Reserpine selectively increases tyrosine hydroxylase and dopamine-β-hydroxylase enzyme protein in central noradrenergic neurons

380

Braht Research, 81 (1974) 380 386 :!:) Elsevier Scientific Publishing Company, Amsterdam • Printed ill The Netherlands

Reserpine selectively increases tyrosine hydroxylase and dopamine/~-hydroxylase enzyme protein in central noradrenergic neurons

D O N A L D J. REIS, T O N G H. JOH, R O B E R T A. ROSS AND V I R G I N I A M. P I C K E L

Laboratory of Neurobiology, Department of Neurology, Cornell University Medical College, New York, N. Y. 10021 (U.S.A.) (Accepted August 19th, 1974)

Over the past several years, it has been established that the activities and amounts of some of the enzymes which synthesize catecholamines in peripheral ganglia and in chromaffin cells of the adrenal medulla are regulated by the background level of nerve impulse activity20. When the discharge of preganglionic fibel s to the sympathetic ganglia or the adlenal medulla is chronically increased by brain stimulation 16, reflexively, e.g. by cold or immobilization stress10, 20, or by the administration of drugs, particularly reserpine 12,14, the activities of the enzymes tyrosine hydroxylase (TH) and dopamine-fl-hydroxylase (DBH) are increased after a latency of several days. Because the effect of such stimuli on enzyme activity depends upon an intact preganglionic innervation, the response has been referred to as trans-synaptic induction 2°. The trans-synaptic induction of TH and DBH appears to be relatively selective, since the activity of the other catecholamine synthesizing enzyme, DOPA decarboxylase (DDC), is not increased in parallel 20. Moreover, the increase in TH activity has been shown immunochemically to be entirely attributable to an accumulation of specific TH enzyme protein and not to activation of pre-existing enzyme molecules 7. Recently, it has been shown in rat that the systemic administration of reserpine will increase the activity of TH in the region of the nucleus locus coeruleus 17,22, a pontine nucleus consisting almost entirely of the cell bodies ofnoradrenergic neurons ~l . In the present study, we have sought to determine if the reserpine-elicited effect on TH activity in the locus coeruleus is due to accumulation of specific enzyme protein or to the activation of existing enzyme molecules, if the effect is paralleled by changes in the regional activity and amount of DBH and the activity of DDC. We have also sought to determine the anatomical specificity of the response by measuring changes of TH activity within terminals of noradrenergic fibers in the hypothalamus and in the dopaminergic cell bodies and terminals of the nigrostriatal system ~1. These studies were performed on adult female Sprague-Dawley rats weighing 150-200 g. Reserpine (Serpasil, Ciba) was injected intraperitoneally and the animals were killed at varying times thereafter by cervical dislocation. The brain was lapidly removed and the region of the locus coeruleus, and, in some experiments, the whole hypothalamus, the region of the substantia nigra, and the caudate nucleus were

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Fig. 1. A: time course of changes in TH and DBH activity in locus coeruleus of rat following a single injection of reserpine (10 mg/kg, i.p). Enzyme activity is expressed as percent of mean activity in locus coeruleus in 6-8 saline injected controls. Control TH activity was 0.99 4- 0.09 nmoles/locus coeruleus/h; DBH activity was 2.46 ~ 0.10 nmoles/locus coeruleus/h. Each point represents mean S.E.M. of 8-12 animals. TH activity is represented in solid circles and DBH activity in open circles. *P < 0.02; ** P < 0.01; *** P < 0.001. B: immunoprecipitation of TH in locus coeruleus from reserpine treated ( ( 3 - - - ( 3 ) and control ( 0 O) rats. Six rats were treated with a single dose (10 mg/kg, i.p.) of reserpine and killed 4 days later. Enzyme activity was increased 2.8 fold. Pooled locus coerulei were homogenized in 10 vol. (w/v) of 50 m M phosphate buffer (pH 7.4) containing 0.1 ~o Triton X-100, and centrifuged. Ten #1 of antibody was added to 10-50/d of homogenate of treated or control animals and final volume adjusted to 60/~1 with buffer. After the mixture was centrifuged, TH activity was assayed in the supernatant. Note that the equivalence point is shifted to the left in locus coeruleus from reserpine-treated animals indicating accumulation of more specific enzyme protein.

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Fig. 2. Immunohistochemical localization of tyrosine hydroxylase in the nucleus locus coeruleus of the rat. Five micron polyethylene glycol sections were incubated with specific TH antisera and stained by the peroxidase-anti-peroxidase method 19. A : section through the nucleus locus coeruleus of an untreated rat. Most cells are stained by diffuse brown precipitate in cytoplasm, but not in nuclei. Several stained neurons located in adjacent nucleus subcoeruleus are identified by arrows. B: section through the nucleus locus coeruleus of a rat treated with reserpine (10 mg/kg, i.p.) 4 days prior to death. The neurons throughout the area contain much greater cytoplasmic peroxidase staining. Two neurons in subcoeruleus are shown by arrows. C: control section through comparable area of locus coeruleus as illustrated in A and B, but reacted with immune rabbit serum from which all TH antibodies were removed by prior adsorption with partially purified TH.

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dissected is. TH activity was assayed by a modification of the method of Coyle2. DBH activity was assayed either by the isotopic dilution method of Molinoff et al. 13 or by the COs trapping procedure of Joh et aLL DDC activity was measured by the method of Lamprecht and Coyle11. Specific antibodies to bovine adrenal TH and DBH were produced as described elsewhere 4-7. Immunochemical titration of TH and DBH was performed according to the adaptation of Joh et al. 7 of the method of Feigelson and Greengard 3. TH was localized immunochemically by modification15 of the technique of Sternberger et al. 19, in which specific antibody was labeled with horseradish peroxidase-anti-horseradish peroxidase conjugate and the peroxidase was visualized by reaction with 3,Y-diaminobenzidine and hydrogen peroxide. Controls for immunochemical specificity consisted of either substitution of pre-immune serum or prior adsorption of antibody with pure antigen. Comparison of reserpine-treated and control animals was made by mounting sections from each on a single slide and carrying them through the staining procedure in parallel. Administration of a single dose of reserpine (10 mg/kg, i.p.) resulted in a greater than 2-fold increase in the activities of TH and DBH in the area of the locus coeruleus (Fig. 1A). Although within the first 24 h TH activity decreased about 15 %, this was followed by a rapid rise in activity, peaking at 48-72 h. Enzyme activity then gradually fell, returning to normal by 21 days. The reserpine-induced response of TH activity was dose-dependent, being elicited at a threshold dose of 0.5 mg/kg. The response of DBH activity in the locus coeruleus was qualitatively similar to that of TH (Fig. 1A). It differed only in that peak activity was reached 24 h later than TH, the maximal response was smaller, and enzyme activity returned to control levels earlier. In contrast to the increase in TH and DBH activities, DDC activity in the region of the locus coeruleus did not change following reserpine administration. We next sought to determine, by immunotitration with highly specific antibodies to TH and DBH, if the increase in enzyme activities was due to increased accumulation of specific enzyme protein. The results of such an immunotitration experiment for TH are shown in Fig. lB. The immunotitration curves are produced by adding increasing amounts of locus coeruleus homogenates from control and reserpine-treated animals to a constant amount of TH antibody. The amounts of antibody are predetermined to be that amount which will precipitate out or inactivate all of the enzyme protein in the smallest amount of tissue used. This procedure results in the disappearance of enzyme activity in the supernatant. As more tissue is added it saturates the available antibody and increasing amounts of active enzyme remain in the supernatant. The amount of tissue enzyme that precipitates all of the antibody is termed the equivalence p o i n t 3 and is graphically identified as the point on the abscissa at which enzyme activity is first detected in the supernatant. Reserpine treatment shifted the equivalence point of the locus coeruleus homogenate to the left of the control. Only 7.8 pl of tissue homogenate from treated animals, in contrast to 22.4/zl from control animals, was required to bind all of the antibody. This indicates that the locus coeruleus from reserpine-treated animals contained about 2.9 times more specific TH enzyme protein than control animals. This increase in enzyme protein in the treated locus coeruleus corresponded almost exactly to the incremental increase in enzyme

384

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TABLE l E F F E C T S OF R E S E R P I N E O N T Y R O S l N E H Y D R O X Y L A S E A C T I V I T Y IN D I F F E R E N T B R A I N R E G I O N S

A n i m a l s were pretreated with reserpine (2 mg/kg, i.p.) for 4 days a n d killed on da3, 5. Tyrosine hydroxylase activity is expressed as either (a) nmoles o f [ ~ C ] D O P A formed/total tissue/h : S.E.M. or (b) n m o l e s o f product/g weight/'h i: S.E.M. n : n u m b e r o f a n i m a l s ; ns not significant. Significance ( P 0.05) determined by Student's t te~t.

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activity (2.8 times) induced by reserpine in the same tissues, lmmunotitration with an antibody to DBH also demonstrated, in a comparable manner, that the increase of locus coeruleus DBH activity induced by reserpine was entirely attributable to an increased accumulation of enzyme protein. The reserpine-evoked increase in TH accumulation in the region of the locus coeruleus was specifically localized to the cell bodies of noradrenergic neurons. As seen in Fig. 2, neurons within the locus coeruleus of reserpine-treated rats (Fig. 2B) show a much more intense labeling with T H antibody than do saline-injected controls (Fig. 2A). This observation shows that in our dissection the reserpine-induced effect is primarily restricted to cell bodies within the locus coeruleus and is additional evidence that reserpine increases the accumulation of T H enzyme protein. While reserpine increased T H activity in the locus coeruleus approximately 3-fold, enzyme activity in the hypothalamus, a region rich in noradrenergic nerve terminals 21, was only increased to 2 0 ~ above control (Table I). Furthermore, T H activity remained unchanged in both substantia nigra and caudate nucleus, in which are respectively located the cell bodies and nerve terminals of the dopaminergic nigrostriatal system 21. Thus, the reserpine-induced response appears to be primarily confined to the cell bodies of noradrenergic neurons. This study confirms and extends the observation that a single injection of reserpine can increase T H activity in noradrenergic neurons of the locus coeruleus 1v,22. In addition, the activity and amounts of DBH are increased in parallel. The reserpineevoked response of these enzymes appears, in most respects, identical to the transsynaptic induction initiated by the drug peripherallyV~,14: it is dose dependent, occurs after a latency of many hours or a few days, is prolonged, is not associated with changes in DDC, and is entirely attributable to accumulation of enzyme protein and not activation of pre-existing enzyme molecules. Whether the increase in enzyme protein in brain or periphery is a consequence of increased synthesis or decreased degradation of enzyme molecules remains to be established. The reserpine effect on T H in the central nervous system is not ubiquitous but

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appears to be restricted to noradrenergic neurons, primarily their cell bodies. Areas containing the terminals of noradrenergic neurons, such as hypothalamus, show only a small response and may explain why others, sampling TH in areas of terminals, found an increase of only 50-60 ~o in response to reserpine 12. The discrepancy between the magnitude of the response in cell bodies and terminals is inexplicable. Conceivably, it might represent either dispersion of enzyme by axoplasmi¢ transport into the widely ramified processes of noradrenergic neurons 18,21 or increased lability of newly accumulated enzyme. The absence of any effect of leserpine on TH in dopaminergic neurons of the substantia nigra is of considerable interest and highlights recent findings of differences in molecular i0rm between TH in central dopaminergic and noradrenergic systems s. Why TH in dopamine neurons fails to be induced by reserpine at the time it is increased in noradrenergic neurons is not known. It cannot be due to a failure of the drug to deplete monoamines in dopamine-containing terminals 1. However, if resetpine's action on locus coeruleus neurons is, as in the periphery, trans-synaptically mediated 20, it is plausible that the drug does not reflexly excite neurons of the substantia nigra in the same manner. Since TH, and to a lesser extent DBH, appears to be rate-limiting in the biosynthesis of norepinephrine in brain as in periphery, our observations suggest that central noradrenergic neurons are endowed with the potential to markedly enhance their capacity to synthesize neurotransmitter by increasing enzyme stores. Such changes may persist long after the initiating stimulus has ceased. This research was supported by grants from NIH (NS 06911, MH 24285) and the Harris Foundation. Robert A. Ross is a recipient of a Fellowship from the Benevolent Foundation of Scottish Rite Freemasonry.

i CARLSSON,A., ROSENGREN,E., BERTLER,,~., AND NILSSON,J., The effect of reserpine on the meta bolism of catecholamines. In S. GARATTINIAND V. GHETTI (Eds.), Psychotropic Drugs, Elsevier, Amsterdam, 1957, pp. 363-372. 2 COYLE, J. T., Tyrosine hydroxylase in rat brain-cofactor requirements, regional and subcellular distribution, Biochem. PharmacoL, 21 (1972) 1935-1944. 3 FEIGELSON,P., AND GREENGARD,O., Immunochemical evidence for increased titers of liver t ryptophan pyrrolase during substrate and hormonal enzyme induction, J. biol. Chem., 237 (1962) 37143717. 4 FUXE, K., GOLDSTEIN, M., HOKFELT, T., AND Jon, T. H., Immunochemical localization of dopamine-fl-hydroxylase in the peripheral and central nervous system, Res. Commun. Chem. Path. Pharmacol., 1 (1970) 627-636. 5 GraBS, J. W., SPECTOR, S., AND UDENFRIEND, S., Production of antibodies to dopamine-flhydroxylase of bovine adrenal medulla, Molec. PharmacoL, 3 (1967) 473-478. 6 HARTMAN,B. K., AND UDENFRIEND,S., The application of immunological techniques to the study of enzymes regulating catecholamine synthesis and degradation, Pharmacol. Rev., 24 (1972) 311-330. 7 JOH, T. H., GEGHMAN,C., AND REIS, O. J., Immunochemical demonstration of increased accumulation of tyrosine hydroxylase protein in sympathetic ganglia and adrenal medulla elicited by reserpine, Proc. nat. Acad. Sci. (Wash.), 70 (1973) 2767-2771. 8 JOH, T. H., AND REIS, D. J., Different forms of tyrosine hydroxylase in noradrenergic and dopaminergic systems in brain, Fed. Proc., 33 (1974) 535.

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9 JOH, T. H., Ross, R. A., AND REts, D. J., A simple and sensitive assay for dopamine-~/-hydroxylase. Anal. Biochem., (1974) in press. 10 KVETf4ANSK?, R., Transsynaptic and humoral regulation of adrenal catecholamine synthesis m stress. In E. USDIN AND S. SNYDER (Eds.), Frontiers in Catecholamine Research, Pergamon, New York, 1973, pp. 223-229. 11 LAMPRECHT, F., AND COYLE, J. T., DOPA decarboxylase in the developing rat brain, Brain Research, 41 (1972) 503-506. 12 MOLINOEF,P. B., BRIMIJOIN,W. S., WEINSHILBOUM,R., AND AXELROD,J., Neurally mediated increase in dopamine-fl-hydroxylase activity, Proc. nat. Acad. Sci. (Wash.), 66 (1970) 453-458. 13 MOLINOFF, P. B., WEINSHmBOUM,R. W., AND AXELROD,J., A sensitive enzymatic assay for dopamine-fl-hydroxylase, J. Pharmacol. exp. Ther., 178 (1971 ) 425-431. 14 MUELLER, R. A., THOENEN, H., AND AXELROD, J., Increase in tyrosine hydroxylase activity after reserpine administration, J. Pharmacol. exp. Ther., 169 (1969) 74-79. 15 PICKEL, V. M., JOB, T. H., FIELD, P. M., BECKER, C. G., AND RE~S, D. J., Cellular localization of tyrosinc hydroxylase by immunohistochemistry, J. Histochem. Cytochem., (1974) in press. 16 RE~S, D. J., MOORHEAD,D. T., RIFKIN, M., JOH, T. H., AND GOLDSTEIN, M., Changes in adrenal enzymes synthesizing catecholamines in attack behavior evoked by hypothalamic stimulation in the cat, Nature (Lond.), 229 (1971) 562-563. 17 REIs, D. J., Ross, R. A., AND Jo14, T. H., Reserpine increases the accumulation of tyrosine hydroxylase and dopamine-fl-hydroxylase enzyme protein in catecholamine neurons of rat brain, Fed. Proc., 33 (1974) 535. 18 ROSS, R. A., AND REIS, D. J., Effects of lesions of locus coeruleus on regional distribution of dopamine-fi-hydroxylase activity in rat brain, Brain Research, 73 (1974) 161-166. 19 STERNBERGER,L. A., HARDY, P. H., AVEYLIS,J. J., AND MYER, H. G., The unlabelled antibody enzyme method of immunochemistry: preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes, J. Histoehem. Cytochem., 18 (1970) 315. 20 THOENEN, H., Comparison between the effect of neuronal activity and nerve growth factor on the enzymes involved in the synthesis of norepinephrine, Pharmacol. Rev., 24 (1972) 255-267. 21 UNGERSTEDT,U., Stereotaxic mapping of the monoamine pathways in the rat brain, Acta physiol. scand., 82, Suppl. 367 (1971) 1-48. 22 ZIGMOND, R. E., SCHON, F., AND 1VERSEN,L. L., Increased tyrosine hydroxylase activity in the locus coeruleus of rat brain stem after reserpine treatment and cold stress, Brain Research, 70 (1974) 547-552.