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Chronic clonidine treatment and its withdrawal: Effects on blood pressure and catecholamine synthesizing enzymes in brain-stem nuclei
European Journal of Pharmacology. 121 (1986) 97-106
97
Elsevier
C H R O N I C C L O N I D I N E T R E A T M E N T A N D ITS WITHDRAWAL: EFFECTS O N B L O O D P R E S S U R E A N D C A T E C H O L A M I N E S Y N T H E S I Z I N G E N Z Y M E S IN B R A I N - S T E M N U C L E I * JEFFREY ATKINSON *, LAURA LAMBAS-SENAS **, MYFANWY PARKER "**, NICOLE BOILLAT, PIERRETTE LUTHI, MIREILLE SONNAY, MICHELE SECCIA ** and BERNARD RENAUD ** lnstitut de Pharmacologie de I'Unwersitb, Rue du Bugnon 21, CH-1011 Lausanne-CHUV. Switzerland and * * Laboratoire de Neuropharmacologie, Facultb de Pharmacie, Unwersitb Claude Bernard, 8 Ave. Rockefeller. 69008 Lyon, France
Received 24 September 1985, accepted 5 November 1985
J. ATKINSON, L. LAMBAS-SENAS, M. PARKER, N. BOILLAT, P. LUTH1, M. SONNAY, M. SECCIA and B. RENAUD, Chronic clonidine treatment and its withdrawal: effects on blood pressure and catecholamine synthesizing enzymes in brain-stem nuclei, European J. Pharmacol. 121 (1986) 97-106. We have studied the effects of withdrawal from chronic clonidine treatment in the adult male spontaneously hypertensive rat (SHR). SHR received clonidine, 0.1 mg. k g - t . d a y - 1 i.v. for 10 days. Clonidine was delivered via osmotic minipumps. After 7 days of treatment there was a 16.5 + 2.5 mm Hg fall in mean arterial pressure. This was accompanied by a decrease in the dopamine-fl-hydroxylase and phenylethanolamine-N-methyl transferase activities of the A1/C1 region. Withdrawal from clonidine was characterized by tachycardia and an increase in mean arterial pressure and heart rate lability. Phenylethanolamine-N-methyl transferase of the A1/C1 region returned to normal ' It the dopamine-,8-hydroxylase activity remained diminished. The dopamine-~-hydroxylase activity of the A 2 / C 2 region was also diminished during withdrawal. We suggest that the blood pressure lowering effect of cionidine is accompanied by a decreased capacity to synthesize adrenaline in the A I / C 1 region where adrenaline could mediate a pressor effect. Increased blood pressure lability during withdrawal is accompanied by a restoration of synthesis of adrenaline in the A I / C 1 region. There is also a decrease in the capacity of synthesis of noradrenaline in the A 2 / C 2 region where adrenaline may mediate a vasodepressor effect. Central nervous system
Clonidine
SHR
Blood pressure
I. Introduction Prolonged t r e a t m e n t with clonidine leads to a fall in b l o o d pressure in the s p o n t a n e o u s l y hypertensive rat ( T h o o l e n et al., 1981a; 1982) and a b r u p t cessation of c l o n i d i n e t r e a t m e n t leads to cardiovascular h y p e r a c t i v i t y (Thoolen et al., 1981a; Parker a n d A t k i n s o n , 1982). The changes in cerebral n e u r o t r a n s m i s s i o n which a c c o m p a n y the * Some of the results reported here were pre~nted at the 15th Annual Meeting of the Swiss Societies for Experimental Biology in Fribourg, Switzerland, March 1983. * To whom all correspondence should be addressed. *** M. Parker was a scholar of the Roche Research Foundation," Basle, Switzerland and L. Lambas-Senas received a fellowship from Laboratoires Hoechst, Paris. France. 0014-2999/86/$03.50 ,~ 1986 Elsevier Science Publishers B.V.
Withdrawal
acute hypotensive effect of clonidine have been d e s c r i b e d in detail ( L a n g e r et al., 1980), but not those after p r o l o n g e d t r e a t m e n t or d u r i n g withdrawal. Several a u t h o r s have raised the interesting possibility that different c a t e c h o l a m i n e r g i c areas of the brain are affected in different ways by prolonged clonidine t r e a t m e n t a n d its w i t h d r a w a l ( N a k a m u r a et al., 1980; D i S t e f a n o et al., 1980). T h e r e has been, however no a t t e m p t to relate changes in c a t e c h o l a m i n e r g i c function in the A 1 / C I and A 2 / C 2 h i n d b r a i n areas directly involved in b l o o d pressure control to changes in b l o o d pressure. W e have c a r r i e d out such a study d u r i n g (1) the p e r i o d of h y p o t e n s i o n p r o d u c e d by p r o l o n g e d t r e a t m e n t with clonidine and (2) the
9~
cardiovascular hyperactivity produced by withdrawal. Recent evidence points to a possible role of hindbrain adrenaline in blood pressure control (Chalmers and West. 1983; Reis et al., 1984a,b: Howe, 1985). We have previously shown that chronic clonidine treatment increases hindbrain adrenaline content, possibly due to decreased release (Atkinson et al., 1985). Moreover, it has been suggested that adrenergic neurons of certain hindbrain areas such as C1 play a key role in the sympathetic vasopressor system (Ross et al., 1984). We now studied possible changes in adrenaline synthesis in the CI area and their link to the cardiovascular effects of clonidine treatment and to its withdrawal. Conversely, some hindbrain noradrenergic systems may play a vasodepressor role (Chalmers and West, 1983; Reis et al., 1984a,b). Decreased noradrenaline synthesis in the A 2 / C 2 area can induce blood pressure lability (Snyder et al., 1978: Talman et al., 1980). Increased blood pressure lability is one of the characteristics of clonidine withdrawal (present paper). Thus we studied possible changes in noradrenaline synthesis in A 2 / C 2 and their link to the cardiovascular effect of clonidine and its withdrawal. Therefore we have followed during and after clonidine treatment the changes occurring in the activities of the catecholamine synthesizing enzymes (tyrosine hydroxylase, dopamine-,8-hydroxylase and phenylethanolamine-N-methyl transferase) in A 1 / C 1 , A 2 / C 2 , locus coeruleus (A6) and substantia nigra (A9) catecholaminergic groups (Dahlstr/3m et al., 1964: H6kfelt ct al., 1974) and in the adrenal medulla. Enzyme activities were measured at two different times: (i) after 7 days of clonidine administration and (ii) during the withdrawal following cessation of 10 days' clonidine treatment.
2. Materials and methods
2.1. Animals and blood pressure recording Male spontaneously hypertensive rats ( S H R / NICo) 3-4 months of age were purchased from
If f a-Credo, SA, 69210 L'Arbreslc, France. Systolic arterial pressure (SAP. mm Hg) was measured by an indirect tail cuff method. Animals (10°~) with a SAP of less than 180 mm Hg were not used. The remainder were fitted with intra-aortic cannula under sodium pentobarbital anesthesia according to the technique of Weeks and Jones (1960). Blood pressure was recorded by connecting the cannula, via a liquid feedthrough swivel, to a strain-gauge transducer. Heart rate (HR) was obtained from a ratemeter driven by the pulse pressure. Blood pressure recording was started after 3 days' recovery and 8 days before minipump implantation. Mean arterial pressure (MAP) and HR were recorded for 15 rain after habituation for 3 h. The individual MAP and HR values of each cardiac cycle of the final 2 rain were averaged, l,ability was calculated as the standard error of this sample expressed as a percentage of the average. On the 10th day following minipump implantation, MAP and HR were recorded for 15 min every h, from 8 h before pump cessation to 20 h after. A final recording was made 30 h after pump cessation. Two groups of rats were used. In the first, blood pressure was recorded in 33 SHR (18 had received ::lonidinc, 0.1 r a g - k g 1. day ~ and 15 were controls). In the second group, 47 SHR without cannula were used for the biochemical studies. On the 7th day after implantation. 24 were killed: 15 had received chmidine (0.1 m g - k g ~• d a y ~) and 9 were controls. The remainder were killed at 16 h after pump cessation: 13 had previously received chmidine (0.1 mg. kg t . day i) and 10 were controls.
2.2. Implantation of osmotic mimpumps Minipump function (Aizet", model 2001, Scientific Marketing Associates. Ltd., London, UK. volume 0.22 ml, flow rate 0.001 ml. h i) was checked in vitro with [5~Cr]ethylene diaminc tetra acetic acid ([51Cr]EDTA). Pumps had a 4 + 1 h start-up time. At the end of the functional life of the pumps their flow rate fell by 75 + 5% in 24 h. Osmotic minipumps were implanted s.c. (under ether anesthesia), in all SttR and the pumps connected to a polyethylene cannula placed in the left external jugular vein. The pumps were filled with
99 [51Cr]EDTA (controls) or with clonidine (0.1 m g . kg 1. d a y - ~) dissolved in [51Cr]EDTA. In the second group pumps were removed from 24 SHR killed on the 7th day following implantation. The in vivo flow rate was determined from the difference in gamma radioactivity with starting counts. In the first group flow rate was checked by counting radioactivity in 0.2 ml plasma samples taken following each MAP session. After pump cessation [51CrIEDTA was no longer present in the circulation. 2.3. Brain punching
The rats were decapitated and the brains, hearts and adrenal medullas rapidly removed. The brains were sectioned in a vertical frontal plane at the anterior hypothalamic level. The caudal part was quick-frozen on dry ice together with the hearts and the adrenal medullas. Each caudal part of the brain was cut into 500/~m thick coronal sections. The A 1 / C I , A 2 / C 2 , locus coeruleus (A6, LC) and substantia nigra (A9) regions were removed with a hollow needle (0.9 mm i.d.) according to a modification (Renaud et al.. 1978) of the technique of Palkovits (1973).
substrate. The incubation time was 25 min. The results were expressed as pmol of dopa formed per h of incubation and per mg of protein for brain structures or per pair of adrenal medullas. DBH activity was determined in 10 ~tl of supernatant by a modification (Denoroy et al., 1981) of the method of Molinoff et al. (1971). The final concentrations of CuSO 4 were 4 × 10 -5 M for LC and A9, 5 × 1 0 5 M for A 1 / C 1 and A 2 / C 2 regions, and 5 x 10 6 M for adrenal medullas. The incubation time was 30 min. The results were expressed as nmol of octopamine formed per h of incubation and per mg of protein for the brain structures or per pair of adrenal medullas. P N M T activity was measured according to a modification (Denoroy et al., 1981) of the method of Saavedra et al. (1974) in 10 ~1 of supernatant. The incubation time was 30 min. The results were expressed as pmol of N-methylphenylethanolamine formed per h of incubation and per mg of protein for brain structures or per pair of adrenal medullas. 2.4. 3. Protein determination Total proteins were estimated in an aliquot of supernatant with the Folin phenol reagent method of Lowry et al. (1951) using bovine serum albumin (Sigma) as a standard.
2.4. Biochemical assays 2.5. Statistics 2.4.1. Tissue homogenization The tissues obtained were disrupted mechanically and by ultrasound (20 KHz, 40 W, 10 s) in 2 mM potassium phosphate buffer (pH 6.0) containing 0.2% Triton X-100 (v/v). The homogenization volume was 300 #1 for the brain nuclei. The adrenal medullas were homogenized in 200 vol. (diluted to 500 vol. for the dopamine-fl-hydroxylase (DBH) and phenylethanolamine-N-methyl transferase (PNMT) assays). The homogenates were centrifuged (2400 x g, 15 min) at 4°C and the supernatants used for the biochemical assays. 2.4.2. Enzymatic assays Tyrosine hydroxylase (TH) activity was determined in 50 ~1 of supernatant by a modification (Renaud et al., 1978) of the radiometric method of Nagatsu et al. (1964) using [3,5-3H]tyrosine as
Results are given as means + S.E.M. Comparison between means were made using the analysis of variance or paired and independent Student's t-tests. Probabilities based on these tests are given as: N S = P > 0 . 0 5 , * P < 0 . 0 5 , ** P <0.01, *** P < 0.001. 2.6. Substances
Clonidine hydrochloride (Catapresan '~) was a gift of Boehringer, Ltd., Ingelheim, FRG. [SICr] ethylene diamine tetra acetic acid was purchased from Amersham International, Amersham, Buckinghamshire, England. The specific activity was 1-2 mCi. m g - I and the radioactivity concentration was 0.1 mCi • m l - 1.
3. Results
I'~,,,on ~tts-ia I
mm~
180
3.1. Changes in body weight, food and water consurnption 170.
Following aortic cannulation, the body weight fell by 5 + 2% by the 4th day, then recovered on the 6th day. Food consumption fell from 69 + 7 g . k g i . d a y - 1 to 2 8 + 6 g . k g - l - d a y - 1 (p< 0.001) on the 1st day following cannulation then recovered by the 7th day. Implantation of osmotic minipumps had no effect on body weight or food intake. Water intake fell following cannulation from 8 3 + 4 t o 4 7 + 7 m l . k g t . d a y - 1 ( P < 0 . 0 1 ) by the 2nd day, then recovered by the 4th day. Implantation of osmotic minipumps caused a fall in water intake of 18 + 4% on the 2nd day, with recovery on the 4th day. There were no differences between the groups of clonidine (0.1 m g . kg day ') treated and control rats. In preliminary experiments, higher doses of clonidine provoked substantial falls in body weight and food and water consumption. By the 4th day a dose of 0.5 m g - k g 1 . d a y i c a u s e d a 3 4 + 10 g drop in body weight, water intake was 15 + 6 ml • kg- ' • day and food i n t a k e 7 + 3 g . k g 1 . d a y i. Ratsreceiv_ ing clonidine doses of 1 and 1.5 m g . kg i. day i neither ate nor drank during the 4 days following implantation. 3.2. Blood pressure and heart rate during clonidine treatmenl Both MAP and H R fell during 3 h habituation by 17 + 3 and 32 + 4% respectively. There was also a 14 + 2% fall in MAP between the stabilized values on the 1st and the 2nd day (fig. 1). Changes in H R were less marked (from 359 + 12 beats per minute (bpm) on the 1st day to 339 + 13 bpm, NS, on the 2nd). Clonidine (0.1 mg. kg i. day ;) induced an average a fall in MAP of 16.5 + 2.5 mm Hg over the first 8 days of infusion (fig. 1). When p u m p function terminated at the end of the 10th day, however, there was no significant difference between controls (153 + 2) and clonidine-treated rats (148 + 3 mm Hg). The hypotensive effect was slow in onset: a significant difference between controls was not obtained until the 4th day follow-
I
160"
150-
130 Of r'-- . . . . . . . . -8.-6 --t,-2 0 . 2 , 4 . , , 6 . 8 DAYS Fig. 1. Effect of c l o n i d i n e on m e a n arterial pressure ( m m Hg) of S H R receiving c l o n i d i n e (full circles, n = 18) at a dose of 0.t mg. kg i. day - 1 i.v. Blood pressure values are given for 8 days before i m p l a n t a t i o n and 8 days after. C o n t r o l S H R (open circles, n = 15) r e c e i v e d m i n i p u m p s which delivered [ ~ I C r ] E D T A only. In both g r o u p s m e a n arterial pressure was m e a s u r e d via a chronically i m p l a n t e d dorsal aortic c a n n u l a following 3 h h a b i t u a t i o n . Asterisks refer to p r o b a b i l i t i e s based on i n d e p e n d e n t t-tests between control S H R and clonidinetreated SHR; * P < 0.05. ** P < 0.01 and *** P < 0.001.
ing implantation. Bl(uad pressure was recorded in 5 out of the 18 clonidine-treated SHR on the day following implantation. None showed a significant difference from the controls. Clonidine had no effect on MAP lability. There was a 30-60 b p m fall in H R following clonidine, but differences from controls were rarely statistically significant: 2nd day: 296 + 8 versus 350 + 12 (P < 0.05), 4th day: 3 0 2 + 8 versus 3 3 1 + 1 4 (NS), 6th day: 3 2 1 + 8 versus 340 + 20 (NS), and 8th day: 301 + 7 versus 338 + 10 (NS). Clonidine had no effect on H R lability. In preliminary experiments, clonidine at a dose of 0.5 m g . k g i. d a y - I produced a fall in MAP from 144 + 6 to 125 + 10 mm Hg (n = 5) by the 4th day. H R fell from 3 4 0 + 1 0 to 2 9 0 + 6 b p m (n = 5). Since (1) this higher dose did not have a greater effect on MAP than the dose of 0.1 m g - k g - ~ - d a y i, and since (2) rats treated with
101 this higher dose neither ate nor drank normally, this treatment was stopped on the 4th day. In S H R (n = 5) given a lower dose (0.05 m g . kg- i . d a y - 1) of clonidine, MAP and H R were the same as the controls. Thus a clonidine dose of 0.1 m g . kg -1. day-1 seemed optimal. This dose produced a fall in MAP with no change in body weight, food or water intake.
following p u m p cessation, SHR previously treated with clonidine showed blood pressure upswings (as described by Thoolen et al. (1982)) in 2.4 + 1.3 periods out of 20. Control S H R did not show upswings. As we did not record blood pressure continuously (but only 15 min every h), we cannot give figures for the absolute frequency of blood pressure upswings during withdrawal.
3.3. Effects of cessation of chronic treatment with clonidine on blood pressure and heart rate
3.4. Heart weights
From 6 h following p u m p cessation, MAP in S H R previously receiving clonidine started to rise and from this time onwards was generally higher than MAP in control S H R but the differences were not statistically significant. The lability of MAP (fig. 2) was similar in both the control and the withdrawal groups up to 10 h following pump cessation. Thereafter lability rose 2-fold in SHR previously receiving clonidine, then fell at 20 h following p u m p cessation and remained unchanged at 30 h. Withdrawal changes in HR were characterized by tachycardia and lability. At 16 h following pump cessation, the values for rate were 380 + 20 versus 320 -t- 10 bpm (P < 0.05) and for lability 7.5 + 2 versus 3.2 + 0.3% (P < 0.05) in previously clonidine-treated SHR and control S H R respectively. During the 20 h recording period
% 6,
-B
-6
-~.
-2
.2
.4
"6
.8
.IO
.12
.It.
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4
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Fig. 2. Changes in lability of mean arterial pressure calculated as standard error of individual heart beat values expressed as a percentage of the average. Values given are for the last 2 rain of each 15 min recording session. Animals were recorded from 8 h before estimated time o f pump cessation to 20 h after. Asterisks
refer to probabilities based on analysis of variance between values for clonidine-treated group (full circles, n =18) and controls (open circles n = 15); ° P < 0.05. *" P < 0.01, *** P < 0.001. Error mean square was 5.2 and F for group was 21.3.
Chronic treatment with clonidine did not change the heart weights of SHR (controls: 4.03 + 0.08, n = 19; clonidine 7 days: 3.83 + 0.12, n = 15; clonidine 10 days and 16 h withdrawal: 3.99 + 0.09 g. kg body weight- t n = 13). Thus the clonidineinduced fall in blood pressure was not accompanied by a decrease in heart weight. 3.5. Enzymes in brain-stem nuclei The only significant changes during clonidine treatment occurred in the A 1 / C I region where there was a 25% decrease in DBH activity (from 3.02 -t- 0.14 to 2.26 + 0.10 units, P < 0.001) and a 31% decrease in P N M T activity (from 32.7 + 2.0 to 22.6 + 2.3 units, P < 0.01 (table 1). No significant changes in TH, DBH or P N M T in either A6, A 2 / C 2 or A9 regions were observed during clonidine treatment (table 1). Sixteen hours after clonidine withdrawal, DBH activity in A 1 / C I region was still significantly reduced (2.39 + 0.14 units, P < 0.01 compared to 3.02 + 0.14 for controls) whereas P N M T activity of this nucleus had returned to a value (34.8 + 1.9 units) not significantly different from that of controls (32.7 + 2.0) (see table 1). DBH activity of the A 2 / C 2 region was also significantly decreased during withdrawal (controls: 4.27 + 0.20, withdrawal: 3.40 + 0.17 units, P < 0.01) (table 1). Apart from the changes noted above, no statistically significant changes in enzyme activities were observed during withdrawal. 3.6. Enzymes in adrenal medullas Chronic clonidine treatment had no effect on catecholamine synthesizing enzyme activities in adrenal medulla (see table 1). However, during
1(12
"I'A BLI:. 1 t'ffects of clonidine treatment a n d clonidine withdrawal o n the activities of the catecholamine synthesizing enzymes in various rat brain catecholaminergic regions and in the adrenal medulla. "IH activity is expressed as follows depending on the tissue: for A I / C I and A 2 / ( ' 2 as pmol of I)OPA formed per h per mg protein: for A6 and A9 regions as nmol of DOPA formed per h per mg protein: for the adrenal medulla as nmol of I)OPA formed per h and per pair of glands. DBIt activity is expressed for A 1 / C I . A2/('2 and A6 regions as nmol of octopamine formed per h per mg protein and for the adrenal medulla as nmol of octopamine for,ned per h and per pair of glands. PN MT activity is expressed for A 1 / ( ' I . A 2 / ( ' 2 and A6 regions as pmol of N-methylphenylethanolamine formed per h per mg of protein and for the adrenal medulla as nmol of N-methylphenylethanolamine formed per h and per pair of glands. " P < 0.05, h p < 0.01, ~ P < 0.001.
Times A 1 / C 1 region C o n t r o l s ( n = 19) Clonidine (n = 151 Withdrawal (n = 131 A 2 / C 2 region Controls (n = 191 ('hmidine (n = 151 Withdrawal (n = 13) A6 region ('ontrols (n -: 19) C k m i d i n e ( n = 151 Withdrawal (n = 131 A9 region Controls (n = 19) Clonidine (n = 15) Withdrawal (n = 13)
TH activit~
I)BH activity
PNMT activits
285 261 330
_+10 4- 16 _+22
3.02_40.14 2.26+-(I.10 • 2.39+0.14 h
32.7 +2.0 22.6 4- 2.3 h 34.8 r l . 9
552 541 514
_+27 ~24 _+32
4.27_+0.20 4.06+_0.31 3.40_+(I.17h
21.5 ~-1.3 18.2 +1.5 19.4 _+1.6
1.32_+ 0.11 1.144- 0.12 1.40_+ 0.11
17.3 ±1.2 14.8 +_1.6 17.8 ~ 1.4
7.89_+0.51 7.15_+0.68 7.3940.74
2.83_+ 0.13 3.17_+ 0.14 2.93+_ 0.18
Adrenal medulla Controls (n = 19) ('lc, nidine (n = 151 Withdrawal (n = 13)
24.9 +_ 1.2 25.6 +- (1.8 31.1 _ 1.5 h
withdrawal there was a 25% increase in TH activity (controls: 24.9 + 1.2, withdrawal: 31.1 + 1.2 units, P < 0 . 0 1 ) and a 13% increase in P N M T activity (controls: 7.55 + 0.27, withdrawal: 8.56 + 0.34 units, P < 0.05) (table 1). DBH activity in adrenal medulla was not modified by either chronic clonidine treatment or its withdrawal (table 1).
99.6 -+3.5 109.8 _+5.6 111.1 _+5.4
7.55 +0.27 8.00_+(I.19 8.56+_0.34"
A 1 / C 1 region. During withdrawal. DBH activity in both A I / C 1 and A 2 / C 2 regions was decreased while P N M T activity in the A 1 / C 1 returned to normal values. Withdrawal was accompanied by an increase in TH and PNMT activities of the adrenal gland.
4.1. Hypotenswe effect of chmidine 4. Discussion Clonidine delivered via osmotic minipump lowered blood pressure and heart rate in the SHR. After withdrawal of clonidine, blood pressure returned to control values, whereas the blood pressure lability increased. There was transient tachycardia and increased HR lability. The blood pressure lowering effect of clonidine was accompanied by a decrease in D B H and P N M T activity in the
We measured blood pressure in the nonanesthetized SHR and gave clonidine over several days via a rate-controlled delivery system (Struyker-Boudier, 1982). Single doses of clonidine acutely lowered MAP under anesthesia but were without effect in awake rats (Parker et al., 19821. The results reported here show a slowly developing fall in MAP of 16.5 + 2.5 mm Hg when clonidine was delivered by osmotic minipumps to awake rats. In experiments with osmotic minipumps,
103 Struyker-Boudier and van Essen (1980) found falls in MAP similar to those we found, but with a higher clonidine dose (0.3 mg • kg- 1 . d a y - l). Lower doses of clonidine did not induce significant falls in MAP in their experiments. They administered clonidine s.c. whereas we administered clonidine i.v. and differences in absorption a n d / o r metabolism may explain the differences observed in the cardiovascular effects. Using a protocol similar to ours, Thoolen et ai. (1982) observed similar falls in MAP but again with a higher dose of clonidine. The lack of a more pronounced hypotensive effect with their 5-fold higher dose may be explained on the basis of stimulation of vascular a-adrenoceptors by clonidine. Thus in SHR, as in man (Wing et al., 1977; Reid et al., 1980) clonidine may have a narrow therapeutic range. There was an apparent delay of 1-2 days in onset of the hypotension in both our experiment and that of Thoolen et al. (1981b). This phenomenon cannot be explained wholly on the basis of a start up time (4 h) for the osmotic minipumps. When a single dose of clonidine was given i.m. blood pressure fell quickly: there was 27% fall in MAP in the h following the injection (Parker et al., 1982). Thus the mechanism of the hypotensive action of clonidine may be different when it is given as a single injection or via a chronic, rate-controlled delivery schedule. Finally at the end of our 10 days" treatment with clonidine there was no significant difference between clonidine-treated and control rats. This weakness of the effect of our chronic clonidine treatment in the long run may be due to tolerance developing to the hypotensive effect of clonidine or to the tailing off of pump flow towards the end of the functional life of the pump.
4.2. Clonidine withdrawal syndrome: experimental protocol and changes in M A P and HR Osmotic minipumps ran until emptied of their contents. Flow rate was checked by measurement of radioactivity. Other dosage schemes have their advantages and disadvantages. Daily injections of clonidine have the advantage of precise determination of the time of the last injection. However, animals may suffer withdrawal crises after each
single daily injection (Salzmann, 1979; Oates et al., 1978). Another alternative is to give clonidine in the diet or in the drinking water. As rats eat and drink little during the light period, once again the possibility arises of daily withdrawal crises. Furthermore, the onset of withdrawal cannot be determined precisely unless consumption is monitored. A final possibility for inducing withdrawal is by surgical removal of the minipump. This has the advantage of precise determination of the time of cessation but the disadvantage that the surgery may modify the withdrawal syndrome. Surgical interventions in the SHR induce falls in food intake and it is known that food deprivation in the rat causes falls in blood pressure (Einhorn et al., 1982). Clonidine withdrawal was characterized by a return of MAP to control values with a slight overshoot of HR. MAP and H R labilities increased. The phenomenon lasted approximately 20 h. Our results are in general agreement with those of Thoolen et al. (1981b) although they observed more pronounced cardiovascular changes. Heart rate overshoot was greater and the changes in MAP and HR lasted longer than in our experiments. Two factors probably explain the difference. Firstly, Thoolen et al. (1981b) gave a higher dose of clonidine and the magnitude of clonidine withdrawal symptoms such as overshoot tachycardia is dependent on dose (DiStefano et al.. 1980). Secondly, they removed their osmotic minipumps surgically, thus the withdrawal syndrome in their experiments was provoked by a combination of withdrawal of clonidine treatment and of the surgery.
4.3. Changes in peripheral and central catecholamine synthesizing enzymes during clonidine treatment and its withdrawal The only significant effect of chronic treatment with clonidine on central enzymatic activities is a decrease in DBH and P N M T activities in the A 1 / C 1 region. Nakamura et al. (1980) did not detect any change in the DBH activity of the A1 region. Their clonidine treatment was shorter (4 days) than ours (7 days). Furthermore in our study the more rostrally located A 1 / C 1 region includes
104
adrenaline neurons (Chamba and Renaud, 1983) which also contain DBH. It is possible that clonidine has a more marked effect on adrenaline than on noradrenaline neurons. Such a possibility is supported by our observation of a decrease in P N M T activity in the same area. Single injections of clonidine decrease adrenaline turnover in the C1 area (Scatton et al., 1979). Using a protocol similar to the present one we showed that following 7 days' treatment with clonidine the only significant change in central monoaminergic function observed was an increase in the adrenaline level of the hindbrain (Atkinson et al.. 1985). The other brain regions we studied were unaffected by clonidine treatment. This confirms e.g. the results of Kane and Johnson (1978) showing that there was no change in TH activity in A6. Furthermore, there was no change in TH activity in A9, a dopaminergic region taken as control area unlikely to be involved in the central control of blood pressure. Prolonged clonidine treatment did not affect catecholamine synthesizing enzyme activities in peripheral tissues such as adrenal medulla. This confirms previous reports of the Johnson group on the tyrosine hydroxylase activity of the adrenal gland (Dix and Johnson, 1977) and superior cervical ganglion or coeliac ganglion (Kane and Johnson, 1978). There was however, an increase in both T H and P N M T activities during withdrawal. Such an increase in T H activity after 24 h withdrawal from clonidine has been previously reported by Dix and Johnson (1977). There are many indications of an increase in sympatho-adrenal activity during withdrawal. Thus increases in T H activity in sympathetic ganglia (Kane and Johnson, 1978; DiStefano et al., 1980) and increases in plasma noradrenaline (Saito, 1981; Thoolen et al., 1981b) have been reported. In our studies, and in other reports, the increased peripheral sympathetic activity occurs during the first 2 days following cessation of clonidine treatment. It thus coincides with the cardiovascular hyperactivity. The increase in peripheral sympatho-adrenal activity during withdrawal is accompanied by changes in central enzymatic activities in both A 1 / C 1 and A 2 / C 2 regions. There is a decrease in DBH activity in the A 2 / C 2 region. As this decrease is not accompa-
nied by a parallel decrease in PNMT, it is possible that it occurs in the noradrenaline neurons rather than in the adrenaline neurons coexisting in the same area. A similar decrease in capacity of synthesis of noradrenaline in this area has been produced by chemical (Snyder et al., 1978) and electrolytic (Talman et al., 1980) lesions. Such lesions are known to induce blood pressure lability. Blood pressure lability was also one of the features of clonidine withdrawal in the SHR in our experiments. DBH activity in the A I / C 1 area remained significantly decreased during withdrawal. P N M T activity, however, which was diminished during treatment, returned to a level comparable to that of the controls. These two results could suggest decreased synthesis of noradrenaline in the A1 part of this area associated with a rebound in synthesis of adrenaline in the CI area. In contrast with the noradrenergic AI neurons, the adrenergic C1 neurons project directly to the intermediolateral column of the spinal cord (Ross et al., 1981) and have been shown by various approaches to be a key element of the sympathetic vasopressor system (Reis et al., 1984a,b). Thus, the return of MAP to control levels is accompanied by restoration of the capacity of synthesis of adrenaline within the vasopressor adrenaline CI neurons. However, a definitive causal relationship between these two phenomena remains to be established.
Acknowledgements This v,'ork was supported by the Swiss National Science Foundation Grant no. 3.759.080: by a grant from the Sandoz Foundation, (Basel, Switzerland). by Boehringer, Switzerland and by a "Jeune Equipe' award from the ' Minist~re de l'Education Nationale (Direction de la Recherche) Paris, France.
References Atkinson, J., N. Boillato B. Renaud, M. Seccia. S.Z. Langer and C. Pimoule, 1983, Clonidine withdrawal in SIqR, Experientia 39. 678. Atkinson, J., M. Sonnay and N. Boillat. 1985, Changes in central monoaminergic function during chronic treatment with clonidine in the spontaneously hypertensive rat, European J. Pharmacol. 106, 613.
105 Chalmers, J.P. and M.J. West, 1983, The neurons system in the pathogenesis of essential hypertension, in: Handbook of Hypertension, Vol. 1. Clinical Aspects of Essential Hypertension, Ed. J.L.S. Robertson (Elsevier, New York) p. 64. Chamba, G. and B. Renaud, 1983, Distribution of tyrosine hydroxylase, dopamine-beta-hydroxylase and phenylethanolamine-N-methyltransferase activities in coronal sections of the rat lower brain stem, Brain Res. 259, 95. Dahlstr~m, A. and K. Fuxe, 1964, Evidence for the existence of monoamine-containing neurons in the central nervous system. i. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta Physiol. Scand. 62 (Suppl.) 232, 1. Denoroy, L., M. Heimburger, B. Renaud, S. Affara, J. Wepierre, Y. Cohen and J. Sassard, 1981, Effects of chronic ,B-blockers treatment on catecholamine-synthesizing enzymes in spontaneously hypertensive rat, Biochem. Pharmacol. 30. 2673. DiStefano, P.G. Fox and E.M. Johnson, Jr., 1980, Characterization of the clonidine withdrawal syndrome in the normotensive rat. J. Pharmacol. Exp. Ther. 214, 263. Dix, R.K. and E.M. Johnson. Jr., 1977, Withdrawal syndrome upon cessation of chronic treatment in rats, European J. Pharmacol. 44, 153. Einhorn, D., J.B. Young and L. Landsberg, 1982, Hypotensive effect of fasting: possible involvement of the sympathetic nervous system and endogenous opiates, Science 217, 727. Hokfelt. T., K. Fuxe, M. Goldstein and O. Johansson, 1974, lmmunohistochemical evidence for the existence of adrenaline neurons in the rat brain, Brain Res. 66, 235. Howe, P.R.C., 1985, Blood pressure control by neurotransmitters in the medulla oblongata and spinal cord, J. Auton. Nerv. Syst. 12, 95. Kane, W.H. and E.M. Johnson, 1978, Effect of chronic clonidine treatment and withdrawal on tyrosine hydroxylase activity in peripheral ganglia and the locus coeruleus, European J. Pharmacol. 51,309. Langer, S.Z., I. Cavero and R. Massingham, 1980. Recent developments in noradrenergic neurotransmission and its relevance to the mechanism of action of certain antihypertensive agents, Hypertension 2, 372. Lowry. O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265. Molinoff, P.B., R. Weinshilboum and J. Axelrod. 1971. A sensitive enzymatic assay for dopamine-beta-hydroxylase, J. Pharmacol. Exp. Ther. 178, 425. Nagatsu, T.. M. Levitt and S. Udenfriend. 1964, A rapid and simple radioassay for tyrosine hydroxylase activity, An. Biochem. 9, 122. Nakamura, K., T. Okada, H. Ishii and K. Nakamura, 1980, Differential effects of alpha-methyldopa, clonidine and hydralazine on norepinephrine and epinephrine synthesizing enzymes in the brainstem nuclei of spontaneously hypertensive rats, Jap. J. Pharmacol. 30, 1. 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 responses, J. Pharmacol. Exp. Ther. 206, 268.
Palkovits, M., 1973, Isolated removal of hypothalamic or other brain nuclei of the rat. Brain Res. 59, 449. Parker, M. and J. Atkinson, 1982, Withdrawal syndromes following cessation of teratment with antihypertensive drugs, Gen. Pharmacol. 13, 79. Parker, M., R. Studer, M. Manganel and J. Atkinson, 1982. Bk~xt pressure overshoot under pentobarbitone anaesthesia following a single dose of clonidine in rats, Clin. Exp. Pharmacol. Physiol. 9, 685. Reid. J.L., N.D. Barber and D.S. Davies, 1980, The clinical pharmacology of clonidine: relationship between plasma concentrati(,,t and pharmacological effect in animals and man. Arch. Int. Pharmacodyn. Ther. (Suppl.I 17. 11. Reis, D.J., A.R. Granata, T.H. Joh. C.A. Ross, D.A. Ruggiero and D.H. Park, 1984a, Brain stem catecholamine mechanisms in tonic and reflex control of bk~xt pressure, Hypertension 6, 11-7. Reis, D.J., ('.A. Ross. D.A. Rugglero, A.R. Granata and T.H. Job, 1984b, Role of adrenaline neurons of the ventrolateral medulla (the CI group) in the tonic and phasic control of arterial pressure. Clin. Exp. Hyper. A6, 221. Renaud, B., S. Fourniere, L. Denoroy, M. Vincent, J.F. Pujol and J. Sassard, 1978. Early increase in phenylethanolamineN-methyltransferase activity in a new strain of spontaneously hypertensive rats. Brain Res. 159, 149. Ross. C.A., D.M. Amstrong, D.A. Ruggiero, V.M. Pickel, T.H. Joh and l).J. Reis. 1981, Adrenaline neurons in the rostral ventrolateral medulla innervate thoracic spinal cord: a combined immunocytochemical and retrograde transport demonstration, Neurosci. Lett. 25, 257. Saavedra, J.M., M. Palkovits, M.J. Brownstein and J. Axelrod, 1974, Localization of phenylethanolamine-N-methyl-tran~ ferase in the rat brain nuclei, Nature 248, 695. Saito, H.. 1981, Clonidine withdrawal hypertension in spontaneously hypertensive rats, Trends Pharmacol. Sci. 2, 176. Salzmann. R., 1979. The effects of treatment and of withdrawal of treatment with guanfacine and clonidine on blood pressure and heart rate in normotensive and renal hypertensive rats, J. Pharmacol. 31,212. Scatton. B., F. Pelayo, M.L. Dubocovich, S.Z. Langer and G. Bartholini, 1979, Effect of clonidine on utilization and potassium-evoked release of adrenaline in rat brain areas, Brain Res. 176, 197. Snyder, D.W.. M.A. Nathan and D.J. Reis, 1978, Chronic lability of arterial pressure produced by selective destruction of the catecholamine innervation of the nucleus tractus solitarii in the rat, Circ. Res. 43, 662. Struyker-Boudier, H.A.J., 1982. Rate-controlled drug delivery: pharmacological, therapeutic and industrial perspectives. Trends Pharmacol. Sci. 3, 162. Struyker-Boudier, H.A.J. and H. Van Essen, 1980, Chronic infusion of clonidine in the spontaneously hypertensive rat, Naunyn-Schmiedeb. Arch. Pharmacol. 311, R49. Talman, W.T., D.W. Snyder and D.J. Reis, 1980. Chronic lability of arterial pressure produced by destruction of A2 catecholaminergic neurons in rat brainstem, Circ. Res. 46, 842. Thoolen, M.J.M.C., P.B.M.W.M. Timmermans and P.A. Van
14)6
Zv, ie|cn. 1981a. 'lhe chmidine v,'ithdrav, al syndrome. Its reproduction and evaluation ill laborator', 3ninlal model~,. (ion. Pharmacol. 12. 31)3. Thoolen. M.J.M.('.. P.B.M.W.M. Tinlmernlans and P.A. Win Z,,~.ietcn. 1981 b. Discontinuation syndrome after continuous, infu~,i~m of chw, idine in tile ~,pontaneou~,ly h~pcrtensive rat. Life .~i. 28. 2103. "l'hoolcn, M.J.M.C.. P.B.M.W.M. "limmcrmanx and P.A. Van Zv, icten. 1982. Guanfitcine and chmidine: antihypcrtcnsivc and v, ithdrav.'al characteristics after continuous infusion
and its interruption in the ~,pont~,neou~,l~,, h~vpertcm, ivc rat normotensi'..'e rat. Naun,,.n-Schmicdeb. Ar,,:h. Ph.:lrnlacol. 319. ;42. Week~,. J.R. and J.A. ,Jone~,. 1960, Routine direct mea~,uerement ~,f arterial pressure in unanesthetized rat. Prt~,_'. Soc. tixp. Biol. Med. 104. 646. Wing. I..M.II...I.U Reid. D.S. Davies. I-.A.M. Neill. P. l'ippett and ('.T. I)ollerv. 1977. I'harmacokinetic and concentration-effect relationships of clonidinc in essential h~,pertension, European J. Clin. Pharmacol. 12. 463.
97
Elsevier
C H R O N I C C L O N I D I N E T R E A T M E N T A N D ITS WITHDRAWAL: EFFECTS O N B L O O D P R E S S U R E A N D C A T E C H O L A M I N E S Y N T H E S I Z I N G E N Z Y M E S IN B R A I N - S T E M N U C L E I * JEFFREY ATKINSON *, LAURA LAMBAS-SENAS **, MYFANWY PARKER "**, NICOLE BOILLAT, PIERRETTE LUTHI, MIREILLE SONNAY, MICHELE SECCIA ** and BERNARD RENAUD ** lnstitut de Pharmacologie de I'Unwersitb, Rue du Bugnon 21, CH-1011 Lausanne-CHUV. Switzerland and * * Laboratoire de Neuropharmacologie, Facultb de Pharmacie, Unwersitb Claude Bernard, 8 Ave. Rockefeller. 69008 Lyon, France
Received 24 September 1985, accepted 5 November 1985
J. ATKINSON, L. LAMBAS-SENAS, M. PARKER, N. BOILLAT, P. LUTH1, M. SONNAY, M. SECCIA and B. RENAUD, Chronic clonidine treatment and its withdrawal: effects on blood pressure and catecholamine synthesizing enzymes in brain-stem nuclei, European J. Pharmacol. 121 (1986) 97-106. We have studied the effects of withdrawal from chronic clonidine treatment in the adult male spontaneously hypertensive rat (SHR). SHR received clonidine, 0.1 mg. k g - t . d a y - 1 i.v. for 10 days. Clonidine was delivered via osmotic minipumps. After 7 days of treatment there was a 16.5 + 2.5 mm Hg fall in mean arterial pressure. This was accompanied by a decrease in the dopamine-fl-hydroxylase and phenylethanolamine-N-methyl transferase activities of the A1/C1 region. Withdrawal from clonidine was characterized by tachycardia and an increase in mean arterial pressure and heart rate lability. Phenylethanolamine-N-methyl transferase of the A1/C1 region returned to normal ' It the dopamine-,8-hydroxylase activity remained diminished. The dopamine-~-hydroxylase activity of the A 2 / C 2 region was also diminished during withdrawal. We suggest that the blood pressure lowering effect of cionidine is accompanied by a decreased capacity to synthesize adrenaline in the A I / C 1 region where adrenaline could mediate a pressor effect. Increased blood pressure lability during withdrawal is accompanied by a restoration of synthesis of adrenaline in the A I / C 1 region. There is also a decrease in the capacity of synthesis of noradrenaline in the A 2 / C 2 region where adrenaline may mediate a vasodepressor effect. Central nervous system
Clonidine
SHR
Blood pressure
I. Introduction Prolonged t r e a t m e n t with clonidine leads to a fall in b l o o d pressure in the s p o n t a n e o u s l y hypertensive rat ( T h o o l e n et al., 1981a; 1982) and a b r u p t cessation of c l o n i d i n e t r e a t m e n t leads to cardiovascular h y p e r a c t i v i t y (Thoolen et al., 1981a; Parker a n d A t k i n s o n , 1982). The changes in cerebral n e u r o t r a n s m i s s i o n which a c c o m p a n y the * Some of the results reported here were pre~nted at the 15th Annual Meeting of the Swiss Societies for Experimental Biology in Fribourg, Switzerland, March 1983. * To whom all correspondence should be addressed. *** M. Parker was a scholar of the Roche Research Foundation," Basle, Switzerland and L. Lambas-Senas received a fellowship from Laboratoires Hoechst, Paris. France. 0014-2999/86/$03.50 ,~ 1986 Elsevier Science Publishers B.V.
Withdrawal
acute hypotensive effect of clonidine have been d e s c r i b e d in detail ( L a n g e r et al., 1980), but not those after p r o l o n g e d t r e a t m e n t or d u r i n g withdrawal. Several a u t h o r s have raised the interesting possibility that different c a t e c h o l a m i n e r g i c areas of the brain are affected in different ways by prolonged clonidine t r e a t m e n t a n d its w i t h d r a w a l ( N a k a m u r a et al., 1980; D i S t e f a n o et al., 1980). T h e r e has been, however no a t t e m p t to relate changes in c a t e c h o l a m i n e r g i c function in the A 1 / C I and A 2 / C 2 h i n d b r a i n areas directly involved in b l o o d pressure control to changes in b l o o d pressure. W e have c a r r i e d out such a study d u r i n g (1) the p e r i o d of h y p o t e n s i o n p r o d u c e d by p r o l o n g e d t r e a t m e n t with clonidine and (2) the
9~
cardiovascular hyperactivity produced by withdrawal. Recent evidence points to a possible role of hindbrain adrenaline in blood pressure control (Chalmers and West. 1983; Reis et al., 1984a,b: Howe, 1985). We have previously shown that chronic clonidine treatment increases hindbrain adrenaline content, possibly due to decreased release (Atkinson et al., 1985). Moreover, it has been suggested that adrenergic neurons of certain hindbrain areas such as C1 play a key role in the sympathetic vasopressor system (Ross et al., 1984). We now studied possible changes in adrenaline synthesis in the CI area and their link to the cardiovascular effects of clonidine treatment and to its withdrawal. Conversely, some hindbrain noradrenergic systems may play a vasodepressor role (Chalmers and West, 1983; Reis et al., 1984a,b). Decreased noradrenaline synthesis in the A 2 / C 2 area can induce blood pressure lability (Snyder et al., 1978: Talman et al., 1980). Increased blood pressure lability is one of the characteristics of clonidine withdrawal (present paper). Thus we studied possible changes in noradrenaline synthesis in A 2 / C 2 and their link to the cardiovascular effect of clonidine and its withdrawal. Therefore we have followed during and after clonidine treatment the changes occurring in the activities of the catecholamine synthesizing enzymes (tyrosine hydroxylase, dopamine-,8-hydroxylase and phenylethanolamine-N-methyl transferase) in A 1 / C 1 , A 2 / C 2 , locus coeruleus (A6) and substantia nigra (A9) catecholaminergic groups (Dahlstr/3m et al., 1964: H6kfelt ct al., 1974) and in the adrenal medulla. Enzyme activities were measured at two different times: (i) after 7 days of clonidine administration and (ii) during the withdrawal following cessation of 10 days' clonidine treatment.
2. Materials and methods
2.1. Animals and blood pressure recording Male spontaneously hypertensive rats ( S H R / NICo) 3-4 months of age were purchased from
If f a-Credo, SA, 69210 L'Arbreslc, France. Systolic arterial pressure (SAP. mm Hg) was measured by an indirect tail cuff method. Animals (10°~) with a SAP of less than 180 mm Hg were not used. The remainder were fitted with intra-aortic cannula under sodium pentobarbital anesthesia according to the technique of Weeks and Jones (1960). Blood pressure was recorded by connecting the cannula, via a liquid feedthrough swivel, to a strain-gauge transducer. Heart rate (HR) was obtained from a ratemeter driven by the pulse pressure. Blood pressure recording was started after 3 days' recovery and 8 days before minipump implantation. Mean arterial pressure (MAP) and HR were recorded for 15 rain after habituation for 3 h. The individual MAP and HR values of each cardiac cycle of the final 2 rain were averaged, l,ability was calculated as the standard error of this sample expressed as a percentage of the average. On the 10th day following minipump implantation, MAP and HR were recorded for 15 min every h, from 8 h before pump cessation to 20 h after. A final recording was made 30 h after pump cessation. Two groups of rats were used. In the first, blood pressure was recorded in 33 SHR (18 had received ::lonidinc, 0.1 r a g - k g 1. day ~ and 15 were controls). In the second group, 47 SHR without cannula were used for the biochemical studies. On the 7th day after implantation. 24 were killed: 15 had received chmidine (0.1 m g - k g ~• d a y ~) and 9 were controls. The remainder were killed at 16 h after pump cessation: 13 had previously received chmidine (0.1 mg. kg t . day i) and 10 were controls.
2.2. Implantation of osmotic mimpumps Minipump function (Aizet", model 2001, Scientific Marketing Associates. Ltd., London, UK. volume 0.22 ml, flow rate 0.001 ml. h i) was checked in vitro with [5~Cr]ethylene diaminc tetra acetic acid ([51Cr]EDTA). Pumps had a 4 + 1 h start-up time. At the end of the functional life of the pumps their flow rate fell by 75 + 5% in 24 h. Osmotic minipumps were implanted s.c. (under ether anesthesia), in all SttR and the pumps connected to a polyethylene cannula placed in the left external jugular vein. The pumps were filled with
99 [51Cr]EDTA (controls) or with clonidine (0.1 m g . kg 1. d a y - ~) dissolved in [51Cr]EDTA. In the second group pumps were removed from 24 SHR killed on the 7th day following implantation. The in vivo flow rate was determined from the difference in gamma radioactivity with starting counts. In the first group flow rate was checked by counting radioactivity in 0.2 ml plasma samples taken following each MAP session. After pump cessation [51CrIEDTA was no longer present in the circulation. 2.3. Brain punching
The rats were decapitated and the brains, hearts and adrenal medullas rapidly removed. The brains were sectioned in a vertical frontal plane at the anterior hypothalamic level. The caudal part was quick-frozen on dry ice together with the hearts and the adrenal medullas. Each caudal part of the brain was cut into 500/~m thick coronal sections. The A 1 / C I , A 2 / C 2 , locus coeruleus (A6, LC) and substantia nigra (A9) regions were removed with a hollow needle (0.9 mm i.d.) according to a modification (Renaud et al.. 1978) of the technique of Palkovits (1973).
substrate. The incubation time was 25 min. The results were expressed as pmol of dopa formed per h of incubation and per mg of protein for brain structures or per pair of adrenal medullas. DBH activity was determined in 10 ~tl of supernatant by a modification (Denoroy et al., 1981) of the method of Molinoff et al. (1971). The final concentrations of CuSO 4 were 4 × 10 -5 M for LC and A9, 5 × 1 0 5 M for A 1 / C 1 and A 2 / C 2 regions, and 5 x 10 6 M for adrenal medullas. The incubation time was 30 min. The results were expressed as nmol of octopamine formed per h of incubation and per mg of protein for the brain structures or per pair of adrenal medullas. P N M T activity was measured according to a modification (Denoroy et al., 1981) of the method of Saavedra et al. (1974) in 10 ~1 of supernatant. The incubation time was 30 min. The results were expressed as pmol of N-methylphenylethanolamine formed per h of incubation and per mg of protein for brain structures or per pair of adrenal medullas. 2.4. 3. Protein determination Total proteins were estimated in an aliquot of supernatant with the Folin phenol reagent method of Lowry et al. (1951) using bovine serum albumin (Sigma) as a standard.
2.4. Biochemical assays 2.5. Statistics 2.4.1. Tissue homogenization The tissues obtained were disrupted mechanically and by ultrasound (20 KHz, 40 W, 10 s) in 2 mM potassium phosphate buffer (pH 6.0) containing 0.2% Triton X-100 (v/v). The homogenization volume was 300 #1 for the brain nuclei. The adrenal medullas were homogenized in 200 vol. (diluted to 500 vol. for the dopamine-fl-hydroxylase (DBH) and phenylethanolamine-N-methyl transferase (PNMT) assays). The homogenates were centrifuged (2400 x g, 15 min) at 4°C and the supernatants used for the biochemical assays. 2.4.2. Enzymatic assays Tyrosine hydroxylase (TH) activity was determined in 50 ~1 of supernatant by a modification (Renaud et al., 1978) of the radiometric method of Nagatsu et al. (1964) using [3,5-3H]tyrosine as
Results are given as means + S.E.M. Comparison between means were made using the analysis of variance or paired and independent Student's t-tests. Probabilities based on these tests are given as: N S = P > 0 . 0 5 , * P < 0 . 0 5 , ** P <0.01, *** P < 0.001. 2.6. Substances
Clonidine hydrochloride (Catapresan '~) was a gift of Boehringer, Ltd., Ingelheim, FRG. [SICr] ethylene diamine tetra acetic acid was purchased from Amersham International, Amersham, Buckinghamshire, England. The specific activity was 1-2 mCi. m g - I and the radioactivity concentration was 0.1 mCi • m l - 1.
3. Results
I'~,,,on ~tts-ia I
mm~
180
3.1. Changes in body weight, food and water consurnption 170.
Following aortic cannulation, the body weight fell by 5 + 2% by the 4th day, then recovered on the 6th day. Food consumption fell from 69 + 7 g . k g i . d a y - 1 to 2 8 + 6 g . k g - l - d a y - 1 (p< 0.001) on the 1st day following cannulation then recovered by the 7th day. Implantation of osmotic minipumps had no effect on body weight or food intake. Water intake fell following cannulation from 8 3 + 4 t o 4 7 + 7 m l . k g t . d a y - 1 ( P < 0 . 0 1 ) by the 2nd day, then recovered by the 4th day. Implantation of osmotic minipumps caused a fall in water intake of 18 + 4% on the 2nd day, with recovery on the 4th day. There were no differences between the groups of clonidine (0.1 m g . kg day ') treated and control rats. In preliminary experiments, higher doses of clonidine provoked substantial falls in body weight and food and water consumption. By the 4th day a dose of 0.5 m g - k g 1 . d a y i c a u s e d a 3 4 + 10 g drop in body weight, water intake was 15 + 6 ml • kg- ' • day and food i n t a k e 7 + 3 g . k g 1 . d a y i. Ratsreceiv_ ing clonidine doses of 1 and 1.5 m g . kg i. day i neither ate nor drank during the 4 days following implantation. 3.2. Blood pressure and heart rate during clonidine treatmenl Both MAP and H R fell during 3 h habituation by 17 + 3 and 32 + 4% respectively. There was also a 14 + 2% fall in MAP between the stabilized values on the 1st and the 2nd day (fig. 1). Changes in H R were less marked (from 359 + 12 beats per minute (bpm) on the 1st day to 339 + 13 bpm, NS, on the 2nd). Clonidine (0.1 mg. kg i. day ;) induced an average a fall in MAP of 16.5 + 2.5 mm Hg over the first 8 days of infusion (fig. 1). When p u m p function terminated at the end of the 10th day, however, there was no significant difference between controls (153 + 2) and clonidine-treated rats (148 + 3 mm Hg). The hypotensive effect was slow in onset: a significant difference between controls was not obtained until the 4th day follow-
I
160"
150-
130 Of r'-- . . . . . . . . -8.-6 --t,-2 0 . 2 , 4 . , , 6 . 8 DAYS Fig. 1. Effect of c l o n i d i n e on m e a n arterial pressure ( m m Hg) of S H R receiving c l o n i d i n e (full circles, n = 18) at a dose of 0.t mg. kg i. day - 1 i.v. Blood pressure values are given for 8 days before i m p l a n t a t i o n and 8 days after. C o n t r o l S H R (open circles, n = 15) r e c e i v e d m i n i p u m p s which delivered [ ~ I C r ] E D T A only. In both g r o u p s m e a n arterial pressure was m e a s u r e d via a chronically i m p l a n t e d dorsal aortic c a n n u l a following 3 h h a b i t u a t i o n . Asterisks refer to p r o b a b i l i t i e s based on i n d e p e n d e n t t-tests between control S H R and clonidinetreated SHR; * P < 0.05. ** P < 0.01 and *** P < 0.001.
ing implantation. Bl(uad pressure was recorded in 5 out of the 18 clonidine-treated SHR on the day following implantation. None showed a significant difference from the controls. Clonidine had no effect on MAP lability. There was a 30-60 b p m fall in H R following clonidine, but differences from controls were rarely statistically significant: 2nd day: 296 + 8 versus 350 + 12 (P < 0.05), 4th day: 3 0 2 + 8 versus 3 3 1 + 1 4 (NS), 6th day: 3 2 1 + 8 versus 340 + 20 (NS), and 8th day: 301 + 7 versus 338 + 10 (NS). Clonidine had no effect on H R lability. In preliminary experiments, clonidine at a dose of 0.5 m g . k g i. d a y - I produced a fall in MAP from 144 + 6 to 125 + 10 mm Hg (n = 5) by the 4th day. H R fell from 3 4 0 + 1 0 to 2 9 0 + 6 b p m (n = 5). Since (1) this higher dose did not have a greater effect on MAP than the dose of 0.1 m g - k g - ~ - d a y i, and since (2) rats treated with
101 this higher dose neither ate nor drank normally, this treatment was stopped on the 4th day. In S H R (n = 5) given a lower dose (0.05 m g . kg- i . d a y - 1) of clonidine, MAP and H R were the same as the controls. Thus a clonidine dose of 0.1 m g . kg -1. day-1 seemed optimal. This dose produced a fall in MAP with no change in body weight, food or water intake.
following p u m p cessation, SHR previously treated with clonidine showed blood pressure upswings (as described by Thoolen et al. (1982)) in 2.4 + 1.3 periods out of 20. Control S H R did not show upswings. As we did not record blood pressure continuously (but only 15 min every h), we cannot give figures for the absolute frequency of blood pressure upswings during withdrawal.
3.3. Effects of cessation of chronic treatment with clonidine on blood pressure and heart rate
3.4. Heart weights
From 6 h following p u m p cessation, MAP in S H R previously receiving clonidine started to rise and from this time onwards was generally higher than MAP in control S H R but the differences were not statistically significant. The lability of MAP (fig. 2) was similar in both the control and the withdrawal groups up to 10 h following pump cessation. Thereafter lability rose 2-fold in SHR previously receiving clonidine, then fell at 20 h following p u m p cessation and remained unchanged at 30 h. Withdrawal changes in HR were characterized by tachycardia and lability. At 16 h following pump cessation, the values for rate were 380 + 20 versus 320 -t- 10 bpm (P < 0.05) and for lability 7.5 + 2 versus 3.2 + 0.3% (P < 0.05) in previously clonidine-treated SHR and control S H R respectively. During the 20 h recording period
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4
.~0
Fig. 2. Changes in lability of mean arterial pressure calculated as standard error of individual heart beat values expressed as a percentage of the average. Values given are for the last 2 rain of each 15 min recording session. Animals were recorded from 8 h before estimated time o f pump cessation to 20 h after. Asterisks
refer to probabilities based on analysis of variance between values for clonidine-treated group (full circles, n =18) and controls (open circles n = 15); ° P < 0.05. *" P < 0.01, *** P < 0.001. Error mean square was 5.2 and F for group was 21.3.
Chronic treatment with clonidine did not change the heart weights of SHR (controls: 4.03 + 0.08, n = 19; clonidine 7 days: 3.83 + 0.12, n = 15; clonidine 10 days and 16 h withdrawal: 3.99 + 0.09 g. kg body weight- t n = 13). Thus the clonidineinduced fall in blood pressure was not accompanied by a decrease in heart weight. 3.5. Enzymes in brain-stem nuclei The only significant changes during clonidine treatment occurred in the A 1 / C I region where there was a 25% decrease in DBH activity (from 3.02 -t- 0.14 to 2.26 + 0.10 units, P < 0.001) and a 31% decrease in P N M T activity (from 32.7 + 2.0 to 22.6 + 2.3 units, P < 0.01 (table 1). No significant changes in TH, DBH or P N M T in either A6, A 2 / C 2 or A9 regions were observed during clonidine treatment (table 1). Sixteen hours after clonidine withdrawal, DBH activity in A 1 / C I region was still significantly reduced (2.39 + 0.14 units, P < 0.01 compared to 3.02 + 0.14 for controls) whereas P N M T activity of this nucleus had returned to a value (34.8 + 1.9 units) not significantly different from that of controls (32.7 + 2.0) (see table 1). DBH activity of the A 2 / C 2 region was also significantly decreased during withdrawal (controls: 4.27 + 0.20, withdrawal: 3.40 + 0.17 units, P < 0.01) (table 1). Apart from the changes noted above, no statistically significant changes in enzyme activities were observed during withdrawal. 3.6. Enzymes in adrenal medullas Chronic clonidine treatment had no effect on catecholamine synthesizing enzyme activities in adrenal medulla (see table 1). However, during
1(12
"I'A BLI:. 1 t'ffects of clonidine treatment a n d clonidine withdrawal o n the activities of the catecholamine synthesizing enzymes in various rat brain catecholaminergic regions and in the adrenal medulla. "IH activity is expressed as follows depending on the tissue: for A I / C I and A 2 / ( ' 2 as pmol of I)OPA formed per h per mg protein: for A6 and A9 regions as nmol of DOPA formed per h per mg protein: for the adrenal medulla as nmol of I)OPA formed per h and per pair of glands. DBIt activity is expressed for A 1 / C I . A2/('2 and A6 regions as nmol of octopamine formed per h per mg protein and for the adrenal medulla as nmol of octopamine for,ned per h and per pair of glands. PN MT activity is expressed for A 1 / ( ' I . A 2 / ( ' 2 and A6 regions as pmol of N-methylphenylethanolamine formed per h per mg of protein and for the adrenal medulla as nmol of N-methylphenylethanolamine formed per h and per pair of glands. " P < 0.05, h p < 0.01, ~ P < 0.001.
Times A 1 / C 1 region C o n t r o l s ( n = 19) Clonidine (n = 151 Withdrawal (n = 131 A 2 / C 2 region Controls (n = 191 ('hmidine (n = 151 Withdrawal (n = 13) A6 region ('ontrols (n -: 19) C k m i d i n e ( n = 151 Withdrawal (n = 131 A9 region Controls (n = 19) Clonidine (n = 15) Withdrawal (n = 13)
TH activit~
I)BH activity
PNMT activits
285 261 330
_+10 4- 16 _+22
3.02_40.14 2.26+-(I.10 • 2.39+0.14 h
32.7 +2.0 22.6 4- 2.3 h 34.8 r l . 9
552 541 514
_+27 ~24 _+32
4.27_+0.20 4.06+_0.31 3.40_+(I.17h
21.5 ~-1.3 18.2 +1.5 19.4 _+1.6
1.32_+ 0.11 1.144- 0.12 1.40_+ 0.11
17.3 ±1.2 14.8 +_1.6 17.8 ~ 1.4
7.89_+0.51 7.15_+0.68 7.3940.74
2.83_+ 0.13 3.17_+ 0.14 2.93+_ 0.18
Adrenal medulla Controls (n = 19) ('lc, nidine (n = 151 Withdrawal (n = 13)
24.9 +_ 1.2 25.6 +- (1.8 31.1 _ 1.5 h
withdrawal there was a 25% increase in TH activity (controls: 24.9 + 1.2, withdrawal: 31.1 + 1.2 units, P < 0 . 0 1 ) and a 13% increase in P N M T activity (controls: 7.55 + 0.27, withdrawal: 8.56 + 0.34 units, P < 0.05) (table 1). DBH activity in adrenal medulla was not modified by either chronic clonidine treatment or its withdrawal (table 1).
99.6 -+3.5 109.8 _+5.6 111.1 _+5.4
7.55 +0.27 8.00_+(I.19 8.56+_0.34"
A 1 / C 1 region. During withdrawal. DBH activity in both A I / C 1 and A 2 / C 2 regions was decreased while P N M T activity in the A 1 / C 1 returned to normal values. Withdrawal was accompanied by an increase in TH and PNMT activities of the adrenal gland.
4.1. Hypotenswe effect of chmidine 4. Discussion Clonidine delivered via osmotic minipump lowered blood pressure and heart rate in the SHR. After withdrawal of clonidine, blood pressure returned to control values, whereas the blood pressure lability increased. There was transient tachycardia and increased HR lability. The blood pressure lowering effect of clonidine was accompanied by a decrease in D B H and P N M T activity in the
We measured blood pressure in the nonanesthetized SHR and gave clonidine over several days via a rate-controlled delivery system (Struyker-Boudier, 1982). Single doses of clonidine acutely lowered MAP under anesthesia but were without effect in awake rats (Parker et al., 19821. The results reported here show a slowly developing fall in MAP of 16.5 + 2.5 mm Hg when clonidine was delivered by osmotic minipumps to awake rats. In experiments with osmotic minipumps,
103 Struyker-Boudier and van Essen (1980) found falls in MAP similar to those we found, but with a higher clonidine dose (0.3 mg • kg- 1 . d a y - l). Lower doses of clonidine did not induce significant falls in MAP in their experiments. They administered clonidine s.c. whereas we administered clonidine i.v. and differences in absorption a n d / o r metabolism may explain the differences observed in the cardiovascular effects. Using a protocol similar to ours, Thoolen et ai. (1982) observed similar falls in MAP but again with a higher dose of clonidine. The lack of a more pronounced hypotensive effect with their 5-fold higher dose may be explained on the basis of stimulation of vascular a-adrenoceptors by clonidine. Thus in SHR, as in man (Wing et al., 1977; Reid et al., 1980) clonidine may have a narrow therapeutic range. There was an apparent delay of 1-2 days in onset of the hypotension in both our experiment and that of Thoolen et al. (1981b). This phenomenon cannot be explained wholly on the basis of a start up time (4 h) for the osmotic minipumps. When a single dose of clonidine was given i.m. blood pressure fell quickly: there was 27% fall in MAP in the h following the injection (Parker et al., 1982). Thus the mechanism of the hypotensive action of clonidine may be different when it is given as a single injection or via a chronic, rate-controlled delivery schedule. Finally at the end of our 10 days" treatment with clonidine there was no significant difference between clonidine-treated and control rats. This weakness of the effect of our chronic clonidine treatment in the long run may be due to tolerance developing to the hypotensive effect of clonidine or to the tailing off of pump flow towards the end of the functional life of the pump.
4.2. Clonidine withdrawal syndrome: experimental protocol and changes in M A P and HR Osmotic minipumps ran until emptied of their contents. Flow rate was checked by measurement of radioactivity. Other dosage schemes have their advantages and disadvantages. Daily injections of clonidine have the advantage of precise determination of the time of the last injection. However, animals may suffer withdrawal crises after each
single daily injection (Salzmann, 1979; Oates et al., 1978). Another alternative is to give clonidine in the diet or in the drinking water. As rats eat and drink little during the light period, once again the possibility arises of daily withdrawal crises. Furthermore, the onset of withdrawal cannot be determined precisely unless consumption is monitored. A final possibility for inducing withdrawal is by surgical removal of the minipump. This has the advantage of precise determination of the time of cessation but the disadvantage that the surgery may modify the withdrawal syndrome. Surgical interventions in the SHR induce falls in food intake and it is known that food deprivation in the rat causes falls in blood pressure (Einhorn et al., 1982). Clonidine withdrawal was characterized by a return of MAP to control values with a slight overshoot of HR. MAP and H R labilities increased. The phenomenon lasted approximately 20 h. Our results are in general agreement with those of Thoolen et al. (1981b) although they observed more pronounced cardiovascular changes. Heart rate overshoot was greater and the changes in MAP and HR lasted longer than in our experiments. Two factors probably explain the difference. Firstly, Thoolen et al. (1981b) gave a higher dose of clonidine and the magnitude of clonidine withdrawal symptoms such as overshoot tachycardia is dependent on dose (DiStefano et al.. 1980). Secondly, they removed their osmotic minipumps surgically, thus the withdrawal syndrome in their experiments was provoked by a combination of withdrawal of clonidine treatment and of the surgery.
4.3. Changes in peripheral and central catecholamine synthesizing enzymes during clonidine treatment and its withdrawal The only significant effect of chronic treatment with clonidine on central enzymatic activities is a decrease in DBH and P N M T activities in the A 1 / C 1 region. Nakamura et al. (1980) did not detect any change in the DBH activity of the A1 region. Their clonidine treatment was shorter (4 days) than ours (7 days). Furthermore in our study the more rostrally located A 1 / C 1 region includes
104
adrenaline neurons (Chamba and Renaud, 1983) which also contain DBH. It is possible that clonidine has a more marked effect on adrenaline than on noradrenaline neurons. Such a possibility is supported by our observation of a decrease in P N M T activity in the same area. Single injections of clonidine decrease adrenaline turnover in the C1 area (Scatton et al., 1979). Using a protocol similar to the present one we showed that following 7 days' treatment with clonidine the only significant change in central monoaminergic function observed was an increase in the adrenaline level of the hindbrain (Atkinson et al.. 1985). The other brain regions we studied were unaffected by clonidine treatment. This confirms e.g. the results of Kane and Johnson (1978) showing that there was no change in TH activity in A6. Furthermore, there was no change in TH activity in A9, a dopaminergic region taken as control area unlikely to be involved in the central control of blood pressure. Prolonged clonidine treatment did not affect catecholamine synthesizing enzyme activities in peripheral tissues such as adrenal medulla. This confirms previous reports of the Johnson group on the tyrosine hydroxylase activity of the adrenal gland (Dix and Johnson, 1977) and superior cervical ganglion or coeliac ganglion (Kane and Johnson, 1978). There was however, an increase in both T H and P N M T activities during withdrawal. Such an increase in T H activity after 24 h withdrawal from clonidine has been previously reported by Dix and Johnson (1977). There are many indications of an increase in sympatho-adrenal activity during withdrawal. Thus increases in T H activity in sympathetic ganglia (Kane and Johnson, 1978; DiStefano et al., 1980) and increases in plasma noradrenaline (Saito, 1981; Thoolen et al., 1981b) have been reported. In our studies, and in other reports, the increased peripheral sympathetic activity occurs during the first 2 days following cessation of clonidine treatment. It thus coincides with the cardiovascular hyperactivity. The increase in peripheral sympatho-adrenal activity during withdrawal is accompanied by changes in central enzymatic activities in both A 1 / C 1 and A 2 / C 2 regions. There is a decrease in DBH activity in the A 2 / C 2 region. As this decrease is not accompa-
nied by a parallel decrease in PNMT, it is possible that it occurs in the noradrenaline neurons rather than in the adrenaline neurons coexisting in the same area. A similar decrease in capacity of synthesis of noradrenaline in this area has been produced by chemical (Snyder et al., 1978) and electrolytic (Talman et al., 1980) lesions. Such lesions are known to induce blood pressure lability. Blood pressure lability was also one of the features of clonidine withdrawal in the SHR in our experiments. DBH activity in the A I / C 1 area remained significantly decreased during withdrawal. P N M T activity, however, which was diminished during treatment, returned to a level comparable to that of the controls. These two results could suggest decreased synthesis of noradrenaline in the A1 part of this area associated with a rebound in synthesis of adrenaline in the CI area. In contrast with the noradrenergic AI neurons, the adrenergic C1 neurons project directly to the intermediolateral column of the spinal cord (Ross et al., 1981) and have been shown by various approaches to be a key element of the sympathetic vasopressor system (Reis et al., 1984a,b). Thus, the return of MAP to control levels is accompanied by restoration of the capacity of synthesis of adrenaline within the vasopressor adrenaline CI neurons. However, a definitive causal relationship between these two phenomena remains to be established.
Acknowledgements This v,'ork was supported by the Swiss National Science Foundation Grant no. 3.759.080: by a grant from the Sandoz Foundation, (Basel, Switzerland). by Boehringer, Switzerland and by a "Jeune Equipe' award from the ' Minist~re de l'Education Nationale (Direction de la Recherche) Paris, France.
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14)6
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