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Changes in enzymes subserving catecholamine metabolism in morphine tolerance and withdrawal in rat
309
BRAIN RESEARCH
Short Communications
Changes in enzymes subserving catecholamine metabolism in morphine tolerance and withdrawal in rat It has been proposed on indirect evidence that the production of tolerance to the chronic administration of morphine to rats results in an increased synthesis of catecholamines in brain and adrenal glandS,7,11. That morphine may produce changes in the activity of some enzymes subserving catecholamine metabolism has not yet been examined. In this study we have investigated the effects of the chronic administration of morphine and of 48 h of abstinence on the activity of several of these enzymes. Changes in the activities of tyrosine hydroxylase, the presumed rate-limiting step in the synthesis of norepinephrine s, monoamine oxidase (MAO), and phenylethanolamine-N-methyl transferase (PNMT), the enzyme catalyzing the conversion of norepinephrine to epinephrine, will be demonstrated. Female Sprague-Dawley rats weighing 235-260 g were housed in groups of 4-6 animals in thermally regulated rooms and fed standard lab chow adlib. The animals were divided into 4 groups of l0 animals each. Two groups were injected daily with morphine sulfate subcutaneously for 15 consecutive days beginning with injections of 5 mg/kg administered twice daily at 8.00 and 16.00 h, and increased incrementally to 95 mg/kg 3 times a day at 8.00, 12.00 and 16.00 h. The tolerant group was killed on day 16 at 10.00 h, 2 h following an injection of 130 mg/kg of morphine; the withdrawal group was killed 48 h after a final dose of 130 mg/kg. Saline controls received daily injections of comparable volumes of saline and were killed on day 16; naive controls were maintained under similar conditions of housing but not handled until their death on the 16th day. After weighing, the animals were killed by decapitation and the brains rapidly removed and dissected. Tyrosine hydroxylase was assayed in the caudate nuclei, pooled from two animals in each group. MAO was measured in the caudate nucleus, hypothalamus and brain stem pooled from two animals. Both adrenal glands from a single animal were pooled for assay of tyrosine hydroxylase, PNMT, and MAO. Samples of brain and adrenal gland were homogenized in 4 vol. of 0.9 % KC1 and 0.25 M sucrose respectively and centrifuged. The supernatants were dialyzed prior to assay for enzyme activities except for an aliquot from the adrenal gland which was assayed undialyzed for tyrosine hydroxylase. Tyrosine hydroxylase was assayed by the method of Nagatsu et al.S; PNMT by the method of Axelrod2; and MAO by the method of Wurtman and Axelrod TM. Changes in the activities of tyrosine hydroxylase, PNMT and MAO in the adrenal gland during tolerance withdrawal cycle to morphine are shown in Table I. Since there is a significant increase in adrenal gland weight during chronic morphine administration consequent to adrenocortical hypertrophyTM enzyme activities are expressed as activity/gland pair rather than by unit weight. Brain Research, 20 (1970) 309-312
3 10
SHORT COMMUNICATIONS
TABLE I ADRENAL G L A N D ENZYME ACTIVITY
Enzyme activity in adrenal gland during tolerance to and after 48 h of abstinence from morphine in rat. NS ~: Not significant (P ;> 0.05) by Student's t test.
Naive control Saline control Change from naive control Tolerant Change from saline control Withdrawal Change from saline control
Adrenal weight (mg/pair 2_ S.E.M.)
Tyrosine hydroxylase (nmoles/pair/ 20 rain 2- S.E.M.)
PNMT (nrnoles/pair/ 20 min 2_ S.E.M.)
MAO ( counts/min • lOa/pair/ 20 rain ~ S.E.M.)
647 + 26 687 4. 32
0.096 ± 0.019 0.089 2- 0.008
9.33 2_ 1.02 8.55 2- 1.73
24.5 ± 6.0 26.6 2- 6.0
(NS) 836 ± 43
(NS) 0.189 2- 0.031
(NS) 6.34 4, 1.43"*
(NS) 23.8 2_ 6.0
(P < 0.05) 815 2_ 20
(P < 0.05) 0.109 2_ 0.023*
(NS) 7.95 2_ 0.88
(NS) 31.3 2_ 4.3***
(P < 0.01)
(NS)
(NS)
(NS)
* Change from tolerant significant (P < 0.02). ** Change from naive control significant (P < 0.01). *** Change from tolerant significant (P < 0.05).
TABLE II ENZYMEACTIVITYIN CAUDATENUCLEUS Enzyme activity in caudate nucleus during tolerance to and after 48 h of abstinence from morphine in rat. NS = Not significant (P > 0.05) by Student's t test.
Naive control Saline control Change from naive control Tolerant Change from saline control Withdrawal Change from saline control
Tyrosine hydroxylase (nmoles/g/20 rain ± S.E.M.)
MA 0 (counts/min/g/20 min 2- S.E.M.)
0.077 -4- 0.013 0.047 4. 0.013 (P < 0.01) 0.110 4- 0.030 (P < 0.05) 0.100 ± 0.037 (NS)
212 2- 15 175 i 23 (NS) 205 4, 36 (NS) 243 4. 18" (P < 0.02)
* Change from tolerant animals significant (P < 0.01).
C h a n g e s in the activities o f all 3 e n z y m e s occur d u r i n g the cycle o f m o r p h i n e t o l e r a n c e a n d withdrawal. D u r i n g t o l e r a n c e there is a significant increase, to a l m o s t 200 ~ o f c o n t r o l , o f t y r o s i n e h y d r o x y l a s e activity in the ad r en al gland w h i ch falls back to n e a r c o n t r o l values 48 h after abstinence. P N M T activity is r ed u ced in t o l e r a n t a n i m a l s when c o m p a r e d to naive but n o t saline injected controls suggesting that there is additive effect o f stress o f i n j e c ti o n and m o r p h i n e in reducing the activity o f this Brain Research, 20 (1970) 309-312
SHORT COMMUNICATIONS
311
enzyme. MAO activity in the adrenal gland does not change during tolerance. During abstinence however, at a time when the animals are manifesting the well recognized clinical signs of withdrawal there is a significant increase in MAO activity. In the brain changes in tyrosine hydroxylase and MAO activities in the caudate nucleus (Table I1) follow, in general, a similar pattern. In brain there is a significant increase in tyrosine hydroxylase activity in tolerance which remains elevated even after 48 h of abstinence. Of particular interest is the fact that saline injections alone result in a significant decrease in tyrosine hydroxylase activity in the caudate nucleus. MAO activity in the caudate but not in hypothalamus or brain stem rose during withdrawal. This study demonstrates that significant changes in the activities of some enzymes involved in the synthesis and degradation of catecholamines occur during the morphine tolerance-withdrawal syndrome in the rat. During tolerance, the primary change is an increase in the activity of tyrosine hydroxylase in both adrenal gland and brain. The increased activity of this enzyme in morphine tolerant rats suggests that the synthesis of catecholamines may be increased and can explain the observations of others: (a) that in tolerance the levels of norepinephrine may be increased in brain1,5, 7 without a change in the release of the aminee,9; (b) that even though catecholamine levels of adrenal gland are unchanged in morphine tolerant animals catecholamine secretion is elevatedS; (c) that the recovery of brain and adrenal catecholamine levels from the depletion induced by reserpine is accelerated by morphine4,5; (d) that brain norepinephrine levels increase to a greater degree in morphine tolerant than in control animals following inhibition of MAO 5,7. The increased activity of tyrosine hydroxylase in the caudate nucleus, a region rich in dopamine, would also support evidence accrued by others that the turnover of this amine is increased, at least acutely, by administration of morphine 6. During tolerance there is a tendency for PNMT levels in the adrenal gland to decrease. Our findings are consistent with the histochemical observations of Ray et al. lo, demonstrating preferential shunting of catecholamine storage in the adrenal medulla from epinephrine to norepinephrine rich areas after morphine, and would support their interpretation that reduced PNMT activity underlies the phenomenon. During withdrawal at a time when tyrosine hydroxylase activity in the adrenal gland, but not in the brain, has returned to control values there is a rise in the activity of MAO in both the caudate nucleus and the adrenal gland. While the mechanism governing this rise is not clear the biological utility of enhanced MAO activity at this time may be to facilitate the destruction of excess biogenic amines resulting from augmented synthesis in tolerance. It is of interest that in morphine tolerance the increase in tyrosine hydroxylase activity in brain is paralleled by a rise in the activity of tryptophan hydroxylase 3, the probable rate-limiting step in serotonin synthesis. It is thus likely that in morphine tolerance, the synthesis mechanisms for 3 putative central neurotransmitters, norepinephrine, dopamine, and serotonin, are all enhanced. The relationship of these changes in pathways subserving the metabolism of biogenic amines for the development of tolerance to morphine or the symptoms of abstinence remains to be elucidated. Brain Research, 21 (1970) 309-312
312
SHORT COMMUNICATIONS T h i s s t u d y was s u p p o r t e d by N I H G r a n t s NS068911, G M 0 1 7 8 9 a n d NS31756. Nancy
M e r l i n o , M a r l e n e R i f k i n and J a n a l g l i c h p r o v i d e d v a l u a b l e t e c h n i c a l
assistance. Department of Neurology, Cornell University Medical College, New York, N.Y. 10021, and Rockefeller University, New York, N.Y. 10021 (U.S.A.)
DONALD J. REIS PAUl, HESS EFRAIN C. AZMITIA, JR.
I AKERA, T., AND BRODY, T. M., The addiction cycle to narcotics in the rat and its relationship to catecholamines, Biochem. Pharmacol., 17 (1968) 675-688. 2 AXEEROD,J., Purification and properties of phenylethanolamine-N-methyl transferase, J. biol. Chem., 237 (1962) 1657-1660. 3 AZMITIA, E. C., JR., HESS, P., AND REIS, D. J., Lile Sci., (1970) in press. 4 FREEDMAN,D. X., FRAM, D. H., AND GIARMAN,N. J., The effect of morphine on the regeneration of brain norepinephrine after reserpine, Fed. Proc., 20 (1961) 321. 5 GUNNE, L.-M., Catecholamines and 5-hydroxytryptamine in morphine tolerance and withdrawal, Acta physiol, scand., 58, Suppl. 204 (1963) 1-91. 6 GUNNE, L.-M., JONSSON, J., AND FUXE, K., Effects of morphine intoxication on brain catecholamine neurons, Europ. J. Pharmacol., 5 (1969) 338-342. 7 MEYNERT,E. W., AND KEINGMAN, G. 1., Tolerance to morphine. I. Effects of catecholamines in the brain and adrenal glands, J. Pharmacol. exp. Ther., 135 (1962) 285-295. 8 NAGATSU,T., LEVITT, M., AND UDENFRIEND,S., Tyrosine hydroxylase. The initial step in norepinephrine synthesis, J. biol. Chem., 239 (1964) 2910-2917. 9 NEEL, M. J., Failure of morphine dependence in rats to influence brain noradrenaline turnover, J. Pharm. Pharmacol., 20 (1968) 951-953. 10 RAY, A. K., MUKHERGI,M., AND GHOSH, J. J., Adrenal catecholamine and related changes during different phases of morphine administration - - a histochemical study, J. Neurochem., 15 (1968) 875-881. I 1 SEOAN, J. W., AND EISENMAN, A. J., Long persisting changes in catecholamine metabolism following addiction to and withdrawal from morphine, Res. Publ. Ass. nerv. ment. Dis., 46 (1968) 96-105. 12 SUNG, C. Y., WAY, E. L., AND SCOTT, K. G., Studies on the relationship of metabolic fate and hormonal effects of DE-methadone to the development of drug tolerance, J. Pharmacol. exp. Ther., 107 (1953) 12-23. 13 WURTMAN, R. J., AND AXEEROD, J., A sensitive and specific assay for the estimation of monoamine oxidase, Biochem. Pharmacol., 12 (1963) 1439-1441. (Accepted April 2nd, 1970)
Brain Research, 20 (1970) 309-312
BRAIN RESEARCH
Short Communications
Changes in enzymes subserving catecholamine metabolism in morphine tolerance and withdrawal in rat It has been proposed on indirect evidence that the production of tolerance to the chronic administration of morphine to rats results in an increased synthesis of catecholamines in brain and adrenal glandS,7,11. That morphine may produce changes in the activity of some enzymes subserving catecholamine metabolism has not yet been examined. In this study we have investigated the effects of the chronic administration of morphine and of 48 h of abstinence on the activity of several of these enzymes. Changes in the activities of tyrosine hydroxylase, the presumed rate-limiting step in the synthesis of norepinephrine s, monoamine oxidase (MAO), and phenylethanolamine-N-methyl transferase (PNMT), the enzyme catalyzing the conversion of norepinephrine to epinephrine, will be demonstrated. Female Sprague-Dawley rats weighing 235-260 g were housed in groups of 4-6 animals in thermally regulated rooms and fed standard lab chow adlib. The animals were divided into 4 groups of l0 animals each. Two groups were injected daily with morphine sulfate subcutaneously for 15 consecutive days beginning with injections of 5 mg/kg administered twice daily at 8.00 and 16.00 h, and increased incrementally to 95 mg/kg 3 times a day at 8.00, 12.00 and 16.00 h. The tolerant group was killed on day 16 at 10.00 h, 2 h following an injection of 130 mg/kg of morphine; the withdrawal group was killed 48 h after a final dose of 130 mg/kg. Saline controls received daily injections of comparable volumes of saline and were killed on day 16; naive controls were maintained under similar conditions of housing but not handled until their death on the 16th day. After weighing, the animals were killed by decapitation and the brains rapidly removed and dissected. Tyrosine hydroxylase was assayed in the caudate nuclei, pooled from two animals in each group. MAO was measured in the caudate nucleus, hypothalamus and brain stem pooled from two animals. Both adrenal glands from a single animal were pooled for assay of tyrosine hydroxylase, PNMT, and MAO. Samples of brain and adrenal gland were homogenized in 4 vol. of 0.9 % KC1 and 0.25 M sucrose respectively and centrifuged. The supernatants were dialyzed prior to assay for enzyme activities except for an aliquot from the adrenal gland which was assayed undialyzed for tyrosine hydroxylase. Tyrosine hydroxylase was assayed by the method of Nagatsu et al.S; PNMT by the method of Axelrod2; and MAO by the method of Wurtman and Axelrod TM. Changes in the activities of tyrosine hydroxylase, PNMT and MAO in the adrenal gland during tolerance withdrawal cycle to morphine are shown in Table I. Since there is a significant increase in adrenal gland weight during chronic morphine administration consequent to adrenocortical hypertrophyTM enzyme activities are expressed as activity/gland pair rather than by unit weight. Brain Research, 20 (1970) 309-312
3 10
SHORT COMMUNICATIONS
TABLE I ADRENAL G L A N D ENZYME ACTIVITY
Enzyme activity in adrenal gland during tolerance to and after 48 h of abstinence from morphine in rat. NS ~: Not significant (P ;> 0.05) by Student's t test.
Naive control Saline control Change from naive control Tolerant Change from saline control Withdrawal Change from saline control
Adrenal weight (mg/pair 2_ S.E.M.)
Tyrosine hydroxylase (nmoles/pair/ 20 rain 2- S.E.M.)
PNMT (nrnoles/pair/ 20 min 2_ S.E.M.)
MAO ( counts/min • lOa/pair/ 20 rain ~ S.E.M.)
647 + 26 687 4. 32
0.096 ± 0.019 0.089 2- 0.008
9.33 2_ 1.02 8.55 2- 1.73
24.5 ± 6.0 26.6 2- 6.0
(NS) 836 ± 43
(NS) 0.189 2- 0.031
(NS) 6.34 4, 1.43"*
(NS) 23.8 2_ 6.0
(P < 0.05) 815 2_ 20
(P < 0.05) 0.109 2_ 0.023*
(NS) 7.95 2_ 0.88
(NS) 31.3 2_ 4.3***
(P < 0.01)
(NS)
(NS)
(NS)
* Change from tolerant significant (P < 0.02). ** Change from naive control significant (P < 0.01). *** Change from tolerant significant (P < 0.05).
TABLE II ENZYMEACTIVITYIN CAUDATENUCLEUS Enzyme activity in caudate nucleus during tolerance to and after 48 h of abstinence from morphine in rat. NS = Not significant (P > 0.05) by Student's t test.
Naive control Saline control Change from naive control Tolerant Change from saline control Withdrawal Change from saline control
Tyrosine hydroxylase (nmoles/g/20 rain ± S.E.M.)
MA 0 (counts/min/g/20 min 2- S.E.M.)
0.077 -4- 0.013 0.047 4. 0.013 (P < 0.01) 0.110 4- 0.030 (P < 0.05) 0.100 ± 0.037 (NS)
212 2- 15 175 i 23 (NS) 205 4, 36 (NS) 243 4. 18" (P < 0.02)
* Change from tolerant animals significant (P < 0.01).
C h a n g e s in the activities o f all 3 e n z y m e s occur d u r i n g the cycle o f m o r p h i n e t o l e r a n c e a n d withdrawal. D u r i n g t o l e r a n c e there is a significant increase, to a l m o s t 200 ~ o f c o n t r o l , o f t y r o s i n e h y d r o x y l a s e activity in the ad r en al gland w h i ch falls back to n e a r c o n t r o l values 48 h after abstinence. P N M T activity is r ed u ced in t o l e r a n t a n i m a l s when c o m p a r e d to naive but n o t saline injected controls suggesting that there is additive effect o f stress o f i n j e c ti o n and m o r p h i n e in reducing the activity o f this Brain Research, 20 (1970) 309-312
SHORT COMMUNICATIONS
311
enzyme. MAO activity in the adrenal gland does not change during tolerance. During abstinence however, at a time when the animals are manifesting the well recognized clinical signs of withdrawal there is a significant increase in MAO activity. In the brain changes in tyrosine hydroxylase and MAO activities in the caudate nucleus (Table I1) follow, in general, a similar pattern. In brain there is a significant increase in tyrosine hydroxylase activity in tolerance which remains elevated even after 48 h of abstinence. Of particular interest is the fact that saline injections alone result in a significant decrease in tyrosine hydroxylase activity in the caudate nucleus. MAO activity in the caudate but not in hypothalamus or brain stem rose during withdrawal. This study demonstrates that significant changes in the activities of some enzymes involved in the synthesis and degradation of catecholamines occur during the morphine tolerance-withdrawal syndrome in the rat. During tolerance, the primary change is an increase in the activity of tyrosine hydroxylase in both adrenal gland and brain. The increased activity of this enzyme in morphine tolerant rats suggests that the synthesis of catecholamines may be increased and can explain the observations of others: (a) that in tolerance the levels of norepinephrine may be increased in brain1,5, 7 without a change in the release of the aminee,9; (b) that even though catecholamine levels of adrenal gland are unchanged in morphine tolerant animals catecholamine secretion is elevatedS; (c) that the recovery of brain and adrenal catecholamine levels from the depletion induced by reserpine is accelerated by morphine4,5; (d) that brain norepinephrine levels increase to a greater degree in morphine tolerant than in control animals following inhibition of MAO 5,7. The increased activity of tyrosine hydroxylase in the caudate nucleus, a region rich in dopamine, would also support evidence accrued by others that the turnover of this amine is increased, at least acutely, by administration of morphine 6. During tolerance there is a tendency for PNMT levels in the adrenal gland to decrease. Our findings are consistent with the histochemical observations of Ray et al. lo, demonstrating preferential shunting of catecholamine storage in the adrenal medulla from epinephrine to norepinephrine rich areas after morphine, and would support their interpretation that reduced PNMT activity underlies the phenomenon. During withdrawal at a time when tyrosine hydroxylase activity in the adrenal gland, but not in the brain, has returned to control values there is a rise in the activity of MAO in both the caudate nucleus and the adrenal gland. While the mechanism governing this rise is not clear the biological utility of enhanced MAO activity at this time may be to facilitate the destruction of excess biogenic amines resulting from augmented synthesis in tolerance. It is of interest that in morphine tolerance the increase in tyrosine hydroxylase activity in brain is paralleled by a rise in the activity of tryptophan hydroxylase 3, the probable rate-limiting step in serotonin synthesis. It is thus likely that in morphine tolerance, the synthesis mechanisms for 3 putative central neurotransmitters, norepinephrine, dopamine, and serotonin, are all enhanced. The relationship of these changes in pathways subserving the metabolism of biogenic amines for the development of tolerance to morphine or the symptoms of abstinence remains to be elucidated. Brain Research, 21 (1970) 309-312
312
SHORT COMMUNICATIONS T h i s s t u d y was s u p p o r t e d by N I H G r a n t s NS068911, G M 0 1 7 8 9 a n d NS31756. Nancy
M e r l i n o , M a r l e n e R i f k i n and J a n a l g l i c h p r o v i d e d v a l u a b l e t e c h n i c a l
assistance. Department of Neurology, Cornell University Medical College, New York, N.Y. 10021, and Rockefeller University, New York, N.Y. 10021 (U.S.A.)
DONALD J. REIS PAUl, HESS EFRAIN C. AZMITIA, JR.
I AKERA, T., AND BRODY, T. M., The addiction cycle to narcotics in the rat and its relationship to catecholamines, Biochem. Pharmacol., 17 (1968) 675-688. 2 AXEEROD,J., Purification and properties of phenylethanolamine-N-methyl transferase, J. biol. Chem., 237 (1962) 1657-1660. 3 AZMITIA, E. C., JR., HESS, P., AND REIS, D. J., Lile Sci., (1970) in press. 4 FREEDMAN,D. X., FRAM, D. H., AND GIARMAN,N. J., The effect of morphine on the regeneration of brain norepinephrine after reserpine, Fed. Proc., 20 (1961) 321. 5 GUNNE, L.-M., Catecholamines and 5-hydroxytryptamine in morphine tolerance and withdrawal, Acta physiol, scand., 58, Suppl. 204 (1963) 1-91. 6 GUNNE, L.-M., JONSSON, J., AND FUXE, K., Effects of morphine intoxication on brain catecholamine neurons, Europ. J. Pharmacol., 5 (1969) 338-342. 7 MEYNERT,E. W., AND KEINGMAN, G. 1., Tolerance to morphine. I. Effects of catecholamines in the brain and adrenal glands, J. Pharmacol. exp. Ther., 135 (1962) 285-295. 8 NAGATSU,T., LEVITT, M., AND UDENFRIEND,S., Tyrosine hydroxylase. The initial step in norepinephrine synthesis, J. biol. Chem., 239 (1964) 2910-2917. 9 NEEL, M. J., Failure of morphine dependence in rats to influence brain noradrenaline turnover, J. Pharm. Pharmacol., 20 (1968) 951-953. 10 RAY, A. K., MUKHERGI,M., AND GHOSH, J. J., Adrenal catecholamine and related changes during different phases of morphine administration - - a histochemical study, J. Neurochem., 15 (1968) 875-881. I 1 SEOAN, J. W., AND EISENMAN, A. J., Long persisting changes in catecholamine metabolism following addiction to and withdrawal from morphine, Res. Publ. Ass. nerv. ment. Dis., 46 (1968) 96-105. 12 SUNG, C. Y., WAY, E. L., AND SCOTT, K. G., Studies on the relationship of metabolic fate and hormonal effects of DE-methadone to the development of drug tolerance, J. Pharmacol. exp. Ther., 107 (1953) 12-23. 13 WURTMAN, R. J., AND AXEEROD, J., A sensitive and specific assay for the estimation of monoamine oxidase, Biochem. Pharmacol., 12 (1963) 1439-1441. (Accepted April 2nd, 1970)
Brain Research, 20 (1970) 309-312