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Effect of chronic clonidine treatment and withdrwal on tyrosine hydroxylase activity in peripheral ganglia and the locus coeruleus
European Journal of Pharmacology, 51 (1978) 309--312 © Elsevier/North-Holland Biomedical Press
309
Short communication EFFECT OF CHRONIC CLONIDINE TREATMENT AND WITHDRAWAL ON TYROSINE HYDROXYLASE ACTIVITY IN PERIPHERAL GANGLIA AND THE LOCUS COERULEUS WILLIAM H. KANE and EUGENE M. JOHNSON, JR. *
Department of Pharmacology, Washington Universtiy Medical School, St. Louis, Missouri 63110, U.S.A. Received 9 June 1978, accepted 25 July 1978
W.H. KANE and E.M. JOHNSON, JR., Effect of chronic clonidine treatment and withdrawal on tyrosine hydroxylase activity in peripheral ganglia and the locus coeruleus, European J. Pharmacol 51 (1978) 309--312. As is observed clinically, cessation of chronic clonidine treatment in the rat results in a syndrome characterized by sympathetic hyperactivity. After three weeks of chronic oral administration of clonidine, tyrosine hydroxylase (TOH) activity was unchanged in superior cervical ganglia and locus coeruleus, but was reduced (45%) in the celiac ganglia. A b r u p t cessation of treatment resulted in increases in TOH activity in superior cervical and celiac ganglia (to 135 and 250% of controls) and in the locus coeruleus (170% of control). These data suggest a selective effect of clonidine treatment and withdrawal on vasomotor fibers. A mechanism explaining physical dependence on clonidine is proposed. Clonidine
Withdrawal
Physical dependence
1. Introduction Ulonidine is an extremely potent centrally acting antihypertensive agent. The mechanism of antihypertensive action appears to involve stimulation of ~-adrenergic receptors in the brain stem (Kobinger and Walland, 1967). Abrubt cessation of clonidine treatment in patients has resulted in a withdrawal syndrome (HSkfelt et al., 1970) characterized by tachycardia, tremor, headache, and occasionally a rebound hypertension of critical magnitude. The general picture is that of sympathetic hyperactivity. We have demonstrated (Dix and Johnson, 1977) that rats experience a similar withdrawal syndrome upon cessation of chronic clonidine treatment. The withdrawal in normotensive rats was characterized by marked tachycardia lasting at least four days. Enhanced sympathetic activity was indicated by an increase (about 75%) in adrenal * Correspondence and proofs to: Dr. Eugene M. Johnson, Jr.
Locus coeruleus
Trans-synaptic induction
tyrosine hydroxylase (TOH) activity upon withdrawal. The increase was dependent on intact innervation of the adrenal and was therefore due to transsynaptic induction caused by increased nerve traffic from higher centers. In this report we demonstrate striking differences in the effect of chronic clonidine treatment and withdrawal on the TOH activity (as an indicator of increased or decreased neuronal activity) in the superior cervical ganglion (SCG) and the celiac ganglion (CG). We also report that withdrawal from clonidine is associated with a rise in TOH activity of the locus coeruleus (LC).
2. Materials and methods
Male Sprague-Dawley rats (225--250 g at start of treatment) were administered clonidine via the drinking water (5/lg/ml; 300-500 pg/kg/day) as previously described (Dix and Johnson, 1977). Control animals received tap water. After three weeks of exposure, ani-
310
W.H. K A N E , E.M. J O H N S O N , JR.
mals were killed (day 0) or switched to tap water containing no drug and killed at various times after cessation of treatment. The area of the LC (Ross and Reis, 1974), the CG, and SCG were removed and frozen on dry ice in small grinding vessels (Kontes). Tissues were homogenized in a Tris buffer (5 mM, pH 7.5) containing 0.2% Triton X-100. TOH activity was determined by the m e t h o d of Phillipson and Sandler (1975) using final concentrations of 40 pM 1-tyrosine and 825 pM DMPH4 and under conditions linear with time and enzyme concentration.
3. Results Clonidine treatment and subsequent withdrawal produced much greater effects on the TOH activity in CG than in SCG (fig. la, b).
After three weeks of clonidine treatment (day 0) TOH activity in SCG was unchanged. After cessation of treatment a modest increase (35-40%) was seen in TOH activity by day 3 and activity returned to control values by day 7. These results are similar to previous (Dix and Johnson, 1977) observations in adrenal except that the increase is less (40% as compared to a b o u t 75% in adrenal). In contrast, TOH activity in CG was reduced (about 45%) after three weeks of clonidine treatment. Upon cessation of treatment, TOH activity increased to a b o u t 250% of control levels by day 5 where it remained for at least three days. Activity remained elevated, though highly variable for at least 15 days. The activity of TOH in the LC (fig. 2) was unchanged by chronic clonidine treatment. Between day 1 and day 3 of withdrawal, enzyme activity increased 60--70% and returned to control levels by day 8. Again, a greater variability was seen in the animals
i
250 !
200
150
lOC
10C
50
50
/ B
01
I I L 345
I
I
I
15 0 345 8 Days after donidine withdrawal
~55
Fig. 1. Effect o f cessation o f chronic clonidine treatm e n t o n t h e tyrosine h y d r o x y l a s e activity in superior cervical ganglia (A) and celiac ganglia (B). Animals were treated for three weeks (5 ~ g / m l in drinking water, 3 0 0 - - 5 0 0 pg/kg/day). Animals were killed on the last day of t r e a t m e n t (day 0) or after switching to tap w a t e r (day 1--15). Control activities were 2.02 + 0.08 nmoles tyrosine o x i d i z e d / p a i r / h (n = 5) for superior cervical ganglia or 0.78 + 0 . 0 6 nmoles/ ganglia/h (n = 14) in celiac ganglia. Values s t a t i s t i c a l l y d i f f e r e n t f r o m controls are d e n o t e d by asterisks (*P < 0.05; **P < 0.01). Each value represents the mean + S.E.M. of 4--7 animals.
I
,
I
Days after clonidine wJthdrQwal
Fig. 2. Tyrosine h y d r o x y l a s e activity in locus coeruleus of animal treated as in fig. 1. Data are combined f r o m t w o experiments. Days 1, 3 and 5 (controls 0.38 + 0.06 nmoles tyrosine o x i d i z e d / p a i r / h n = 7) were d o n e in one e x p e r i m e n t and day 4, 8 and 15 (controls 0.51 + 0.10 nmoles of tyrosine oxidized/pair/h n = 6) in the second e x p e r i m e n t . Values statistically different f r o m control are d e n o t e d by asterisks (*P < 0.05; **P < 0.01). Each value represents m e a n + S.E.M. of 4--7 animals.
TYROSINE HYDROXYLASE ACTIVITY IN CLONIDINE WITHDRAWAL 15 days post-clonidine. An increase in the TOH activity of the LC after cessation of clonidine treatment has been observed in four separate experiments.
4. D i s c u s s i o n
Much greater effects of clonidine treatment and withdrawal are seen on the TOH activity in the CG, as compared to the SCG. This difference probably reflects a much greater effect of clonidine treatment and withdrawal on the preganglionic nervous input to these structures. Our results therefore support the conclusion of Haeusler (1974) that clonidine selectively inhibits preganglionic input to adrenergic vasomotor fibers, and suggests that withdrawal results in a selective increase in vasomotor traffic. Several lines of experimental evidence are consistent with the idea that the increase in the TOH activity of the locus coeruleus, as in the periphery, reflects increased activity of the neurons (e.g. Lewander et al., 1977). The LC is the major nucleus of noradrenergic perikarya in the brain. Clonidine has been shown to have a direct inhibitory effect on the neurons of the LC (Cedarbaum and Aghajanian, 1976) which appears to be mediated by presynaptic s-receptors. Electrophysiological data (Aghjanian et al., 1977) indicates that the neurons of the LC have collateral branches which synapse on themselves or other neurons of the LC and hence clonidine m a y be mimicking the normal autoinhibitory activity of the neurons. These observations suggest a simple mechanism to explain the physical dependence on clonidine. Clonidine would reduce the activity of the LC by acting on autoinhibitory s-receptors. The constant exposure of the inhibitory receptors to high concentration of clonidine may produce a subsensitivity of these receptors analogous to that seen in peripheral systems (Langer and LuchelliFortis, 1977). Abrupt cessation of clonidine therapy would therefore result in a failure of
311
the normal inhibition of the cells due to the subsensitivity; the neurons would fire at an excessive rate and TOH activity would increase. The firing rate of the neurons of the LC and the enzyme activity would return to basal levels as the normal c o m p l e m e n t of functional inhibitory receptors are restored. The time course of the increase of TOH in the LC is, indeed, very similar to the time course of the tachycardia seen upon clonidine withdrawal (Dix and Johnson, 1977). Consistent with this suggestion are the recent demonstrations that electrical stimulation of the LC in rats results in an increase in norepinephrine metabolites in blood (Crawley et al., 1978). Direct electrical stimulation of the area of the LC in the rhesus m o n k e y produces tachycardia and an increase in blood pressure (M. Raichle, personal communication) and tachycardia in the rat ( K a w a m u r a et al., 1978). Hence, the increased neuronal activity in the LC, suggested by our results, might be expected to produce s y m p t o m s characteristic of clonidine withdrawal. Our data and this working hypothesis d o not, of course, preclude the possibility that similar changes occur in other noradrenergic cell groups which may be important in the withdrawal syndrome. In any event, our results demonstrate that readily quantifiable peripheral and central biochemical parameters may be monitored in conjunction with the physiological changes associated with this phenomenon. The clonidine withdrawal syndrome may prove valuable as a general model of drug dependence. Acknowledgements We thank Richard Macia and Sarah Oldham for their technical assistance. This work was supported by The National Foundation, March of Dimes (Basil O'Connor Grant), by NIH grant HL-20604 and by NIH training grant 5 T32 GM07200. References Aghajanian, G.K., J.M. Cedarbaum and R.Y. Wang, 1977, Evidence for norepinephrine-mediated colla-
312 teral inhibition of locus coeruleus neurons, Brain Res. 136, 570. Cedarbaum, J.M. and G.K. Aghajanian, 1976, Noradrenergic neurons of the locus coeruleus: inhibition by epinephrine and actNation by the s-antagonist piperoxane, Brain Res. 112, 413. Crawley, J.N., S.E. Hattox, J.W. Maas and R.H. Roth, 1978, 3-Methoxy-4-hydroxyphenylethyleneglycol increases in plasma after stimulation of the locus coeruleus, Brain Res. 1 4 1 , 3 8 0 . Dix, R.K. and E.M. Johnson, 1977, Withdrawal syndrome upon cessation of chronic elonidine treatment in rats, European J. Pharmacol. 4 4 , 1 5 3 . Haeusler, G., 1974, Further similarities between the action of clonidine and a central activation of the depressor baroreceptor reflex, Naunyn-Schmiedeb. Arch. Pharmacol. 285, 1. H6kfelt, B., H. Hedeland and J.E. Dymling, 1970, Studies in catecholamines, renin, and aldosterone following Cata presan (2-(2,6-dichlorophenylamine)-2-imidazoline hydrochloride) in hypertensive patients, European J. Pharmacol. 10,389. Kawamura, H., D.G. Gunn and E.D. Frolich, 1978, Cardiovascular alteration by nucleus locus coeruleus in spontaneously hypertensive rat, Brain Res. 140,137.
W.H. KANE, E.M. JOHNSON, JR. Kobinger, W. and A. Walland, 1967, Investigations into the mechanism of the hypotensive effect of 2-(2,6-dichlorophenylamino)-2-imidazoline HC1, European J. Pharmacol. 2 , 1 5 5 . Langer, S.Z. and M.A. Luchelli-Fortis, 1977, Subsensitivity of the presynaptic ~-adrenoceptors after short-term surgical denervation of the cat nictitating membrane, J. Pharmacol. Exptt. Therap. 202,610. Lewander, T., T.H. Joh and D.J. Reis, 1977, Tyrosine hydroxylase: delayed activation in central noradrenergic neurons and induction in adrenal medulla elicited by stimulation of central cholinergic receptors, J. Pharmacol. Exptl. Therap. 200~ 523. Phillipson, O.T. and M. Sandler, 1975, The effect of hydrocortisone and adrenocorticotrophic hormone on monoamine oxidase and tyrosine hydroxylase in explant cultures of embryonic chick sympathetic ganglion, Brain Res. 9 0 , 2 8 3 . Ross, R.A. and D.J.Reis, 1974, Effect of lesions of locus coeruleus on regional distribution of dopamine-~-hydroxylase activity in rat brain, Brain Res. 7 3 , 1 6 1 .
309
Short communication EFFECT OF CHRONIC CLONIDINE TREATMENT AND WITHDRAWAL ON TYROSINE HYDROXYLASE ACTIVITY IN PERIPHERAL GANGLIA AND THE LOCUS COERULEUS WILLIAM H. KANE and EUGENE M. JOHNSON, JR. *
Department of Pharmacology, Washington Universtiy Medical School, St. Louis, Missouri 63110, U.S.A. Received 9 June 1978, accepted 25 July 1978
W.H. KANE and E.M. JOHNSON, JR., Effect of chronic clonidine treatment and withdrawal on tyrosine hydroxylase activity in peripheral ganglia and the locus coeruleus, European J. Pharmacol 51 (1978) 309--312. As is observed clinically, cessation of chronic clonidine treatment in the rat results in a syndrome characterized by sympathetic hyperactivity. After three weeks of chronic oral administration of clonidine, tyrosine hydroxylase (TOH) activity was unchanged in superior cervical ganglia and locus coeruleus, but was reduced (45%) in the celiac ganglia. A b r u p t cessation of treatment resulted in increases in TOH activity in superior cervical and celiac ganglia (to 135 and 250% of controls) and in the locus coeruleus (170% of control). These data suggest a selective effect of clonidine treatment and withdrawal on vasomotor fibers. A mechanism explaining physical dependence on clonidine is proposed. Clonidine
Withdrawal
Physical dependence
1. Introduction Ulonidine is an extremely potent centrally acting antihypertensive agent. The mechanism of antihypertensive action appears to involve stimulation of ~-adrenergic receptors in the brain stem (Kobinger and Walland, 1967). Abrubt cessation of clonidine treatment in patients has resulted in a withdrawal syndrome (HSkfelt et al., 1970) characterized by tachycardia, tremor, headache, and occasionally a rebound hypertension of critical magnitude. The general picture is that of sympathetic hyperactivity. We have demonstrated (Dix and Johnson, 1977) that rats experience a similar withdrawal syndrome upon cessation of chronic clonidine treatment. The withdrawal in normotensive rats was characterized by marked tachycardia lasting at least four days. Enhanced sympathetic activity was indicated by an increase (about 75%) in adrenal * Correspondence and proofs to: Dr. Eugene M. Johnson, Jr.
Locus coeruleus
Trans-synaptic induction
tyrosine hydroxylase (TOH) activity upon withdrawal. The increase was dependent on intact innervation of the adrenal and was therefore due to transsynaptic induction caused by increased nerve traffic from higher centers. In this report we demonstrate striking differences in the effect of chronic clonidine treatment and withdrawal on the TOH activity (as an indicator of increased or decreased neuronal activity) in the superior cervical ganglion (SCG) and the celiac ganglion (CG). We also report that withdrawal from clonidine is associated with a rise in TOH activity of the locus coeruleus (LC).
2. Materials and methods
Male Sprague-Dawley rats (225--250 g at start of treatment) were administered clonidine via the drinking water (5/lg/ml; 300-500 pg/kg/day) as previously described (Dix and Johnson, 1977). Control animals received tap water. After three weeks of exposure, ani-
310
W.H. K A N E , E.M. J O H N S O N , JR.
mals were killed (day 0) or switched to tap water containing no drug and killed at various times after cessation of treatment. The area of the LC (Ross and Reis, 1974), the CG, and SCG were removed and frozen on dry ice in small grinding vessels (Kontes). Tissues were homogenized in a Tris buffer (5 mM, pH 7.5) containing 0.2% Triton X-100. TOH activity was determined by the m e t h o d of Phillipson and Sandler (1975) using final concentrations of 40 pM 1-tyrosine and 825 pM DMPH4 and under conditions linear with time and enzyme concentration.
3. Results Clonidine treatment and subsequent withdrawal produced much greater effects on the TOH activity in CG than in SCG (fig. la, b).
After three weeks of clonidine treatment (day 0) TOH activity in SCG was unchanged. After cessation of treatment a modest increase (35-40%) was seen in TOH activity by day 3 and activity returned to control values by day 7. These results are similar to previous (Dix and Johnson, 1977) observations in adrenal except that the increase is less (40% as compared to a b o u t 75% in adrenal). In contrast, TOH activity in CG was reduced (about 45%) after three weeks of clonidine treatment. Upon cessation of treatment, TOH activity increased to a b o u t 250% of control levels by day 5 where it remained for at least three days. Activity remained elevated, though highly variable for at least 15 days. The activity of TOH in the LC (fig. 2) was unchanged by chronic clonidine treatment. Between day 1 and day 3 of withdrawal, enzyme activity increased 60--70% and returned to control levels by day 8. Again, a greater variability was seen in the animals
i
250 !
200
150
lOC
10C
50
50
/ B
01
I I L 345
I
I
I
15 0 345 8 Days after donidine withdrawal
~55
Fig. 1. Effect o f cessation o f chronic clonidine treatm e n t o n t h e tyrosine h y d r o x y l a s e activity in superior cervical ganglia (A) and celiac ganglia (B). Animals were treated for three weeks (5 ~ g / m l in drinking water, 3 0 0 - - 5 0 0 pg/kg/day). Animals were killed on the last day of t r e a t m e n t (day 0) or after switching to tap w a t e r (day 1--15). Control activities were 2.02 + 0.08 nmoles tyrosine o x i d i z e d / p a i r / h (n = 5) for superior cervical ganglia or 0.78 + 0 . 0 6 nmoles/ ganglia/h (n = 14) in celiac ganglia. Values s t a t i s t i c a l l y d i f f e r e n t f r o m controls are d e n o t e d by asterisks (*P < 0.05; **P < 0.01). Each value represents the mean + S.E.M. of 4--7 animals.
I
,
I
Days after clonidine wJthdrQwal
Fig. 2. Tyrosine h y d r o x y l a s e activity in locus coeruleus of animal treated as in fig. 1. Data are combined f r o m t w o experiments. Days 1, 3 and 5 (controls 0.38 + 0.06 nmoles tyrosine o x i d i z e d / p a i r / h n = 7) were d o n e in one e x p e r i m e n t and day 4, 8 and 15 (controls 0.51 + 0.10 nmoles of tyrosine oxidized/pair/h n = 6) in the second e x p e r i m e n t . Values statistically different f r o m control are d e n o t e d by asterisks (*P < 0.05; **P < 0.01). Each value represents m e a n + S.E.M. of 4--7 animals.
TYROSINE HYDROXYLASE ACTIVITY IN CLONIDINE WITHDRAWAL 15 days post-clonidine. An increase in the TOH activity of the LC after cessation of clonidine treatment has been observed in four separate experiments.
4. D i s c u s s i o n
Much greater effects of clonidine treatment and withdrawal are seen on the TOH activity in the CG, as compared to the SCG. This difference probably reflects a much greater effect of clonidine treatment and withdrawal on the preganglionic nervous input to these structures. Our results therefore support the conclusion of Haeusler (1974) that clonidine selectively inhibits preganglionic input to adrenergic vasomotor fibers, and suggests that withdrawal results in a selective increase in vasomotor traffic. Several lines of experimental evidence are consistent with the idea that the increase in the TOH activity of the locus coeruleus, as in the periphery, reflects increased activity of the neurons (e.g. Lewander et al., 1977). The LC is the major nucleus of noradrenergic perikarya in the brain. Clonidine has been shown to have a direct inhibitory effect on the neurons of the LC (Cedarbaum and Aghajanian, 1976) which appears to be mediated by presynaptic s-receptors. Electrophysiological data (Aghjanian et al., 1977) indicates that the neurons of the LC have collateral branches which synapse on themselves or other neurons of the LC and hence clonidine m a y be mimicking the normal autoinhibitory activity of the neurons. These observations suggest a simple mechanism to explain the physical dependence on clonidine. Clonidine would reduce the activity of the LC by acting on autoinhibitory s-receptors. The constant exposure of the inhibitory receptors to high concentration of clonidine may produce a subsensitivity of these receptors analogous to that seen in peripheral systems (Langer and LuchelliFortis, 1977). Abrupt cessation of clonidine therapy would therefore result in a failure of
311
the normal inhibition of the cells due to the subsensitivity; the neurons would fire at an excessive rate and TOH activity would increase. The firing rate of the neurons of the LC and the enzyme activity would return to basal levels as the normal c o m p l e m e n t of functional inhibitory receptors are restored. The time course of the increase of TOH in the LC is, indeed, very similar to the time course of the tachycardia seen upon clonidine withdrawal (Dix and Johnson, 1977). Consistent with this suggestion are the recent demonstrations that electrical stimulation of the LC in rats results in an increase in norepinephrine metabolites in blood (Crawley et al., 1978). Direct electrical stimulation of the area of the LC in the rhesus m o n k e y produces tachycardia and an increase in blood pressure (M. Raichle, personal communication) and tachycardia in the rat ( K a w a m u r a et al., 1978). Hence, the increased neuronal activity in the LC, suggested by our results, might be expected to produce s y m p t o m s characteristic of clonidine withdrawal. Our data and this working hypothesis d o not, of course, preclude the possibility that similar changes occur in other noradrenergic cell groups which may be important in the withdrawal syndrome. In any event, our results demonstrate that readily quantifiable peripheral and central biochemical parameters may be monitored in conjunction with the physiological changes associated with this phenomenon. The clonidine withdrawal syndrome may prove valuable as a general model of drug dependence. Acknowledgements We thank Richard Macia and Sarah Oldham for their technical assistance. This work was supported by The National Foundation, March of Dimes (Basil O'Connor Grant), by NIH grant HL-20604 and by NIH training grant 5 T32 GM07200. References Aghajanian, G.K., J.M. Cedarbaum and R.Y. Wang, 1977, Evidence for norepinephrine-mediated colla-
312 teral inhibition of locus coeruleus neurons, Brain Res. 136, 570. Cedarbaum, J.M. and G.K. Aghajanian, 1976, Noradrenergic neurons of the locus coeruleus: inhibition by epinephrine and actNation by the s-antagonist piperoxane, Brain Res. 112, 413. Crawley, J.N., S.E. Hattox, J.W. Maas and R.H. Roth, 1978, 3-Methoxy-4-hydroxyphenylethyleneglycol increases in plasma after stimulation of the locus coeruleus, Brain Res. 1 4 1 , 3 8 0 . Dix, R.K. and E.M. Johnson, 1977, Withdrawal syndrome upon cessation of chronic elonidine treatment in rats, European J. Pharmacol. 4 4 , 1 5 3 . Haeusler, G., 1974, Further similarities between the action of clonidine and a central activation of the depressor baroreceptor reflex, Naunyn-Schmiedeb. Arch. Pharmacol. 285, 1. H6kfelt, B., H. Hedeland and J.E. Dymling, 1970, Studies in catecholamines, renin, and aldosterone following Cata presan (2-(2,6-dichlorophenylamine)-2-imidazoline hydrochloride) in hypertensive patients, European J. Pharmacol. 10,389. Kawamura, H., D.G. Gunn and E.D. Frolich, 1978, Cardiovascular alteration by nucleus locus coeruleus in spontaneously hypertensive rat, Brain Res. 140,137.
W.H. KANE, E.M. JOHNSON, JR. Kobinger, W. and A. Walland, 1967, Investigations into the mechanism of the hypotensive effect of 2-(2,6-dichlorophenylamino)-2-imidazoline HC1, European J. Pharmacol. 2 , 1 5 5 . Langer, S.Z. and M.A. Luchelli-Fortis, 1977, Subsensitivity of the presynaptic ~-adrenoceptors after short-term surgical denervation of the cat nictitating membrane, J. Pharmacol. Exptt. Therap. 202,610. Lewander, T., T.H. Joh and D.J. Reis, 1977, Tyrosine hydroxylase: delayed activation in central noradrenergic neurons and induction in adrenal medulla elicited by stimulation of central cholinergic receptors, J. Pharmacol. Exptl. Therap. 200~ 523. Phillipson, O.T. and M. Sandler, 1975, The effect of hydrocortisone and adrenocorticotrophic hormone on monoamine oxidase and tyrosine hydroxylase in explant cultures of embryonic chick sympathetic ganglion, Brain Res. 9 0 , 2 8 3 . Ross, R.A. and D.J.Reis, 1974, Effect of lesions of locus coeruleus on regional distribution of dopamine-~-hydroxylase activity in rat brain, Brain Res. 7 3 , 1 6 1 .