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Chronic clonidine treatment produces desensitisation of post but not presynaptic α2-adrenoceptors
European Journal of Pharmacology, 138 (1987) 95-100
95
Elsevier EJP 00780
Chronic clonidine treatment produces desensitisation of postbut not presynaptic a2-adrenoceptors John P.M. Finberg * and I.J. Kopin National Institute of Neurological and Communicatioe Disorders and Stroke, National Institutes of Health, Bethesda, MD 20205, U.S.A.
Received 2 March 1987, accepted 24 March 1987
Rats were treated chronically with clonidine by osmotic minipump (500 #g/kg daily for 14 days). One day after the end of the infusion period, the animals were pithed, and a2-presynaptic and -postsynaptic adrenoceptor responses evaluated using inhibition of cardioaccelerator response to sympathetic stimulation by guanabenz, and pressor response to guanabenz respectively. Chronic clonidine treatment had no effect on the presynaptic effect of guanabenz, but the postsynaptic response to this agonist was decreased. In a separate group of pithed rats treated chronically with clonidine, there was no alteration in the increment in plasma noradrenaline concentration produced by sympathetic stimulation, and no alteration in the yohimbine-induced enhancement of plasma noradrenaline response to sympathetic stimulation. These data argue against desensitisation of peripheral presynaptic a2-adrenoceptors by chronic administration of a2-adrenoceptor agonist. Sympathetic hyperactivity following withdrawal of chronic clonidine treatment may be mediated by down-regulation of postsynaptic a2-adrenoceptors in the CNS. Clonidine; Pithed rats; Guanabenz; Plasma noradrenaline; a2-Adrenoceptors
1. Introduction
One proposed mechanism of antidepressant drug action is that increased synaptic levels of neurotransmitter noradrenaline (following M A O inhibition or neuronal amine uptake blockade) cause down-regulation of presynaptic inhibitory a2-adrenoceptors. Reduced inhibitory presynaptic control would result in increased neurotransmitter release and antidepressant effect (Cohen et al., 1980). Attempts to test this hypothesis by receptor binding techniques have produced contradictory results, some workers finding reduced (Cohen et al., 1982) and others increased (Asakura et al., 1982) binding of a2-adrenoceptor ligands in the
* To whom all correspondence should be addressed at present address: Rappaport Institute for Medical Research, Technion, Haifa, P.O. Box 9697, Israel.
CNS. However, current receptor binding techniques cannot differentiate between presynaptic and postsynaptic a2-adrenoceptor sites. Reduced responsiveness to clonidine on chronic antidepressant drug administration has been demonstrated by a variety of behavioural and electrophysiological techniques (Svensson and Usdin, 1978; Spyraki and Fibiger, 1980), but similarly, these techniques cannot distinguish between a true presynaptic effect of clonidine (i.e. at receptors situated on terminal varicosities) and a postsynaptic effect (at receptors situated on cell bodies or dendrites). An essential condition for the a2-adrenoceptor down-regulation theory is that presynaptic a 2adrenoceptors down-regulate in the presence of chronically elevated agonist concentrations. We decided, therefore, to test the ability of chronic clonidine treatment to alter peripheral a2-presynaptic adrenoceptor function, since in the sym-
0014-2999/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
96 pathetic nervous system the possibility exists to differentiate between presynaptic and postsynaptic effects. Evidence has been produced showing desensitisation of peripheral a2-adrenoceptors on chronic antidepressant administration, similarly to that postulated to occur in the CNS (Crews and Smith, 1978; Finberg and Tal, 1985). Sudden withdrawal of long-term clonidine treatment in human subjects may result in a syndrome typical of sympathetic hyperactivity, including tachycardia, lability of blood pressure, nervousness, sweating and increased catecholamine secretion (H~Skfelt et al., 1970; Hansson et al., 1973). This syndrome has been reproduced in rats by chronic (2-3 weeks) treatment with clonidine (Dix and Johnson, 1977; Oates et al., 1978; 1979). Recently, Thoolen et al. (1982) infused clonidine chronically in rats using osmotic minipumps. Sympathetic overactivity, as evidence by tachycardia and upswings of arterial pressure, occurred over the 24 h period following surgical extirpation of the pumps. This increased sympathetic discharge is believed to be of central origin, since it can be blocked by ganglion blockade (Oates et al., 1979) or centrally administered a2-adrenoceptor agonist (Jonkman et al., 1985). We now describe the activity of peripheral presynaptic and postsynaptic a2-adrenoceptors in rats treated chronically with clonidine. Stimulation of the whole sympathetic nervous system in the pithed rat caused increase in plasma noradrenaline level, which can be enhanced by az-adrenoceptor blockade, showing that it is under presynaptic inhibitory control (Yamaguchi and Kopin, 1979). Down-regulation of presynaptic receptors should, therefore, increase plasma noradrenaline levels towards those seen in the presence of a2-adrenoceptor blockade. In the present study, a modification of this technique was used, in which only the pressor region of the spinal cord was stimulated, and desmethylimipramine (DMI) was administered to increase neurotransmitter overflow and a2-adrenoceptor activation (Zukowska-Grojec et al., 1983). Inhibition of cardioaccelerator response to sympathetic stimulation by guanabenz was used as a physiological indicator of a2-presynaptic adrenoceptor function, and guanabenz pressor response was
measured as an index of postsynaptic a2-adrenoceptor stimulation.
2. Materials and methods 2.1. Chronic clonidine treatment
Osmotic minipumps (Alzet ®, 2ML2) containing either saline or clonidine (in a concentration calculated to release 500 /Lg/kg daily) were implanted subcutaneously under halothane anesthesia in male Sprague-Dawley rats (body weight 270-340 g). Fourteen days later, the pumps were removed under brief halothane anesthesia, and pithing carried out 16 h later. Animals were allowed access to standard rat pellets and water ad libitum until pithing. Pithing experiments were carried out in pairs; one control and one clonidine-treated rats. 2.2. Pithed rat preparations
Rats were anesthetised with halothane and pithed via the left orbit. Artificial respiration was immediately commenced at 1 ml/100 g body weight at a rate of 60 per min. The stainless steel pithing rod was connected to a Grass square-wave stimulator. For stimulation of noradrenaline release, the pithing rod was coated with high impedance shellac except for a 1 cm length corresponding to vertebrate T7 to T9 (see Gillespie et al., 1970). For cardioaccelerator nerve stimulation, the exposed length of the rod corresponded to C6 to T1. A second stainless steel rod under the skin of the back was used as the opposite electrode. Cannulas were inserted into both carotid arteries, for recording of arterial pressure and blood sampling, and a jugular vein, for intravenous (i.v.) injection of drugs. Heart rate was recorded by cardiotachometer triggered from the arterial pulse. Body temperature was maintained at 37 ° C using a thermostatically controlled heating system. All animals were injected with gallamine (20 m g / k g i.v.) to suppress skeletal muscle contractions during stimulation.
97 2.3. Cardioaccelerator stimulation Following an equilibration period of 40 min after pithing, single stimuli (50 V, 0.5 ms) were applied at intervals of 1.5 rain. When 4-5 consistent control responses (increase in heart rate of 15-25 beats per min above control) had been obtained, guanabenz was injected starting at 2 /~g/kg i.v., and cardioaccelerator stimulation repeated 5 min later. When blood pressure had returned to control levels, the next dose of guanabenz was injected, and heart rate and blood pressure measurements repeated as before. Following the highest dose of guanabenz, pressor responses to angiotensin II (0.025-0.25 n m o l / k g i.v.) were determined. 2.4. Pressor stimulation After equilibration as above, rats were injected with D M I (1 m g / k g i.v.) and left to equilibrate for a further 30 min. An arterial blood sample (0.25 ml) was then taken, and pressor stimulation commenced (50 V, 1 ms) at 1 Hz. Four minutes later, a second blood sample was taken, and stimulation frequency increased to 3 Hz for a further 4 min period with blood sample at end. The stimulator was then switched off, and yohimbine (1 m g / k g i.v.) injected. Thirty minutes later, blood sampling and stimulation was repeated as above. All blood samples Were replaced immediately with the same volume of heparinised rat donor blood.
during electrical stimulation was expressed as increment over corresponding control values. Statistical significance was calculated using Student's t-test (two-tailed).
3. Results Clonidine-treated rats did not gain weight over the treatment period, as opposed to control rats, which gained on the average 50 g. Following removal of minipumps, clonidine-treated rats showed irritability, pilo-erection and diarrhoea. Resting blood pressure following pithing was slightly lower in clonidine-treated than in control
~1HR %Control 80- L
50-- ~ 20--
~"ABP50,,c
2.5. Plasma catecholamine assay Blood samples were kept on ice, centrifuged rapidly and plasma noradrenaline concentration determined by radio-enzymatic assay as described by Yamaguchi and Kopin (1979).
/
2c
L
I
2
5
Guanabenz
2.6. Treatment of data Pressor responses were measured as peak increase in diastolic or mean blood pressure above control. Cardioaccelerator responses were expressed as percentage of control responses (before guanabenz). Plasma noradrenaline concentration
10
(pg/kg)
Fig. 1. Cardiovascular activity of guanabenz in pithed rats treated chronically with clonidine. Upper section: inhibition of cardioaccelerator response to spinal cord stimulation (C6 to T1); AHR=increase in heart rate to single pulse. Lower section: increase in mean arterial pressure (AMABP, mm Hg). (e), (A): controls (chronic saline); (©), (zx): chronic clonidine (500 /xg/kg daily for 14 days). Means+S.E.M. of 6 experiments. * P < 0.05 for difference from control.
98 rats (46.5 + 1.52 m m Hg versus 54.5 + 3.2 m m Hg, mean + S.E.M., P < 0.05), whereas heart rate was similar (350 + 8.7 versus 359 + 11 beats per min). Inhibition of cardioacceleration by guanabenz was identical in clonidine-treated and control rats (see fig. 1), whereas the extent of the pressor response to guanabenz was less in clonidine-treated rats. In order to compare the dose-response curves for guanabenz pressor effect, the data shown in fig. 1 were first linearized by plotting log dose versus reciprocal of pressor response. The resuiting straight lines (P < 0.01 for correlation coefficient) showed a non-significant difference (P > 0.05) for slope but a significant difference (P < 0.05) for intercept on the y-axis. Comparison of the individual data points showed a significant difference (P < 0.05) at the 2 /~g/kg dose, but non-significant difference (P = 0.1-0.2) at the other two dose levels. Pressor responses to angiotensin II (0.1 n m o l / k g ) were identical in clonidine-treated and control rats ( + 82.2 + 5.7 and + 86.6 _+ 4.9 m m Hg increase in mean blood pressure respectively). Resting plasma noradrenaline concentration following pithing was similar in both groups of APlllsma [NA] nM
CHRONIC
SALINE
CHRONIC
CLONIDINE
60
50
/
/
40
/
/
/
/
30
/
/
/
Z
20
10
k
I
I
J
1
3
1
3
HZ
Fig. 2. The effect of chronic clonidine treatment on the increase in plasma noradrenaline concentration (Aplasma NA) caused by spinal cord stimulation (T7 to T9). (O), (A) before yohimbine. (O), (zx) following yohimbine (1 m g / k g i.v.). M e a n s _ S.E.M. of 5 experiments at each chronic treatment.
rats (clonidine 3.7 _+ 0.45 nM, control 3.2 _+ 0.40 nM). The increment in plasma noradrenaline levels following stimulation at 1 and 3 Hz was also similar in both groups of animals (fig. 2) as were the corresponding changes following yohimbine. Yohimbine caused an approximately 3-fold increase in plasma noradrenaline levels during nerve stimulation over the corresponding values in its absence.
4. Discussion
A number of studies of chronic ~lonidine administration in rats have shown down regulation of effects mediated by central a2-adrenoceptors. Maura et al. (1985) showed that following 12 days administration of clonidine (100 btg/day) there was a reduction in the potency of noradrenaline to inhibit KCl-induced release of [3H]noradrenaline and [3H]5HT from brain synaptosomes. Rochette et al. (1982) found that the ability of clonidine to reduce noradrenaline turnover in brainstem and hypothalamus was reduced by daily injections of clonidine (0.2 m g / k g ) for 2 weeks. Engberg et al. (1982) measured locus coeruleus activity and found an increased rate of firing on withdrawal from chronic clonidine (100-500 /zg/kg daily for 2 weeks) with a reduced sensitivity of the cells to inhibition by systemic or iontophoretically applied clonidine. On the other hand, other workers did not see any change in various biochemical effects of cionidine following chronic treatment. Svensson and Str~Smbom (1977) found that the reduction in brain catecholamine synthesis in the mouse produced by clonidine persisted during chronic treatment, and Tang et al. (1978) similarly reported a consistent decrease in rat brain total 3methoxy-4-hydroxyphenylglycol by clonidine during chronic treatment. In the latter case, however, whole brain tissue was used. Villeneuve et al. (1985) found no change in the ability of clonidine to inhibit lipolytic activity of isolated fat cells taken from golden hamsters treated chronically with clonidine. These findings are in agreement with the clinical observation (Lund-Johansen, 1974) that the hemodynamic effects of clonidine
99 in h u m a n patients are similar after a single dose and one year's treatment with the drug. Desensitisation of az-adrenoceptors following brief exposure to high concentrations of agonist has been reported in isolated tissue preparations. Langer and D u b o c o v i c h (1977) observed subsensitivity of presynaptic a2-adrenoceptors in the spleen following perfusion of the organ with noradrenaline, and Ball et al. (1982) showed a similar effect in mouse vas deferens. Acute exposure to clonidine decreased presynaptic a-adrenoceptor sensitivity in isolated guinea-pig ileum but not in isolated rat vas deferens (Ilhan and long, 1985). I n c u b a t i o n of isolated h u m a n platelets with clonidine, or administration of clonidine to h u m a n p a t i e n t s for 7 days, c a u s e d r e d u c t i o n in [3H]yohimbine binding sites (Brodde et al., 1982). In the present study, no evidence was seen for down-regulation of presynaptic a2-adrenoceptors in the rat following chronic clonidine administration, using both heart-rate measurements, and biochemical determination of noradrenaline release. This observation is in contrast to that of Majewski and Story (1979) who observed subsensitivity of presynaptic a-adrenoceptors in isolated rat atria. This difference could be explained b y the use of an in vivo model in the present study. The fact that postsynaptic a2-adrenoce ptors did show a reduced sensitivity (as shown b y reduced pressor response to a low dose of guanabenz) appears to point to a difference between presynaptic and postsynaptic receptors in their response to sustained high agonist concentrations. Since clonidine is believed to exert its antihypertensive effects in the C N S by action on a postsynaptic receptor (Kobinger and Pichler, 1974), it would appear reasonable to think that, following chronic administration, the central postsynaptic receptor would be the one showing subsensitivity, in keeping with the present findings. It should be emphasised that the central az-adrenoceptormediating reduced noradrenafine turnover m a y be a postsynaptic receptor, if it is situated on dendrons or cell bodies of neurones secreting noradrenaline. The lack of ability of presynaptic a2-adrenoceptors to down-regulate on chronic exposure to clonidine casts some d o u b t on the hypotheses of
antidepressant action based on a2-adrenoceptor d o w n regulation. Receptors m a y adapt differently, however, to sustained presence of imidazoline and phenylethylamine-type agonists (Ruffolo et al., 1977). Thus this hypothesis must be tested further using chronic treatment with phenylethylamine agonists. It is interesting that Campbell and McK e r n a n (1986) recently observed that chronic treatment of rats with desipramine and clorgyline did not caused any alteration in the ability of clonidine to reduce [3 H]noradrenaline release from cortical synaptosomes, and concluded that the observed reduction in [3H]clonidine binding indicated a reduction in postsynaptic but not presynaptic a2-adrenoceptor number.
References Asakura, M., T. Tsukamoto and K. Hasegawa, 1982, Modulation of rat brain a 2 and fl adrenergic receptor sensitivity following long term treatment with antidepressants, Brain Res. 235, 192. Ball, N., J.L. Danks, S. Dorudi and P.A. Nasmyth, 1982, Desensitization by noradrenaline of responses to stimulation of pre- and postsynaptic adrenoceptors, Br. J. Pharmacol. 76, 201. Brodde, O.E., M. Anlauf, N. Graben and K.D. Bock, 1982, In vitro and in vivo down-regulation of human platelet a 2adrenoceptors by clonidine, European J. Clin. Pharmacol. 23, 403. Campbell, I.C. and R.M. McKernan, 1986, Clorgyline and desipramine alter the sensitivity of [3H]-noradrenaline release to calcium but not to clonidine, Brain Res. 372, 253. Cohen, R.M., I.C. Campbell, M.R. Cohen, T. Torda, D. Pickar, L.J. Siever and D.L. Murphy, 1980, Presynaptic noradrenergic regulation during depression and antidepressant drug treatment, J. Psychiat. Res. 3, 93. Cohen, R.M., I.C. Campbell, M. Dauphin, J.F. Tallman and D.L. Murphy, 1982, Changes in a and fl receptor densities in rat brain as a result of treatment with monoamine oxidase inhibiting antidepressants, Neuropharmacology 21, 293. Crews, F.T. and C.B. Smith, 1978, Presynaptic alpha-receptor subsensitivity after long term antidepressant treatment, Science 202, 322. Dix, R.K. and E.M. Johnson, 1977, Withdrawal syndrome upon cessation of chronic clonidine treatment in rats, European J. Pharmacol. 44, 153. Engberg, G., M. Elam and T.H. Svensson, 1982, Clonidine withdrawal: activation of brain noradrenergic neurons with specifically reduced a2-receptor sensitivity, Life Sci. 30, 235. Finberg, J.P.M. and A. Tal, 1985, Reduced peripheral presyn-
100 aptic adrenoceptor sensitivity following chronic antidepressant treatment in rats, Br. J. Pharmacol. 84, 609. Gillespie, J.S., A. Maclaren and D. Pollock, 1970, A method of stimulating different segments of the autonomic outflow from the spinal column to various organs in the pithed cat and rat, Br. J. Pharmacol. 40, 257. Hansson, L., S.N. Hunyor, S. Julius and S.W. Hoobler, 1973, Blood pressure crisis following withdrawal of clonidine (Catapres, Catapresan) with special reference to arterial and urinary catecholamine levels and suggestions for acute management, Am. Heart J. 85, 605. Ht~kfelt, B., H. Hedeland and J. Dymling, 1970, Studies on catecholamines, renin and aldosterone following catapresan (2-(2,5-dicklorophenylamine)-2-imidazoline hydrochloride) in hypertensive patients, European J. Pharmacol. 10, 389. Ilhan, M. and J.P. Long, 1985, Clonidine-induced desensitization differentiates presynaptic alpha-2 adrenoceptors of the guinea-pig ileum and rat vas deferens, Arch. Int. Pharmacodyn. Ther. 275, 199. Jonkman, F., A.M., P.W. Man, M.J.M.C. Thoolen and P.A. Van Zwieten, 1985, Location of the mechanism of the clonidine withdrawal tachycardia in rats, J. Pharm. Pharmacol. 37, 580. Kobinger, W. and L. Pickler, 1974, Evidence for direct aadrenoceptor stimulation of effector neurons in cardiovascular centers by clonidine, European J. Pharmacol. 22, 151. Langer, S.Z. and M.L. Dubocovich, 1977, Subsensitivity of presynaptic a-adrenoceptors after exposure to noradrenaline, European J. Pharmacol. 41, 87. Lund-Johansen, P., 1974, Hemodynamic changes at rest and during exercise in long-term clonidine therapy of essential hypertension, Acta Med. Scand. 195, 111. Majewski, H. and D.F. Story, 1979, Effects of withdrawal of clonidine treatment on pre-junctional a-adrenoceptor sensitivity in rat atria, Clin. Exp. Pharmacol. Physiol. 6, 205. Maura, G., G. Bonanno and M. Raiteri, 1985, Chronic clonidine induces functional down-regulation of presynaptic aadrenoceptors regulating [3H]noradrenaline and [3H]5-hydroxytryptamine release in the rat brain, European J. Pharmacol. 112, 105. Oates, H.F., L.M. Stoker, J.C. Monaghan and G.S. Stokes, 1978, Withdrawal of clonidine-effects of varying dosage or duration of treatment on subsequent blood pressure and heart rate repsonses, J. Pharmacol. Exp. Ther. 206, 268.
Oates, H.F., L.M. Stoker and G.S. Stokes, 1979, Studies in the rat on the haemodynamic overshoot response to withdrawal of guanfacine or clonidine treatment, Clin. Exp. Pharmacol. Physiol. 6, 61. Rochette, L., A.-M. Bralet and J. Bralet, 1982, Effect of chronic clonidine treatment on the turnover of noradrenaline and dopamine in various regions of the rat brain, Naunyn-Schmiedeb. Arch. Pharmacol. 319, 40. Ruffolo, R.R., B.S. Turowski and P.N. Patil, 1977, Lack of cross-desensitization between structurally dissimilar aadrenoceptor agonists, J. Pharm. Pharmacol. 29, 378. Spyraki, C. and H.C. Fibiger, 1980, Functional evidence for subsensitivity of noradrenergic a2-receptors after chronic desipramine treatment, Life Sci. 27, 1863. Svensson, T.H. and U. Str~Smbom, 1977, Discontinuation of chronic clonidine treatment: evidence for facilitated brain noradrenergic transmission, Naunyn-Schmiedeb. Arch. Pharmacol. 299, 83. Svensson, J.H. and R. Usdin, 1978, Feedback inhibition of brain noradrenaline neurons by tricyclic antidepressants: a-receptor mediation, Science 202, 1089. Tang, S.W., D.M. Helmeste and H.C. Stancer, 1978, The effect of acute and chronic desipramine and amitriptyline treatment on rat brain total 3-methoxy-4-hydroxyphenylglycol, Naunyn-Schmiedeb. Arch. Pharmacol. 305, 207. Thoolen, M.J.M.C., P.B.M.W.M. Timmermans and P.A. Van Zwieten, 1982, Guanfacine and clonidine: antihypertensive and withdrawal characteristics after continuous infusion and its interruption in the spontaneously hypertensive and normotensive rat, Naunyn-Schmiedeb. Arch. Pharmacol. 319, 82. Villeneuve, A., C. Carpene, M. Berlan and M. Lafontan, 1985, Lack of desensitisation of alpha-2 mediated inhibition of lipolysis in fat cells after acute and chronic treatment with clonidine, J. Pharmacol. Exp. Ther. 233, 433. Yamaguchi, I. and I.J. Kopin, 1979, Plasma catecholamine and blood pressure responses to sympathetic stimulation in pithed rats, Am. J. Physiol. 237, H305. Zukowska-Grojec, Z., M.A. Bayorh and 1.J. Kopin, 1983, Effect of desipramine on the effects of a-adrenoceptor inhibitors on pressor responses and release of norepinephrine into plasma of pithed rats, J. Cardiovasc. Pharmacol. 5, 297.
95
Elsevier EJP 00780
Chronic clonidine treatment produces desensitisation of postbut not presynaptic a2-adrenoceptors John P.M. Finberg * and I.J. Kopin National Institute of Neurological and Communicatioe Disorders and Stroke, National Institutes of Health, Bethesda, MD 20205, U.S.A.
Received 2 March 1987, accepted 24 March 1987
Rats were treated chronically with clonidine by osmotic minipump (500 #g/kg daily for 14 days). One day after the end of the infusion period, the animals were pithed, and a2-presynaptic and -postsynaptic adrenoceptor responses evaluated using inhibition of cardioaccelerator response to sympathetic stimulation by guanabenz, and pressor response to guanabenz respectively. Chronic clonidine treatment had no effect on the presynaptic effect of guanabenz, but the postsynaptic response to this agonist was decreased. In a separate group of pithed rats treated chronically with clonidine, there was no alteration in the increment in plasma noradrenaline concentration produced by sympathetic stimulation, and no alteration in the yohimbine-induced enhancement of plasma noradrenaline response to sympathetic stimulation. These data argue against desensitisation of peripheral presynaptic a2-adrenoceptors by chronic administration of a2-adrenoceptor agonist. Sympathetic hyperactivity following withdrawal of chronic clonidine treatment may be mediated by down-regulation of postsynaptic a2-adrenoceptors in the CNS. Clonidine; Pithed rats; Guanabenz; Plasma noradrenaline; a2-Adrenoceptors
1. Introduction
One proposed mechanism of antidepressant drug action is that increased synaptic levels of neurotransmitter noradrenaline (following M A O inhibition or neuronal amine uptake blockade) cause down-regulation of presynaptic inhibitory a2-adrenoceptors. Reduced inhibitory presynaptic control would result in increased neurotransmitter release and antidepressant effect (Cohen et al., 1980). Attempts to test this hypothesis by receptor binding techniques have produced contradictory results, some workers finding reduced (Cohen et al., 1982) and others increased (Asakura et al., 1982) binding of a2-adrenoceptor ligands in the
* To whom all correspondence should be addressed at present address: Rappaport Institute for Medical Research, Technion, Haifa, P.O. Box 9697, Israel.
CNS. However, current receptor binding techniques cannot differentiate between presynaptic and postsynaptic a2-adrenoceptor sites. Reduced responsiveness to clonidine on chronic antidepressant drug administration has been demonstrated by a variety of behavioural and electrophysiological techniques (Svensson and Usdin, 1978; Spyraki and Fibiger, 1980), but similarly, these techniques cannot distinguish between a true presynaptic effect of clonidine (i.e. at receptors situated on terminal varicosities) and a postsynaptic effect (at receptors situated on cell bodies or dendrites). An essential condition for the a2-adrenoceptor down-regulation theory is that presynaptic a 2adrenoceptors down-regulate in the presence of chronically elevated agonist concentrations. We decided, therefore, to test the ability of chronic clonidine treatment to alter peripheral a2-presynaptic adrenoceptor function, since in the sym-
0014-2999/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
96 pathetic nervous system the possibility exists to differentiate between presynaptic and postsynaptic effects. Evidence has been produced showing desensitisation of peripheral a2-adrenoceptors on chronic antidepressant administration, similarly to that postulated to occur in the CNS (Crews and Smith, 1978; Finberg and Tal, 1985). Sudden withdrawal of long-term clonidine treatment in human subjects may result in a syndrome typical of sympathetic hyperactivity, including tachycardia, lability of blood pressure, nervousness, sweating and increased catecholamine secretion (H~Skfelt et al., 1970; Hansson et al., 1973). This syndrome has been reproduced in rats by chronic (2-3 weeks) treatment with clonidine (Dix and Johnson, 1977; Oates et al., 1978; 1979). Recently, Thoolen et al. (1982) infused clonidine chronically in rats using osmotic minipumps. Sympathetic overactivity, as evidence by tachycardia and upswings of arterial pressure, occurred over the 24 h period following surgical extirpation of the pumps. This increased sympathetic discharge is believed to be of central origin, since it can be blocked by ganglion blockade (Oates et al., 1979) or centrally administered a2-adrenoceptor agonist (Jonkman et al., 1985). We now describe the activity of peripheral presynaptic and postsynaptic a2-adrenoceptors in rats treated chronically with clonidine. Stimulation of the whole sympathetic nervous system in the pithed rat caused increase in plasma noradrenaline level, which can be enhanced by az-adrenoceptor blockade, showing that it is under presynaptic inhibitory control (Yamaguchi and Kopin, 1979). Down-regulation of presynaptic receptors should, therefore, increase plasma noradrenaline levels towards those seen in the presence of a2-adrenoceptor blockade. In the present study, a modification of this technique was used, in which only the pressor region of the spinal cord was stimulated, and desmethylimipramine (DMI) was administered to increase neurotransmitter overflow and a2-adrenoceptor activation (Zukowska-Grojec et al., 1983). Inhibition of cardioaccelerator response to sympathetic stimulation by guanabenz was used as a physiological indicator of a2-presynaptic adrenoceptor function, and guanabenz pressor response was
measured as an index of postsynaptic a2-adrenoceptor stimulation.
2. Materials and methods 2.1. Chronic clonidine treatment
Osmotic minipumps (Alzet ®, 2ML2) containing either saline or clonidine (in a concentration calculated to release 500 /Lg/kg daily) were implanted subcutaneously under halothane anesthesia in male Sprague-Dawley rats (body weight 270-340 g). Fourteen days later, the pumps were removed under brief halothane anesthesia, and pithing carried out 16 h later. Animals were allowed access to standard rat pellets and water ad libitum until pithing. Pithing experiments were carried out in pairs; one control and one clonidine-treated rats. 2.2. Pithed rat preparations
Rats were anesthetised with halothane and pithed via the left orbit. Artificial respiration was immediately commenced at 1 ml/100 g body weight at a rate of 60 per min. The stainless steel pithing rod was connected to a Grass square-wave stimulator. For stimulation of noradrenaline release, the pithing rod was coated with high impedance shellac except for a 1 cm length corresponding to vertebrate T7 to T9 (see Gillespie et al., 1970). For cardioaccelerator nerve stimulation, the exposed length of the rod corresponded to C6 to T1. A second stainless steel rod under the skin of the back was used as the opposite electrode. Cannulas were inserted into both carotid arteries, for recording of arterial pressure and blood sampling, and a jugular vein, for intravenous (i.v.) injection of drugs. Heart rate was recorded by cardiotachometer triggered from the arterial pulse. Body temperature was maintained at 37 ° C using a thermostatically controlled heating system. All animals were injected with gallamine (20 m g / k g i.v.) to suppress skeletal muscle contractions during stimulation.
97 2.3. Cardioaccelerator stimulation Following an equilibration period of 40 min after pithing, single stimuli (50 V, 0.5 ms) were applied at intervals of 1.5 rain. When 4-5 consistent control responses (increase in heart rate of 15-25 beats per min above control) had been obtained, guanabenz was injected starting at 2 /~g/kg i.v., and cardioaccelerator stimulation repeated 5 min later. When blood pressure had returned to control levels, the next dose of guanabenz was injected, and heart rate and blood pressure measurements repeated as before. Following the highest dose of guanabenz, pressor responses to angiotensin II (0.025-0.25 n m o l / k g i.v.) were determined. 2.4. Pressor stimulation After equilibration as above, rats were injected with D M I (1 m g / k g i.v.) and left to equilibrate for a further 30 min. An arterial blood sample (0.25 ml) was then taken, and pressor stimulation commenced (50 V, 1 ms) at 1 Hz. Four minutes later, a second blood sample was taken, and stimulation frequency increased to 3 Hz for a further 4 min period with blood sample at end. The stimulator was then switched off, and yohimbine (1 m g / k g i.v.) injected. Thirty minutes later, blood sampling and stimulation was repeated as above. All blood samples Were replaced immediately with the same volume of heparinised rat donor blood.
during electrical stimulation was expressed as increment over corresponding control values. Statistical significance was calculated using Student's t-test (two-tailed).
3. Results Clonidine-treated rats did not gain weight over the treatment period, as opposed to control rats, which gained on the average 50 g. Following removal of minipumps, clonidine-treated rats showed irritability, pilo-erection and diarrhoea. Resting blood pressure following pithing was slightly lower in clonidine-treated than in control
~1HR %Control 80- L
50-- ~ 20--
~"ABP50,,c
2.5. Plasma catecholamine assay Blood samples were kept on ice, centrifuged rapidly and plasma noradrenaline concentration determined by radio-enzymatic assay as described by Yamaguchi and Kopin (1979).
/
2c
L
I
2
5
Guanabenz
2.6. Treatment of data Pressor responses were measured as peak increase in diastolic or mean blood pressure above control. Cardioaccelerator responses were expressed as percentage of control responses (before guanabenz). Plasma noradrenaline concentration
10
(pg/kg)
Fig. 1. Cardiovascular activity of guanabenz in pithed rats treated chronically with clonidine. Upper section: inhibition of cardioaccelerator response to spinal cord stimulation (C6 to T1); AHR=increase in heart rate to single pulse. Lower section: increase in mean arterial pressure (AMABP, mm Hg). (e), (A): controls (chronic saline); (©), (zx): chronic clonidine (500 /xg/kg daily for 14 days). Means+S.E.M. of 6 experiments. * P < 0.05 for difference from control.
98 rats (46.5 + 1.52 m m Hg versus 54.5 + 3.2 m m Hg, mean + S.E.M., P < 0.05), whereas heart rate was similar (350 + 8.7 versus 359 + 11 beats per min). Inhibition of cardioacceleration by guanabenz was identical in clonidine-treated and control rats (see fig. 1), whereas the extent of the pressor response to guanabenz was less in clonidine-treated rats. In order to compare the dose-response curves for guanabenz pressor effect, the data shown in fig. 1 were first linearized by plotting log dose versus reciprocal of pressor response. The resuiting straight lines (P < 0.01 for correlation coefficient) showed a non-significant difference (P > 0.05) for slope but a significant difference (P < 0.05) for intercept on the y-axis. Comparison of the individual data points showed a significant difference (P < 0.05) at the 2 /~g/kg dose, but non-significant difference (P = 0.1-0.2) at the other two dose levels. Pressor responses to angiotensin II (0.1 n m o l / k g ) were identical in clonidine-treated and control rats ( + 82.2 + 5.7 and + 86.6 _+ 4.9 m m Hg increase in mean blood pressure respectively). Resting plasma noradrenaline concentration following pithing was similar in both groups of APlllsma [NA] nM
CHRONIC
SALINE
CHRONIC
CLONIDINE
60
50
/
/
40
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/
30
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Fig. 2. The effect of chronic clonidine treatment on the increase in plasma noradrenaline concentration (Aplasma NA) caused by spinal cord stimulation (T7 to T9). (O), (A) before yohimbine. (O), (zx) following yohimbine (1 m g / k g i.v.). M e a n s _ S.E.M. of 5 experiments at each chronic treatment.
rats (clonidine 3.7 _+ 0.45 nM, control 3.2 _+ 0.40 nM). The increment in plasma noradrenaline levels following stimulation at 1 and 3 Hz was also similar in both groups of animals (fig. 2) as were the corresponding changes following yohimbine. Yohimbine caused an approximately 3-fold increase in plasma noradrenaline levels during nerve stimulation over the corresponding values in its absence.
4. Discussion
A number of studies of chronic ~lonidine administration in rats have shown down regulation of effects mediated by central a2-adrenoceptors. Maura et al. (1985) showed that following 12 days administration of clonidine (100 btg/day) there was a reduction in the potency of noradrenaline to inhibit KCl-induced release of [3H]noradrenaline and [3H]5HT from brain synaptosomes. Rochette et al. (1982) found that the ability of clonidine to reduce noradrenaline turnover in brainstem and hypothalamus was reduced by daily injections of clonidine (0.2 m g / k g ) for 2 weeks. Engberg et al. (1982) measured locus coeruleus activity and found an increased rate of firing on withdrawal from chronic clonidine (100-500 /zg/kg daily for 2 weeks) with a reduced sensitivity of the cells to inhibition by systemic or iontophoretically applied clonidine. On the other hand, other workers did not see any change in various biochemical effects of cionidine following chronic treatment. Svensson and Str~Smbom (1977) found that the reduction in brain catecholamine synthesis in the mouse produced by clonidine persisted during chronic treatment, and Tang et al. (1978) similarly reported a consistent decrease in rat brain total 3methoxy-4-hydroxyphenylglycol by clonidine during chronic treatment. In the latter case, however, whole brain tissue was used. Villeneuve et al. (1985) found no change in the ability of clonidine to inhibit lipolytic activity of isolated fat cells taken from golden hamsters treated chronically with clonidine. These findings are in agreement with the clinical observation (Lund-Johansen, 1974) that the hemodynamic effects of clonidine
99 in h u m a n patients are similar after a single dose and one year's treatment with the drug. Desensitisation of az-adrenoceptors following brief exposure to high concentrations of agonist has been reported in isolated tissue preparations. Langer and D u b o c o v i c h (1977) observed subsensitivity of presynaptic a2-adrenoceptors in the spleen following perfusion of the organ with noradrenaline, and Ball et al. (1982) showed a similar effect in mouse vas deferens. Acute exposure to clonidine decreased presynaptic a-adrenoceptor sensitivity in isolated guinea-pig ileum but not in isolated rat vas deferens (Ilhan and long, 1985). I n c u b a t i o n of isolated h u m a n platelets with clonidine, or administration of clonidine to h u m a n p a t i e n t s for 7 days, c a u s e d r e d u c t i o n in [3H]yohimbine binding sites (Brodde et al., 1982). In the present study, no evidence was seen for down-regulation of presynaptic a2-adrenoceptors in the rat following chronic clonidine administration, using both heart-rate measurements, and biochemical determination of noradrenaline release. This observation is in contrast to that of Majewski and Story (1979) who observed subsensitivity of presynaptic a-adrenoceptors in isolated rat atria. This difference could be explained b y the use of an in vivo model in the present study. The fact that postsynaptic a2-adrenoce ptors did show a reduced sensitivity (as shown b y reduced pressor response to a low dose of guanabenz) appears to point to a difference between presynaptic and postsynaptic receptors in their response to sustained high agonist concentrations. Since clonidine is believed to exert its antihypertensive effects in the C N S by action on a postsynaptic receptor (Kobinger and Pichler, 1974), it would appear reasonable to think that, following chronic administration, the central postsynaptic receptor would be the one showing subsensitivity, in keeping with the present findings. It should be emphasised that the central az-adrenoceptormediating reduced noradrenafine turnover m a y be a postsynaptic receptor, if it is situated on dendrons or cell bodies of neurones secreting noradrenaline. The lack of ability of presynaptic a2-adrenoceptors to down-regulate on chronic exposure to clonidine casts some d o u b t on the hypotheses of
antidepressant action based on a2-adrenoceptor d o w n regulation. Receptors m a y adapt differently, however, to sustained presence of imidazoline and phenylethylamine-type agonists (Ruffolo et al., 1977). Thus this hypothesis must be tested further using chronic treatment with phenylethylamine agonists. It is interesting that Campbell and McK e r n a n (1986) recently observed that chronic treatment of rats with desipramine and clorgyline did not caused any alteration in the ability of clonidine to reduce [3 H]noradrenaline release from cortical synaptosomes, and concluded that the observed reduction in [3H]clonidine binding indicated a reduction in postsynaptic but not presynaptic a2-adrenoceptor number.
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