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Intrathecal coadministration of clonidine with serotonin receptor agonists produces supra-additive visceral antinociception in the rat
Brain Research, 555 (1991) 35-42 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939116816Y
35
BRES 16816
Intrathecal coadministration of clonidine with serotonin receptor agonists produces supra-additive visceral antinociception in the rat R.M. Danzebrink and G.F. Gebhart Department of Pharmacology, The University of Iowa, Collegeof Medicine, Iowa City, 1A 52242 (U.S.A.)
(Accepted 26 February 1991) Key words: Antinociception; Clonidine; DOI; Intrathecal; RU-24969; Visceral; Isobolographic
The intrathecal (i.t.) coadministration of sub-antinociceptive doses of clonidine, an a2-adrenoceptor agonist, with DO I or RU-24969 (5-HT2 or 5-HTm receptor agonists, respectively) produced dose-dependent supra-additive antinociceptive effects in a model of visceral pain. The enhanced attenuation of responses to noxious colorectal distension produced by the coadministration of these drugs is evidenced by significant leftward shifts in the dose-response curves as compared to those of each drug alone and by isobolographic analysis. The supra-additive antinociceptive effects produced following the i.t. coadministration of clonidine with RU-24969 were antagonized by i.t. pretreatment with phentolamine; the coadministration of phentolamine with methysergide produced no greater antagonism of effects. The supra-additive antinociceptive effects produced by i.t. coadministration of clonidine with DOI were antagonized by i.t. pretreatment with methysergide; the coadministration of methysergide with yohimbine produced no greater antagonism of effects. These data suggest that receptors acted upon by descending bulbospinal neurons interact to modulate the rostrad transmission of visceral nociceptive transmission.
INTRODUCTION The involvement of descending pathways in the modulation of nociceptive transmission in the spinal cord is well established. Electrical stimulation in the nucleus raphe magnus, a major source of serotonin (5-HT) terminals in the lumbar dorsal horn, inhibits the responses of spinal dorsal horn neurons to noxious cutaneous stimulation 4,9,13. Central serotonergic pathways are believed to be important in spinal nociceptive processing since drugs which directly stimulate 5-HT receptors (e.g., 5-HT, MK-212, quipazine) elevate nociceptive thresholds in cutaneous and visceral models of pain 6'31. Other pharmacological treatments that increase the availability of 5-HT (e.g., administration of 5-HT or a 5-HT-releasing agent such as p-chloroamphetamine) also inhibit nociceptive reflexes 12. Furthermore, decreased functioning of descending 5-HT pathways can enhance nociceptive responses (e.g. ref. 24, but see ref. 11). There also is compelling evidence to support the role of spinal adrenoceptors in the modulation of nociceptive transmission. Focal electrical ~5 and chemical 14 activation of norepinephrine (NE)-containing cells in the nucleus locus coeruleus inhibit the responses of dorsal horn
neurons to noxious peripheral stimulation. Furthermore, intrathecally (i.t.) administered a2-adrenoceptor agonists dose-dependently elevate nociceptive thresholds in cutaneous 25'27 and visceral6 models of pain; these effects are antagonized by i.t. administered a2- , but not aladrenoceptor antagonists. The possible spinal interaction between these descending bulbospinal systems has been implied 32. Recently, Minor et al. 2°, and Archer et al. 2, reported that the antinociceptive efficacy of i.t. administered 5-HT was completely antagonized by i.t. pretreatment with the NE-depleting agent N-2-chloroethyl-N-ethyl-2-bromobenzylamine (DSP4) in tail-flick, hot-plate and shock titration tests in rats. Central noradrenergic depletion (DSP4 or 6-hydroxydopamine (6-OHDA)) also abolished the antinociceptive effects produced following the i.t. administration of 5-methoxy-N,N-dimethyltryptamine (5MeODMT) or p-chloroamphetamine in hot plate, tail flick and shock titration tests 3. The antinociceptive effects of 5-MeODMT that were abolished by i.t. pretreatment with 6 - O H D A were restored by i.t. administration of NE just prior to 5-MeODMT administration 19. Furthermore, peripheral noradrenergic depletion had no effect on the antinociception produced by systemic 5-MeODMT, suggesting that central but not
Correspondence: R.M. Danzebrink, Department of Pharmacology, The University of Iowa, College of Medicine, Iowa City, IA 52242, U.S.A. Fax: (1) (319) 335-7903.
36 p e r i p h e r a l n o r a d r e n e r g i c m e c h a n i s m s are i n v o l v e d in the a n t i n o c i c e p t i v e effects p r o d u c e d by 5 - M e O D M T 18. T h e aim of the p r e s e n t study was to e x a m i n e the interaction
between
descending
catecholamine-con-
taining b u l b o s p i n a l n e u r o n s in a m o d e l of visceral pain. Since n o c i c e p t i v e r e s p o n s e s to c o l o r e c t a l distension can be easily m e a s u r e d
in a w a k e ,
unanesthetized
rats, a
p h a r m a c o l o g i c a l investigation of the i n t e r a c t i o n b e t w e e n receptors
acted
upon
by d e s c e n d i n g
monoaminergic
achieved by preparing each drug in a concentration which allowed delivery in 3.75/d such that the combination of both drug solutions just prior to injection resulted in delivery of the desired doses in 75 /d. Antagonists were administered in a similar manner. The first i.t. injection was begun 1 min following the last baseline colorectal distension. Subsequent i.t. injections were spaced 12 min apart, thus permitting 4 colorectal distensions per dose of drug or vehicle (i.e., at 2, 5, 8, and 11 min after each i.t. injection). In all experiments the first i.t. injection was vehicle and the first dose of drug was given 12 min later. Adrenoceptor or serotonin receptor antagonists, when studied, were given as a single pretreatment 12 min prior to the administration of the agonists.
systems was c o n d u c t e d in intact animals. A p r e l i m i n a r y r e p o r t of t h e s e d a t a has b e e n m a d e 7. MATERIALS AND METHODS
Subjects and stimuli Experiments were conducted in male Sprague-Dawley rats (Biolab, St. Paul, MN) weighing 350-450 g. Distension of the descending colon and rectum was achieved by pressure-controllled air inflation of a 7-8 cm flexible, latex balloon. The noxious quality of colorectal distension has been characterized 22 and the method described 6'7'22. Briefly, the balloon was inserted intra-anally such that the end of the balloon was 1 cm anterior to the anus. An in-line pressure transducer (PX136 Omega Engineering, Inc., Stamford, CT) continuously monitored pressure within the balloon; pressure was controlled by a device described previously 1. Colorectal distension in awake rats produces an increase in mean arterial pressure (MAP), a readily quantifiable pseudaffective response that involves supraspinal integration 22. To measure the change in MAP (AMAP) in these experiments, phasic colorectal distension (80 mm Hg, 20 s) was the noxious stimulus. This cardiovascular response has been previously characterized 6'7'22.
Surgical preparation All surgical procedures were performed under sodium pentobarbital anesthesia (50 mg/kg) administered i.p. Femoral arterial and i.t. catheters were implanted for measurement of arterial pressure and drug administration. Intrathecal catheters (PE 10 tubing, 8.5 cm long) were inserted through the atlanto-occipital membrane into the subarachnoid space and directed down the spinal cord until the tip was at the lumbar enlargement 3°. The catheter was then surgically anchored to the musculature of the back of the neck to preserve its position in the i.t. space. Location of the distal end of the catheter was verified at the end of an experiment by injection of Fast green dye and post mortem examination of the spinal cord. The arterial catheter was guided s.c. for dorsal exteriorization at the neck, just caudal to the i.t. catheter, to provide long-term access and protection from dislocation. Rats were treated post-operatively with 30,000 U of Penicillin G (Flo-Cillin, Bristol Labs) i.m. to prevent bacterial infection, and with 20 ml Dextrose 5% in 0.9% saline s.c. to prevent dehydration and weight loss. Animals were housed individually following surgery with free access to food and water and were allowed to recover 1 week prior to use.
Experimental protocol On the day of an experiment a flexible latex balloon was positioned in the descending colon and rectum of an awake, unanesthetized rat. For insertion, the balloon was lightly coated with a surgical lubricant (Surgilube, Fougera & Co.). Each animal was tested on multiple days (not more than 4), but never received the same drug twice, and recovered 3 days between experimental tests. Colorectal distensions were given every 3 min. Three baseline values for AMAP in response to colorectal distension were determined before drug administration was initiated. All drugs, as well as the control dose of vehicle, were given i.t. in equal volumes (7.5/~l). The coadministration of two drugs was
Drugs The az-adrenoceptor agonist used in this study was clonidine HCI (a generous gift of Boehringer Ingelheim Ltd., Elmsford, NY). Adrenoceptor antagonists used were yohimbine HC1 and idazoxan HCI (Sigma Chemical Co., St. Louis, MO.), prazosin HCI (a generous gift of Pfizer Inc., New York, NY) and phentolamine HCI (a generous gift of Ciba-Geigy, Summit, NJ). The opioid receptor antagonist employed was naloxone HCI (Sigma Chemical Co., St. Louis, MO). The 5-HT m receptor agonist was RU-24969 succinate (a generous gift of Roussel-Uclaf, Romainville, France) and the 5-HT 2 receptor agonist was DOI (1-(2,5-dimethoxy-4-iodophenyl)2-aminopropane HCI; Research Biochemieals, Inc., Natick, MA). The 5-HT receptor antagonist administered was methysergide maleate (a generous gift of Sandoz Pharmaceuticals, East Hanover, NJ). All drugs were freshly prepared in deionized water in concentrations that allowed i.t. injections to be made in volumes of 7.5/~1. Because of solubility limitations, moderate heating and stirring were required to dissolve methysergide, yohimbine and prazosin. All i.t. injections were made by hand and followed by a 7.5/~1 flush of water to clear the catheter and insure complete drug delivery. The drug or vehicle injection as well as the water flush were delivered over a 1 min period. All i.t. drug doses are presented as ag of the salt forms described above.
Data statistics The mean data from cumulative doses were used to determine linearity and deviation from parallelism by a one-way ANOVA. EDs0 values were calculated by a separate EDso program. Data for AMAP are reported as % control. The EDso is defined as the dose of drug which produces a half maximum response (i.e., the dose of drug that reduced the AMAP to 50% of the maximum increase in response to an 80 mm Hg, 20 s distension). Isobolographic analysis for drug-drug interactions were conducted following the procedure of Tallarida et al. 29. RESULTS T h e i.t. a d m i n i s t r a t i o n of c l o n i d i n e ( 2 - 8 p g ) p r o d u c e d a d o s e - d e p e n d e n t a t t e n u a t i o n of t h e A M A P in r e s p o n s e to n o x i o u s c o l o r e c t a l distension with an E D s o of 5 ± 2 p g (Fig. 1, original d a t a of d o s e - r e l a t e d a n t i n o c i c e p t i v e effects p r o d u c e d by c l o n i d i n e are d e r i v e d f r o m D a n z e brink and G e b h a r t 6 ) . I n t r a t h e c a l l y a d m i n i s t e r e d R U 24969 (60-240 # g ) , a 5 - H T m r e c e p t o r agonist, and D O I (25-100 p g ) , a 5 - H T 2 r e c e p t o r agonist, e a c h p r o d u c e d d o s e - d e p e n d e n t a n t i n o c i c e p t i v e effects as e v i d e n c e d by attenuation
of the A M A P
in r e s p o n s e
to c o l o r e c t a i
distension (Fig. 2). T h e EDsos of R U - 2 4 9 6 9 and D O I w e r e d e t e r m i n e d to be 122 ± 44 p g and 54 + 10 p g , r e s p e c t i v e l y (original d a t a d e r i v e d f r o m D a n z e b r i n k and GebhartX).
37 Comparison of cumulative doses of clonidine with half the EDso of RU-24969 or DOI to cumulative doses of clonidine alone The i.t. coadministration of sub-antinociceptive doses of clonidine (0.5-2/~g) with half the EDs0 of RU-24969 (61/~g) produced antinociceptive effects that were equivalent to those produced by greater doses of clonidine given alone (Fig. 1A). The potent interaction of the aE-adrenoceptor agonist with the 5-HTla receptor agonist which resulted in enhanced antinociceptive effects was dose-related. Further enhancement of antinociceptive effects was not produced following the coadministration of the EDs0 of clonidine (4 #g) with half the EDso of RU-24969. The i.t. coadministration of sub-antinociceptive doses of clonidine (1.0-2 gg) with half the ED50 of DOI (28 ktg) also produced antinociceptive effects that were equivalent to those produced by greater doses of clonidine given alone (Fig. 1B). These enhanced antinociceptive effects also were dose-related.
A
Comparison of the interaction of cumulative doses of RU-24969 or DOI with 1/4 or 1/2 the EDso of clonidine to cumulative doses of RU-24969 or DOI alone The antinociceptive effects produced by RU-24969 (61-244 ~g) alone and in combination with clonidine (1-2 gg) are presented in Fig. 2A. The two lesser doses of RU-24969 (30 and 61 ktg) did not produce significant antinociceptive effects. However, coadministration of sub-antinociceptive doses of clonidine (1 or 2/~g -- 1/4 or 1/2; EDs0) with RU-24969 (30-122 gg) resulted in a significant leftward parallel shift of the dose-response curve. These data reveal that: (i) the administration of lesser doses of clonidine with doses of RU-24969 less than or equal to the EDs0 enhance the antinociceptive effects produced by similar doses of RU-24969 alone; (it) the enhanced antinociceptive effects of RU-24969 are maximal following coadministration with the least dose of clonidine tested (i.e., the effects produced following the coadministration of 1 ~g or 2 pg clonidine with RU-24969
A control 120
control 120
0 clonidine • clonldlne + 1 / 2 ED-50 RU-24969
• RU-24969 • RU-24969 + 1 / 4 ED-50 clonldlne 0 RU-24969 + 1 / 2 ED-50 ¢lonldine
100
100 -r E
80
-,E E
~
6o
o.
a.
•~
80 60 40
40 2O
20 O O
i
i
i
i
i
0.1
0.3
1
3
10
i
i
0
30
1O0
Dose R U - 2 4 9 6 9
300
(#g)
Dose Clonidlne ( # g )
B
B % control 120
control 120
0 clonidine • clonidine + I / 2 £D-50 DOI
• ooI • DOI + 1/4 ED-50 clonldlne 0 DOI + 1/2 ED-50 clonldlne
100 100
"~ -r E
80
~
60
~ ~
60
o.
o.
~
8o
E
"5
40
40 20
20 O 0.1
i
i
i
0.3
3
10
Dose Clonldine (/~g)
Fig. 1. A: dose-dependent attenuation of the AMAP by i.t. cumulative doses of clonidine (2-8/lg) and by i.t. coadministration of clonidine (0.5-4/~g) with RU-24969 (61/~g = 1/2 EDs0). Data are mean values + S.E.M. as % control. B: dose-depenaent attenuation of the zIMAP by i.t. cumulative doses of clonidine (2-8/~g) and by i.t. coadministration of clonidine (0.5-4 #g) with DOI (26 gg = 1/2 EDs0 ). Data are mean values _+ S.E.M. as % control.
0
30
100
300
Dose DOI ( # g )
Fig. 2. A: dose-dependent attenuation of the AMAP by i.t. cumulative doses of RU-24969 (30-200 gg) and by i.t. coadministration of RU-24969 (30-120 gg) with clonidine (1/~g -- 1/4 EDs0 or 2~g = 1/2 EDso). Data are mean values + S.E.M. as % control. B: dose-dependent attenuation of the AMAP by i.t. cumulative doses of DOI (14-100 gg) and by i.t. coadministration of DOI (14-56pg) with clonidine (1/~g = 1/4 EDso, 2#g = 1/2 EDso). Data are mean values _+ S.E.M. as % control.
38 with clonidine (1 or 2/~g), which also was not expected to be antinociceptive, attenuated the AMAP to 46 + 4 or 36 + 11% of control. Furthermore, coadministration of the EDs0 dose of RU-24969 (122/zg) with clonidine (1 or 2/(g) attenuated responses to distension to 23 + 15% and 17 + 8% of control, respectively, whereas a 50% reduction would have been expected based on additivity. The enhanced antinociception produced by the coadministration of D O I with clonidine is presented in comparison to the effects produced by clonidine alone in Fig. 3B. When coadministered with 1 or 2 ~g clonidine, the EDs0 dose of D O I (56/~g) reduced the AMAP to noxious colorectal distension to 27 + 6 or 27 + 8% of control, respectively. These antinociceptive effects are greater than would be expected by additivity since the EDs0 dose of D O I in combination with 1 or 2 /~g clonidine would be expected to attenuate the AMAP to 50% of control. Supra-additive attenuation of the AMAP also is produced following the coadministration of 14/~g (54 + 12 or 50 + 14% of control) or 28/tg (58 + 20 or 32 + 7% of control) of DOI with 1 or 2 / t g clonidine, respectively. Since 14 or 28 ~g D O I do not attenuate the AMAP to noxious colorectal distension, antinociception would not have been expected to result from the coadministration of these doses of D O I with subantinociceptive doses of clonidine. Yet, these combinations attenuated the AMAP to approximately 50% of control.
are not statistically different); and (iii) the mechanism of the antinociception produced by the interaction may be similar to that of either clonidine or RU-24969 given alone since the combination of these drugs resulted in a parallel, leftward shift of the dose-response curve. The i.t. coadministration of 1 or 2/~g clonidine (1/4 or 1/2 EDs0 ) with D O I (1/4 EDs0 , 1/2 EDs0 or EDs0 ) resulted in enhanced antinociceptive effects as evidenced by a significant leftward, yet non-parallel, shift of the dose-response curve of D O I (Fig. 2B). For example, doses of D O I which alone do not produce significant antinociceptive effects (14-28 #g) were shown to produce significant attenuation of the increase in MAP to colorectal distension when administered in combination with sub-antinociceptive doses of cionidine. The antinociceptive effect produced following administration of the EDs0 dose of D O I (54/(g) also was enhanced when coadministered with clonidine. This interaction, like the clonidine-RU-24969 interaction, was maximal at a dose of 1/~g clonidine.
Comparison of effects produced by the interaction of clonidine with cumulative doses of a 5-HT receptor agonist to clonidine alone Fig. 3 compares the interactions of clonidine with RU-24969 or with D O I to clonidine alone. As is shown in Fig. 3A, sub-antinociceptive doses of RU-24969 produced significant attenuation of the AMAP produced by noxious colorectal distension when coadministered with sub-antinociceptive doses of clonidine. The combination of 30/~g RU-24969 with 1 or 2 ~g clonidine, which would not be expected to alter nociceptive responses based on additivity, attenuated the AMAP to noxious colorectal distension to 52 + 12 or 58 + 12% of control, respectively. Another combination of RU-24969 (61/tg)
A: Q C:
clonidine
•jvehlcle ~¢*ntrol (1 ~ ~. 1==,~
® r-
I=..t,., (= ,.g .i..~l..)
0%
B" I
clonldln.)
_o
,,, I + " ,', ! ÷ ','
The dose-response curves representing the coadministration of clonidine with RU-24969 (open circles) or with D O I (filled circles) in fixed ratios are shown in Fig. 4A; the dose of the mixture is represented on the abscissa. The dose-response curves of these interactions are not
+ RU-24969
•
= o
Isobolographic analysis
I '*
control (1 /~g ¢lonldlne) + 14 big
+ 28/~g + 56/~g
,*
J
'""
1
.u-=,,..
,
50%
&MAP (mrnHg, % control)
+ DOI
vehlcle
i
,.
clonidine
~---4e
J
t* I DOI
I--~*
J~ntrol (2 /~g clonldln*) ~+ 14/=g l + 28/ag ~--4. I + S6~lg J~-i.
I
t
100% 0%
50%
DOl
100%
AMAP (mmHg, % control)
Fig. 3. Attenuation of the AMAP produced following i.t. coadministration of clonidine (1/~g, top panel or 2 #g, bottom panel) with a serotonin receptor agonist. A: effects of clonidine given in combination with RU-24969 (30-122 k(g)- B: effects of clonidine given in combination with DOI (14-56 pg). Vehicle is water pretreatment. Control represents effects of cionidine (1 or 2/~g) alone. Data are mean values + S.E.M. as % control. *, Significant difference from control (P < 0.05).
39
A parallel (F = 19), suggesting that the mechanism for the interaction of clonidine with RU-24969 is different than that of clonidine with DOI. Data from the 'mix' dose-response curves were used to generate the isobolograms in Figs. 4B,C. In the isobologram, the ED50 dose of clonidine is represented on the abscissa and that of RU-24969 or D O I on the ordinate. The theoretical additive line is illustrated by a solid line connecting the EDs0 doses of clonidine with the 5-HT receptor agonists. The EDs0 for the combination of clonidine with D O I or RU-24969 in a ratio of 1:14 or 1:30, respectively, is calculated by the method of Tallarida et al. 29. Variances and confidence limits for this point were calculated from the variances of each component alone (e.g., clonidine or RU-24969 or DOI). The confidence limits for each component of the EDso of the mixture are represented such that the confidence limits of clonidine are shown as horizontal lines while those of the 5-HT receptor agonists are shown as vertical lines. The experimentally determined EDso for the combination of clonidine with D O I in a ratio of 1:14 was comprised of 1.5 _+ 0.5 #g clonidine and 20.5 + 6.5/~g DOI. This point is plotted as (1.5, 20.5) on the clonidine-DOI isobolograph (Fig. 4B). The theoretical additive EDs0 was calculated to be 2 + 0.05/zg clonidine and 29 _+ 0.65/zg DOI. The confidence limits of these points do not overlap and the potency ratio was found to be significant (P < 0.05). These data clearly show a supra-additive interaction. The experimentally determined EDso for the combination of clonidine with RU-24969 in a ratio of 1:30 is 1.5 + 0.5 a g clonidine and 47.5 _+ 15.5/zg RU-24969. This point is plotted as (1.5,47.5) on the clonidine-RU-24969 isobolograph (Fig. 4C) and was compared to the theoretical additive EDs0 (calculated to be 2 + 0.02 pg clonidine and 68 + 0.7/zg RU-24969) by a t-test for relative potency. The potency ratio was found to be significant (P < 0.05), indicating that the interaction is
A:
clonidine + DOI
• clonldlne + DOI (1:14) o clonldine + R U - 2 4 9 6 9 (1:30)
?, control 80
" • 60 E E
40
tt
20
<3
B
i
i
=
i
0
50
I O0
150
DOSemtx (/zg)
6O
~
40
0
.=
20
o r~
o
i
i
i
C
i
"~ 15o ::L
== loo o1 I
-~
50
= o
,'~
0
0 1 2 3 4 5 6 Dose clonldlne (#g) Fig. 4. A: dose-dependent attenuation of the AMAP following the i.t. coadministration of clonidine (1-4/~g) with DOI (14-56/~g, filled circles; ratio of clonidine:DOI = 1:14) or RU-24969 (30-122 pg, open circles; ratio of clonidine:RU-24969 = 1:30). Data are mean values +_ S.E.M. as % control. Isoholograms for the effective doses for i.t. coadministration of donidine with DOI (B) or RU-24969 (C) to attenuate to 50% the d M A P in response to colorectal distension. The solid line represents the additive line constructed by joining the EDs0 dose of clonidine with that of DOI (B) or RU-24969 (C). The confidence limits for the clonidine (horizontal) and DOI or RU-24969 (vertical) components of each EDs0 are indicated for the actual mixture (solid circle) and for the theoretical additive point (open circle). The isobol points are determined from the EDso doses of ratios of clonidine to DOI (1:14) and to RU-24969 (1:30).
B:
clonidine + R U - 2 4 9 6 9
vehicle
vehicle control (clon. + DOI)
J
control (clon. + R U ) p
i
I
+ phentolamlne
+ phentol. ~
+ prazoeln
I
+ yohimbine
J
i
I
'
+ yohlmblne + methyserglde
+ methyserglde
+ methysergide + yohlmbine
+
i
50% Z~MAP (mmHg, % control)
I
'
I
=
+ rnethyserglde + phentolamlne
+ naloxone
0%
i
0 1 2 3 4 5 6 Dose clonldlne (p,g)
100~
0~0
Idazoxan
l
50~0
I00~
&MAP (mmHg, ~ control)
Fig. 5. Effects of antagonist pretreatment on attenuation of the ,dMAP produced by the i.t. coadministration of clonidine (1 #g = 1/4 ED50) with DOI (28/~g = 1/2 ED50; A) or with RU-24969 (61/~g = 1/2 EDso; B). Data are mean values S.E.M. as % control. *, Significant difference from control (P < 0.05, control = 1/zg clonidine + 28/,g DOI or 61/,g RU-24969). Vehicle = water pretreatment = 100%. Each antagonist was administered as an i.t. pretreatment in a 30/~g dose. Phentol. = phentolamine.
40 different from additivity (i.e., synergy). This is illustrated by the fact that the confidence limits of the points on the isobol do not overlap.
Antagonist pretreatment The possible mechanisms underlying the enhanced antinociceptive effects produced following the i.t. coadministration of 1/~g clonidine with 1/2 the EDso of DOI or of RU-24969 were tested by i.t. pretreatment with various receptor-selective antagonists. As is shown in Fig. 5A, the interaction between clonidine and DOI, which attenuated the AMAP to noxious colorectal distension to 58% of control, is antagonized to a similar extent by pretreatment with methysergide (30/~g) alone or by the combination of methysergide (30 #g) with yohimbine (30 /~g). Significant antagonism of this interaction was not achieved following pretreatment with phentolamine (30 /~g), yohimbine (30/~g) or naloxone (15 ~g). The interaction of clonidine with RU-24969, which attenuated the AMAP to colorectal distension to 46% of control, was significantly antagonized by i.t. pretreatment with phentolamine (30/~g) alone or by the coadministration of methysergide (30/~g) with phentolamine (30/~g), but not by pretreatment with prazosin (30 #g), yohimbine (30/~g), methysergide (30/~g) or idazoxan (30 /~g; Fig. 5B). DISCUSSION The results of the present study provide clear evidence for a functional interaction between descending noradrenergic and serotonergic systems in the modulation of visceral nociception. This interaction involves, at least in part, spinal aE-adrenoceptors, 5-HTla and 5-HT2 receptors. Isobolographic analysis reveals that the i.t. coadministration of sub-antinociceptive doses of clonidine with either RU-24969 or DOI produces supra-additive antinociceptive effects in response to noxious colorectal distension.
Clonidine and RU-24969 The coadministration of clonidine with RU-24969 produces a parallel, significant leftward shift of the dose-response curve of clonidine or RU-24969. This suggests that the mechanism responsible for the interaction (i.e., the enhanced antinociceptive effects) is similar to the mechanism underlying the antinociceptive effects of clonidine or RU-24969 when either drug is administered alone. A heterogeneity of aE-adrenoceptors in rat has been suggested. Determination of the amino acid sequence 16 and molecular weight 17 of the aE-adrenoceptor have led to the identification of two structurally distinct a2-adrenoceptor subtypes. Cloning and gene
expression studies have revealed that the genes for these two a2-adrenoceptor subtypes are located on different chromosomes 16'26. Recently, Brown et al. 5 showed that the binding of yohimbine to azb-adrenoceptors could be competed for by RU-24969 or methysergide in a complex manner. It remains to be determined whether RU-24969 and methysergide inhibit binding of yohimbine to this site competetively or allosterically. Nevertheless, a possible mechanism for the interaction of clonidine with RU24969 is a positive cooperativity of binding to the a2b-adrenoceptor subtype. The fact that the interaction described in the present study is antagonized by pretreatment with phentolamine suggests the likely involvement of a-adrenoceptors, al-Adrenoceptor and imidazoline receptors are not involved since the enhanced antinociceptive effects resultant from coadministration of clonidine and RU-24969 were not antagonized by prazosin or idazoxan, respectively. Methysergide also did not antagonize the interaction of clonidine with RU-24969. These data provide more evidence for a functional interaction between 5-HT and az-adrenoceptors. Both 5-HT 1 receptors 23 and az-adrenoceptors 1° have been shown to be linked to a guanine nucleotide, specifically G i. That these receptors are coupled to the same effector system provides an alternate level at which the interaction of clonidine with RU-24969 may occur. For example, clonidine and RU-24969 may interact to activate G i to a greater extent than could be achieved if either drug was administered alone. To date, characterization of the spinal interaction of 5-HT receptors with adrenoceptors has been largely accomplished using neurotoxins. For example, Minor et al. 2° first reported that the antinociceptive effect of i.t. administered 5-HT was blocked by pretreatment with the NE depleting agent DSP4. Since then it has been well described that the antinociception resultant from i.t. administration of 5-HT or 5-HT 1 receptor agonists is dependent on an intact noradrenergic system. Data from the present study provide pharmacological evidence in support of this hypothesis. Rather than destruction of the noradrenergic system, a2-receptors were antagonized in the present study by phentolamine. Presumably, this i.t. pretreatment attenuates the influence of the noradrenergic system at post-synaptic sites without altering the anatomical basis for the interaction. Thus, the noradrenergic system remains intact and receptor supersensitivity does not develop. The present report documented that i.t. pretreatment with phentolamine antagonizes the antinociceptive effects produced by coadministration of clonidine with RU-24969, suggesting that attenuation of the antinociceptive efficacy of 5-HT receptor agonists may in fact be related to compromised a2-mediated effects.
41 Clonidine and D O I The i.t. coadministration of DOI with clonidine produced a non-parallel, yet significant leftward shift of the DOI dose-response curve. These data suggest that the mechanism for interaction between 5-HT 2 receptors and aE-adrenoceptors is different from that responsible for the RU-24969-clonidine interaction. Studies involving antagonist pretreatment confirm this hypothesis since methysergide antagonized the interaction between clonidine and DOI whereas pretreatment With phentolamine, yohimbine or naloxone did not. It has been suggested that endogenous opioids may be released by catecholamines in the lumbar spinal cord and that the antinociceptive effects of noradrenergic agonists, for example, may in part be due to opioidergic mechanisms 33. Pretreatment with i.t. naloxone was used to evaluate the involvement of opioid receptors in the enhanced antinociceptive effects produced by the clonidine-DOI interaction in the present study. Naloxone did not antagonize the effects resulting from this interaction. Thus, opioid receptors apparently are not significantly involved in the interaction of 5-HT 2 receptors with a2-adrenoreceptors. The nature of the interaction between 5-HT2 receptors and a2-adrenoceptors is not easily defined. While clonidine is known to exert its effects through activation of the G i guanine nucleotide, DOI and other 5-HT2 receptor agonists are believed to be linked to phosphoinositide hydrolysis23. That these receptors are coupled to different effector systems suggests that the interaction produced by activation of 5-HT 2 receptors and a2-adrenoceptors occurs at a different cellular level. Blood pressure At the dosages employed in this study, the i.t. administration of DOI produces significant increases in resting blood pressure while clonidine or RU-24969 are without effect on resting MAP 6,s. The coadministration of clonidine with DOI or RU-24969 did not produce any effects on resting MAP in the present study that differed from what is produced by administration of either drug alone (data not shown). Thus, effects on resting cardiovascular tone were not produced and supra-additive antinociceptive effects occurred independently of changes in resting blood pressure.
SUMMARY These data provide the first pharmacological evidence for a functional interaction between spinal adrenoceptors and serotonin receptors in the modulation of visceral nociception. In behavioral studies, the antinociceptive efficacy of selective adrenoceptor or serotonin receptor
agonists in response to noxious cutaneous stimulation is known. However, the interaction between serotonin receptor agonists and NE has been implied only in response to noxious cutaneous stimuli; the subtype(s) of serotonin or adrenergic receptors were not identified. Recently, however, serotonin and clonidine were shown to interact synergistically to inhibit the noxious-evoked activity of wide dynamic range neurons in the dorsal horn of the spinal cord, suggesting the involvement of a 2adrenoceptors 21. The present study defines the role of two serotonin receptor subtypes in the modulation of visceral nociception by an aE-adrenoceptor agonist. The nature of the mechanism responsible for the defined interactions also was investigated. The nature of the interaction between 5-HT 2 receptors and a2-adrenoce ptors is unclear, but is apparently 'serotonin-like' since methysergide antagonized the effects produced by coadministration of clonidine with DOI; no further antagonism was produced by combined pretreatment with methysergide and phentolamine. The interaction of a2-adrenoceptors and 5-HT1B receptors is 'a-like' since the effects produced following the coadministration of clonidine with RU-24969 were antagonized by phentolamine; no further antagonism was produced by combined pretreatment with phentolamine and methysergide. That the mechanisms responsible for these interactions are different may be related to the pre- vs post-synaptic location of the receptor subtypes (e.g., 5-HT1B and a2-adrenoceptors may modulate the release of neurotransmitter from nerve terminals2S), modification of binding to the receptor (e.g., RU-24969 and clonidine may compete for the same receptor) or the manner in which the interaction activates intracellular responses (e.g., a 2adrenoceptors and 5-HT m receptors are coupled to the same effector system). Importantly, a significant interaction between two descending monoaminergic systems known to be involved in the modulation of nociceptive transmission is defined. That 5-HT receptors and a2-adrenoceptors communicate in some way to inhibit visceral nociception is relevant to the study of pain since little is known about the mechanisms responsible for visceral pain. Indeed the precise nature of interactions involved in nociceptive processing are hard to define in light of the complex neurochemistry, neuroanatomy and modulatory influences described in the dorsal horn of the spinal cord. Not addressed in this study is the possible involvement of other putative modulatory systems (e.g., adenosine, 7-aminobutyric acid, acetylcholine). That the described interaction may result from multisynaptic activity between different laminae of the dorsal horn of the spinal cord also is not addressed. Furthermore, the tonic activity of local circuit and descending neurons which
42 m o d u l a t e the release o f n e u r o t r a n s m i t t e r s and n e u r o m o d u l a t o r s is not i n v e s t i g a t e d here. A n u n d e r s t a n d i n g of h o w t h e s e factors i n t e r r e l a t e are critical to the u n d e r s t a n d i n g of n e u r a l m e c h a n i s m s i n v o l v e d in the m o d u l a -
Acknowledgments. We would like to thank Mike Burcham for production of the graphics and Marilynn Kirkpatrick for secretarial assistance. Supported by NIH Grants T32 GM 07069 (R.M.D.) and NS 19912 and an unrestricted pain research grant from BristolMyers Squibb Co.
tion o f spinal n o c i c e p t i v e transmission.
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17 Lanier, S.M., Homcy, C.J., Patenaude, C. and Graham, R.M.. Identification of structurally distinct a2-adrenergic receptors, J. Biol. Chem., 263 (1988) 14491-14496. 18 Minor, B.G., Archer, T., Post, C., Jonsson, G. and Mohammed, A.R., 5-HT agonist induced analgesia modulated by central but not peripheral noradrenaline depletion in rats, J. Neural Trans., 66 (1986) 243-259. 19 Minor, B.G., Persson, M.L., Post, C., Jonsson, G. and Archer, T., Intrathecal noradrenaline restores 5-methoxy-N,N-dimethyltryptamine-induced antinociception abolished by intrathecal 6-hydroxytryptamine, J. Neural Trans., 72 (1988) 107-120. 20 Minor, B.G., Post, C. and Archer, T., Blockade of intrathecal 5-hydroxytryptamine-induced antinociception in rats by noradrenaline depletion, Neurosci. Lett., 54 (1985) 39-44. 21 Nakagawa, I., Omote, K., Kitahata, L.M., Collins, J.G. and Murata, K., Serotonergic mediation of spinal analgesia and its interaction with noradrenergic systems, Anesthesiology, 73 (1990) 474-478. 22 Ness, T.J. and Gebhart, G.E, Colorectal distension as a noxious visceral stimulus: Physiologic and pharmacologic characterization of pseudoaffective reflexes in the rat, Brain Research, 450 (1988) 153-169. 23 Peroutka, S.J., 5-Hydroxytryptamine receptor subtypes: molecular, biochemical and physiological characterization, Trends Neurosci., 11 (1988) 496-500. 24 Proudfit, H.K., Reversible inactivation of raphe magnus neurons: effects of nociceptive threshold and morphine-induced analgesia, Brain Research, 201 (1980) 459-464. 25 Reddy, S.V., Maderdrut, J.L. and Yaksh, T.L., Spinal cord pharmacology of adrenergic agonist-mediated antinociception, J. Pharmacol. Exp. Ther., 213 (1980) 525-533. 26 Regan, J.W., Kobilka, T.S., Yang-Feng, T.L., Caron, M.G., Lefkowitz, R.J. and Kobilka, B.K., Cloning and expression of a human kidney cDNA for an a2-adrenergic receptor subtype, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 6301-6305. 27 Solomon, R.E., Brody, M.J. and Gebhart, G.F., Pharmacological characterization of a-adrenoceptors involved in the antinociceptive and cardiovascular effects of intrathecally administered clonidine, J. Pharmacol. Exp. Ther., 251 (1989) 27-38. 28 Starke, K., Regulation of noradrenaline release by presynaptic receptor systems, Rev. Physiol. Biochem. Pharmacol., 77 (1977) 1-124. 29 Tallarida, R.J., Porreca, E and Cowan, A., Statistical analysis of drug-drug and site-site interactions with isobolograms, Life Sci., 45 (1989) 947-961. 30 Yaksh, T.L. and Rudy, T.A., Chronic catheterization of the spinal subarachnoid space, Physiol. Behav., 17 (1976) 10311036. 31 Yaksh, T.L. and Wilson, P.R., Spinal serotonin terminal system mediates antinociception, J. Pharmacol. Exp. Ther., 208 (1979) 446-453. 32 Zemlan, EP., Corrigan, S.A. and Pfaff, D.W., Noradrenergic and serotonergic mediation of spinal analgesia mechanisms, Eur. J. Pharmacol., 61 (1980) 111-124. 33 Zorman, G., Belcher, G., Adams, J.E. and Fields, H.L., Lumbar intrathecal naloxone blocks analgesia produced by microstimulation of the ventro-medial medulla in the rat, Brain Research, 236 (1982) 77-84.
35
BRES 16816
Intrathecal coadministration of clonidine with serotonin receptor agonists produces supra-additive visceral antinociception in the rat R.M. Danzebrink and G.F. Gebhart Department of Pharmacology, The University of Iowa, Collegeof Medicine, Iowa City, 1A 52242 (U.S.A.)
(Accepted 26 February 1991) Key words: Antinociception; Clonidine; DOI; Intrathecal; RU-24969; Visceral; Isobolographic
The intrathecal (i.t.) coadministration of sub-antinociceptive doses of clonidine, an a2-adrenoceptor agonist, with DO I or RU-24969 (5-HT2 or 5-HTm receptor agonists, respectively) produced dose-dependent supra-additive antinociceptive effects in a model of visceral pain. The enhanced attenuation of responses to noxious colorectal distension produced by the coadministration of these drugs is evidenced by significant leftward shifts in the dose-response curves as compared to those of each drug alone and by isobolographic analysis. The supra-additive antinociceptive effects produced following the i.t. coadministration of clonidine with RU-24969 were antagonized by i.t. pretreatment with phentolamine; the coadministration of phentolamine with methysergide produced no greater antagonism of effects. The supra-additive antinociceptive effects produced by i.t. coadministration of clonidine with DOI were antagonized by i.t. pretreatment with methysergide; the coadministration of methysergide with yohimbine produced no greater antagonism of effects. These data suggest that receptors acted upon by descending bulbospinal neurons interact to modulate the rostrad transmission of visceral nociceptive transmission.
INTRODUCTION The involvement of descending pathways in the modulation of nociceptive transmission in the spinal cord is well established. Electrical stimulation in the nucleus raphe magnus, a major source of serotonin (5-HT) terminals in the lumbar dorsal horn, inhibits the responses of spinal dorsal horn neurons to noxious cutaneous stimulation 4,9,13. Central serotonergic pathways are believed to be important in spinal nociceptive processing since drugs which directly stimulate 5-HT receptors (e.g., 5-HT, MK-212, quipazine) elevate nociceptive thresholds in cutaneous and visceral models of pain 6'31. Other pharmacological treatments that increase the availability of 5-HT (e.g., administration of 5-HT or a 5-HT-releasing agent such as p-chloroamphetamine) also inhibit nociceptive reflexes 12. Furthermore, decreased functioning of descending 5-HT pathways can enhance nociceptive responses (e.g. ref. 24, but see ref. 11). There also is compelling evidence to support the role of spinal adrenoceptors in the modulation of nociceptive transmission. Focal electrical ~5 and chemical 14 activation of norepinephrine (NE)-containing cells in the nucleus locus coeruleus inhibit the responses of dorsal horn
neurons to noxious peripheral stimulation. Furthermore, intrathecally (i.t.) administered a2-adrenoceptor agonists dose-dependently elevate nociceptive thresholds in cutaneous 25'27 and visceral6 models of pain; these effects are antagonized by i.t. administered a2- , but not aladrenoceptor antagonists. The possible spinal interaction between these descending bulbospinal systems has been implied 32. Recently, Minor et al. 2°, and Archer et al. 2, reported that the antinociceptive efficacy of i.t. administered 5-HT was completely antagonized by i.t. pretreatment with the NE-depleting agent N-2-chloroethyl-N-ethyl-2-bromobenzylamine (DSP4) in tail-flick, hot-plate and shock titration tests in rats. Central noradrenergic depletion (DSP4 or 6-hydroxydopamine (6-OHDA)) also abolished the antinociceptive effects produced following the i.t. administration of 5-methoxy-N,N-dimethyltryptamine (5MeODMT) or p-chloroamphetamine in hot plate, tail flick and shock titration tests 3. The antinociceptive effects of 5-MeODMT that were abolished by i.t. pretreatment with 6 - O H D A were restored by i.t. administration of NE just prior to 5-MeODMT administration 19. Furthermore, peripheral noradrenergic depletion had no effect on the antinociception produced by systemic 5-MeODMT, suggesting that central but not
Correspondence: R.M. Danzebrink, Department of Pharmacology, The University of Iowa, College of Medicine, Iowa City, IA 52242, U.S.A. Fax: (1) (319) 335-7903.
36 p e r i p h e r a l n o r a d r e n e r g i c m e c h a n i s m s are i n v o l v e d in the a n t i n o c i c e p t i v e effects p r o d u c e d by 5 - M e O D M T 18. T h e aim of the p r e s e n t study was to e x a m i n e the interaction
between
descending
catecholamine-con-
taining b u l b o s p i n a l n e u r o n s in a m o d e l of visceral pain. Since n o c i c e p t i v e r e s p o n s e s to c o l o r e c t a l distension can be easily m e a s u r e d
in a w a k e ,
unanesthetized
rats, a
p h a r m a c o l o g i c a l investigation of the i n t e r a c t i o n b e t w e e n receptors
acted
upon
by d e s c e n d i n g
monoaminergic
achieved by preparing each drug in a concentration which allowed delivery in 3.75/d such that the combination of both drug solutions just prior to injection resulted in delivery of the desired doses in 75 /d. Antagonists were administered in a similar manner. The first i.t. injection was begun 1 min following the last baseline colorectal distension. Subsequent i.t. injections were spaced 12 min apart, thus permitting 4 colorectal distensions per dose of drug or vehicle (i.e., at 2, 5, 8, and 11 min after each i.t. injection). In all experiments the first i.t. injection was vehicle and the first dose of drug was given 12 min later. Adrenoceptor or serotonin receptor antagonists, when studied, were given as a single pretreatment 12 min prior to the administration of the agonists.
systems was c o n d u c t e d in intact animals. A p r e l i m i n a r y r e p o r t of t h e s e d a t a has b e e n m a d e 7. MATERIALS AND METHODS
Subjects and stimuli Experiments were conducted in male Sprague-Dawley rats (Biolab, St. Paul, MN) weighing 350-450 g. Distension of the descending colon and rectum was achieved by pressure-controllled air inflation of a 7-8 cm flexible, latex balloon. The noxious quality of colorectal distension has been characterized 22 and the method described 6'7'22. Briefly, the balloon was inserted intra-anally such that the end of the balloon was 1 cm anterior to the anus. An in-line pressure transducer (PX136 Omega Engineering, Inc., Stamford, CT) continuously monitored pressure within the balloon; pressure was controlled by a device described previously 1. Colorectal distension in awake rats produces an increase in mean arterial pressure (MAP), a readily quantifiable pseudaffective response that involves supraspinal integration 22. To measure the change in MAP (AMAP) in these experiments, phasic colorectal distension (80 mm Hg, 20 s) was the noxious stimulus. This cardiovascular response has been previously characterized 6'7'22.
Surgical preparation All surgical procedures were performed under sodium pentobarbital anesthesia (50 mg/kg) administered i.p. Femoral arterial and i.t. catheters were implanted for measurement of arterial pressure and drug administration. Intrathecal catheters (PE 10 tubing, 8.5 cm long) were inserted through the atlanto-occipital membrane into the subarachnoid space and directed down the spinal cord until the tip was at the lumbar enlargement 3°. The catheter was then surgically anchored to the musculature of the back of the neck to preserve its position in the i.t. space. Location of the distal end of the catheter was verified at the end of an experiment by injection of Fast green dye and post mortem examination of the spinal cord. The arterial catheter was guided s.c. for dorsal exteriorization at the neck, just caudal to the i.t. catheter, to provide long-term access and protection from dislocation. Rats were treated post-operatively with 30,000 U of Penicillin G (Flo-Cillin, Bristol Labs) i.m. to prevent bacterial infection, and with 20 ml Dextrose 5% in 0.9% saline s.c. to prevent dehydration and weight loss. Animals were housed individually following surgery with free access to food and water and were allowed to recover 1 week prior to use.
Experimental protocol On the day of an experiment a flexible latex balloon was positioned in the descending colon and rectum of an awake, unanesthetized rat. For insertion, the balloon was lightly coated with a surgical lubricant (Surgilube, Fougera & Co.). Each animal was tested on multiple days (not more than 4), but never received the same drug twice, and recovered 3 days between experimental tests. Colorectal distensions were given every 3 min. Three baseline values for AMAP in response to colorectal distension were determined before drug administration was initiated. All drugs, as well as the control dose of vehicle, were given i.t. in equal volumes (7.5/~l). The coadministration of two drugs was
Drugs The az-adrenoceptor agonist used in this study was clonidine HCI (a generous gift of Boehringer Ingelheim Ltd., Elmsford, NY). Adrenoceptor antagonists used were yohimbine HC1 and idazoxan HCI (Sigma Chemical Co., St. Louis, MO.), prazosin HCI (a generous gift of Pfizer Inc., New York, NY) and phentolamine HCI (a generous gift of Ciba-Geigy, Summit, NJ). The opioid receptor antagonist employed was naloxone HCI (Sigma Chemical Co., St. Louis, MO). The 5-HT m receptor agonist was RU-24969 succinate (a generous gift of Roussel-Uclaf, Romainville, France) and the 5-HT 2 receptor agonist was DOI (1-(2,5-dimethoxy-4-iodophenyl)2-aminopropane HCI; Research Biochemieals, Inc., Natick, MA). The 5-HT receptor antagonist administered was methysergide maleate (a generous gift of Sandoz Pharmaceuticals, East Hanover, NJ). All drugs were freshly prepared in deionized water in concentrations that allowed i.t. injections to be made in volumes of 7.5/~1. Because of solubility limitations, moderate heating and stirring were required to dissolve methysergide, yohimbine and prazosin. All i.t. injections were made by hand and followed by a 7.5/~1 flush of water to clear the catheter and insure complete drug delivery. The drug or vehicle injection as well as the water flush were delivered over a 1 min period. All i.t. drug doses are presented as ag of the salt forms described above.
Data statistics The mean data from cumulative doses were used to determine linearity and deviation from parallelism by a one-way ANOVA. EDs0 values were calculated by a separate EDso program. Data for AMAP are reported as % control. The EDso is defined as the dose of drug which produces a half maximum response (i.e., the dose of drug that reduced the AMAP to 50% of the maximum increase in response to an 80 mm Hg, 20 s distension). Isobolographic analysis for drug-drug interactions were conducted following the procedure of Tallarida et al. 29. RESULTS T h e i.t. a d m i n i s t r a t i o n of c l o n i d i n e ( 2 - 8 p g ) p r o d u c e d a d o s e - d e p e n d e n t a t t e n u a t i o n of t h e A M A P in r e s p o n s e to n o x i o u s c o l o r e c t a l distension with an E D s o of 5 ± 2 p g (Fig. 1, original d a t a of d o s e - r e l a t e d a n t i n o c i c e p t i v e effects p r o d u c e d by c l o n i d i n e are d e r i v e d f r o m D a n z e brink and G e b h a r t 6 ) . I n t r a t h e c a l l y a d m i n i s t e r e d R U 24969 (60-240 # g ) , a 5 - H T m r e c e p t o r agonist, and D O I (25-100 p g ) , a 5 - H T 2 r e c e p t o r agonist, e a c h p r o d u c e d d o s e - d e p e n d e n t a n t i n o c i c e p t i v e effects as e v i d e n c e d by attenuation
of the A M A P
in r e s p o n s e
to c o l o r e c t a i
distension (Fig. 2). T h e EDsos of R U - 2 4 9 6 9 and D O I w e r e d e t e r m i n e d to be 122 ± 44 p g and 54 + 10 p g , r e s p e c t i v e l y (original d a t a d e r i v e d f r o m D a n z e b r i n k and GebhartX).
37 Comparison of cumulative doses of clonidine with half the EDso of RU-24969 or DOI to cumulative doses of clonidine alone The i.t. coadministration of sub-antinociceptive doses of clonidine (0.5-2/~g) with half the EDs0 of RU-24969 (61/~g) produced antinociceptive effects that were equivalent to those produced by greater doses of clonidine given alone (Fig. 1A). The potent interaction of the aE-adrenoceptor agonist with the 5-HTla receptor agonist which resulted in enhanced antinociceptive effects was dose-related. Further enhancement of antinociceptive effects was not produced following the coadministration of the EDs0 of clonidine (4 #g) with half the EDso of RU-24969. The i.t. coadministration of sub-antinociceptive doses of clonidine (1.0-2 gg) with half the ED50 of DOI (28 ktg) also produced antinociceptive effects that were equivalent to those produced by greater doses of clonidine given alone (Fig. 1B). These enhanced antinociceptive effects also were dose-related.
A
Comparison of the interaction of cumulative doses of RU-24969 or DOI with 1/4 or 1/2 the EDso of clonidine to cumulative doses of RU-24969 or DOI alone The antinociceptive effects produced by RU-24969 (61-244 ~g) alone and in combination with clonidine (1-2 gg) are presented in Fig. 2A. The two lesser doses of RU-24969 (30 and 61 ktg) did not produce significant antinociceptive effects. However, coadministration of sub-antinociceptive doses of clonidine (1 or 2/~g -- 1/4 or 1/2; EDs0) with RU-24969 (30-122 gg) resulted in a significant leftward parallel shift of the dose-response curve. These data reveal that: (i) the administration of lesser doses of clonidine with doses of RU-24969 less than or equal to the EDs0 enhance the antinociceptive effects produced by similar doses of RU-24969 alone; (it) the enhanced antinociceptive effects of RU-24969 are maximal following coadministration with the least dose of clonidine tested (i.e., the effects produced following the coadministration of 1 ~g or 2 pg clonidine with RU-24969
A control 120
control 120
0 clonidine • clonldlne + 1 / 2 ED-50 RU-24969
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(#g)
Dose Clonidlne ( # g )
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0 clonidine • clonidine + I / 2 £D-50 DOI
• ooI • DOI + 1/4 ED-50 clonldlne 0 DOI + 1/2 ED-50 clonldlne
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Fig. 1. A: dose-dependent attenuation of the AMAP by i.t. cumulative doses of clonidine (2-8/lg) and by i.t. coadministration of clonidine (0.5-4/~g) with RU-24969 (61/~g = 1/2 EDs0). Data are mean values + S.E.M. as % control. B: dose-depenaent attenuation of the zIMAP by i.t. cumulative doses of clonidine (2-8/~g) and by i.t. coadministration of clonidine (0.5-4 #g) with DOI (26 gg = 1/2 EDs0 ). Data are mean values _+ S.E.M. as % control.
0
30
100
300
Dose DOI ( # g )
Fig. 2. A: dose-dependent attenuation of the AMAP by i.t. cumulative doses of RU-24969 (30-200 gg) and by i.t. coadministration of RU-24969 (30-120 gg) with clonidine (1/~g -- 1/4 EDs0 or 2~g = 1/2 EDso). Data are mean values + S.E.M. as % control. B: dose-dependent attenuation of the AMAP by i.t. cumulative doses of DOI (14-100 gg) and by i.t. coadministration of DOI (14-56pg) with clonidine (1/~g = 1/4 EDso, 2#g = 1/2 EDso). Data are mean values _+ S.E.M. as % control.
38 with clonidine (1 or 2/~g), which also was not expected to be antinociceptive, attenuated the AMAP to 46 + 4 or 36 + 11% of control. Furthermore, coadministration of the EDs0 dose of RU-24969 (122/zg) with clonidine (1 or 2/(g) attenuated responses to distension to 23 + 15% and 17 + 8% of control, respectively, whereas a 50% reduction would have been expected based on additivity. The enhanced antinociception produced by the coadministration of D O I with clonidine is presented in comparison to the effects produced by clonidine alone in Fig. 3B. When coadministered with 1 or 2 ~g clonidine, the EDs0 dose of D O I (56/~g) reduced the AMAP to noxious colorectal distension to 27 + 6 or 27 + 8% of control, respectively. These antinociceptive effects are greater than would be expected by additivity since the EDs0 dose of D O I in combination with 1 or 2 /~g clonidine would be expected to attenuate the AMAP to 50% of control. Supra-additive attenuation of the AMAP also is produced following the coadministration of 14/~g (54 + 12 or 50 + 14% of control) or 28/tg (58 + 20 or 32 + 7% of control) of DOI with 1 or 2 / t g clonidine, respectively. Since 14 or 28 ~g D O I do not attenuate the AMAP to noxious colorectal distension, antinociception would not have been expected to result from the coadministration of these doses of D O I with subantinociceptive doses of clonidine. Yet, these combinations attenuated the AMAP to approximately 50% of control.
are not statistically different); and (iii) the mechanism of the antinociception produced by the interaction may be similar to that of either clonidine or RU-24969 given alone since the combination of these drugs resulted in a parallel, leftward shift of the dose-response curve. The i.t. coadministration of 1 or 2/~g clonidine (1/4 or 1/2 EDs0 ) with D O I (1/4 EDs0 , 1/2 EDs0 or EDs0 ) resulted in enhanced antinociceptive effects as evidenced by a significant leftward, yet non-parallel, shift of the dose-response curve of D O I (Fig. 2B). For example, doses of D O I which alone do not produce significant antinociceptive effects (14-28 #g) were shown to produce significant attenuation of the increase in MAP to colorectal distension when administered in combination with sub-antinociceptive doses of cionidine. The antinociceptive effect produced following administration of the EDs0 dose of D O I (54/(g) also was enhanced when coadministered with clonidine. This interaction, like the clonidine-RU-24969 interaction, was maximal at a dose of 1/~g clonidine.
Comparison of effects produced by the interaction of clonidine with cumulative doses of a 5-HT receptor agonist to clonidine alone Fig. 3 compares the interactions of clonidine with RU-24969 or with D O I to clonidine alone. As is shown in Fig. 3A, sub-antinociceptive doses of RU-24969 produced significant attenuation of the AMAP produced by noxious colorectal distension when coadministered with sub-antinociceptive doses of clonidine. The combination of 30/~g RU-24969 with 1 or 2 ~g clonidine, which would not be expected to alter nociceptive responses based on additivity, attenuated the AMAP to noxious colorectal distension to 52 + 12 or 58 + 12% of control, respectively. Another combination of RU-24969 (61/tg)
A: Q C:
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The dose-response curves representing the coadministration of clonidine with RU-24969 (open circles) or with D O I (filled circles) in fixed ratios are shown in Fig. 4A; the dose of the mixture is represented on the abscissa. The dose-response curves of these interactions are not
+ RU-24969
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Fig. 3. Attenuation of the AMAP produced following i.t. coadministration of clonidine (1/~g, top panel or 2 #g, bottom panel) with a serotonin receptor agonist. A: effects of clonidine given in combination with RU-24969 (30-122 k(g)- B: effects of clonidine given in combination with DOI (14-56 pg). Vehicle is water pretreatment. Control represents effects of cionidine (1 or 2/~g) alone. Data are mean values + S.E.M. as % control. *, Significant difference from control (P < 0.05).
39
A parallel (F = 19), suggesting that the mechanism for the interaction of clonidine with RU-24969 is different than that of clonidine with DOI. Data from the 'mix' dose-response curves were used to generate the isobolograms in Figs. 4B,C. In the isobologram, the ED50 dose of clonidine is represented on the abscissa and that of RU-24969 or D O I on the ordinate. The theoretical additive line is illustrated by a solid line connecting the EDs0 doses of clonidine with the 5-HT receptor agonists. The EDs0 for the combination of clonidine with D O I or RU-24969 in a ratio of 1:14 or 1:30, respectively, is calculated by the method of Tallarida et al. 29. Variances and confidence limits for this point were calculated from the variances of each component alone (e.g., clonidine or RU-24969 or DOI). The confidence limits for each component of the EDso of the mixture are represented such that the confidence limits of clonidine are shown as horizontal lines while those of the 5-HT receptor agonists are shown as vertical lines. The experimentally determined EDso for the combination of clonidine with D O I in a ratio of 1:14 was comprised of 1.5 _+ 0.5 #g clonidine and 20.5 + 6.5/~g DOI. This point is plotted as (1.5, 20.5) on the clonidine-DOI isobolograph (Fig. 4B). The theoretical additive EDs0 was calculated to be 2 + 0.05/zg clonidine and 29 _+ 0.65/zg DOI. The confidence limits of these points do not overlap and the potency ratio was found to be significant (P < 0.05). These data clearly show a supra-additive interaction. The experimentally determined EDso for the combination of clonidine with RU-24969 in a ratio of 1:30 is 1.5 + 0.5 a g clonidine and 47.5 _+ 15.5/zg RU-24969. This point is plotted as (1.5,47.5) on the clonidine-RU-24969 isobolograph (Fig. 4C) and was compared to the theoretical additive EDs0 (calculated to be 2 + 0.02 pg clonidine and 68 + 0.7/zg RU-24969) by a t-test for relative potency. The potency ratio was found to be significant (P < 0.05), indicating that the interaction is
A:
clonidine + DOI
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0 1 2 3 4 5 6 Dose clonldlne (#g) Fig. 4. A: dose-dependent attenuation of the AMAP following the i.t. coadministration of clonidine (1-4/~g) with DOI (14-56/~g, filled circles; ratio of clonidine:DOI = 1:14) or RU-24969 (30-122 pg, open circles; ratio of clonidine:RU-24969 = 1:30). Data are mean values +_ S.E.M. as % control. Isoholograms for the effective doses for i.t. coadministration of donidine with DOI (B) or RU-24969 (C) to attenuate to 50% the d M A P in response to colorectal distension. The solid line represents the additive line constructed by joining the EDs0 dose of clonidine with that of DOI (B) or RU-24969 (C). The confidence limits for the clonidine (horizontal) and DOI or RU-24969 (vertical) components of each EDs0 are indicated for the actual mixture (solid circle) and for the theoretical additive point (open circle). The isobol points are determined from the EDso doses of ratios of clonidine to DOI (1:14) and to RU-24969 (1:30).
B:
clonidine + R U - 2 4 9 6 9
vehicle
vehicle control (clon. + DOI)
J
control (clon. + R U ) p
i
I
+ phentolamlne
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i
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+ naloxone
0%
i
0 1 2 3 4 5 6 Dose clonldlne (p,g)
100~
0~0
Idazoxan
l
50~0
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Fig. 5. Effects of antagonist pretreatment on attenuation of the ,dMAP produced by the i.t. coadministration of clonidine (1 #g = 1/4 ED50) with DOI (28/~g = 1/2 ED50; A) or with RU-24969 (61/~g = 1/2 EDso; B). Data are mean values S.E.M. as % control. *, Significant difference from control (P < 0.05, control = 1/zg clonidine + 28/,g DOI or 61/,g RU-24969). Vehicle = water pretreatment = 100%. Each antagonist was administered as an i.t. pretreatment in a 30/~g dose. Phentol. = phentolamine.
40 different from additivity (i.e., synergy). This is illustrated by the fact that the confidence limits of the points on the isobol do not overlap.
Antagonist pretreatment The possible mechanisms underlying the enhanced antinociceptive effects produced following the i.t. coadministration of 1/~g clonidine with 1/2 the EDso of DOI or of RU-24969 were tested by i.t. pretreatment with various receptor-selective antagonists. As is shown in Fig. 5A, the interaction between clonidine and DOI, which attenuated the AMAP to noxious colorectal distension to 58% of control, is antagonized to a similar extent by pretreatment with methysergide (30/~g) alone or by the combination of methysergide (30 #g) with yohimbine (30 /~g). Significant antagonism of this interaction was not achieved following pretreatment with phentolamine (30 /~g), yohimbine (30/~g) or naloxone (15 ~g). The interaction of clonidine with RU-24969, which attenuated the AMAP to colorectal distension to 46% of control, was significantly antagonized by i.t. pretreatment with phentolamine (30/~g) alone or by the coadministration of methysergide (30/~g) with phentolamine (30/~g), but not by pretreatment with prazosin (30 #g), yohimbine (30/~g), methysergide (30/~g) or idazoxan (30 /~g; Fig. 5B). DISCUSSION The results of the present study provide clear evidence for a functional interaction between descending noradrenergic and serotonergic systems in the modulation of visceral nociception. This interaction involves, at least in part, spinal aE-adrenoceptors, 5-HTla and 5-HT2 receptors. Isobolographic analysis reveals that the i.t. coadministration of sub-antinociceptive doses of clonidine with either RU-24969 or DOI produces supra-additive antinociceptive effects in response to noxious colorectal distension.
Clonidine and RU-24969 The coadministration of clonidine with RU-24969 produces a parallel, significant leftward shift of the dose-response curve of clonidine or RU-24969. This suggests that the mechanism responsible for the interaction (i.e., the enhanced antinociceptive effects) is similar to the mechanism underlying the antinociceptive effects of clonidine or RU-24969 when either drug is administered alone. A heterogeneity of aE-adrenoceptors in rat has been suggested. Determination of the amino acid sequence 16 and molecular weight 17 of the aE-adrenoceptor have led to the identification of two structurally distinct a2-adrenoceptor subtypes. Cloning and gene
expression studies have revealed that the genes for these two a2-adrenoceptor subtypes are located on different chromosomes 16'26. Recently, Brown et al. 5 showed that the binding of yohimbine to azb-adrenoceptors could be competed for by RU-24969 or methysergide in a complex manner. It remains to be determined whether RU-24969 and methysergide inhibit binding of yohimbine to this site competetively or allosterically. Nevertheless, a possible mechanism for the interaction of clonidine with RU24969 is a positive cooperativity of binding to the a2b-adrenoceptor subtype. The fact that the interaction described in the present study is antagonized by pretreatment with phentolamine suggests the likely involvement of a-adrenoceptors, al-Adrenoceptor and imidazoline receptors are not involved since the enhanced antinociceptive effects resultant from coadministration of clonidine and RU-24969 were not antagonized by prazosin or idazoxan, respectively. Methysergide also did not antagonize the interaction of clonidine with RU-24969. These data provide more evidence for a functional interaction between 5-HT and az-adrenoceptors. Both 5-HT 1 receptors 23 and az-adrenoceptors 1° have been shown to be linked to a guanine nucleotide, specifically G i. That these receptors are coupled to the same effector system provides an alternate level at which the interaction of clonidine with RU-24969 may occur. For example, clonidine and RU-24969 may interact to activate G i to a greater extent than could be achieved if either drug was administered alone. To date, characterization of the spinal interaction of 5-HT receptors with adrenoceptors has been largely accomplished using neurotoxins. For example, Minor et al. 2° first reported that the antinociceptive effect of i.t. administered 5-HT was blocked by pretreatment with the NE depleting agent DSP4. Since then it has been well described that the antinociception resultant from i.t. administration of 5-HT or 5-HT 1 receptor agonists is dependent on an intact noradrenergic system. Data from the present study provide pharmacological evidence in support of this hypothesis. Rather than destruction of the noradrenergic system, a2-receptors were antagonized in the present study by phentolamine. Presumably, this i.t. pretreatment attenuates the influence of the noradrenergic system at post-synaptic sites without altering the anatomical basis for the interaction. Thus, the noradrenergic system remains intact and receptor supersensitivity does not develop. The present report documented that i.t. pretreatment with phentolamine antagonizes the antinociceptive effects produced by coadministration of clonidine with RU-24969, suggesting that attenuation of the antinociceptive efficacy of 5-HT receptor agonists may in fact be related to compromised a2-mediated effects.
41 Clonidine and D O I The i.t. coadministration of DOI with clonidine produced a non-parallel, yet significant leftward shift of the DOI dose-response curve. These data suggest that the mechanism for interaction between 5-HT 2 receptors and aE-adrenoceptors is different from that responsible for the RU-24969-clonidine interaction. Studies involving antagonist pretreatment confirm this hypothesis since methysergide antagonized the interaction between clonidine and DOI whereas pretreatment With phentolamine, yohimbine or naloxone did not. It has been suggested that endogenous opioids may be released by catecholamines in the lumbar spinal cord and that the antinociceptive effects of noradrenergic agonists, for example, may in part be due to opioidergic mechanisms 33. Pretreatment with i.t. naloxone was used to evaluate the involvement of opioid receptors in the enhanced antinociceptive effects produced by the clonidine-DOI interaction in the present study. Naloxone did not antagonize the effects resulting from this interaction. Thus, opioid receptors apparently are not significantly involved in the interaction of 5-HT 2 receptors with a2-adrenoreceptors. The nature of the interaction between 5-HT2 receptors and a2-adrenoceptors is not easily defined. While clonidine is known to exert its effects through activation of the G i guanine nucleotide, DOI and other 5-HT2 receptor agonists are believed to be linked to phosphoinositide hydrolysis23. That these receptors are coupled to different effector systems suggests that the interaction produced by activation of 5-HT 2 receptors and a2-adrenoceptors occurs at a different cellular level. Blood pressure At the dosages employed in this study, the i.t. administration of DOI produces significant increases in resting blood pressure while clonidine or RU-24969 are without effect on resting MAP 6,s. The coadministration of clonidine with DOI or RU-24969 did not produce any effects on resting MAP in the present study that differed from what is produced by administration of either drug alone (data not shown). Thus, effects on resting cardiovascular tone were not produced and supra-additive antinociceptive effects occurred independently of changes in resting blood pressure.
SUMMARY These data provide the first pharmacological evidence for a functional interaction between spinal adrenoceptors and serotonin receptors in the modulation of visceral nociception. In behavioral studies, the antinociceptive efficacy of selective adrenoceptor or serotonin receptor
agonists in response to noxious cutaneous stimulation is known. However, the interaction between serotonin receptor agonists and NE has been implied only in response to noxious cutaneous stimuli; the subtype(s) of serotonin or adrenergic receptors were not identified. Recently, however, serotonin and clonidine were shown to interact synergistically to inhibit the noxious-evoked activity of wide dynamic range neurons in the dorsal horn of the spinal cord, suggesting the involvement of a 2adrenoceptors 21. The present study defines the role of two serotonin receptor subtypes in the modulation of visceral nociception by an aE-adrenoceptor agonist. The nature of the mechanism responsible for the defined interactions also was investigated. The nature of the interaction between 5-HT 2 receptors and a2-adrenoce ptors is unclear, but is apparently 'serotonin-like' since methysergide antagonized the effects produced by coadministration of clonidine with DOI; no further antagonism was produced by combined pretreatment with methysergide and phentolamine. The interaction of a2-adrenoceptors and 5-HT1B receptors is 'a-like' since the effects produced following the coadministration of clonidine with RU-24969 were antagonized by phentolamine; no further antagonism was produced by combined pretreatment with phentolamine and methysergide. That the mechanisms responsible for these interactions are different may be related to the pre- vs post-synaptic location of the receptor subtypes (e.g., 5-HT1B and a2-adrenoceptors may modulate the release of neurotransmitter from nerve terminals2S), modification of binding to the receptor (e.g., RU-24969 and clonidine may compete for the same receptor) or the manner in which the interaction activates intracellular responses (e.g., a 2adrenoceptors and 5-HT m receptors are coupled to the same effector system). Importantly, a significant interaction between two descending monoaminergic systems known to be involved in the modulation of nociceptive transmission is defined. That 5-HT receptors and a2-adrenoceptors communicate in some way to inhibit visceral nociception is relevant to the study of pain since little is known about the mechanisms responsible for visceral pain. Indeed the precise nature of interactions involved in nociceptive processing are hard to define in light of the complex neurochemistry, neuroanatomy and modulatory influences described in the dorsal horn of the spinal cord. Not addressed in this study is the possible involvement of other putative modulatory systems (e.g., adenosine, 7-aminobutyric acid, acetylcholine). That the described interaction may result from multisynaptic activity between different laminae of the dorsal horn of the spinal cord also is not addressed. Furthermore, the tonic activity of local circuit and descending neurons which
42 m o d u l a t e the release o f n e u r o t r a n s m i t t e r s and n e u r o m o d u l a t o r s is not i n v e s t i g a t e d here. A n u n d e r s t a n d i n g of h o w t h e s e factors i n t e r r e l a t e are critical to the u n d e r s t a n d i n g of n e u r a l m e c h a n i s m s i n v o l v e d in the m o d u l a -
Acknowledgments. We would like to thank Mike Burcham for production of the graphics and Marilynn Kirkpatrick for secretarial assistance. Supported by NIH Grants T32 GM 07069 (R.M.D.) and NS 19912 and an unrestricted pain research grant from BristolMyers Squibb Co.
tion o f spinal n o c i c e p t i v e transmission.
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