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Adrenal influence on tyrosine hydroxylase activity in superior cervical ganglion
Brain Research, 202 (1980) 347-356
347
' 9 Elsevier/North-Holland Biomedical Press
ADRENAL INFLUENCE ON TYROSINE HYDROXYLASE ACTIVITY IN SUPERIOR CERVICAL GANGLION
KEITH A. MARKEY* and PAUL Y. SZE**
Department of Biobehavioral Sciences, The University of Connecticut, Storrs, Conn. 06268 (U.S.A.) (Accepted June 12th, 1980)
Key words : tyrosine hydroxylase - - superior cervical ganglion - - glucocorticoids - - epinephrine
SUMMARY
The involvement of adrenal hormones as regulatory factors in maintaining physiological levels of tyrosine hydroxylase (TH) was examined in mouse superior cervical ganglion. Following bilateral adrenalectomy, TH activity in the ganglion fell at a slow but steady rate, reaching 60-65 % of the control levels after 2 weeks. Decentralization is known also to reduce TH activity in the ganglion. The effects of adrenalectomy and decentralization were therefore compared, and they were found to be additive, indicating different mechanisms in the two cases. The reduction of TH activity following adrenalectomy was not prevented by replacement with corticosterone (0.5 mg/kg, daily). However, replacement with epinephrine (4 mg/kg, daily) completely prevented the fall of TH activity in adrenalectomized animals. Isoproterenol, a fl-adrenergic receptor agonist, was as effective as epinephrine in preventing the reduction of TH activity following adrenalectomy. Furthermore, in intact animals, chronic administration of SKF 64139, an inhibitor of adrenal PNMT which depletes circulating epinephrine levels, also reduced ganglionic TH activity to the same level as that after adrenalectomy. These results indicate that epinephrine, but not corticosterone, is the adrenal factor required for physiological maintenance of normal levels of TH in the superior cervical ganglion. INTRODUCTION
Several studies indicated the possibility of a regulatory role of glucocorticoids in the induction of tyrosine hydroxylase (TH) in the superior cervical ganglion. First, the * Present address: Department of Psychiatry, New York University Medical Center, New York, N.Y. 10016, U.S.A. ** To whom reprint requests should be addressed.
348 inducibility of ganglionic T H by cold stress appeared to follow a c~rcadian rla5 thin that correlated with that of circulating glucocorticoids 16. Only during periods of high steroid levels could T H activity be elevated by the cold exposure. Pre-treatmcnt x~ith dexamcthasone during periods of low endogenous steroid levels permitted the elevation of enzyme activity. Second. induction of ganglionic T H by reserpine could be blocked with the glucocorticoid antagonist cortexolone 7. Third. in the induction ~ f I ' H by net,re growth factor (NGF) in cultured ganglion, the effect of N G F was potentiated b~ addition of dexamethasone to the culture mediu m~7. Fourth, TH activity in the ganglion was increased following administration of a pharmacological dose of dexamethasone a,7. The dexamethasone effect here. however, seemed to involve a pre-ganglionic mechanism, since the effect was abolished in decentralized ganglion, Epinephrine, the other major adrenal hormone, also appeared to have a role tn the regulation of T H in the superior cervical ganglion. Pharmacological doses ol the hormone were found to increase T H activity in the ganglion through a mechanism that did not seem to involve pre-ganglionic input. Instead. the effect of epinephrine could be blocked by propranolol, a fi-adrenergic receptor antagonist ~-7 In spite of the above findings from pharmacological effects and effects m wtro. no clear understanding has emerged as whether the adrenal hormones are indced involved physiologically as regulatory factors for the enzyme. Using the classical approach of adrenalectomy and hormone replacement, the present study was designed to examine the significance of the adrenal influence in the regulation of physiological levels of ganglionic TH activity in vivo, MATERIALS AND METHODS Animals and surgel3, Male C57BL/10Bg mice (2-4 months old) were bred and maintained in our specific pathogen-free colony. The pathogen-free condition was used in order to minimize subclinical diseases which may serve as a source of stress in altering normal adrenal activity. The mice were maintained on a t2 h light-dark cycle under controlled temperature and humidity. Bilateral adrenalectomy and decentralization of the superior cervical ganglion were performed under chloral hydrate anesthesia. Adrenalectomized animals received 0.9 ~o NaCI in drinking water. Enzyme assays T H activity was assayed by measuring the formation of ZHg.O alter hydroxylation of [aH]tyrosine according to the procedure of Nagatsu et al. 1~ with modifications. The individual ganglion was dispersed by freezing and thawing twice in 20 #1 distilled HzO. The reaction was initiated by adding 20 #1 of a substrate mixture containing: 800 mM sodium phosphate (pH 6.0); 40 # m L-[3,5-ZH]tyrosine (New England Nuclear. spec. act. 4 mCi/16.6 #mol); 2.0 mM 2_amino-4-hydroxy-6-methyl-5.6.%8-tetrahydropteridine (6-MPH4); 50 m M 2-mercaptoethanot. Alter incubation at 37"C for 20 min (linear rates within this time period), the reaction was stopped by the addition of 0.1 N NaOH. Tritiated H20 was isolated on an ion exchange column consisting of Bio-Rad
349 100'
0
80-
t~
~
60-
,~
40-
~(6) T H
E ~
N C W
20'
0
I 0
i 7
Days
after
I 14
-! 21
Adx.
Fig. I. T H activity and DOPA decarboxylase (DDC) activity in superior cervical ganglia following adrenalectomy (Adx). Controls (0 day) were sham-operated. * P < 0.01; ** P < 0.005; compared with 0 day control.
A G 50 W-X8 cation exchange resin and Bio-Rex 9 anion exchange resin. The use of
bilayer cation and anion exchange resins substantially reduced the blank value. DOPA decarboxylase activity was assayed by measuring the formation of 14CO2 from carboxyl-L-[14C]dihydroxyphenylalanineaccording to the procedure of Sims et al z0 with modifications. The individual ganglion was dispersed by freezing and thawing twice in 50/~1 of 0.1 M sodium phosphate buffer (pH 6.8). The reaction was initiated by adding 50 ,ul of a substrate mixture containing l0 mM [1-14C]dihydroxyphenylalanine (New England Nuclear; spec. act. 1.0 mCi/5.0 retool). After incubation at 37 °C for 20 rain, the reaction was stopped by the addition of sulfuric acid. The 14CO2 produced was absorbed in hyamine hydroxide.
Statistical analysis Significance of difference between groups was determined by Student's t-test. All values were shown as mean j: S.E.M., with the number of animals in each group indicated in parentheses. Lineweaver-Burk plots were constructed using least square fit. Coefficients of correlation were equal to or greater than 0.92. RESULTS
Efl. ects of adrenalectomy on T H activit), To determine the influence of adrenal function on TH in the superior cervical ganglion, the enzyme activity was measured in adrenalectomized animals. Fig. 1 shows that following bilateral adrenalectomy TH activity fell to 70 ~ of control within one week, reaching 60-65 ~ by the second week. DOPA decarboxylase activity, monitored
350 in the g a n g l i o n as a reference, was n o t altered t h r o u g h o u t the entire period. These resuits indicate that n o r m a l a d r e n a l function is required for m a i n t a i n i n g p h y s i o l o g i c a l levels o f T H activity in the ganglion. Since D O P A d e c a r b o x y l a s e activity r e m a i n e d unchanged, the reduction o f T H activity was unlikely due to the loss o f s y m p a t h e t i c n e u r o n s in the ganglion o r o t h e r non-specific changes f o l l o w i n g a d r e n a l e c t o m y .
%
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~
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~
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f
i~ 40
~10 II
-:~
~
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,o
Z
o
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lb
l's
1 6 - M P H 4 , mM
Fig. 2. Lineweaver-Burk plots of TH activity vs (A) tyrosine concentrations and (B) 6-MPH4 concentrations. Ganglionic TIFFwas from intact controls ( 0 0 ) or from mice adrenalectomized for 2 weeks ((3 -©). Pooled ganglia from 5-6 animals were used to construct each line. A: the concentration of tyrosine was 20 ctM. B : the concentration of 6-MPH4 was held constant at 1.0 raM. A shortened incubation time (5 rain) was used for the enzyme assay. TH activity is expressed as pmol[5 rain/ganglion. Each point is the average of duplicate determinations.
351 The reduction of ganglionic TH activity was characterized by kinetic analyses (Fig. 2). Lineweaver-Burk plots reveal that only the Vmax of TH was decreased following adrenalectomy. The Km of the enzyme for either tyrosine or 6-MPH4 was not altered. (The K,,~ values were estimated as 70 #M tyrosine and 280/~M for 6-MPH4.) From the kinetic data, it becomes possible to suggest that the decrease of TH activity following adrenalectomy may be due to a reduction of the steady-state levels of the enzyme.
Comparison of adrenal and pre-ganglionic effects on TH activity Pre-ganglionic transection ('decentralization') is known to result also in a reduction of ganglionic TH activity2,3,6,s,9,1a,24. The effects of adrenalectomy and decentralization were therefore compared. As shown in Fig. 3, TH activity was reduced to 52 ~ of control two weeks after decentralization. In the ganglion that was decentralized, adrenalectomy produced a further reduction of TH activity, reaching only 30 o/ /o of control or 60 ~ of the level from decentralization alone. Thus, the effects of adrenalectomy and decentralization were additive, indicating that the adrenal effect could occur independent of the central cholinergic innervation.
Hormone replacementfollowing adrenalectomy To identify the hormone(s) involved in the adrenal effect, adrenalectomized animals were given replacement injections of either corticosterone or epinephrine. The
100"
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°~
' ~ 40'
a~
-r I-" 20'
INTACT
ADX
DE(
ADX
(6)
(8)
(5)
D~*c (4)
Fig. 3. Effects of adrenalectomy and decentralization on T H activity in superior cervical ganglia. Mice were adrenalectomized for 2 weeks (ADX); with their ganglia decentralized for 2 weeks (DEC); both adrenalectomized and decentralized for 2 weeks (ADX ÷ DEC); or sham-operated (INTACT). Decentralization of ganglia in all animals was performed unilaterally; ganglia of sham-operated animals were used for comparison. ** P -< 0.005 compared with intact control.
352 TABLE I
Effects of hormone replacement on ganglionic TH activity following adrenalectomv
Adrenalectomized or intacl control mice were given daily subcutaneous injections of the hormone tin sesame oil suspension)for 2 weeks.Each value is mean -~ S.E,M. from the indicated number of animals. Treatment
TH activity (pmol/k/ganglion)
Intact control c o r t i c o s t e r o n e (0.5 m g / k g ) e p i n e p h r i n e (4 m g / k g l
106 • 8.4 109 i 9.0 101 -1 3.2
(61 (o1 (6~
Adrenalectomized --- c o r t i c o s t e r o n e (0.5 m g / k g ) e p i n e p h r i n e (4 m g / k g ) isoproterenol (5.4mg/kg)
66 70 112 101
(~ (8) (61 (6)
z_ -i -~ r
3.6** 4.6 ~ 7.6 8.2
* P ~-- 0.01. ** P < 0.005.
replacement was initiated immediately after surgery and continued daily throughout the entire 2 week period. As shown in Table I. daily injections of corticosterone (0.5 mg/kg) did not prevent the decrease of T H activity caused by adrenalectomy, nor did the treatment have an effect on normal enzyme activity in intact animals. Higher doses of the steroid were used (up to 10 mg/kg, daily), but the adrenalectomy effect on TH activity was still not prevented (data not presented). In contrast to corticosterone, replacement with epinephrine fully prevented the effect of adrenatectomy in reducing ganglionic TH activity (Table 1). It should be noted that with the small doses used here (4 mg/kg), epinephrine had no effect on normal enzyme activity in intact animals, although pharmacological doses (50 rng/kg) could increase ganglionic T H activity~. Yet. such small doses were sufficient to prevent the effect of adrenalectomy. Furthermore. the ability of epinephrine to prevent the adrenalectomy effect could be mimicked by isoproterenol, indicating the involvemenl of fl-adrenergic receptor. From Lineweaver-Burk analyses (varying tyrosine and 6-MPH4 concentrations as in Fig. 2), the kinetic parameters of T H from intact and epinephrine-replaced adrenalectomized animals were found to be identical (data not shown). Replacement with epinephrine restored the Vmax of TH. which was reduced in adrenalectomized animals (see Fig. 2). Thus. it appears that hormone replacement reversed the effect of adrenalectomy and resulted in an enzyme kinetically identical to that from normal animals.
TH activity after inhibition of adrenal P N M T To support the finding that the reduction of ganglionic T H activity following adrenalectomy was due to epinephrine, the enzyme activity was further determined in animals with their circulating epinephrine depleted by SKF 64139, a specific inhibitor of adrenal phenylethanolamine N-methyl-transferase (PNMT) activity. Table II shows that after chronic administration of the adrenal P N M T inhibitor for two weeks, T H
353 TABLE II
Effect o f S K F 64139 on ganglionic T H activity Mice were given SKF 64139 orally at the indicated dose twice daily for 2 weeks. Each value is mean ~S.E.M. from the indicated number of animals.
Treatment
T H activity (pmol/h/ganglion)
Control SKF 64139 (100 mg/kg)
110 ± 4.0 (6) 70 ± 3.5* (7)
* P -; 0.005.
activity in the ganglion was reduced to 70 ~ of control. At the dose used (100 mg/kg, twice daily), SKF 64139 was known to deplete epinephrine in adrenal glands to as low as 25 O//oof normal 19. Clearly, the reduction of ganglionic TH activity following pharmacological depletion of circulating epinephrine was comparable to that found after adrenalectomy.
Reserpine induction of TH after adrenalectomy Since corticosterone did not appear to be a regulatory component in maintaining physiological levels of TH activity in the ganglion, the possibility was tested that the steroid might serve as a required factor in order for the induction of the enzyme to occur. In this experiment, the effect of reserpine in inducing ganglion TH was examined in adrenalectomized animals. In intact animals, administration of reserpine is well
160
140
120c 100-
>~ .m > ,m
-~
Z
80I
60.
< ~
p-
40. 20-
INTACT CONTROL (6)
INTACT * RES (4)
ADX (8)
AOX + RES (9)
Fig. 4. Induction of ganglionic TH by reserpine in adrenalectomized mice. Animals were intact or adrenalectomized (A DX) for 2 weeks. Reserpine (RES) was administered (5 mg/kg, s.c. in saline) 2 days before sacrifice. * P -- 0.01 ; ** P < 0.005 compared with intact saline control.
354 known to produce an induction of ganglionic T H through a trans-synapt~c mechanism z4. As shown in Fig. 4. the induction of ganglionic T H by reserpine could occur completely in adrenalectomized animals. Thus, the result indicates that trans synaptic induction of T H in the ganglion could proceed in the absence of glucocorticoids. The result also indicates that the enzyme induction by reserpine was independent of adrenal epinephrine. DISCUSSION Research for more than a decade has clearly established that cholinergtc activity from pre-ganglionic innervation is an important factor in the regulation of T H in the superior cervical ganglion 1-4,9-t2,24,25. The present study indicates that adrenal epinephrine is another regulatory factor in maintaining physiological levels of ganglionic T H activity. As in the case of decentralization, adrenalectomy led to a marked reduction of T H activity in the ganglion. Replacement with corticosterone, even at pharmacological doses, failed to prevent reduction of enzyme activity in adrenalectomized animals. However, replacement with epinephrine, at small doses that showed no apparent effect on normal T H activity in intact animals, could fully prevent the reduction of enzyme activity caused by adrenalectomy. Moreover, the effect of adrenalectomy could be mimicked by pharmacological depletion of circulating epinephrine following chronic administration of an adrenal P N M T inhibitor. Thus, it has become clear that the effect of adrenalectomy in reducing ganglionic T H activity was due to depletion of epinephrine. The adrenal influence on ganglionic T H appears to be independent ~,[ t he cholinergic influence from pre-ganglionic innervation. This is indicated by the additive effects of adrenalectomy and decentralization. Since isoproterenol was as effective as epinephrine, the hormone action appears to involve fl-adrenergic receptor. This is consistent with the previous finding that the induction of ganglionic T H by pharmacological doses of epinephrine was blocked by propranolol, a fl-adrenergic receptor antagonist 6. Using adrenalectomy as our approach, our findings on corticosterone do not support the contention that glucocorticoids are a regulatory factor in vivo for ganglionic TH. both in physiological maintenance of the enzyme and in trans-synaptic induction of the enzyme, at least by reserpine. It may be noted that glucocorticoids are also not :involved in trans-synaptic induction of T H in adrenal medulla by reserpine, since the reserpine effect is not prevented by hypophysectomy22. We have confirmed the previous finding that administration of dexamethasone can induce T H in the ganglion ~',7. However, in a study comparing 4 glucocorticoids (dexamethasone, corficosterone, llydrocortisone, and triamcinolone), only the synthetic steroid dexamethasone was found to be effective2L Corticosterone, the principal glucocorticoid in the mouse and rat. failed to produce an inductive effect on ganglionic T H even at doses as high as 50 mg/kg. Another study from our laboratory has characterized the cytosol glucocorticoid receptor in the superior cervical ganglion, and found that the steroid receptor exhibits "transcortin-like' properties, with no binding affinity for dexamethasone 23. There-
355 fore, the effect of dexamethasone on T H cannot involve an intraneuronal steroid receptor-mediated mechanism. Moreover, decentralization immediately prior to dexamethasone administration did not prevent the increase of ganglionic T H activity, although earlier decentralization (24 h or longer) abolished the steroid effect ls,zl, in such newly decentralized ganglion, the increase o f enzyme activity by dexamethasone was still blocked by chlorisondamine, a cholinergic receptor antagonistZL Since synaptic activity is k n o w n to continue for a brief period following nerve transection, these findings suggest that the primary site of the dexamethasone effect may be the pre-ganglionic terminals. Indeed, a recent study in our laboratory has shown a marked elevation of choline and acetylcholine levels in the ganglion following dexamethasone administration, and also an increase o f high-affinity ganglionic [3H]choline uptake in vitro by the steroid (Marchi et al., in preparation). Significantly, the elevation o f choline and acetylcholine levels was not found in the ganglion after administration o f triamcinolohe, a glucocorticoid which is ineffective in producing an increase o f ganglionic T H activity. In light of these recent findings, it has become clear that the use of the synthetic steroid dexamethasone, as in several previous studies on sympathetic ganglion, does not reflect the hormonal actions o f glucocorticoids, physiologically. In summary, an assessment of the significance o f adrenal influence on ganglionic TH has revealed that epinephrine, but not corticosterone, is an important regulatory component in maintaining physiological levels of T H activity in the superior cervical ganglion. In the regulation o f ganglionic TH, the hormonal component from epinephrine appears to be as important as the neural component from the cholinergic innerration. ACKNOWLEDGEMENT This research was supported by G r a n t MH29237 from the U.S. Public Health Service.
REFERENCES I Black, I. B., Hendry, I. A. and Iversen, L. L., Effects of surgical decentralization and nerve growth factor on the maturation of adrenergic neurons in a mouse sympathetic ganglia, J. Neurochem., 19 (1972) 1367-1377. 2 Black, 1. B., Job, I. H. and Reis, D. J., Accumulation of tyrosine hydroxylase molecules during growth and development of superior cervical ganglion, Brain Research, 75 (1974) 133 144. 3 Black, I. B. and Mytilineou, C., Trans-synaptic regulation of the development of end organ innervation by sympathetic neurons, Brain Research, 101 (1976) 503 521. 4 Hamill, R. W., Bloom, E. M. and Black, I. B., The effect of spinal cord transection on the development of cholinergic and adrenergic sympathetic neurons, Brain Research, 134 (1977) 269 278. 5 Hanbauer, I., Guidotti, A. and Costa, E., Dexamethasone induces tyrosine hydroxylase in sympathetic ganglia but not in adrenal medulla, Brain Research, 85 (1975) 527 531. 6 Hanbauer, 1., Kopin, I. J., Guidotti, A. and Costa, E., Induction of tyrosine hydroxylase by beta adrenergic receptor agonists in normal and decentralized sympathetic ganglia: role of 3',5'-cyclic adenosine monophosphate, J. Pharmacol. exp. Ther., 193 (1975) 95-104. 7 Hanbauer, 1., Lovenberg, W., Guidotti, A. and Costa, E., Role of cholinergic and glucocorticosteroid receptors in the tyrosine hydroxylase induction elicited by reserpine in superior cervical ganglion, Brain Research, 96 (1975) 197-200.
356 8 Hanbauer, I. and Costa, E., Induction of tyrosine hydroxylase in superior cervical gangJia of rats opposite influence of muscarinic and nicotinic receptor agonists+ Neuropharmacoh)gy. 15 [1976) 85-90. 9 Hendry, 1. A., Trans-synaptic regulation of tyrosine hydroxylase activity in u dewlopmg s~mpathetic ganglion: effects of nerve growth factor (NGF), NGF-antiserum, and pempidine. Brain Research, 56 (1973) 313--320. 10 Mackay, A. V. P. and Iversen, E L.. Trans-synaptic regulation of tyrosine hydroxylase activity in adrenergic neurons: effects of potassium concentration on cultured sympathetic ganglia, Nat#o,nSchmiedberg's Arch. exp. Path. Pharmak., 272 (1972) 225 -229. 11 Mueller, R. A., Thoenen, H. and Axelrod. J.. Increase in tyrosine hydroxylase after reserpine aclministration, J. Pharmaeol. exp. Ther., 169 (19691 74-79. 12 Mueller. R. A., Thoenen, H. and Axelrod, J .. Inhibition of neuronaUy induced tyrosine h5 droxytase by nicotinic receptor blockade. Europ. J. Pharmacol.. 10 {1970J 5 l-56. 13 Mytilineou, C. and Black. I. B,. Regeneration of sympathetic neurons : effects of decentralization. Brain Research, 109 (1976J 382~ 386. 14 Nagaiah, K.. MacDonald. P. and Guroff. G., Induction of tyrosine hydroxytase synthesis in rat superior cervical gangia in vitro by nerve growth factor and dexamethasone. Biochem. Biophys. Res. Commtm., 75 (1977~ 832--837. 15 Nagatsu, T., Levitt, M. and Udenfriend. S.. A rapid and simple radioassay for t~r~ine b3 drox} la~c activity, Analyt. Biochem., 9 (1964) 122 126. 16 Otten, U. and Thoenen, H., Circadian rhythm and tyrosine hydroxylase induction t~y short-tet m cold stress - - modulating action of gl ucocorticoids in new born and adult rats, Proc. m~t. A cad. Sci. W a s h . . 7211975) 1415 t419. 17 Otten, U. and Thoenen, H.. Modulatory rote of glucocorticoids on nerve growth factor mediated enzyme induction m organ cultures of sympathetic ganglia, Brain Research. I I l (1976) 438 441. 18 Otten. U. and Thoenen, H., Selective induction of tyrosine hydroxylase and dopamine beta-hydroxylase in sympathetic ganglia in organ culture - role of glucocorticoids as modulators. Molec. Pharmacol.. 12 f1976~ 353 -361. 19 Pendleton. R G., Kaiser, C. and Gessner. G.. Studies on adrenal phenylethanolamine N-meth~Ltransferase ~PNMT~ with SKF 64139. a selective inhibitor. J. Pharmacol. exp. Ther+. 197 (1976~ 623 632. 20 Sims. K. L.. Davis, G. A. and Bloom. F. I~.. Activities of 3,4-dihydroxy-L-phc+wlatanine and 5hydroxy-L-tryptophan decarbox~lases in rat brain: assay characteristics and distribution. J. Nenrochem.. 20 (1973) 449 -464 21 Sze. P. Y. and Hedrick. B. J., Dexamethasone effect on ganglionic tyrosine hydroxylase activity+ Trans. Amer. Soc. Neurochem., IO (1979) 163+ 22 fhoenen. H.. Mueller, R. A and Axelrod. J., Trans-s3 naptic induction of adrenal ~ rosine hydroxylase..I. Pharmacol. exp Ther.. 169 (1969} 249- 254. 23 Towle, A. C.. Sze. P. Y. and Lauder, J. M., Cytosol glucocorticoid receptors ua morloammergtc ceil groups, ~).ans. Amer. Soc. Neurochem.. I 0 ( 1979 J 119. 24 Zigmond. R. E.. Mackay. A. V. P. and [ver~en. L. L.. Minimum duration of tran~-synaptic stimulation required for the induction of tyrosine hydroxylase by reserpine i~ the rat supertor cervical ganglion. J. Neurochem.. 23 (1974) 355+ 358+ 25 Zigmond. R. E. and Chalazonitis. A.. Long-term effects of preganglionic nerve stimulation on tyrosine hydroxylase activity in the rat superior cervical ganglion. Brain Research. t 64 (1979) 137 152.
347
' 9 Elsevier/North-Holland Biomedical Press
ADRENAL INFLUENCE ON TYROSINE HYDROXYLASE ACTIVITY IN SUPERIOR CERVICAL GANGLION
KEITH A. MARKEY* and PAUL Y. SZE**
Department of Biobehavioral Sciences, The University of Connecticut, Storrs, Conn. 06268 (U.S.A.) (Accepted June 12th, 1980)
Key words : tyrosine hydroxylase - - superior cervical ganglion - - glucocorticoids - - epinephrine
SUMMARY
The involvement of adrenal hormones as regulatory factors in maintaining physiological levels of tyrosine hydroxylase (TH) was examined in mouse superior cervical ganglion. Following bilateral adrenalectomy, TH activity in the ganglion fell at a slow but steady rate, reaching 60-65 % of the control levels after 2 weeks. Decentralization is known also to reduce TH activity in the ganglion. The effects of adrenalectomy and decentralization were therefore compared, and they were found to be additive, indicating different mechanisms in the two cases. The reduction of TH activity following adrenalectomy was not prevented by replacement with corticosterone (0.5 mg/kg, daily). However, replacement with epinephrine (4 mg/kg, daily) completely prevented the fall of TH activity in adrenalectomized animals. Isoproterenol, a fl-adrenergic receptor agonist, was as effective as epinephrine in preventing the reduction of TH activity following adrenalectomy. Furthermore, in intact animals, chronic administration of SKF 64139, an inhibitor of adrenal PNMT which depletes circulating epinephrine levels, also reduced ganglionic TH activity to the same level as that after adrenalectomy. These results indicate that epinephrine, but not corticosterone, is the adrenal factor required for physiological maintenance of normal levels of TH in the superior cervical ganglion. INTRODUCTION
Several studies indicated the possibility of a regulatory role of glucocorticoids in the induction of tyrosine hydroxylase (TH) in the superior cervical ganglion. First, the * Present address: Department of Psychiatry, New York University Medical Center, New York, N.Y. 10016, U.S.A. ** To whom reprint requests should be addressed.
348 inducibility of ganglionic T H by cold stress appeared to follow a c~rcadian rla5 thin that correlated with that of circulating glucocorticoids 16. Only during periods of high steroid levels could T H activity be elevated by the cold exposure. Pre-treatmcnt x~ith dexamcthasone during periods of low endogenous steroid levels permitted the elevation of enzyme activity. Second. induction of ganglionic T H by reserpine could be blocked with the glucocorticoid antagonist cortexolone 7. Third. in the induction ~ f I ' H by net,re growth factor (NGF) in cultured ganglion, the effect of N G F was potentiated b~ addition of dexamethasone to the culture mediu m~7. Fourth, TH activity in the ganglion was increased following administration of a pharmacological dose of dexamethasone a,7. The dexamethasone effect here. however, seemed to involve a pre-ganglionic mechanism, since the effect was abolished in decentralized ganglion, Epinephrine, the other major adrenal hormone, also appeared to have a role tn the regulation of T H in the superior cervical ganglion. Pharmacological doses ol the hormone were found to increase T H activity in the ganglion through a mechanism that did not seem to involve pre-ganglionic input. Instead. the effect of epinephrine could be blocked by propranolol, a fi-adrenergic receptor antagonist ~-7 In spite of the above findings from pharmacological effects and effects m wtro. no clear understanding has emerged as whether the adrenal hormones are indced involved physiologically as regulatory factors for the enzyme. Using the classical approach of adrenalectomy and hormone replacement, the present study was designed to examine the significance of the adrenal influence in the regulation of physiological levels of ganglionic TH activity in vivo, MATERIALS AND METHODS Animals and surgel3, Male C57BL/10Bg mice (2-4 months old) were bred and maintained in our specific pathogen-free colony. The pathogen-free condition was used in order to minimize subclinical diseases which may serve as a source of stress in altering normal adrenal activity. The mice were maintained on a t2 h light-dark cycle under controlled temperature and humidity. Bilateral adrenalectomy and decentralization of the superior cervical ganglion were performed under chloral hydrate anesthesia. Adrenalectomized animals received 0.9 ~o NaCI in drinking water. Enzyme assays T H activity was assayed by measuring the formation of ZHg.O alter hydroxylation of [aH]tyrosine according to the procedure of Nagatsu et al. 1~ with modifications. The individual ganglion was dispersed by freezing and thawing twice in 20 #1 distilled HzO. The reaction was initiated by adding 20 #1 of a substrate mixture containing: 800 mM sodium phosphate (pH 6.0); 40 # m L-[3,5-ZH]tyrosine (New England Nuclear. spec. act. 4 mCi/16.6 #mol); 2.0 mM 2_amino-4-hydroxy-6-methyl-5.6.%8-tetrahydropteridine (6-MPH4); 50 m M 2-mercaptoethanot. Alter incubation at 37"C for 20 min (linear rates within this time period), the reaction was stopped by the addition of 0.1 N NaOH. Tritiated H20 was isolated on an ion exchange column consisting of Bio-Rad
349 100'
0
80-
t~
~
60-
,~
40-
~(6) T H
E ~
N C W
20'
0
I 0
i 7
Days
after
I 14
-! 21
Adx.
Fig. I. T H activity and DOPA decarboxylase (DDC) activity in superior cervical ganglia following adrenalectomy (Adx). Controls (0 day) were sham-operated. * P < 0.01; ** P < 0.005; compared with 0 day control.
A G 50 W-X8 cation exchange resin and Bio-Rex 9 anion exchange resin. The use of
bilayer cation and anion exchange resins substantially reduced the blank value. DOPA decarboxylase activity was assayed by measuring the formation of 14CO2 from carboxyl-L-[14C]dihydroxyphenylalanineaccording to the procedure of Sims et al z0 with modifications. The individual ganglion was dispersed by freezing and thawing twice in 50/~1 of 0.1 M sodium phosphate buffer (pH 6.8). The reaction was initiated by adding 50 ,ul of a substrate mixture containing l0 mM [1-14C]dihydroxyphenylalanine (New England Nuclear; spec. act. 1.0 mCi/5.0 retool). After incubation at 37 °C for 20 rain, the reaction was stopped by the addition of sulfuric acid. The 14CO2 produced was absorbed in hyamine hydroxide.
Statistical analysis Significance of difference between groups was determined by Student's t-test. All values were shown as mean j: S.E.M., with the number of animals in each group indicated in parentheses. Lineweaver-Burk plots were constructed using least square fit. Coefficients of correlation were equal to or greater than 0.92. RESULTS
Efl. ects of adrenalectomy on T H activit), To determine the influence of adrenal function on TH in the superior cervical ganglion, the enzyme activity was measured in adrenalectomized animals. Fig. 1 shows that following bilateral adrenalectomy TH activity fell to 70 ~ of control within one week, reaching 60-65 ~ by the second week. DOPA decarboxylase activity, monitored
350 in the g a n g l i o n as a reference, was n o t altered t h r o u g h o u t the entire period. These resuits indicate that n o r m a l a d r e n a l function is required for m a i n t a i n i n g p h y s i o l o g i c a l levels o f T H activity in the ganglion. Since D O P A d e c a r b o x y l a s e activity r e m a i n e d unchanged, the reduction o f T H activity was unlikely due to the loss o f s y m p a t h e t i c n e u r o n s in the ganglion o r o t h e r non-specific changes f o l l o w i n g a d r e n a l e c t o m y .
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Fig. 2. Lineweaver-Burk plots of TH activity vs (A) tyrosine concentrations and (B) 6-MPH4 concentrations. Ganglionic TIFFwas from intact controls ( 0 0 ) or from mice adrenalectomized for 2 weeks ((3 -©). Pooled ganglia from 5-6 animals were used to construct each line. A: the concentration of tyrosine was 20 ctM. B : the concentration of 6-MPH4 was held constant at 1.0 raM. A shortened incubation time (5 rain) was used for the enzyme assay. TH activity is expressed as pmol[5 rain/ganglion. Each point is the average of duplicate determinations.
351 The reduction of ganglionic TH activity was characterized by kinetic analyses (Fig. 2). Lineweaver-Burk plots reveal that only the Vmax of TH was decreased following adrenalectomy. The Km of the enzyme for either tyrosine or 6-MPH4 was not altered. (The K,,~ values were estimated as 70 #M tyrosine and 280/~M for 6-MPH4.) From the kinetic data, it becomes possible to suggest that the decrease of TH activity following adrenalectomy may be due to a reduction of the steady-state levels of the enzyme.
Comparison of adrenal and pre-ganglionic effects on TH activity Pre-ganglionic transection ('decentralization') is known to result also in a reduction of ganglionic TH activity2,3,6,s,9,1a,24. The effects of adrenalectomy and decentralization were therefore compared. As shown in Fig. 3, TH activity was reduced to 52 ~ of control two weeks after decentralization. In the ganglion that was decentralized, adrenalectomy produced a further reduction of TH activity, reaching only 30 o/ /o of control or 60 ~ of the level from decentralization alone. Thus, the effects of adrenalectomy and decentralization were additive, indicating that the adrenal effect could occur independent of the central cholinergic innervation.
Hormone replacementfollowing adrenalectomy To identify the hormone(s) involved in the adrenal effect, adrenalectomized animals were given replacement injections of either corticosterone or epinephrine. The
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INTACT
ADX
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(8)
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Fig. 3. Effects of adrenalectomy and decentralization on T H activity in superior cervical ganglia. Mice were adrenalectomized for 2 weeks (ADX); with their ganglia decentralized for 2 weeks (DEC); both adrenalectomized and decentralized for 2 weeks (ADX ÷ DEC); or sham-operated (INTACT). Decentralization of ganglia in all animals was performed unilaterally; ganglia of sham-operated animals were used for comparison. ** P -< 0.005 compared with intact control.
352 TABLE I
Effects of hormone replacement on ganglionic TH activity following adrenalectomv
Adrenalectomized or intacl control mice were given daily subcutaneous injections of the hormone tin sesame oil suspension)for 2 weeks.Each value is mean -~ S.E,M. from the indicated number of animals. Treatment
TH activity (pmol/k/ganglion)
Intact control c o r t i c o s t e r o n e (0.5 m g / k g ) e p i n e p h r i n e (4 m g / k g l
106 • 8.4 109 i 9.0 101 -1 3.2
(61 (o1 (6~
Adrenalectomized --- c o r t i c o s t e r o n e (0.5 m g / k g ) e p i n e p h r i n e (4 m g / k g ) isoproterenol (5.4mg/kg)
66 70 112 101
(~ (8) (61 (6)
z_ -i -~ r
3.6** 4.6 ~ 7.6 8.2
* P ~-- 0.01. ** P < 0.005.
replacement was initiated immediately after surgery and continued daily throughout the entire 2 week period. As shown in Table I. daily injections of corticosterone (0.5 mg/kg) did not prevent the decrease of T H activity caused by adrenalectomy, nor did the treatment have an effect on normal enzyme activity in intact animals. Higher doses of the steroid were used (up to 10 mg/kg, daily), but the adrenalectomy effect on TH activity was still not prevented (data not presented). In contrast to corticosterone, replacement with epinephrine fully prevented the effect of adrenatectomy in reducing ganglionic TH activity (Table 1). It should be noted that with the small doses used here (4 mg/kg), epinephrine had no effect on normal enzyme activity in intact animals, although pharmacological doses (50 rng/kg) could increase ganglionic T H activity~. Yet. such small doses were sufficient to prevent the effect of adrenalectomy. Furthermore. the ability of epinephrine to prevent the adrenalectomy effect could be mimicked by isoproterenol, indicating the involvemenl of fl-adrenergic receptor. From Lineweaver-Burk analyses (varying tyrosine and 6-MPH4 concentrations as in Fig. 2), the kinetic parameters of T H from intact and epinephrine-replaced adrenalectomized animals were found to be identical (data not shown). Replacement with epinephrine restored the Vmax of TH. which was reduced in adrenalectomized animals (see Fig. 2). Thus. it appears that hormone replacement reversed the effect of adrenalectomy and resulted in an enzyme kinetically identical to that from normal animals.
TH activity after inhibition of adrenal P N M T To support the finding that the reduction of ganglionic T H activity following adrenalectomy was due to epinephrine, the enzyme activity was further determined in animals with their circulating epinephrine depleted by SKF 64139, a specific inhibitor of adrenal phenylethanolamine N-methyl-transferase (PNMT) activity. Table II shows that after chronic administration of the adrenal P N M T inhibitor for two weeks, T H
353 TABLE II
Effect o f S K F 64139 on ganglionic T H activity Mice were given SKF 64139 orally at the indicated dose twice daily for 2 weeks. Each value is mean ~S.E.M. from the indicated number of animals.
Treatment
T H activity (pmol/h/ganglion)
Control SKF 64139 (100 mg/kg)
110 ± 4.0 (6) 70 ± 3.5* (7)
* P -; 0.005.
activity in the ganglion was reduced to 70 ~ of control. At the dose used (100 mg/kg, twice daily), SKF 64139 was known to deplete epinephrine in adrenal glands to as low as 25 O//oof normal 19. Clearly, the reduction of ganglionic TH activity following pharmacological depletion of circulating epinephrine was comparable to that found after adrenalectomy.
Reserpine induction of TH after adrenalectomy Since corticosterone did not appear to be a regulatory component in maintaining physiological levels of TH activity in the ganglion, the possibility was tested that the steroid might serve as a required factor in order for the induction of the enzyme to occur. In this experiment, the effect of reserpine in inducing ganglion TH was examined in adrenalectomized animals. In intact animals, administration of reserpine is well
160
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60.
< ~
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40. 20-
INTACT CONTROL (6)
INTACT * RES (4)
ADX (8)
AOX + RES (9)
Fig. 4. Induction of ganglionic TH by reserpine in adrenalectomized mice. Animals were intact or adrenalectomized (A DX) for 2 weeks. Reserpine (RES) was administered (5 mg/kg, s.c. in saline) 2 days before sacrifice. * P -- 0.01 ; ** P < 0.005 compared with intact saline control.
354 known to produce an induction of ganglionic T H through a trans-synapt~c mechanism z4. As shown in Fig. 4. the induction of ganglionic T H by reserpine could occur completely in adrenalectomized animals. Thus, the result indicates that trans synaptic induction of T H in the ganglion could proceed in the absence of glucocorticoids. The result also indicates that the enzyme induction by reserpine was independent of adrenal epinephrine. DISCUSSION Research for more than a decade has clearly established that cholinergtc activity from pre-ganglionic innervation is an important factor in the regulation of T H in the superior cervical ganglion 1-4,9-t2,24,25. The present study indicates that adrenal epinephrine is another regulatory factor in maintaining physiological levels of ganglionic T H activity. As in the case of decentralization, adrenalectomy led to a marked reduction of T H activity in the ganglion. Replacement with corticosterone, even at pharmacological doses, failed to prevent reduction of enzyme activity in adrenalectomized animals. However, replacement with epinephrine, at small doses that showed no apparent effect on normal T H activity in intact animals, could fully prevent the reduction of enzyme activity caused by adrenalectomy. Moreover, the effect of adrenalectomy could be mimicked by pharmacological depletion of circulating epinephrine following chronic administration of an adrenal P N M T inhibitor. Thus, it has become clear that the effect of adrenalectomy in reducing ganglionic T H activity was due to depletion of epinephrine. The adrenal influence on ganglionic T H appears to be independent ~,[ t he cholinergic influence from pre-ganglionic innervation. This is indicated by the additive effects of adrenalectomy and decentralization. Since isoproterenol was as effective as epinephrine, the hormone action appears to involve fl-adrenergic receptor. This is consistent with the previous finding that the induction of ganglionic T H by pharmacological doses of epinephrine was blocked by propranolol, a fl-adrenergic receptor antagonist 6. Using adrenalectomy as our approach, our findings on corticosterone do not support the contention that glucocorticoids are a regulatory factor in vivo for ganglionic TH. both in physiological maintenance of the enzyme and in trans-synaptic induction of the enzyme, at least by reserpine. It may be noted that glucocorticoids are also not :involved in trans-synaptic induction of T H in adrenal medulla by reserpine, since the reserpine effect is not prevented by hypophysectomy22. We have confirmed the previous finding that administration of dexamethasone can induce T H in the ganglion ~',7. However, in a study comparing 4 glucocorticoids (dexamethasone, corficosterone, llydrocortisone, and triamcinolone), only the synthetic steroid dexamethasone was found to be effective2L Corticosterone, the principal glucocorticoid in the mouse and rat. failed to produce an inductive effect on ganglionic T H even at doses as high as 50 mg/kg. Another study from our laboratory has characterized the cytosol glucocorticoid receptor in the superior cervical ganglion, and found that the steroid receptor exhibits "transcortin-like' properties, with no binding affinity for dexamethasone 23. There-
355 fore, the effect of dexamethasone on T H cannot involve an intraneuronal steroid receptor-mediated mechanism. Moreover, decentralization immediately prior to dexamethasone administration did not prevent the increase of ganglionic T H activity, although earlier decentralization (24 h or longer) abolished the steroid effect ls,zl, in such newly decentralized ganglion, the increase o f enzyme activity by dexamethasone was still blocked by chlorisondamine, a cholinergic receptor antagonistZL Since synaptic activity is k n o w n to continue for a brief period following nerve transection, these findings suggest that the primary site of the dexamethasone effect may be the pre-ganglionic terminals. Indeed, a recent study in our laboratory has shown a marked elevation of choline and acetylcholine levels in the ganglion following dexamethasone administration, and also an increase o f high-affinity ganglionic [3H]choline uptake in vitro by the steroid (Marchi et al., in preparation). Significantly, the elevation o f choline and acetylcholine levels was not found in the ganglion after administration o f triamcinolohe, a glucocorticoid which is ineffective in producing an increase o f ganglionic T H activity. In light of these recent findings, it has become clear that the use of the synthetic steroid dexamethasone, as in several previous studies on sympathetic ganglion, does not reflect the hormonal actions o f glucocorticoids, physiologically. In summary, an assessment of the significance o f adrenal influence on ganglionic TH has revealed that epinephrine, but not corticosterone, is an important regulatory component in maintaining physiological levels of T H activity in the superior cervical ganglion. In the regulation o f ganglionic TH, the hormonal component from epinephrine appears to be as important as the neural component from the cholinergic innerration. ACKNOWLEDGEMENT This research was supported by G r a n t MH29237 from the U.S. Public Health Service.
REFERENCES I Black, I. B., Hendry, I. A. and Iversen, L. L., Effects of surgical decentralization and nerve growth factor on the maturation of adrenergic neurons in a mouse sympathetic ganglia, J. Neurochem., 19 (1972) 1367-1377. 2 Black, 1. B., Job, I. H. and Reis, D. J., Accumulation of tyrosine hydroxylase molecules during growth and development of superior cervical ganglion, Brain Research, 75 (1974) 133 144. 3 Black, I. B. and Mytilineou, C., Trans-synaptic regulation of the development of end organ innervation by sympathetic neurons, Brain Research, 101 (1976) 503 521. 4 Hamill, R. W., Bloom, E. M. and Black, I. B., The effect of spinal cord transection on the development of cholinergic and adrenergic sympathetic neurons, Brain Research, 134 (1977) 269 278. 5 Hanbauer, I., Guidotti, A. and Costa, E., Dexamethasone induces tyrosine hydroxylase in sympathetic ganglia but not in adrenal medulla, Brain Research, 85 (1975) 527 531. 6 Hanbauer, 1., Kopin, I. J., Guidotti, A. and Costa, E., Induction of tyrosine hydroxylase by beta adrenergic receptor agonists in normal and decentralized sympathetic ganglia: role of 3',5'-cyclic adenosine monophosphate, J. Pharmacol. exp. Ther., 193 (1975) 95-104. 7 Hanbauer, 1., Lovenberg, W., Guidotti, A. and Costa, E., Role of cholinergic and glucocorticosteroid receptors in the tyrosine hydroxylase induction elicited by reserpine in superior cervical ganglion, Brain Research, 96 (1975) 197-200.
356 8 Hanbauer, I. and Costa, E., Induction of tyrosine hydroxylase in superior cervical gangJia of rats opposite influence of muscarinic and nicotinic receptor agonists+ Neuropharmacoh)gy. 15 [1976) 85-90. 9 Hendry, 1. A., Trans-synaptic regulation of tyrosine hydroxylase activity in u dewlopmg s~mpathetic ganglion: effects of nerve growth factor (NGF), NGF-antiserum, and pempidine. Brain Research, 56 (1973) 313--320. 10 Mackay, A. V. P. and Iversen, E L.. Trans-synaptic regulation of tyrosine hydroxylase activity in adrenergic neurons: effects of potassium concentration on cultured sympathetic ganglia, Nat#o,nSchmiedberg's Arch. exp. Path. Pharmak., 272 (1972) 225 -229. 11 Mueller, R. A., Thoenen, H. and Axelrod. J.. Increase in tyrosine hydroxylase after reserpine aclministration, J. Pharmaeol. exp. Ther., 169 (19691 74-79. 12 Mueller. R. A., Thoenen, H. and Axelrod, J .. Inhibition of neuronaUy induced tyrosine h5 droxytase by nicotinic receptor blockade. Europ. J. Pharmacol.. 10 {1970J 5 l-56. 13 Mytilineou, C. and Black. I. B,. Regeneration of sympathetic neurons : effects of decentralization. Brain Research, 109 (1976J 382~ 386. 14 Nagaiah, K.. MacDonald. P. and Guroff. G., Induction of tyrosine hydroxytase synthesis in rat superior cervical gangia in vitro by nerve growth factor and dexamethasone. Biochem. Biophys. Res. Commtm., 75 (1977~ 832--837. 15 Nagatsu, T., Levitt, M. and Udenfriend. S.. A rapid and simple radioassay for t~r~ine b3 drox} la~c activity, Analyt. Biochem., 9 (1964) 122 126. 16 Otten, U. and Thoenen, H., Circadian rhythm and tyrosine hydroxylase induction t~y short-tet m cold stress - - modulating action of gl ucocorticoids in new born and adult rats, Proc. m~t. A cad. Sci. W a s h . . 7211975) 1415 t419. 17 Otten, U. and Thoenen, H.. Modulatory rote of glucocorticoids on nerve growth factor mediated enzyme induction m organ cultures of sympathetic ganglia, Brain Research. I I l (1976) 438 441. 18 Otten. U. and Thoenen, H., Selective induction of tyrosine hydroxylase and dopamine beta-hydroxylase in sympathetic ganglia in organ culture - role of glucocorticoids as modulators. Molec. Pharmacol.. 12 f1976~ 353 -361. 19 Pendleton. R G., Kaiser, C. and Gessner. G.. Studies on adrenal phenylethanolamine N-meth~Ltransferase ~PNMT~ with SKF 64139. a selective inhibitor. J. Pharmacol. exp. Ther+. 197 (1976~ 623 632. 20 Sims. K. L.. Davis, G. A. and Bloom. F. I~.. Activities of 3,4-dihydroxy-L-phc+wlatanine and 5hydroxy-L-tryptophan decarbox~lases in rat brain: assay characteristics and distribution. J. Nenrochem.. 20 (1973) 449 -464 21 Sze. P. Y. and Hedrick. B. J., Dexamethasone effect on ganglionic tyrosine hydroxylase activity+ Trans. Amer. Soc. Neurochem., IO (1979) 163+ 22 fhoenen. H.. Mueller, R. A and Axelrod. J., Trans-s3 naptic induction of adrenal ~ rosine hydroxylase..I. Pharmacol. exp Ther.. 169 (1969} 249- 254. 23 Towle, A. C.. Sze. P. Y. and Lauder, J. M., Cytosol glucocorticoid receptors ua morloammergtc ceil groups, ~).ans. Amer. Soc. Neurochem.. I 0 ( 1979 J 119. 24 Zigmond. R. E.. Mackay. A. V. P. and [ver~en. L. L.. Minimum duration of tran~-synaptic stimulation required for the induction of tyrosine hydroxylase by reserpine i~ the rat supertor cervical ganglion. J. Neurochem.. 23 (1974) 355+ 358+ 25 Zigmond. R. E. and Chalazonitis. A.. Long-term effects of preganglionic nerve stimulation on tyrosine hydroxylase activity in the rat superior cervical ganglion. Brain Research. t 64 (1979) 137 152.