It is not function as a nicotinic antagonist in the central nervous system . clear from our results whether mecamylamine exerts its antagonistic action by blocklng a nicotinic or an acetylcholine binding site . Acknowledgements Supported by NSF grant ~PCM 76-03876, NIDA grant #5 ROl DA 00755 and Research Development Award i~5 K02 MH 00028 to WWM . The authors gratefully acknowledge the advice of Mr . Larrel Harris, Edgewood Arsenal, MD 21010, who recommended the dosage and route of administration of mecamylamine . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 .
L . AHTEE and S . KAAKKOLA, Br . J . Pharmac . 62, 213-218 (1978) . B . J . MORLEY, J . F . LORDEN, G . B . BROWN, G.E . KEMP and R . J . BRADLEY, Brain Research 134, 161-166 (1977) . M . PALKOVITS, M~ROWNSTEIN, J . M . SAAVEDRA and J . AXELROD, Brain Research 77, 137-149 (1974) . R . F . SOK~L and F . J . ROHLF, Biometry , W . H . Freeman and Company, San Francisco, pp . 208-209 (1969) . R . F . SOKAL and F . J . ROHLF, Biometry , W . H . Freeman and Company, San Francisco, pp . 242-245 (1969) . C . A . STONE, M . L . TORCHIANA, A . NAVARRO and K . H . BEYER, J . Pharmac . Exp . Ther . 117, 169-183 (1956) . S . UEKI, K .~K KETSU and E . E . DOMINO, Exp . Neurol . 3, 141-148 (1961) . C . A . STONE, K . L . MECKENBURG and M . L . TORCHIANA, Arch . Intem . Pharmacodyn . 117, 419-434 (1958) . G . H . HALL and6. M . TURNER, Biochem . Phannac . 21, 1829-1838 (1972) . B . BHAGAT, S . Z . KRAMER and J . SEIFTER, Eur . J .~harmac . _2, 234-235 (1967) .
Life Sciences, Vol . 24, pp . 417-420 Printed in the U .S .A .
MECAMYLAMINE BLOCKADE OF NICOTINE ENHANCED NORADRENALINE TURNOVER IN RAT BRAIN William W . Morgan and Karla A . Pfeil Department of Anatomy The University of Texas Health Science Center at San Antonio 7703 Floyd Curl Drive San Antonio, Texas 78284 U .S .A . (Received in final form December 13, 1978) Summa~ The administration of nicotine significantly enhanced the depletion in noradrenaline (NA) observed in the brains of male Sprague-Dawley rats following alpha methyl-pare-tyrosine (aMPT) administration . These data indicate that nicotine enhances the turnover of NA in the rat brain . This effect of nicotine was completely blocked by mecamylamine administration while mecamylamine alone had no observed effect on NA content or turnover . These data are consistent with the action of mecamylamine as an effective antagonist of the action of nicotine in the rat brain . There has been recent conflicting evidence as to whether mecamylamine is effective as a blocker of the action of nicotine in the central nervous system (1,2) . Since we had recently observed that the administration of nicotine increases the turnover of noradrenaline (NA) in the rat brain, the present study was undertaken to determine if mecamylamine could block this action of nicotine . Methods Adult male Sprague-Dawley rats (150-180 g ; Simonsen, California) were housed in our animal quarters for one week before experimentation . The animals were housed 4 per cage in an animal room equipped with a regulated 14 :10 light-dark lighting regimen with the lights on from 0600 to 2000 local time . Food and water were available ad libitum . Rats were assigned to experimental subgroups utilizing a completely randomized schedule which was also employed to determine the time of drug treatment and subsequent sacrifice of each animal . Drug treatments were not initiated before 1200 and all sacrifices were completed by 1800 . In the initial study nicotine was administered intraperitoneally (i .p .) at dosages of 0 .5 mg or 1 mg/kg at hourly intervals for 2 hours . In the second experiment mecamylamine HC1 was administered at a dosage of 5 mg/kg intravenously (via the tail vein) 30 minutes before each of the 2 hourly i .p . administrations of nicotine (1 mg/kg) . In all the experiments, control animals received a saline injection via the same route for each drug injection into the experimental animals . In a third study rats received i .v . 0300-9653/79/0129-041702 .00/0 Copyright (c) 1979 Pergamon Press
41 8
Nicotine Enhanced NA Turnover
Vol . 24, No . 5, 1979
administrations of mecamylamine HC1 (5 mg/kg) 30 minutes before each of 2 hourly administrations of saline . In all studies some of the animals in each control or experimental subgroup received a single injection of alpha-methyl-p-tyrosine (aMPT, 250 mg/kg, i .p ., methyl ester, Regis), a potent inhibitor of tyrosine hydroxylase, 2 hours before sacrifice . As with all other drugs, each animal received «MPT for the exact same time interval . The decline in NA content observed in the brain following aMPT administration served as an indirect measure of the turnover of this amine . Since we have previously observed that the decline in NA produced by aMPT is linear in the brain area of interest for at least 4 hours (submitted for publication), in this study NA levels were determined only 2 hours following uMPT . The rats were sacrificed by decapitation, and the brain was removed in less than 1 minute . A coronal section was made at the anterior margin of the pons, and the brain tissue rostral to this cut was immediately frozen on dry ice and stored . The dissected brain samples were homogenized in 5000 pL of 0 .1 N perchloric acid, and the levels of NA were determined in duplicate 25 uL aliquots of each homogenate following the procedure of Palkovits et al . (3) . The statistical significance of differences in the same parameter among the experimental groups was determined first with the analysis of variance (4) . If significant differences were demonstrated, the data were subjected to further statistical evaluation with the Student Newman Keuls test (5) . Results Two dosages of either 0 .5 or 1 mg/kg nicotine given 1 hour apart did not significantly affect the concentration of NA in the rat brain . On the other hand, the decline in NA levels in the brain following the administration of aMPT was significantly greater (p<0 .01) in those animals that received either dosage of nicotine when compared to the same parameter in rats which received saline (Table I) . These data are consistent with an increase in NA turnover in the brains of the nicotine-treated rats . TABLE I Effect of Nicotine on Noradrenaline in the Rat Brain . Treatment
ng/gm Noradrenaline ± S .E .M .b No aMPT
aMPT
Saline
201111(8)
1601 8(8)
Nicotine (0 .5 mg/kg)
1861 9(8)
13118(7) a
Nicotine (1 mg/kg)
193112(8)
12715(8) a
a -_-s~n
sal
from iné, rea e p< . significance of differences among nMPT-treated groups p<0 .01 b - standard error of the mean
Vol . 24, No . 5, 1979
Nicotine Enhanced NA 1~rnover
419
When mecamylamine HC1 was administered 30 minutes before each of 2 dosages of nicotine (1 mg/kg), the mecamylamine was effective in completely blocking the effect of nicotine on the a-MPT-induced decline in NA in the brain (Table II) . When mecamylamine was administered alone but by the same time schedule, this drug had no effect on either NA concentration or the decline in NA observed following aMPT administration (Table III) . TABLE II Ability of Meca~ylamine in Two Dosages to Block the Effect of Nicotine on Noradrenaline in the Rat Brain . Treatment
ng/gm Noradrenaline t S .E .M . No aMPT
aMPT
Saline
244± 9(8)
196± 8(8)
Nicotine
223±10(8)
151±10(8)a
Mecamylamine + Nicotine
226±15(8)
184± 8(8)
a -s~n3~an~rom sâlinë, aM~<~ Ö~ statistical significance among aMPT groups p<0.001 TABLE III Effect of Mecamylamine in Two Dosages on Noradrenaline in the Rat Brain . Treatment
ng/gm Noradrenaline t S .E .M . No aMPT
aMPT
Saline
211112(6)
146±8(6)
Mecar~lamine
195±13(6)
156±8(6)
Discussion Mecamylamine has been shown to possess the properties of a ganglionic blocking drug with no important qualitative differences from the quaternary ammonium ganglionic blocking agents (6) . Further, mecamylamine has a potent blocking action on the golgi recurrent collateral-Renshaw cell synapse in the spinal cord (7) . This particular action illustrates the nicotinic blocking action of mecamylamine and also the ability of this drug to penetrate the blood-brain barrier at least in small amounts . Since mecamylamine is structurally a secondary amine, the ability of this drug to cross the bloodbrain barrier would be predicted . Meca~ylamine is also a potent antagonist
42 0
Nicotine Enhanced NA Turnover
Vol, 24, No . 5, 1979
of nicotine induced convulsions which are presumably initiated through a Recently, Ahtee and Kaakkola (1) concluded central action of nicotine (8) . that mecargylamine decreased the turnover of striatal dopamine probably by a mechanism opposite to that of nicotine . Contrary to the evidence that suggests that mecamylamine is an effective nicotinic antagonist in the central nervous system, Morley and others (2) found that mecamylamine was ineffective in reducing the binding of radioactively labeled a-bungarotoxin, a specific nicotinic antagonist, in a number of brain regions of the rat . Considering the conflicting evidence as to the mode of action of mecamylamine in the central nervous system, it was decided to test the effect of this compound on the nicotine induced increase in NA turnover in the rat brain . The data presented show that nicotine administration increased the depletion of NA produced in the brain of rats following aMPT administration . These data are consistent with a nicotine-induced increase in NA turnover in the rat brain . A similar effect of nicotine has been observed previously (9, 10) . Mecamylamine pretreatment completely antagonized the action of nicotine on NA turnover while mecamylamine alone had no demonstrable effect on this same parameter . These data provide positive evidence that mecamylamine may It is not function as a nicotinic antagonist in the central nervous system . clear from our results whether mecamylamine exerts its antagonistic action by blocklng a nicotinic or an acetylcholine binding site . Acknowledgements Supported by NSF grant ~PCM 76-03876, NIDA grant #5 ROl DA 00755 and Research Development Award i~5 K02 MH 00028 to WWM . The authors gratefully acknowledge the advice of Mr . Larrel Harris, Edgewood Arsenal, MD 21010, who recommended the dosage and route of administration of mecamylamine . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 .
L . AHTEE and S . KAAKKOLA, Br . J . Pharmac . 62, 213-218 (1978) . B . J . MORLEY, J . F . LORDEN, G . B . BROWN, G.E . KEMP and R . J . BRADLEY, Brain Research 134, 161-166 (1977) . M . PALKOVITS, M~ROWNSTEIN, J . M . SAAVEDRA and J . AXELROD, Brain Research 77, 137-149 (1974) . R . F . SOK~L and F . J . ROHLF, Biometry , W . H . Freeman and Company, San Francisco, pp . 208-209 (1969) . R . F . SOKAL and F . J . ROHLF, Biometry , W . H . Freeman and Company, San Francisco, pp . 242-245 (1969) . C . A . STONE, M . L . TORCHIANA, A . NAVARRO and K . H . BEYER, J . Pharmac . Exp . Ther . 117, 169-183 (1956) . S . UEKI, K .~K KETSU and E . E . DOMINO, Exp . Neurol . 3, 141-148 (1961) . C . A . STONE, K . L . MECKENBURG and M . L . TORCHIANA, Arch . Intem . Pharmacodyn . 117, 419-434 (1958) . G . H . HALL and6. M . TURNER, Biochem . Phannac . 21, 1829-1838 (1972) . B . BHAGAT, S . Z . KRAMER and J . SEIFTER, Eur . J .~harmac . _2, 234-235 (1967) .