532
Brain Research, 85 (1975) 532-534 Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - Printed in T h e N e t h e r l a n d s
Differential rates of increase in pontine tyrosine hydroxylase and dopaminefl-hydroxylase activities after reserpine
S H E I L A M. B R O O K E AND H A N S C. F I B I G E R
Division of" Neurological Sciences. Department of Psychiatry, University of British Colmnbia, Vancouver (Canada) (Accepted N o v e m b e r 25th, 1974}
Increases in the activities of tyrosine hydroxylase and dopamine-~-hydr0xylase. two enzymes involved in the biosynthesis of noradrenaline, have been reported in peripheral sympathetic neurons after reserpine4-6, 9-1 t. It has also been demonstrated that reserpine can increase tyrosine hydroxylase activity in the brain stem and in particular in the locus coeruleus, a pontine nucleus which contains noradrenergic cell bodies lz. Reis and Molinoff 7 found, however, that reserpine did not increase dopamine-6-hydroxylase activity in the brain stem. hypothalamus or cortex of rats 24 h after the last of 3 daily reserpine injections. As has been pointed out by Zigmond et al. t2. however, compared with the peripheral nervous system the effects of reserpine on the activity of tyrosine hydroxylase in brain are generally smaller and require more prolonged administration of the drug. The present experiments were undertaken therefore to determine if longer periods of time are required in order to pharmacologically influence dopamine-/%hydroxylase activity in the central nervous system. Male Wistar rats (Woodlyn Farms. Guelph, Ont.), weighing 450-500 g at the start of the experiment received daily subcutaneous injections of reserpine (2.5 mg/kg, Serpasil) for 3 days. The controls were injected with vehicle. The animals were housed individually and maintained on ad libitum food and water. At various intervals after the last injection (1, 5 and 12 days) the animals were killed by cervical fracture after which the brain was removed and dissected on ice. A section of the pons. which contained the locus coeruleus, was obtained by removing the cerebellum and making one cut through the pons at the level of the sulci limitantes and a second cut 1.5 m m rostral to the first. This dissection has been described in more detail by Zigmond et al. ~ . The tissue was weighed and homogenized in 40 vol. of cold Tris buffer (0.005 M. pH 6.0) 0.15% Triton X-100. Tyrosine hydroxylase was measured using D M P H 4 cofactor according to the method of MeGeer et al. 3. Dopamine-fl-hydroxylase was measured by the method of Coyle and AxelrodL Assays were carried out on 0.2 ml of whole homogenate and the final Cu 2+ concentration in each incubation mixture was 4.0 10 -~ M. Incubation mixtures containing boiled tissue plus known quantities oH octopamine were used as standards for determining the dopamine-fl-hydroxylase activity. In accord with previous reports, reserpine significantly increased tyrosine
533 TABLE I EFFECT OF RESERPINE (2.5 mg/kg/day, for 3 days) ON PONTINE TYROSINEHYDROXYLASEAND DOPAMINE-/% HYDROXYLASE ACTIVITIES D a t a represent m e a n s ( ± S.E.M.) of 5-6 animals in each group. Control Vm~x for tyrosine hydroxylase was 4.6 _- 0.3 n m o l e s D O P A / g tissue/h. Control Vmax for dopamine-fl-hydroxylase was 173 ~ 12 n m o l e s o c t o p a m i n e / g tissue/h.
Days after last Tyrosine hydroxylase reserpine injection ( °o...of control value)
Dopamine-[3-hydroxylase ( ,°/oof control value)
1 5 12
105ill 182 ± 16" 1 7 3 ~ 15"
2 5 7 5 15" 214 ~_ 11" 1 6 6 ~ 10"
* Significantly different f r o m control values, P < 0.01.
hydroxylase activity in the brain stem8,1L The increase was greatest (257 ~ above control levels) 1 day after the last reserpine injection and appeared to decline gradually thereafter (Table 1). The pontine tyrosine hydroxylase activity was still significantly elevated 12 days after the last injection however. The results obtained with dopaminefl-hydroxylase are also shown in Table I. We confirmed the observation by Reis and Molinoff 7 that dopamine-fl-hydroxylase activity was not significantly different from controls l day after the last of 3 daily reserpine injections. Five days after the last reserpine injection, however, pontine dopamine-fl-hydroxylase activity was significantly elevated and this elevation was also apparent 12 days after the reserpine regimen. The present experiments demonstrate that dopamine-fl-hydroxylase activity in the brain can, like tyrosine hydroxylase, be increased by reserpine. However, the latency of the reserpine-induced increase in dopamine-fl-hydroxylase activity was significantly greater than that required to increase tyrosine hydroxylase activity. There are several alternative mechanisms by which reserpine may produce these differential increases in the activities of pontine tyrosine hydroxylase and dopamine-fl-hydroxylase in the pons. One possibility is that different factors determine the rate of synthesis or the turnover of tyrosine hydroxylase and dopamine-fl-hydroxylase in the central noradrenergic neurons. If this is the case, it appears that regulatory differences may exist between central and peripheral noradrenergic neurons insofar as reserpine appears to increase the activities of tyrosine hydroxylase and dopamine-fl-hydroxylase at similar rates in peripheral sympathetic ganglia4, 6. Alternatively, the reserpineinduced increases in the activities of these two enzymes may occur in different populations of neurons in the pons. Thus, while Zigmond e t al. 12 demonstrated that the greatest reserpine-induced increase in pontine tyrosine hydroxylase occurred in the locus coeruleus, further experiments are required to determine the particular pontine dopamine-/4-hydroxylase-containing neurons on which reserpine has its most pronounced effects. It is possible for example that the delayed increase in dopamine-flhydroxylase activity in the pons represents the arrival by axoplasmic transport of increased levels of this enzyme in nerve endings of catecholaminergic neurons which
534 terminate in the puns but whose cell bodies lie outside this structure. Finally, in view of the fact that adrenergic neurons have recently been described in the brain stem'L these must also be considered as possible target cells for these actions of reserpine. NOTE ADDED IN PROOF
Since submission of this manuscript, Reis et al. have also reported on reserpineinduced increases in tyroslne hydroxytase and dopamine-/3-hydroxylase activities in the ports (REIS. D. J., JoH, T. H., Ross, R. A.. AND PICKEL, V. M., Reserpine selectively increases tyrosine hydroxylase and dopamine-/:~-hydroxylase enzyme proteinin central noradrenergic neurons, Brain Research, 81 (1974)380-386.). These workers demonstrated that the increased enzyme activities are observed primarily in the cell bodies of the locus coeruleus and that these increases are due to an enhanced accumulation of enzyme protein molecules rather than to an activation of pre-existing molecules. We would like to thank H. P. Fibiger for her excellent technical assistance. Supported by the Medical Research Council. H. C. Fibiger is an M R C Scholar.
1 COYLE,J., AND AXELROD, J., Dopamine-/5-hydroxylase in rat brain: developmental characteristics, J. Neurochem., 19 11972) 449-459. 2 H(3KFELT, T.. FUXE, K., AND GOLDSTEIN. M., lmmunohistochemicat studies on monoammecontaining cell systems, Brain Research. 62 (1973) 461-469 3 McGEER, E., MCGEER, P., AND WADA, J., Distribution of tyrosine hydroxytase ill human and animal brain, J. Neurochem.. 18 (1971) 1647-1658. 4 MOLINOFF, P. B., BRIMLIOIN, S.. AND AXELROD, J., Induction of dopamine-~-hydroxylase and tyrosine hydroxylase in rat hearts and sympathetic ganglion, J. PharmacoL exp. Ther., ] 82 (1972~ 1 ] 6-129. 5 MOLINOFF, P. B., BRIMI.IOIN, S., WEINSHILBOUM, R., AND AXELROD,J., Neural mediated increases in dopamine-fl-hydroxylase, Proc. nat. Acad. Sci. (Wash.), 66 11970) 543-458. 6 MUELLER, R. A., THOENEN, H., AND AXELROD, J., Increase in tyrosine hydroxylase activity after reserpine administration, J. Pharmacol. exp. Ther., 169 (1969) 74-79. 7 REIS, D. J., AND MOLINOFF, P. B., Brain dopamine-fi-hydroxytase : regional distribution a n d effects of lesions and 6-hydroxydopamine on activity, J. Neurochem, 19 11972) 195-204. 8 SEGAL, D. S.. SULLIVAN,J. L.. KUCZENSKI,R. l., AND MANDELL.A. J., Effects oflong-termreserpine treatment on brain tyrosine hydroxylase and behavioral activity, Science. 173 (1971) 847-849. 9 THOENEN, H. H., MUELLER,R. A., AND AXELROD,J., Phase differences in the induction o1Ltyrosine hydroxylase in cell body and nerve terminals of sympathetic neurons. Proc. nat. Acad. Sci. ? Wash. ). 65 (1970) 58-62. l0 V1VEROS,O. H.~ ARQUEROS,L.. CONNET, R. J., AND KIRSHNER, H., Mechanism of secretion l rum the adrenal medulla. IV. The fate of the storage vesicles following insulin and reserpine administration, Molec. Pharmacol., 5 (1969) 69-82. 11 ZIGMONO,R. E.. AND MACKAY, A. V. P., Dissociation ofstimulatory and synthetic phases in the induction of tyrosine hydroxylase, Nature (Lond.), 247 (1974) 112-113. 12 Z1GMOND, R. E., SCHON, F., AND IVERSEN, t . L., Increased tyrosine hydroxyfase activity in the locus coeruleus of rat brain stern after reserpine treatment and cold stress. Brain Research. 70 (1974) 547-552.
Brain Research, 85 (1975) 532-534 Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - Printed in T h e N e t h e r l a n d s
Differential rates of increase in pontine tyrosine hydroxylase and dopaminefl-hydroxylase activities after reserpine
S H E I L A M. B R O O K E AND H A N S C. F I B I G E R
Division of" Neurological Sciences. Department of Psychiatry, University of British Colmnbia, Vancouver (Canada) (Accepted N o v e m b e r 25th, 1974}
Increases in the activities of tyrosine hydroxylase and dopamine-~-hydr0xylase. two enzymes involved in the biosynthesis of noradrenaline, have been reported in peripheral sympathetic neurons after reserpine4-6, 9-1 t. It has also been demonstrated that reserpine can increase tyrosine hydroxylase activity in the brain stem and in particular in the locus coeruleus, a pontine nucleus which contains noradrenergic cell bodies lz. Reis and Molinoff 7 found, however, that reserpine did not increase dopamine-6-hydroxylase activity in the brain stem. hypothalamus or cortex of rats 24 h after the last of 3 daily reserpine injections. As has been pointed out by Zigmond et al. t2. however, compared with the peripheral nervous system the effects of reserpine on the activity of tyrosine hydroxylase in brain are generally smaller and require more prolonged administration of the drug. The present experiments were undertaken therefore to determine if longer periods of time are required in order to pharmacologically influence dopamine-/%hydroxylase activity in the central nervous system. Male Wistar rats (Woodlyn Farms. Guelph, Ont.), weighing 450-500 g at the start of the experiment received daily subcutaneous injections of reserpine (2.5 mg/kg, Serpasil) for 3 days. The controls were injected with vehicle. The animals were housed individually and maintained on ad libitum food and water. At various intervals after the last injection (1, 5 and 12 days) the animals were killed by cervical fracture after which the brain was removed and dissected on ice. A section of the pons. which contained the locus coeruleus, was obtained by removing the cerebellum and making one cut through the pons at the level of the sulci limitantes and a second cut 1.5 m m rostral to the first. This dissection has been described in more detail by Zigmond et al. ~ . The tissue was weighed and homogenized in 40 vol. of cold Tris buffer (0.005 M. pH 6.0) 0.15% Triton X-100. Tyrosine hydroxylase was measured using D M P H 4 cofactor according to the method of MeGeer et al. 3. Dopamine-fl-hydroxylase was measured by the method of Coyle and AxelrodL Assays were carried out on 0.2 ml of whole homogenate and the final Cu 2+ concentration in each incubation mixture was 4.0 10 -~ M. Incubation mixtures containing boiled tissue plus known quantities oH octopamine were used as standards for determining the dopamine-fl-hydroxylase activity. In accord with previous reports, reserpine significantly increased tyrosine
533 TABLE I EFFECT OF RESERPINE (2.5 mg/kg/day, for 3 days) ON PONTINE TYROSINEHYDROXYLASEAND DOPAMINE-/% HYDROXYLASE ACTIVITIES D a t a represent m e a n s ( ± S.E.M.) of 5-6 animals in each group. Control Vm~x for tyrosine hydroxylase was 4.6 _- 0.3 n m o l e s D O P A / g tissue/h. Control Vmax for dopamine-fl-hydroxylase was 173 ~ 12 n m o l e s o c t o p a m i n e / g tissue/h.
Days after last Tyrosine hydroxylase reserpine injection ( °o...of control value)
Dopamine-[3-hydroxylase ( ,°/oof control value)
1 5 12
105ill 182 ± 16" 1 7 3 ~ 15"
2 5 7 5 15" 214 ~_ 11" 1 6 6 ~ 10"
* Significantly different f r o m control values, P < 0.01.
hydroxylase activity in the brain stem8,1L The increase was greatest (257 ~ above control levels) 1 day after the last reserpine injection and appeared to decline gradually thereafter (Table 1). The pontine tyrosine hydroxylase activity was still significantly elevated 12 days after the last injection however. The results obtained with dopaminefl-hydroxylase are also shown in Table I. We confirmed the observation by Reis and Molinoff 7 that dopamine-fl-hydroxylase activity was not significantly different from controls l day after the last of 3 daily reserpine injections. Five days after the last reserpine injection, however, pontine dopamine-fl-hydroxylase activity was significantly elevated and this elevation was also apparent 12 days after the reserpine regimen. The present experiments demonstrate that dopamine-fl-hydroxylase activity in the brain can, like tyrosine hydroxylase, be increased by reserpine. However, the latency of the reserpine-induced increase in dopamine-fl-hydroxylase activity was significantly greater than that required to increase tyrosine hydroxylase activity. There are several alternative mechanisms by which reserpine may produce these differential increases in the activities of pontine tyrosine hydroxylase and dopamine-fl-hydroxylase in the pons. One possibility is that different factors determine the rate of synthesis or the turnover of tyrosine hydroxylase and dopamine-fl-hydroxylase in the central noradrenergic neurons. If this is the case, it appears that regulatory differences may exist between central and peripheral noradrenergic neurons insofar as reserpine appears to increase the activities of tyrosine hydroxylase and dopamine-fl-hydroxylase at similar rates in peripheral sympathetic ganglia4, 6. Alternatively, the reserpineinduced increases in the activities of these two enzymes may occur in different populations of neurons in the pons. Thus, while Zigmond e t al. 12 demonstrated that the greatest reserpine-induced increase in pontine tyrosine hydroxylase occurred in the locus coeruleus, further experiments are required to determine the particular pontine dopamine-/4-hydroxylase-containing neurons on which reserpine has its most pronounced effects. It is possible for example that the delayed increase in dopamine-flhydroxylase activity in the pons represents the arrival by axoplasmic transport of increased levels of this enzyme in nerve endings of catecholaminergic neurons which
534 terminate in the puns but whose cell bodies lie outside this structure. Finally, in view of the fact that adrenergic neurons have recently been described in the brain stem'L these must also be considered as possible target cells for these actions of reserpine. NOTE ADDED IN PROOF
Since submission of this manuscript, Reis et al. have also reported on reserpineinduced increases in tyroslne hydroxytase and dopamine-/3-hydroxylase activities in the ports (REIS. D. J., JoH, T. H., Ross, R. A.. AND PICKEL, V. M., Reserpine selectively increases tyrosine hydroxylase and dopamine-/:~-hydroxylase enzyme proteinin central noradrenergic neurons, Brain Research, 81 (1974)380-386.). These workers demonstrated that the increased enzyme activities are observed primarily in the cell bodies of the locus coeruleus and that these increases are due to an enhanced accumulation of enzyme protein molecules rather than to an activation of pre-existing molecules. We would like to thank H. P. Fibiger for her excellent technical assistance. Supported by the Medical Research Council. H. C. Fibiger is an M R C Scholar.
1 COYLE,J., AND AXELROD, J., Dopamine-/5-hydroxylase in rat brain: developmental characteristics, J. Neurochem., 19 11972) 449-459. 2 H(3KFELT, T.. FUXE, K., AND GOLDSTEIN. M., lmmunohistochemicat studies on monoammecontaining cell systems, Brain Research. 62 (1973) 461-469 3 McGEER, E., MCGEER, P., AND WADA, J., Distribution of tyrosine hydroxytase ill human and animal brain, J. Neurochem.. 18 (1971) 1647-1658. 4 MOLINOFF, P. B., BRIMLIOIN, S.. AND AXELROD, J., Induction of dopamine-~-hydroxylase and tyrosine hydroxylase in rat hearts and sympathetic ganglion, J. PharmacoL exp. Ther., ] 82 (1972~ 1 ] 6-129. 5 MOLINOFF, P. B., BRIMI.IOIN, S., WEINSHILBOUM, R., AND AXELROD,J., Neural mediated increases in dopamine-fl-hydroxylase, Proc. nat. Acad. Sci. (Wash.), 66 11970) 543-458. 6 MUELLER, R. A., THOENEN, H., AND AXELROD, J., Increase in tyrosine hydroxylase activity after reserpine administration, J. Pharmacol. exp. Ther., 169 (1969) 74-79. 7 REIS, D. J., AND MOLINOFF, P. B., Brain dopamine-fi-hydroxytase : regional distribution a n d effects of lesions and 6-hydroxydopamine on activity, J. Neurochem, 19 11972) 195-204. 8 SEGAL, D. S.. SULLIVAN,J. L.. KUCZENSKI,R. l., AND MANDELL.A. J., Effects oflong-termreserpine treatment on brain tyrosine hydroxylase and behavioral activity, Science. 173 (1971) 847-849. 9 THOENEN, H. H., MUELLER,R. A., AND AXELROD,J., Phase differences in the induction o1Ltyrosine hydroxylase in cell body and nerve terminals of sympathetic neurons. Proc. nat. Acad. Sci. ? Wash. ). 65 (1970) 58-62. l0 V1VEROS,O. H.~ ARQUEROS,L.. CONNET, R. J., AND KIRSHNER, H., Mechanism of secretion l rum the adrenal medulla. IV. The fate of the storage vesicles following insulin and reserpine administration, Molec. Pharmacol., 5 (1969) 69-82. 11 ZIGMONO,R. E.. AND MACKAY, A. V. P., Dissociation ofstimulatory and synthetic phases in the induction of tyrosine hydroxylase, Nature (Lond.), 247 (1974) 112-113. 12 Z1GMOND, R. E., SCHON, F., AND IVERSEN, t . L., Increased tyrosine hydroxyfase activity in the locus coeruleus of rat brain stern after reserpine treatment and cold stress. Brain Research. 70 (1974) 547-552.
