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Induction of tyrosine hydroxylase in the superior cervical ganglia of rats: Opposite influence of muscarinic and nicotinic receptor agonists
INDUCTION OF TYROSINE HYDROXYLASE IN THE SUPERIOR CERVICAL GANGLIA OF RATS: OPPOSITE INFLUENCE OF MUSCARINIC AND NTCOTTNIC RECEPTOR AGONISTS I. HANBAUEK Section
on Biochemical Pharmacology. Hypertension-Endocrine National Heart and Lung Institute, Bethesda. Maryland
Branch. 2001J
and E. C~STA Laboratory
of Preclinical Pharmacology, Saint EliTabeths Hospital.
National Institute of Mental Washington. D.C. 20032
Health.
Summary~-Nicotine induces tqrosine hydroxylase in superior cervical ganglia. This action is facilitated by a blockade of muscarinic receptors and is inhibited by an activation of these receptors. In ganglia. an endogcnous store of catecholamines which is released by muscarinic receptor agonists. mediates the inhibition of tyrosine hydroxylase induction elicited by methacholine. Since apomorphinc inhibits the tyrosine hydroxylasc induction by nicotine or rescrpine, but methacholine only inhibits the action of nicotine. it is suggested that the inhibition of tyrosin. hydroxylase induction elicited by methacholinc is mediated by dopamine. Nicotine fails to induce tyrosine hydroxylase in decentralized ganglia. When atropinc IS associated with nicotine, the latter can alcq induce tyrosine hydroxylasc in decentralized ganglia. It is concluded that activation of muscarinic and nicotinic receptors exerts a mutually antagonistic influence in the control of tyrosine hydroxylase biosynthesis in sympathetic ganglia.
In adrenal medulla a sustained stimulation of nicotinic receptors lasting I 2 hr. induces tyrosino hydroxylase after a time interval of about 12 14 hr (GUIDOTTI and COSTA.1973. 1974; COSTA.GLIIDOTTI and HANI~ALXR. 1974). In adrenal medulla the stimulation of nicotinic receptors increases the 3’S’-cyclic adenosinc monophosphate (cyclic AMP) content. whereas stimulation
at 20 hr and returns to control lcvcls at 36 hr (CHLIANC; and COSTA, 1974). While in medulla. the induction of tyrosinc hydroxylase requires an increase of cyclic AMP conccntration which activates a cytosol histone kinase. in ganglia tyrosine hydroxylase induction is not always preceded by an increase in cyclic AMP content (Cosof the muscarinic receptors causes an increase of TA ct cd.. 1974: HANRAL.I.R. Gr IDOTTI and COSTA. 3’.5’-cyclic guanosino monophosphate (cyclic GMP) 1976; OTTEN, OESCH and THOENEN, 1973). When cyc(GLIIDOTTI. HANIIAIII.K and COSTA. 1975a). While an lic AMP content of sympathetic ganglia is increased increase of 5 to IO-fold in the cyclic AMP content for longer than 4 hr tyrosinc hydroxylase is induced of medulla lasting I hr or longer always leads to tyro48 hr later (HANI~AIJFR. KOPIN.GL IIIOTTI and COSTA. sine hydroxylase induction. the elevation of cyclic 1975a). Activation of the adrenergic beta receptors GMP content fails to be associated with a change is an inducing stimulus for ganglionic tyrosine hydin tyrosinc hydroxylase activity. The increase of meroxylase which is dependent on an early increase of dullary cyclic AMP content is coupled with an increcyclic AMP content and does not require innervation. ment in the activity of a soluble cyclic AMP indepenDexamethasone can also induce tyrosinc hydroxylasc dent protein kinase (GUIDOIX KLIROSAWA.CHLIANG in ganglia. but unlike the adrenergic beta rcceptol and COSTA.197%). The increase of this enzyme activity agonists it does not increase cyclic AMP and requires preganglionic innervation (HANRAI I R. GI. IIXITTI and outlasts by 3S-4hr the increment in cyclic AMP content. The activated cnryme has some of the attributes of COSIA. 1975b). histone kinase and it is translocatcd from cytosol to The present report concerns the participation of a particulate fraction which contains adrenal medulnicotinic and muscarinic receptors in the rcgulatior. lary nuclei (KUROSAWA. GLIII~O~~I and COSTA, 1976). of tyrosine hydroxylase in superior cervical ganglia. In this structure the translocation precedes by 1 2 This paper shows that nicotinic receptor agonists inhr the increment in ribonucleic acid synthesis. The duce tyrosine hydroxylase in normal and decentralired synthesis rate of tyrosine hydroxylase is augmented ganglia. The blockade of muscarinic receptors faciliat 8 hr following stimulus application. This rate peaks tates tyrosine hydroxylase induction by nicotinic rc8.5
86
I. HANEAL I K and E. COSTA
ceptor agonists, whereas the activation of the muscarinic receptors inhibits the tyrosine hydroxylasc induction elicited by nicotinic receptor activation. The intermediation of dopamine receptors in the inhibitory action of muscarinic receptor activation is suggested.
MATERIALS
AND METHODS
Malt Sprague-Dawlcy rats (Zivic Miller. Allison Park. Pa.) weighing 1OG 120 g were used 3 to 4 days after arrival at our animal quarters. During this time. they were kept in circadian artificial illumination (12 hr cycles) at 23 ‘C and fed Purina rat chow. Rats were injected with various drugs (see Results) and 48 hr after drug treatment the animals were killed by cervical dislocation. Both superior cervical ganglia were removed and homogenized in 0.5 ml 0,067 M phosphate buffer, pH 6.2. The tyrosine hydroxylase activity was assayed as previously described (HANBAUEK vr al.. 1975b) using one ganglion per assay and 6-methyltetrahydropterine as cofactor. Unilateral decentralization was performed in the laboratory under sodium methohexital anaesthesia. 3-4 days before drug injection. The following drugs were used: sodium methohexital (Brevital@ sodium, Eli Lilly Co.. Indianapolis. Ind.); nicotine salicylate (K & K Labs., Plainview, New York); carbamylcholine (Aldrich Chemical Co.. Milwaukee. Wis.); atropine sulphate H,O (Schwartz and Mann. Division of Becton. Dickinson and Co.. Orangeburg. New York); acetyl-betamcthylcholine (mcthacholinc. Merck and Co.. Inc.. Rahway. N.J.): Serpasil@ (Ciba-Geigy. Summit, N.J.); apomorphine HCl (S.B. Penik. N.Y.). RESULTS
The tyrosine hydroxylase activity of intact superior cervical ganglia was increased 48 hr after 50 /tmol/kg nicotine administered subcutaneously (Table 1). This
Table I. Induction of ganglionic tyrosine hydroxylase by nicotine and inhibition by simultaneous muscarinic receptor stimulation
Drugs (/lmol!kg) Saline Nicotine (2.5 s.c.) Nicotine (50 s.c.) Carbachol (8.1 i.p.) Methacholine (42 i.p.) Methacholine (42 i.p.) + Nicotine (50 s.c.)
Table
2. The c&t of carhamylcholine and nicotine tyrosinc hqdroxylase activity in adrenal medulla
Tyrosine hydroxylase (nmol DOPA, pair adrenal medullae per hr)
Drugs (jtmol;kg) Saline Carbamylcholinc Nicotine (40 s.c.) Methacholine (42 Methacholinc (42 + Nicotine (40
on
(X.1 i.p.) i.p.) i.p.) s.c.)
19.5 33.7 27.x IY.0 31.0
* P < 0925 (II = 6). Methacholinc was illjectcd IO min bcforc rats wcrc killed 24 hr after drug treatment.
* 2 * * i
2.0 2.6* 24* 2.0 3.7*
nicotine.
The
dose appears to be close to the threshold for tyrosine hydroxylase induction (25 /tmol/kg S.C. did not induce tyrosine hydroxylase). However. in decentralized ganglia neither of the two nicotine doses increased tyrosine hydroxylase activity. The increase of tyrosine hydroxylase activity elicited by nicotine can not be attributed to a change in the kinetic state of the enzyme because the Michaelis constant for the pteridine cofactor is not changed as a result of nicotine injection and the increase of tyrosine hydroxylase occurs after a time interval of 3&48 hr. When 42 Itmol/kg of mcthacholine was injected into-aperitoneally. tyrosine hydroxylase activity was not increased. Moreover. when methacholine was injected together with an inducing dose of nicotine it prevented the action of nicotine on ganglionic tyrosine hydroxylase (Table I ). In the doses tested. carbamylcholinc failed to induce tyrosine hydroxylase in superior cervical ganglia (Table I). Both carbamylcholine and nicotine cause 21delayed increase in the tyrosine hydroxylase activity of adrenal medulla (Table 2) which was due to an increase in the number of tyrosine hydroxylase molecules. Methacholinc neither induced adrenal tyrosine hydroxylase nor prevented the induction elicited by nicotine (Table 2). These results suggest that in adrenal medulla the simultaneous activation of nicotinic and muscarinic receptors is compatible with tyrosine hydroxylase induction. whereas in ganglia the activation of both receptors prevents the induction of tyrosine hydroxylase elicited by nicotinic receptor agonists.
Tyrosine hydroxylasc (nmol DOPA,‘ganglion per hr) Intact Decentralized 3.1 2.9 4.4 2.x 3.0 2.9
* + f * * k
0.20 @I6 0.19* 0.22 0.22 0.16
2.1 + 0.24 2.0 * 0.17 2.2 f 0.19
2.1 * 0.17
*P < 0.01. Each mean + S.E.M. refers to 68 experiments. Methacholine was mjectcd i.p. IO min before nicotine. The rats were decentralircd 48 hr before the drug treatment.
The antagonistic relationship between nicotinic and muscarinic receptor activation in inducing ganglionic tyrosine hydroxylasc is upheld by the results reported in Table 3. They show that 43 ,umol/kg atropine administered intraperitoneally failed to change tyrosine hydroxylase activity in intact and decentralized ganglia. However, in rats injected with atropine the successive subcutaneous injection of 25 iimol/kg of nicotine. which failed to induce tyrosine hqdroxylase when
ACh receptors Table
3. Atropine
and tyrosine
hydroxylasc
facilitation of tyrosine hydroxylase induction and decentralized superior cervical ganglia
+mol/kg) i.p.
SC.
3.2 3.4 2.9 4.2 4%
43 25 25 50
43 43
hq nicotine
in intact
Tyrosine hydroxqlasc (nmol DOPA:ganglion per hr) Decentralized Intact 3 days 7 days
Nicotine
Atropine
x7
induction
+ + f + *
0.30 0.37 0.16 0,14* @14*
2.5 f 0.10 2.0 + 0, I7 3.4 * 0.17* 3.4 * 0, I’)*
2.3 + 0.30 2.0 i_ 0.12 3.1 * o.I6+ ___
* P < 001 ; t P < 0.05 01 = 6). Three or seven days after monolateral decentralization of the superior ccr\ ~cal ganglion. rats received atropine IO min before nicotine. The animals Mcrc killed 3X ht after drug treatment.
given alone (Table 3), could induce tyrosine hydroxylase in intact and decentralized ganglia. The data of Table 3 also show that 50 pmol/kg nicotine injected subcutaneously, which was inactive in decentralized ganglia (Table 1). increased tyrosine hydroxylase activity in this tissue after a pretreatment with atropine. Moreover, the data presented in Table 3 show that atropine can also facilitate the action of subthreshold doses of nicotine in ganglia decentralized one week before the experiment.
Mediation tnethucholinr
by dopamine on thr
of
the
induction
h_vdroxyla.sr hi* nicotinic
inhibitory
actiotl
of yanylionic
wcrptor
of
t~~rosinr
agonists
It has been reported
that the induction of ganglionic tyrosine hydroxylase elicited by reserpine can be antagonized by nicotinic receptor blockers (MLILLLLIK. THO~NEN and Axf:LROf). 1969). The data reported in Table 4 show that methacholine blocked the induction of tyrosine hydroxylase elicited by nicotine in superior cervical ganglia but it failed to block the tyrosine hydroxylase induction elicited by reserpine. To ascertain whether the different responsiveTable 4. The effect of muscarinic and dopamine agonists on the induction of tyrosine hydroxylase by nicotine or reserpine Drugs (pmol/kg)
receptor elicited
Tyrosine hydroxylase (nmol DOPA/ganglion per hr)
Saline Methacholine 40 Reserpine 12.3 Methacholine 40 + reserpine 12.3 Apomorphine 31 + reserpine 12.3 Nicotine 50 Apomorphine 31 + nicotine 50 *P < 0.05; tP < @Ol. Each mean + S.E.M. refers to and apomorphine were injected before subcutaneous injection of rats were killed 48 hr after drug
2.4 2.7 3.7 3.8
k f k f
0.18 0.11 0,26+ @32+
2.4 + 0.20 3.3 & 0,26* 2.6 + 0.21
8 ganglia. Methacholine. intraperitoneally 10 min nicotine or reserpine. The treatment.
ness of the reserpine induction to mcthacholine was due to the absence of the adrenergic transmitter in the ganglionic intcrneuroncs. the action of apomorphine on the induction of tyrosine hydroxylase elicited by reserpine and nicotine was compared. The results of these experiments arc reported in Table 4. They show that apomorphine inhibited the induction of ganglionic tyrosine hydroxylase elicited by nicotine and reserpine. To corroborate the participation of dopaminergic receptors in the inhibition of tyrosine hydroxylase induction elicited by muscarinic receptor stimulation, the action of subthrcshold doses of nicotine on the induction of tyrosinc hydroxylase in bilaterally decentralized ganglia of rats treated with rescrpine was also tested. Table 5 shows that depletion of catecholamines by rcserpine facilitated the induction of ganglionic tyrosine hydroxylase activity elicited bq nicotine.
DISC'CSSION Neuronal communication is transacted at sqnapscs and the resulting interaction between receptor and Table 5. The effect of nicotine and rcscrpinc on tlrosine hydroxylase activity m bilaterally decentralized superior cervical ganglia
Treatment Saline Nicotine (20 PmolFkg s.c.) Rescrpine (I 2.3 ltmol kg s.c.) Reserpinc (12.3 pmol;kg s.c.) + Nicotine (20 Atmol,!kg s.c.)
TqroGne hydroxylasc (nmol DOPA ganglion per hr) 2.x 5 0.15 2.9 * 0. I6 3. I
f 0.IO
3.x + 0.74*
*P < 0.01 when compared with saline or nicotine treated groups. The mean i: S.E.M. refers to 6 ganglia. The rats were bilaterally decentralized 4 days before drug treatment. Rcserpine was injected 4 hr before nicotine and the rats were killed 44 hr thereafter.
88
I. HANISAUI K and E. COSTA
transmitters influences the regulation of protein synthesis in postsynaptic cells. We have discussed previously that the trans-synaptic induction of tyrosinc hydroxylase is a model to study how stimulation of receptors located in ncuronal membranes can influence the synthesis of specific proteins (GtIIIX)TTI et ul., 1975b: KUKOSAWA ct ul.. 1975). Tyrosinc hydroxylasc can be localized immunohistochemically in perikarya of catecholaminergic neurones where the membranes of the endoplasmic reticulum, the Golgi apparatus and the neurotubules react specifically with the antibody directed to tyrosine hydroxylase (PICIXL, JOH and RI:IS, 1975). The regulation of tyrosine hydroxylase synthesis offers an idcal model to study how various receptors located in ncuronal membranes can interact with each other and control the synthesis of a specific protein. In superior cervical ganglia, the activity of noradrencrgic neurones is regulated by a number of synaptic inputs which operate on nicotinic, muscarinic and dopamincrgic receptors (L~ur:r and TOSAKA. 1970; VOLL~.. 1966). It is currently believed that the function of these membrane receptors expresses itself as a regulation of noradrenergic neuronc excitability: activation of nicotinic or muscarinic receptors in ganglion cell bodies causes depolarization (HALFILY. 197421.b) and also ganglionic interncuroncs (small intcnscly Huorescent cells) arc depolarized by muscarinic rcccptor activation (LIIET and OWMAN. 1974). Howcvcr. the depolarization of ganglionic intcrncuroncs elicits catecholaminc secretion. which by acting on specific ncuronal receptors. decrcascs the excitability of noradrcncrgic ncurones (COSTA. RI.VZIN. KI’NXMAN. SWCTOR and BIUIII. 1961). The nicotinic receptors mediate the fast excitatory potentials in postsynaptic cells (HAI-FI:LJ,. 1974a). A late excitatory potential is generated by activation of muscarinic rcccptors. whcreas the true inhibitory synaptic potential requires the activation of muscarinic receptors in gdnglionic interneurones and the release of a catecholamine. presumably dopamine from these interncuroncs (LIKT and OWMAN. 1974). Our results show that the activation of nicotinic receptors can cause a delayed increase of tyrosine hydroxylasc activity. Since this increase is not due to a change in the kinetic properties of tyrosine hydroxylase and it requires DNA transcription and protein synthesis. it rcfccts a trans-synaptic induction of tyrosine hydroxylasc synthesis. Our cxpcriments reveal that carbachol and nicotine induce tyrosine hydroxylase in adrenal medulla but only nicotine can induce tyrosine hydroxylase in sympathetic ganglia. While nicotinic receptor agonists arc equally active in inducing tyrosine hydroxylase in intact and denervated adrenal medulla (HANI~ALII.Kand GI,IIX)TTI. 1975) the present experiments show that nicotine can induce tyrosine hydroxylasc in intact but not in decentralized sympathetic ganglia. The reason for this difference can be inferred only indirectly from experiments in which nicotine and atropinc injections were combined. Atropine appears to lower the nicotine
threshold to induct tyrosinc hydroxylase in intact sympathetic ganglia and facilitates the induction of tyrosine hydroxylase by nicotine in decentralized ganglia. Thus, it appears that in decentralized ganglia, the injection of high doses of nicotine stimulates muscarinic rcccptors which. in turn. cause an antagonistic action on tyrosine hydroxqlasc induction. When this muscarinic stimulation is abolished by atropine. nicotine also induces tyrosine hydroxylase in decentralized ganglia. We have tested whether stimulation of muscarinic receptors antagonizes the induction of ganglionic tyrosine hydroxylasc elicited by nicotine or reserpine. Methacholinc. a selcctivc muscarinic receptor agonist abolishes the tyrosine hydroxqlase induction by nicotine but not that by reserpine. On one hand, this tinding contirmed the direct participation of muscarinic and nicotinic receptors in the trans-synaptic regulation of throsine hydroxylase in ganglia and on the other hand, it suggested that the inhibition of tyrosine hydroxylase induction mediated by muscarinic receptor activation requires the availability of catecholamine stores. The participation of catecholamines was tested by studying the ell’ects of npomorphine on the tyrosine hydroxylase induction elicited by reserpine and nicotine. Apomorphine inhibits the tyrosine hydroxylase induction elicited by reserpine and nicotine. That the stimulation of beta adrcnergic receptors can induce tyrosinc hydroxylase was demonstrated by previous cxperimcnts with intact and decentralized ganglia (HASBAI I K ct ul.. 1975a). Since norepinephrine. as well as epincphrine and dopamine are also stored in sympathetic ganglia of rats (Kos~ow. Bmmvrc’ and C~STA. 1975). it is possible to entertain the hypothesis that either of these catecholamine rcceptors participate in the regulation of ganglionic tyrosinc hydroxylasc. It is currently bclicvcd that dopamine release mediates the true inhibitory hyperpolariration elicited by trans-synaptic muscarinic activation (GIWI.NC;AKIIand KI:HARIAN.1974). The present expcrimcnts dcmonstratc that this type of regulation is opcrativc in tho inhibitory effects of methacholine on the tyrosine hydroxylase induction elicited by nicotine. lsoprotcrcnol was shown by OTTEN. MU:LLFK, 01 SCH and THOI.NIN (I 974) to cause an increase of cyclic AMP content in sympathetic ganglion cells due to an increased cyclic AMP synthesis. Moreover. Dt GROAT and VOLLI. (1966) have shown that isoproterenol enhances the ganglionic responses to the stimulator) action of muscarinic agents. Finally. HAI.FI:LL (1969) has demonstrated that a single injection of isoprotcrcnol dcpolari/cs the sympathetic ganglion cells for about 30 min. Thus. isoprotcrenol diminishes the post-tetanic hyperpolarisation and enhances postsynaptic activity in noradrcnergic cells. Our results suggest that dopaminc and isoproterenol (HANIIAUK ~‘1(I/.. 197521)hale opposite effects in the regulation of tyrosinc hydrox!lase synthesis: stimulation of dopaminc rcccptors limits the induction of tyrosine hydroxylase clicitcd bq nicotine. and stimulation of beta adrenergic rcccptorh is a direct stimulus for tyrosinc
ACh
receptors
and tyrosine
hydroxylasc induction. Perhaps, in some situations beta adrcnergic receptors may serve a function. In adrenal demedullated rats exposed to cold. the transsynaptic induction of tyrosine hydroxylase appears to be mediated by a mechanism which involves a facilitation through an activation of beta adrcnergic rcceptors (GUIIX)TTI rt ul.. 1975a). Whether some ganglionic intcrneuroncs store exclusivel) cpincphrine or whether all ganglionic intcrneuroncs store and secrete dopamine and cpinephrinc is not understood at the present time (Kos~ow c’f ~1.. 1975). Our results do not clarify whether dopaminc secretion is mediated through nicotinic or muscarinic receptors. but the cvidcnce available suggests that dopamine must be considered as one of the cndogenous regulatory substances for ganglionic tyrosine hydroxllase synthesis. In conclusion. the present experiments and our previous work (G~IIIX)TTI (‘f trl.. 1975a) allow us to propose the following scheme describing the participation of various transmitters in the regulation of tyrosine hydroxylasc synthesis in sympathetic ganglia of rats. (I) Glucocorticoids are an important permissive factor for tyrosinc hydroxylase induction. This facilitation requires glucocorticoid receptor activation which is operative only in the presence of an intact synaptic circuit (HASISAILK cl trl., 1975b). In fact, dexamethasone induces tyrosine h!droxylase only if the prcganglionic nerves are intact. (2) Beta adrenergic receptor activation can induce tyrosine hydroxylase in the absence and in the presence of affcrent nerves. This induction involves an early increase of cyclic AMP and may bc related to the sustained dcpolarization elicited by beta adrenergic receptor agonists. In adrenal dcmedullated rats. the induction of ganglionic tyrosine hydroxylase during short term (4 hr) exposure to cold may involve a participation of the physiological activation of these receptors. Endogenous ganglionic stores of epincphrine may be preferentially mobili/cd to elicit the tyrosine hydroxylast activation after cold exposure of adrenal demedullated rats. In adrenal demedullated rats the transsynaptic induction of ganglionic tyrosine hydroxylase elicited by cold differs from that observed in normal rats because it involves an immediate increase of ganglionic cyclic AMP content and the stimulus threshold is reduced from 24 hr in normal rats to 4 hr in demedullated rats. (3) Nicotinic receptor activation causes depolarization of ganglion cells which is dissociated from an early increase of cyclic AMP content. This action is counteracted by muscarinic receptor activation. In dccentraliled ganglia. nicotinic receptor activation induces tyrosine hydroxylase only when muscarinic receptors are blocked by atropine. This blockade lowers the threshold for tyrosine hydroxylase induction by nicotine also in intact ganglia. (4) Muscarinic receptor activation inhibits the tyrosine hydroxylase induction elicited by nicotinic rcceptor activation through the release of endogenous dopamine from ganglionic interneurones. Since this action can be mimicked by apomorphine. WC propose
hydroxylase
induction
89
that the inhibitory action on tyrosine hydroxylase induction by muscarinic receptor activation is mediated by endogenous dopamine. REFERENCES C~rr ANG, D. M. and COS~A. E. (1974).
Biosynthesis of tyrosine hydroxqlase in rat adrenal medulla after cold expo-
Naril. Acud. Sci. 1;S.A. 71 : 4570-4574. A. and HANHAUI K. I. (1974). Do cyclic nuclcotidcs promote the trans-synaptic induction of tqrosine hqdroxylase? Lift Sci. 14: 1169 11X8. COS~A. E.. RI VLIN. A. M.. KL’NT~‘MAV.R., SPI CTOK, S. and BKOIXI:. B. B. (1961). Role for ganglionic norcpinephrine in sympathetic synaptic transmission. Scic,nce N.Y. 133: 1822 1823. DI GROAT. W. C. and VOLLI, R. L. (1966). Interaction hetwccn the catecholamines and ganglionic stimulating agents in sympathetic ganglia. J. Phur~~uc. tsp. Thrr. ls4: 2OU 215. GKI I NC;AKI). P. and KI.IIAI~IAN.J. W. (I 974). Role of cyclic AMP in synaptic transmission in the mammalian peripheral nervous system. F<,dn. Proc. Fcd~. rl~r. Soca. c’u,‘. Bfol. 33: I O59m 1067. G[ II~OTTI.A. and COSTA. E. (1973). Involvement of adenosine 3’S’-monophosphate in the activation of tyrosine hydroxylasc elicited by drugs. .‘jci<,t~cc,. N.Y. 179: 902 904. GL~IIXXTI. A. and COSTA. E. (1974). A role for nicotinic receptors in the regulation of the adenylate cyclase of adrenal medulla. J. Phurmac. ~‘z~. Thrr. 189: 665-675. G~‘II)OTTI. A.. HA~I~ALII.K. 1. and ~OSTA. E. (1975a). Role of cyclic nucleotides in the induction of tyrosinc hydroxylase. In: Atlruwc~s in Clrlic Ntrcl~otitlc Rcsrurch, Vol. 5. (DKL%ZIONI). G. 1.. GUI I NGAKI). P. and ROHISON, G. A.. Eds.) pp. 619 639. New York. Raven Press. GI’IIX>T~I. A.. KL.KOSAWA. A.. CH~ ANG. D. M. and COSTA. E. (1975h). Protein kinase activation as an early event in the tram-synaptic induction of tyrosine-%mono-oxygenase in adrenal medulla. Proc. mtt~. ACM/. Sri. U.S.A. 72: 1152~1156. HAI TVLY, W. (1969). Effects of catecholamines in the rat superior cervical ganglion and their postulated role as physiological modulators of ganglionic transmission. Ploy. Bruin Rm. 31: 61-72. HAt FILI. W. (1974a). The effects of various nicotine-like agents in the cat superior cervical ganglion itt sitlt. sure. Proc.
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93- I i7. HAI WLY. W. (l974b). Muscarinic postsynaptic events in the cat superior cervical ganglion irt situ. Nmt~!~~-Schmicdchwys ilrch. crp. Purh. Phu).wk. 281: I19 143. HANI~AI EK. I., GUIIX~TTI, A. and COSTA. E. (1976). Involvement of cyclic nucleotides m the long term induction of tyrosinc hydroxylase. In: Procwdirtys of thr IX Congress of t/w Collqitml qicwrt. Paris. France
Irltc~rtiutiodc~
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and Picrro?‘. press). HAXI~AU K. I.. KOI’IN. 1. J.. Gl’II)OTTl. A. and COS~A. E. (1975a). Induction of tyrosinc hydroxylase elicited by I~~[i~-(~~il-~~ii~rgic rccsptor agonists in normal and deccntrah/cd sympathetic ganglia: role of cyclic 3’S’-adenosine monophosphate. J. Phurmac. cd\-p. Thw 193: 95- 104. HANHAIJ~R, 1.. GL’IDoTTI. A. and COSTA. E. (1975h). Dexamethasone induces tyrosine hydroxylase in sympathetic ganglion but not in adrenal medulla. Brain Res. 85: 527 531.
HANI~AI I K. I. and GL,IlX)T71. A. (1975). Further evidence for a CAMP dependent regulation of tyrosine-3-monooxygenasc induction in adrenal medulla: effect of denervatton. Natr/t~~n-Schrrtictl~,l~~~~.s Arch. top. Path. Pharrmk. 287: 213-217.
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Kos~ow. S. H.. Bn GWIC. M. and COS~A, E. (1975). Catechobdmines in sympathetic gangha of rat: effects of dexamethasonc and rcserpine. J. N~IW~/XWI. 24: 277 281. Kr KOSAWA. A.. G~~IIX~TTI. A. and COS~A. E. (19761. Induction of tvrosme 3-mono-oxygenasc elicited b) carbamylcholine in intact and denervated adrenal medulla: role of histonc kinase activation and translocation. Molrc. Phar~~c. (In press). LIIQT. B. and TOSAKA. T. (19701. Dopaminc as a synaptic transmitter and modulator in sympathetic ganglia: a diffcrent mode of synaptic action. Proc. ttut/l. Acad. Sci. C’.S.A. 67: 667 673. LII~I r. B. and OWMAN, CH. (1974). Concomitant changes in rormaldehyde-induced tluorcsccnce of dopamine intcrneurons and in slow inhibitory postsqnaptic potentials of the rabbit superior cervical ganglion induced by stimulation of the preganglionic nerve or by a muscarmic agent. d. Ph~~iol., Low/. 237: 635-662. Mr I t.Li it. R. A.. THAI NI N. H. and AXI ILKOI). J. ( 1969). In-
E. COSTA crease of tyrosine
hydroxylase activity after reserpine administration. J. Pharmuc. e.wp. Thrr. 169: 7479. OTTIN. U., MUI.LLIK. R. A.. OIXH, F. and THOEN~N. H. (1974). Location of an isoproterenol responsive cyclic AMP pool in adrenergic nerve cell bodies and its relationship to tyrosine-3-mono-oxygenase induction. Proc. natn. Acad. Sci. U.S.A. 71: 2217--2221. OTTf N. V.. OI~SCH. F. and THOENEN. H. (1973). Dissociation between changes in CAMP and subsequent induction of TH in the rat superior cervical ganglion and adrenal medulla. Naun!n-Schnlic,drhrrys Arch. cup Path. Pharmak. 280: 129- 140. PICIWL. V. M.. JOH, T. H. and RHS. D. J. (1975). Immunohistochemical localization of tyrosine hydroxylase in brain by light and electron microscopy. Brain Rrs. 85: 295-300. VOLLI, R. L. (1966). Modification by drugs of synaptic processes in sympathetic ganglia. Pharrnac. Rcr. 18: 839m 869.
on Biochemical Pharmacology. Hypertension-Endocrine National Heart and Lung Institute, Bethesda. Maryland
Branch. 2001J
and E. C~STA Laboratory
of Preclinical Pharmacology, Saint EliTabeths Hospital.
National Institute of Mental Washington. D.C. 20032
Health.
Summary~-Nicotine induces tqrosine hydroxylase in superior cervical ganglia. This action is facilitated by a blockade of muscarinic receptors and is inhibited by an activation of these receptors. In ganglia. an endogcnous store of catecholamines which is released by muscarinic receptor agonists. mediates the inhibition of tyrosine hydroxylase induction elicited by methacholine. Since apomorphinc inhibits the tyrosine hydroxylasc induction by nicotine or rescrpine, but methacholine only inhibits the action of nicotine. it is suggested that the inhibition of tyrosin. hydroxylase induction elicited by methacholinc is mediated by dopamine. Nicotine fails to induce tyrosine hydroxylase in decentralized ganglia. When atropinc IS associated with nicotine, the latter can alcq induce tyrosine hydroxylasc in decentralized ganglia. It is concluded that activation of muscarinic and nicotinic receptors exerts a mutually antagonistic influence in the control of tyrosine hydroxylase biosynthesis in sympathetic ganglia.
In adrenal medulla a sustained stimulation of nicotinic receptors lasting I 2 hr. induces tyrosino hydroxylase after a time interval of about 12 14 hr (GUIDOTTI and COSTA.1973. 1974; COSTA.GLIIDOTTI and HANI~ALXR. 1974). In adrenal medulla the stimulation of nicotinic receptors increases the 3’S’-cyclic adenosinc monophosphate (cyclic AMP) content. whereas stimulation
at 20 hr and returns to control lcvcls at 36 hr (CHLIANC; and COSTA, 1974). While in medulla. the induction of tyrosinc hydroxylase requires an increase of cyclic AMP conccntration which activates a cytosol histone kinase. in ganglia tyrosine hydroxylase induction is not always preceded by an increase in cyclic AMP content (Cosof the muscarinic receptors causes an increase of TA ct cd.. 1974: HANRAL.I.R. Gr IDOTTI and COSTA. 3’.5’-cyclic guanosino monophosphate (cyclic GMP) 1976; OTTEN, OESCH and THOENEN, 1973). When cyc(GLIIDOTTI. HANIIAIII.K and COSTA. 1975a). While an lic AMP content of sympathetic ganglia is increased increase of 5 to IO-fold in the cyclic AMP content for longer than 4 hr tyrosinc hydroxylase is induced of medulla lasting I hr or longer always leads to tyro48 hr later (HANI~AIJFR. KOPIN.GL IIIOTTI and COSTA. sine hydroxylase induction. the elevation of cyclic 1975a). Activation of the adrenergic beta receptors GMP content fails to be associated with a change is an inducing stimulus for ganglionic tyrosine hydin tyrosinc hydroxylase activity. The increase of meroxylase which is dependent on an early increase of dullary cyclic AMP content is coupled with an increcyclic AMP content and does not require innervation. ment in the activity of a soluble cyclic AMP indepenDexamethasone can also induce tyrosinc hydroxylasc dent protein kinase (GUIDOIX KLIROSAWA.CHLIANG in ganglia. but unlike the adrenergic beta rcceptol and COSTA.197%). The increase of this enzyme activity agonists it does not increase cyclic AMP and requires preganglionic innervation (HANRAI I R. GI. IIXITTI and outlasts by 3S-4hr the increment in cyclic AMP content. The activated cnryme has some of the attributes of COSIA. 1975b). histone kinase and it is translocatcd from cytosol to The present report concerns the participation of a particulate fraction which contains adrenal medulnicotinic and muscarinic receptors in the rcgulatior. lary nuclei (KUROSAWA. GLIII~O~~I and COSTA, 1976). of tyrosine hydroxylase in superior cervical ganglia. In this structure the translocation precedes by 1 2 This paper shows that nicotinic receptor agonists inhr the increment in ribonucleic acid synthesis. The duce tyrosine hydroxylase in normal and decentralired synthesis rate of tyrosine hydroxylase is augmented ganglia. The blockade of muscarinic receptors faciliat 8 hr following stimulus application. This rate peaks tates tyrosine hydroxylase induction by nicotinic rc8.5
86
I. HANEAL I K and E. COSTA
ceptor agonists, whereas the activation of the muscarinic receptors inhibits the tyrosine hydroxylasc induction elicited by nicotinic receptor activation. The intermediation of dopamine receptors in the inhibitory action of muscarinic receptor activation is suggested.
MATERIALS
AND METHODS
Malt Sprague-Dawlcy rats (Zivic Miller. Allison Park. Pa.) weighing 1OG 120 g were used 3 to 4 days after arrival at our animal quarters. During this time. they were kept in circadian artificial illumination (12 hr cycles) at 23 ‘C and fed Purina rat chow. Rats were injected with various drugs (see Results) and 48 hr after drug treatment the animals were killed by cervical dislocation. Both superior cervical ganglia were removed and homogenized in 0.5 ml 0,067 M phosphate buffer, pH 6.2. The tyrosine hydroxylase activity was assayed as previously described (HANBAUEK vr al.. 1975b) using one ganglion per assay and 6-methyltetrahydropterine as cofactor. Unilateral decentralization was performed in the laboratory under sodium methohexital anaesthesia. 3-4 days before drug injection. The following drugs were used: sodium methohexital (Brevital@ sodium, Eli Lilly Co.. Indianapolis. Ind.); nicotine salicylate (K & K Labs., Plainview, New York); carbamylcholine (Aldrich Chemical Co.. Milwaukee. Wis.); atropine sulphate H,O (Schwartz and Mann. Division of Becton. Dickinson and Co.. Orangeburg. New York); acetyl-betamcthylcholine (mcthacholinc. Merck and Co.. Inc.. Rahway. N.J.): Serpasil@ (Ciba-Geigy. Summit, N.J.); apomorphine HCl (S.B. Penik. N.Y.). RESULTS
The tyrosine hydroxylase activity of intact superior cervical ganglia was increased 48 hr after 50 /tmol/kg nicotine administered subcutaneously (Table 1). This
Table I. Induction of ganglionic tyrosine hydroxylase by nicotine and inhibition by simultaneous muscarinic receptor stimulation
Drugs (/lmol!kg) Saline Nicotine (2.5 s.c.) Nicotine (50 s.c.) Carbachol (8.1 i.p.) Methacholine (42 i.p.) Methacholine (42 i.p.) + Nicotine (50 s.c.)
Table
2. The c&t of carhamylcholine and nicotine tyrosinc hqdroxylase activity in adrenal medulla
Tyrosine hydroxylase (nmol DOPA, pair adrenal medullae per hr)
Drugs (jtmol;kg) Saline Carbamylcholinc Nicotine (40 s.c.) Methacholine (42 Methacholinc (42 + Nicotine (40
on
(X.1 i.p.) i.p.) i.p.) s.c.)
19.5 33.7 27.x IY.0 31.0
* P < 0925 (II = 6). Methacholinc was illjectcd IO min bcforc rats wcrc killed 24 hr after drug treatment.
* 2 * * i
2.0 2.6* 24* 2.0 3.7*
nicotine.
The
dose appears to be close to the threshold for tyrosine hydroxylase induction (25 /tmol/kg S.C. did not induce tyrosine hydroxylase). However. in decentralized ganglia neither of the two nicotine doses increased tyrosine hydroxylase activity. The increase of tyrosine hydroxylase activity elicited by nicotine can not be attributed to a change in the kinetic state of the enzyme because the Michaelis constant for the pteridine cofactor is not changed as a result of nicotine injection and the increase of tyrosine hydroxylase occurs after a time interval of 3&48 hr. When 42 Itmol/kg of mcthacholine was injected into-aperitoneally. tyrosine hydroxylase activity was not increased. Moreover. when methacholine was injected together with an inducing dose of nicotine it prevented the action of nicotine on ganglionic tyrosine hydroxylase (Table I ). In the doses tested. carbamylcholinc failed to induce tyrosine hydroxylase in superior cervical ganglia (Table I). Both carbamylcholine and nicotine cause 21delayed increase in the tyrosine hydroxylase activity of adrenal medulla (Table 2) which was due to an increase in the number of tyrosine hydroxylase molecules. Methacholinc neither induced adrenal tyrosine hydroxylase nor prevented the induction elicited by nicotine (Table 2). These results suggest that in adrenal medulla the simultaneous activation of nicotinic and muscarinic receptors is compatible with tyrosine hydroxylase induction. whereas in ganglia the activation of both receptors prevents the induction of tyrosine hydroxylase elicited by nicotinic receptor agonists.
Tyrosine hydroxylasc (nmol DOPA,‘ganglion per hr) Intact Decentralized 3.1 2.9 4.4 2.x 3.0 2.9
* + f * * k
0.20 @I6 0.19* 0.22 0.22 0.16
2.1 + 0.24 2.0 * 0.17 2.2 f 0.19
2.1 * 0.17
*P < 0.01. Each mean + S.E.M. refers to 68 experiments. Methacholine was mjectcd i.p. IO min before nicotine. The rats were decentralircd 48 hr before the drug treatment.
The antagonistic relationship between nicotinic and muscarinic receptor activation in inducing ganglionic tyrosine hydroxylasc is upheld by the results reported in Table 3. They show that 43 ,umol/kg atropine administered intraperitoneally failed to change tyrosine hydroxylase activity in intact and decentralized ganglia. However, in rats injected with atropine the successive subcutaneous injection of 25 iimol/kg of nicotine. which failed to induce tyrosine hqdroxylase when
ACh receptors Table
3. Atropine
and tyrosine
hydroxylasc
facilitation of tyrosine hydroxylase induction and decentralized superior cervical ganglia
+mol/kg) i.p.
SC.
3.2 3.4 2.9 4.2 4%
43 25 25 50
43 43
hq nicotine
in intact
Tyrosine hydroxqlasc (nmol DOPA:ganglion per hr) Decentralized Intact 3 days 7 days
Nicotine
Atropine
x7
induction
+ + f + *
0.30 0.37 0.16 0,14* @14*
2.5 f 0.10 2.0 + 0, I7 3.4 * 0.17* 3.4 * 0, I’)*
2.3 + 0.30 2.0 i_ 0.12 3.1 * o.I6+ ___
* P < 001 ; t P < 0.05 01 = 6). Three or seven days after monolateral decentralization of the superior ccr\ ~cal ganglion. rats received atropine IO min before nicotine. The animals Mcrc killed 3X ht after drug treatment.
given alone (Table 3), could induce tyrosine hydroxylase in intact and decentralized ganglia. The data of Table 3 also show that 50 pmol/kg nicotine injected subcutaneously, which was inactive in decentralized ganglia (Table 1). increased tyrosine hydroxylase activity in this tissue after a pretreatment with atropine. Moreover, the data presented in Table 3 show that atropine can also facilitate the action of subthreshold doses of nicotine in ganglia decentralized one week before the experiment.
Mediation tnethucholinr
by dopamine on thr
of
the
induction
h_vdroxyla.sr hi* nicotinic
inhibitory
actiotl
of yanylionic
wcrptor
of
t~~rosinr
agonists
It has been reported
that the induction of ganglionic tyrosine hydroxylase elicited by reserpine can be antagonized by nicotinic receptor blockers (MLILLLLIK. THO~NEN and Axf:LROf). 1969). The data reported in Table 4 show that methacholine blocked the induction of tyrosine hydroxylase elicited by nicotine in superior cervical ganglia but it failed to block the tyrosine hydroxylase induction elicited by reserpine. To ascertain whether the different responsiveTable 4. The effect of muscarinic and dopamine agonists on the induction of tyrosine hydroxylase by nicotine or reserpine Drugs (pmol/kg)
receptor elicited
Tyrosine hydroxylase (nmol DOPA/ganglion per hr)
Saline Methacholine 40 Reserpine 12.3 Methacholine 40 + reserpine 12.3 Apomorphine 31 + reserpine 12.3 Nicotine 50 Apomorphine 31 + nicotine 50 *P < 0.05; tP < @Ol. Each mean + S.E.M. refers to and apomorphine were injected before subcutaneous injection of rats were killed 48 hr after drug
2.4 2.7 3.7 3.8
k f k f
0.18 0.11 0,26+ @32+
2.4 + 0.20 3.3 & 0,26* 2.6 + 0.21
8 ganglia. Methacholine. intraperitoneally 10 min nicotine or reserpine. The treatment.
ness of the reserpine induction to mcthacholine was due to the absence of the adrenergic transmitter in the ganglionic intcrneuroncs. the action of apomorphine on the induction of tyrosine hydroxylase elicited by reserpine and nicotine was compared. The results of these experiments arc reported in Table 4. They show that apomorphine inhibited the induction of ganglionic tyrosine hydroxylase elicited by nicotine and reserpine. To corroborate the participation of dopaminergic receptors in the inhibition of tyrosine hydroxylase induction elicited by muscarinic receptor stimulation, the action of subthrcshold doses of nicotine on the induction of tyrosinc hydroxylase in bilaterally decentralized ganglia of rats treated with rescrpine was also tested. Table 5 shows that depletion of catecholamines by rcserpine facilitated the induction of ganglionic tyrosine hydroxylase activity elicited bq nicotine.
DISC'CSSION Neuronal communication is transacted at sqnapscs and the resulting interaction between receptor and Table 5. The effect of nicotine and rcscrpinc on tlrosine hydroxylase activity m bilaterally decentralized superior cervical ganglia
Treatment Saline Nicotine (20 PmolFkg s.c.) Rescrpine (I 2.3 ltmol kg s.c.) Reserpinc (12.3 pmol;kg s.c.) + Nicotine (20 Atmol,!kg s.c.)
TqroGne hydroxylasc (nmol DOPA ganglion per hr) 2.x 5 0.15 2.9 * 0. I6 3. I
f 0.IO
3.x + 0.74*
*P < 0.01 when compared with saline or nicotine treated groups. The mean i: S.E.M. refers to 6 ganglia. The rats were bilaterally decentralized 4 days before drug treatment. Rcserpine was injected 4 hr before nicotine and the rats were killed 44 hr thereafter.
88
I. HANISAUI K and E. COSTA
transmitters influences the regulation of protein synthesis in postsynaptic cells. We have discussed previously that the trans-synaptic induction of tyrosinc hydroxylase is a model to study how stimulation of receptors located in ncuronal membranes can influence the synthesis of specific proteins (GtIIIX)TTI et ul., 1975b: KUKOSAWA ct ul.. 1975). Tyrosinc hydroxylasc can be localized immunohistochemically in perikarya of catecholaminergic neurones where the membranes of the endoplasmic reticulum, the Golgi apparatus and the neurotubules react specifically with the antibody directed to tyrosine hydroxylase (PICIXL, JOH and RI:IS, 1975). The regulation of tyrosine hydroxylase synthesis offers an idcal model to study how various receptors located in ncuronal membranes can interact with each other and control the synthesis of a specific protein. In superior cervical ganglia, the activity of noradrencrgic neurones is regulated by a number of synaptic inputs which operate on nicotinic, muscarinic and dopamincrgic receptors (L~ur:r and TOSAKA. 1970; VOLL~.. 1966). It is currently believed that the function of these membrane receptors expresses itself as a regulation of noradrenergic neuronc excitability: activation of nicotinic or muscarinic receptors in ganglion cell bodies causes depolarization (HALFILY. 197421.b) and also ganglionic interncuroncs (small intcnscly Huorescent cells) arc depolarized by muscarinic rcccptor activation (LIIET and OWMAN. 1974). Howcvcr. the depolarization of ganglionic intcrncuroncs elicits catecholaminc secretion. which by acting on specific ncuronal receptors. decrcascs the excitability of noradrcncrgic ncurones (COSTA. RI.VZIN. KI’NXMAN. SWCTOR and BIUIII. 1961). The nicotinic receptors mediate the fast excitatory potentials in postsynaptic cells (HAI-FI:LJ,. 1974a). A late excitatory potential is generated by activation of muscarinic rcccptors. whcreas the true inhibitory synaptic potential requires the activation of muscarinic receptors in gdnglionic interneurones and the release of a catecholamine. presumably dopamine from these interncuroncs (LIKT and OWMAN. 1974). Our results show that the activation of nicotinic receptors can cause a delayed increase of tyrosine hydroxylasc activity. Since this increase is not due to a change in the kinetic properties of tyrosine hydroxylase and it requires DNA transcription and protein synthesis. it rcfccts a trans-synaptic induction of tyrosine hydroxylasc synthesis. Our cxpcriments reveal that carbachol and nicotine induce tyrosine hydroxylase in adrenal medulla but only nicotine can induce tyrosine hydroxylase in sympathetic ganglia. While nicotinic receptor agonists arc equally active in inducing tyrosine hydroxylase in intact and denervated adrenal medulla (HANI~ALII.Kand GI,IIX)TTI. 1975) the present experiments show that nicotine can induce tyrosine hydroxylasc in intact but not in decentralized sympathetic ganglia. The reason for this difference can be inferred only indirectly from experiments in which nicotine and atropinc injections were combined. Atropine appears to lower the nicotine
threshold to induct tyrosinc hydroxylase in intact sympathetic ganglia and facilitates the induction of tyrosine hydroxylase by nicotine in decentralized ganglia. Thus, it appears that in decentralized ganglia, the injection of high doses of nicotine stimulates muscarinic rcccptors which. in turn. cause an antagonistic action on tyrosine hydroxqlasc induction. When this muscarinic stimulation is abolished by atropine. nicotine also induces tyrosine hydroxylase in decentralized ganglia. We have tested whether stimulation of muscarinic receptors antagonizes the induction of ganglionic tyrosine hydroxylasc elicited by nicotine or reserpine. Methacholinc. a selcctivc muscarinic receptor agonist abolishes the tyrosine hydroxqlase induction by nicotine but not that by reserpine. On one hand, this tinding contirmed the direct participation of muscarinic and nicotinic receptors in the trans-synaptic regulation of throsine hydroxylase in ganglia and on the other hand, it suggested that the inhibition of tyrosine hydroxylase induction mediated by muscarinic receptor activation requires the availability of catecholamine stores. The participation of catecholamines was tested by studying the ell’ects of npomorphine on the tyrosine hydroxylase induction elicited by reserpine and nicotine. Apomorphine inhibits the tyrosine hydroxylase induction elicited by reserpine and nicotine. That the stimulation of beta adrcnergic receptors can induce tyrosinc hydroxylase was demonstrated by previous cxperimcnts with intact and decentralized ganglia (HASBAI I K ct ul.. 1975a). Since norepinephrine. as well as epincphrine and dopamine are also stored in sympathetic ganglia of rats (Kos~ow. Bmmvrc’ and C~STA. 1975). it is possible to entertain the hypothesis that either of these catecholamine rcceptors participate in the regulation of ganglionic tyrosinc hydroxylasc. It is currently bclicvcd that dopamine release mediates the true inhibitory hyperpolariration elicited by trans-synaptic muscarinic activation (GIWI.NC;AKIIand KI:HARIAN.1974). The present expcrimcnts dcmonstratc that this type of regulation is opcrativc in tho inhibitory effects of methacholine on the tyrosine hydroxylase induction elicited by nicotine. lsoprotcrcnol was shown by OTTEN. MU:LLFK, 01 SCH and THOI.NIN (I 974) to cause an increase of cyclic AMP content in sympathetic ganglion cells due to an increased cyclic AMP synthesis. Moreover. Dt GROAT and VOLLI. (1966) have shown that isoproterenol enhances the ganglionic responses to the stimulator) action of muscarinic agents. Finally. HAI.FI:LL (1969) has demonstrated that a single injection of isoprotcrcnol dcpolari/cs the sympathetic ganglion cells for about 30 min. Thus. isoprotcrenol diminishes the post-tetanic hyperpolarisation and enhances postsynaptic activity in noradrcnergic cells. Our results suggest that dopaminc and isoproterenol (HANIIAUK ~‘1(I/.. 197521)hale opposite effects in the regulation of tyrosinc hydrox!lase synthesis: stimulation of dopaminc rcccptors limits the induction of tyrosine hydroxylase clicitcd bq nicotine. and stimulation of beta adrenergic rcccptorh is a direct stimulus for tyrosinc
ACh
receptors
and tyrosine
hydroxylasc induction. Perhaps, in some situations beta adrcnergic receptors may serve a function. In adrenal demedullated rats exposed to cold. the transsynaptic induction of tyrosine hydroxylase appears to be mediated by a mechanism which involves a facilitation through an activation of beta adrcnergic rcceptors (GUIIX)TTI rt ul.. 1975a). Whether some ganglionic intcrneuroncs store exclusivel) cpincphrine or whether all ganglionic intcrneuroncs store and secrete dopamine and cpinephrinc is not understood at the present time (Kos~ow c’f ~1.. 1975). Our results do not clarify whether dopaminc secretion is mediated through nicotinic or muscarinic receptors. but the cvidcnce available suggests that dopamine must be considered as one of the cndogenous regulatory substances for ganglionic tyrosine hydroxllase synthesis. In conclusion. the present experiments and our previous work (G~IIIX)TTI (‘f trl.. 1975a) allow us to propose the following scheme describing the participation of various transmitters in the regulation of tyrosine hydroxylasc synthesis in sympathetic ganglia of rats. (I) Glucocorticoids are an important permissive factor for tyrosinc hydroxylase induction. This facilitation requires glucocorticoid receptor activation which is operative only in the presence of an intact synaptic circuit (HASISAILK cl trl., 1975b). In fact, dexamethasone induces tyrosine h!droxylase only if the prcganglionic nerves are intact. (2) Beta adrenergic receptor activation can induce tyrosine hydroxylase in the absence and in the presence of affcrent nerves. This induction involves an early increase of cyclic AMP and may bc related to the sustained dcpolarization elicited by beta adrenergic receptor agonists. In adrenal dcmedullated rats. the induction of ganglionic tyrosine hydroxylase during short term (4 hr) exposure to cold may involve a participation of the physiological activation of these receptors. Endogenous ganglionic stores of epincphrine may be preferentially mobili/cd to elicit the tyrosine hydroxylast activation after cold exposure of adrenal demedullated rats. In adrenal demedullated rats the transsynaptic induction of ganglionic tyrosine hydroxylase elicited by cold differs from that observed in normal rats because it involves an immediate increase of ganglionic cyclic AMP content and the stimulus threshold is reduced from 24 hr in normal rats to 4 hr in demedullated rats. (3) Nicotinic receptor activation causes depolarization of ganglion cells which is dissociated from an early increase of cyclic AMP content. This action is counteracted by muscarinic receptor activation. In dccentraliled ganglia. nicotinic receptor activation induces tyrosine hydroxylase only when muscarinic receptors are blocked by atropine. This blockade lowers the threshold for tyrosine hydroxylase induction by nicotine also in intact ganglia. (4) Muscarinic receptor activation inhibits the tyrosine hydroxylase induction elicited by nicotinic rcceptor activation through the release of endogenous dopamine from ganglionic interneurones. Since this action can be mimicked by apomorphine. WC propose
hydroxylase
induction
89
that the inhibitory action on tyrosine hydroxylase induction by muscarinic receptor activation is mediated by endogenous dopamine. REFERENCES C~rr ANG, D. M. and COS~A. E. (1974).
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