Brain Research, 581 (1992) 59-66 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
59
BRES 17747
Imipramine inhibits intrathecal substance P-induced behavior and blocks spinal cord substance P receptors in mice Tatsuro Iwashita and Takao Shimizu Department of Pharmacology, School of Medicine, Kagoshima University, Kagoshima (Japan) (Accepted 31 December 1991) Key words: Antinociception; Imipramine; Substance P; Intrathecal administration; Behavioral response; [3H]SP binding; Spinal cord
The mechanism of the antinociceptive effect of the tricyclic antidepressant imipramine was investigated in mice. Intrathecal (i.t.) administration of imipramine produced dose-dependent antinociception in the tail-pinch and tail-flick tests with EDso values (95% confidence limit) of 27.5 (17.0-43.9) and 20.2 (12.6-32.2) nmol, respectively. In substance P (SP)-induced nociceptive behavior, imipramine (i.t.) also produced dose-dependent antinociception with EDs0 value of 20.2 (16.1-25.2) nmol. Tissue concentration of imipramine was between 55.2 and 104.4 nmol/g tissue when these EDs0 values of imipramine were i.t. administered. In the SP-induced behavior, the antinociceptive effect of 31.6 nmol of imipramine was not antagonized by the a-adrenergic receptor antagonist phentolamine, the serotonergic receptor antagonist methysergide, or the opioid receptor antagonist naloxone. In vitro study, imipramine dose-dependently inhibited specific [3H]SP binding in the spinal cord homogenate with IC50 value of 2.37 × 10-4 M and this value corresponds to 8.6/~mol/g tissue concentration. These data indicate that imipramine produces antinociceptive effect at about 100 times lower dose than SP receptor blockade. INTRODUCTION
depressants is antagonized by the opioid antagonist naloxone 3,7-9,20,30,35.
Antidepressants applied for the treatment of chronic pain in humans and prospective studies with amitriptyline suggest that this effect is independent of an antidepressant effect 5'25'43. In experimental animals, many investigators also have reported that the antidepressants amitryptyline v,35,39,41, clomipramine 9,3°,35, desipramine 3, 14,30,39 and imipramine L7'2°'39'4~ have an antinociceptive
Substance P (SP) is generally thought to be a sensory neurotransmitter of pain 36. Intrathecally (i.t.) administered SP produces caudally directed biting, licking and scratching behavior in mice ~8"28 and this behavior resembles the behavior produced by cutaneous irritants tS. Previously, SP-induced behavior has been shown to be reversed by i.t. opioids, norepinephrine (NE) 19, and 5-HT 17 like other noxious stimuli. Therefore, this SP-induced behavior is thought to be a nociceptive response. Antidepressants are also reported to reduce the SP-induced behavior 12'14.
effect on various nociceptive stimuli. Imipramine is also reported to have an antinociceptive effect in experimental pain with human volunteers 4. Although the antinociceptive mechanism of antidepressants is not still clearly understood, two possible mechanisms are suggested. The first possible mechanism is the influence of antidepressants on the serotonergic (5-HT) system. Several bodies of evidence exist showing that the bulbospinal serotonergic system inhibits nociceptive transmission 2'26'32. Antidepressants are known to inhibit the monoamine uptake mechanism to the nerve endings 33'34'37, and the antinociceptive effect of some antidepressants is reported to be reversed by the 5-HT antagonist methysergide 7'9 or metergoline 35. A n o t h e r possible mechanism is the interaction with opioid receptors. Antidepressants are known to bind to the opioid receptors x'3'2° and many studies demonstrate that antinociceptive effect of anti-
In the present study, we have observed the antinociceptive effect of tricyclic antidepressant imipramine on thermal, mechanical noxious stimuli and SP-induced behavior in mice. We have examined the site of antinociceptive effect of imipramine by comparing the tissue concentration of imipramine between i.t. administration and systemic administration. The possible mechanisms of the antinociceptive effect of imipramine were investigated, whether the effect of imipramine on SP-induced behavior is reversed by the administration of various neurotransmitter antagonists, or whether imipramine also affects SP receptors.
Correspondence: T. Iwashita, Department of Pharmacology, School of Medicine, Kagoshima University, Kagoshima 890, Japan. Fax: (81) (992) 65-8567.
6O MATERIALS AND METHODS
Animals Male adult ddY mice weighing 26-40 g (6-9 weeks of age) were used. Mice were fed at the Institute of Laboratory Animal Sciences, Kagoshima University, at a room temperature of 22-24°C and humidity of 60-70%. They were housed in colony cages with free access to food and water in 12/12 light-dark cycle.
(LC-4B, Bioanalytical SystemsJ set at a potential oi +11.8 V versa~ an Ag/AgC1 reference electrode and a computing integrator-primer (Waters 740, Waters Associates). The analytical column temperature was controlled at 34°(;. The mobile phase consisted of 40% acetonitrile containing 0.05 M KzHPO 4 adjusted to pH 3.50 with 10% phosphoric acid, and was pumped through the column at a rate of l ml/min.
[~H]SP binding assay Drug administration Intrathecal injections were made in conscious mice by the Hylden and Wilcox 16 method. The lumbar puncture was made approximately at the fifth and sixth lumbar intervertebral space using a 26-gauge needle on a 10 pl Hamilton syringe. Puncture of the dura was reliably indicated by a flick of the tail, 5 ktl of the drug was injected.
Antinociceptive assay Tail-flick test. The latency for a mouse to flick its tail away from a source of radiant heat 6 was measured with a thermal analgesimeter (Natsume, KN-205E). Mouse was restrained in a plastic holder and its tail blackened and placed beneath a focussed spotlight lamp (heat source). A foot-switch simultaneously activated the heat source and a digital timer. When the tail flicked away from the heat source, the timer was stopped and tall flick latency was recorded. A baseline tail-flick latency (pre-value) was between 2.0 and 7.0 s. A 25 s cutoff time was used as a maximal effect for protection from tissue injury. Tail-pinch test. The quantitative tall-pinch test by Takagi et al. 4° was used with a small modification. The base of the mouse tail was pinched with artery clips. The pressure ranged from 100 to 600 g in 50 g increments. Pain sensitivity was assessed when a mouse turned its head to its tail and bit the artery clip within 5 s. The decreased sensitivity to pain was scored from 1 to 7. A pre-value of the sensitivity to pain was between 2.0 and 3.5. A 7 as cutoff value was used when mouse did not bite the 600 g pressure artery clip. Following drug administration, the alternative rate of the nociceptive response was calculated as a maximal possible effect (MPE).
M P E (%) =
post-value - pre-value
x 100
cutoff value - pre-value
SP-induced behavior. Mice were i.t. injected with 6 pmol of SP and caudally directed biting, licking and scratching behavior was counted for 1 min after injection. Values for 50% effective dose (EDs0) were determined by the Litchfield and Wilcoxon 23 method. In this test, an antinociceptive effect was defined as 50% MPE in the tall-flick and tall-pinch test, and 50% of the control response in the SP-induced behavior.
Measurement of imipramine content in the spinal cord and the whole brain Mice were injected intraperitoneally (i.p.) with imipramine, 20 mg/kg or 15.8 nmol, 31.6 nmol i.t., and decapitated 30 rain after i.p. injection or 5 min after i.t. injection. The whole brain and the spinal cord were removed and weighed, then frozen on dry-ice and stored at -4&C until the assay. The tissue sample was homogenized in 10 vol. (w/v) of methanol, using an ultrasonic cell disruptor (40% pulsed power for 30 s; Model 185, Branson), and centrifuged at 26,000 x g for 20 rain at 4°(2 (Kubota, KR-2000(Yr). The supernatant was filtered through a 0.45/~m membrane filter (LC3A, Gelman Sciences) and a 100/A aliquot of the filtered solution was injected into a high-performance liquid chromatography (HPLC) system. The HPLC system consisted of a delivery pump (Waters 510, Waters Associates), a sample injector (WISP 710B, Waters Associates), a reverse-phase column (250 mm length × 4.6 mm i.d.; Eicompak 5-ODS, Eicom Co.), an electrochemical detector
[3H]SP binding assay was performed by the Perrone et al.-': method with a small modification. [3H]SP (42.6 Ci/mmol specific activity) was obtained from New England Nuclear (Boston). Mice were decapitated and the spinal cord was rapidly removed and weighed. The tissue samples were homogenized in 10 vol. (w/v) of ice-cold 50 mM Tris buffer (Trizma; Sigma Chemical Co.) (pH 7.7 at 20°C) using a Polytron homogenizer (setting 6 for 30 s; Kinematical. The homogenate was centrifuged twice at 12,000 × g for 20 min at 0°C (Kubota, KR-20000T) with intermediate rehomogenization of the pellet in fresh buffer following the first centrifugation. The resultant pellet was resuspended in 5 vol. (w/v) of assay buffer consisting of 50 mM Tris buffer (pH 7.7 at 20°C), 2 mM CaC12, 2 mM MgCI 2, 4 ktg/ml leupeptin (Peptide Institute, Osaka, Japan), 2 #g/ml chymostatin (Peptide Institute), 40 pg/ml bacitracin (Sigma Chemical Co., St. Louis, MO) and 0.02% bovine serum albumin (Sigma Chemical Co.), then frozen and stored at -40°C until binding assay. For the binding assay, the tissue preparation was diluted with an assay buffer to 2 mg/ml protein concentration and a 100 kd aliquot of this tissue suspension was incubated at 20°C in a final volume of 500 #1 of the assay buffer with [3H]SP for 60 rain. Specific binding was defined as that displaced by 1/~M unlabeled SP. Incubation was terminated by filtration under reduced pressure over GF/F filter (Whatman Int. UK) that had been presoaked with 0.1% polyethyleneimine (Nacalai Tesque, Kyoto, Japan) for 3 h. The filters were rinsed with 3 × 3 ml ice-cold Tris buffer (pH 7.7 at 20°C) and emulsified by shaking with 10 ml Biofluor (New England Nuclear, Boston) for 30 min. Samples were counted by liquid scintillation spectrometry (Packard, TRI-CARB 4430). Protein was determined by the Lowry et al. 24 method. To investigate the effect of unlabeled SP or imipramine on [3H]SP specific binding, various concentrations of unlabeled SP or imipramine were co-incubated with 0.84 nM [3H]SP and the displacement curves were drawn. Values for 50% inhibitory concentration (IC50) of drugs against [3H]SP binding were determined from linear regression analysis of Hill plots.
Drugs Imipramine hydrochloride, norepinephrine hydrochloride, 5-hydroxytryptamine creatinine sulfate and naloxone hydrochloride were purchased from Sigma Chemical Co. (St. Louis, MO); morphine hydrochloride was from Takeda Chemical Industries (Osaka, Japan); phentolamine mesylate was from Ciba-Geigy Japan Co. (Takarazuka, Japan); methysergide maleate was from Sandoz (Basel, Switzerland); substance P was from Peptide Institute (Osaka, Japan). All drugs administered i.t. were dissolved in sterile artificial cerebrospinal fluid (NaCI 138 mM, KCI 3 mM, CaCI 2 1.25 mM, MgCI 2 1 mM and D-glucose 1 mM) and made up to a volume of 5 pl. Imipramine administered i.p. was dissolved in sterile 0.9% NaC1 and made in a volume of 10 ml/kg.
Statistical analysis The results were analysed by the Kruskal-Wallis test followed by the Wilcoxon U-test. All values are expressed as the mean +__ S.E.M.
RESULTS
Antinociceptiye effect o f imipramine Tail-pinch test. I m i p r a m i n e a d m i n i s t e r e d i.t. o r i.p.
61 (%)
p r o d u c e d a d o s e - d e p e n d e n t antinociceptive effect (Fig. 1). M a x i m u m effects were o b t a i n e d 5 min after i.t. injection (Fig. 1A) and 15-30 min after i.p. injection (Fig.
120
c 0 (z ¢0
100
==
MPE
(%) c 0 o
100
c o 50
50
0 I
-20
0 5
I
roll
15 30 Time a f t e r injection
I
60
7.9
15.8
23.7
31.6
i 47.3
Imipramine (nmol) min
MPE
(%)
**
lOO
lii I
G.
Fig. 2. Antinociceptive effect of imipramine on intrathecally administered SP-induced behavior. Each column represents the mean + S.E.M. (n = 8) of the percentage of control response. Imipramine was i.t. administered 5 min before SP administration. Control response indicates the number of the SP-induced behavior 5 min after i.t. administration of 5 pl artificial CSF (vehicle). *'**Indicate significant difference from the control response, P < 0.05 or P < 0,01, respectively.
50
0 -20
,, 0
i i i 15 30 45 Time a f t e r injection
I-" 6 0 min
MPE
(%)
C
100 ~ * *
**
50
0 -20
I I 0 5
l i 15 30 T i m e a f t e r injection
I 60
min
Fig. 1. Time-course of antinociceptive effect of imipramine on tailpinch test (A,B) and tail-flick test (C) in mice. The ordinate shows the antinociceptive effect expressed as MPE (%) indicated in Materials and Methods, and the abscissa shows time after drug administration. Each vertical bar is the mean + S.E.M. from at least 8 animals. A,C: mice were intrathecally administered 5/A volume of artificial cerebrospinal fluid (CSF) (o) or imipramine (15.8 nmol, O; 31.6 nmol, A; 63.1 nmol, II). B: mice were intraperitoneally administered 10 ml/kg volume of saline ((3) or imipramine (12.5 mg/kg, O; 25.0 mg/kg; a ; 50.0 mg/kg, II). *'** Indicate significant difference from the vehicle control group, P < 0.05 or P < 0.01, respectively.
1B). The EDs0 value (95% confidence limit) for 5 min after i.t. injection was 27.5 (17.0-43.9) nmol, and the EDs0 for 30 min after i.p. injection was 19.5 (9.8-39.0) mg/kg. Tail-flick test. I m i p r a m i n e injected i.t. p r o d u c e d a d o s e - d e p e n d e n t antinociceptive effect (Fig. 1C) and the m a x i m u m effect was observed 5-15 min after i.t. injection. The EDs0 value for 5 min after i.t. injection was 20.2 (12.6-32.2) nmol. SP-induced behavior. In control animals, i.t. injection of SP p r o d u c e d the nociceptive b e h a v i o r of biting, licking and scratching within 1 min of injection. A v e r a g e total count of the behavior was 64.4 + 7.8 counts/min (mean + S . E . M . , n = 8). Since i.t. administered imip r a m i n e p r o d u c e d its m a x i m u m antinociceptive effect 5 min after injection in both the tail-pinch and tail-flick tests, imipramine was injected 5 min before SP administration. I m i p r a m i n e p r o d u c e d a d o s e - d e p e n d e n t inhibition of SP-induced behavior (Fig. 2), with an EDs0 value of 20.2 (16.1-25.2) nmol.
Influences of several neurotransmitter antagonists on the effect of imipramine in SP-treated mice a-Adrenergic receptor antagonist. N E (40 pmol) i.t. coadministered with SP r e d u c e d SP-induced behavior to 29.6 + 13.4% of the control response. P h e n t o l a m i n e (10 nmol) i.t. co-administered with SP did not significantly increase SP-induced b e h a v i o r (114.8 + 28.9% of the con-
62
A
B
(%)
(%)
2oo
"
O
2OO
150
150
tt
¢o
==
-6 i. ,-
100
O o
+
100
c
~
50 0.
o NE Phentolamine
-
÷
-
÷
-
-
~
+
Imipramine Phentolamine
-
*
-
+
-
-
*
+
Fig. 3. Antinociceptive effect of NE (A) or imipramine (B) on SPinduced behavior, and antagonism of this effect by a-adrenergic receptor antagonist phentolamine. Each column represents the mean + S.E.M. (n = 8) of the percentage of control response. Control response indicates the number of the SP-induced behavior i.t. coadministered with vehicle for NE, phentolamine or given the vehicle for imipramine 5 min before SP administration. A: control response was 41.9 + 2.6 counts/rain. NE (40 pmol) was i.t. co-administered with SP. Phentolamine (10 nmol) was also i.t. coadministered with SP. B: control response was 45.0 + 13.2 counts/min. Imipramine (31.6 nmol) was i.t. administered 5, min before SP administration. Phentolamine (10 nmol) was i.t. coadministered with SP. *'**Indicate significant difference from the control response, P < 0.05 or P < 0.01, respectively. **Indicates significant difference between NE group and NE + Phentolamine group, P < 0.01.
trol response), but reversed the effect of N E on SP-induced b e h a v i o r (123.9 + 11.5% of the control response) (Fig. 3A). I m i p r a m i n e (31.6 nmol) injected i.t. 5 min before SP administration also r e d u c e d SP-induced behavior to 16.2 + 6.4% of the control response, but phentolamine (10 nmol) i.t. co-administered with SP did not significantly reverse the effect of i m i p r a m i n e on SP-induced b e h a v i o r (41.3 + 16.4% of the control response)
(Fig. 3B). Serotonergic receptor antagonist. 5-HT (6.5 nmol) i.t. co-administered with SP r e d u c e d the SP-induced behavior to 14.0 _ 12.3% of the control response. A l t h o u g h i.t. injection of 65.0 nmol 5-HT p r o d u c e d scratching behavior, the administration of 6.5 nmol 5 - H T did not produce scratching b e h a v i o r by itself. Methysergide (40 nmol) i.t. co-administered with SP did not significantly increase SP-induced b e h a v i o r (130.8 + 20.5% of the control response) but reversed the effect of 5-HT on SP-induced b e h a v i o r (165.3 _+ 33.3% of the control response) (Fig. 4A). I m i p r a m i n e (31.6 nmol) injected i.t. 5 min before SP administration also r e d u c e d SP-induced be-
havior to 13.2 + 3.8% of the control response, but methysergide (40 nmol) i.t. co-administered with SP did not reverse the effect of imipramine on SP-induced behavior (26.9 _+ 10.5% of the control response) (Fig. 4B). Opioid receptor antagonist. M o r p h i n e (0.125 nmol) i.t. co-administered with SP reduced SP-induced behavior to 38.4 + 13.9% of the control response. Naloxone (4 nmol) i.t. co-administered with SP did not significantly increase SP-induced behavior (98.9 + 22.7% of the control response), but reversed the effect of morphine on SP-induced behavior (125.1 _+ 18.4% of the control response) (Fig. 5A). I m i p r a m i n e (31.6 nmol) injected i.t. 5 min before SP administration reduced SPinduced behavior to 4.6 + 3.2% of the control response, but naloxone (4 nmol) i.t. co-administered with SP did not reverse the effect of imipramine on SP-induced behavior (6.3 + 3.6% of the control response) (Fig. 5B).
Measurement of imipramine in the spinal cord and the whole brain The imipramine contents in the spinal cord and the whole brain were m e a s u r e d 30 min after the i.p. injec-
A
B (%) 200
t t
(%)
I
200
]too
+
,oo f c
g.
g.
l i
50
o_;i 5-HT
Methysergide
-
+
-
+
Imipramlne
-
+
-
-
+
+
Methyserglde
-
-
+
+
+
Fig. 4. Antinociceptive effect of 5-HT (A) or imipramine (B) on SP-induced behavior, and antagonism of this effect by serotonergic receptor antagonist methysergide. Each column represents the mean + S.E.M. (n = 8) of the percentage of control response. Control response means the same as described in Fig. 3. A: control response was 41.5 + 9.0 counts/min, 5-HT (6,5 nmol) was i.t. co-administered with SP. Methysergide (40 nmol) was also i.t. coadministered with SP. B: control response was 53.1 + 8.0 counts/ min. Imipramine (31.6 nmol) was i.t. administered 5 min before SP administration. Methysergide (40 nmol) was i.t. coadministered with SP. **Indicates significant difference from the control response, P < 0,01. ttIndicates significant difference between 5-HT group and 5-HT + methysergide group, P < 0,01.
63 A
TABLE I
Contents of imipramine in the spinal cord and the whole brain after intraperitoneal or intrathecal administration of imipramine
30
All values are expressed as the mean + S.E.M. of data obtained in 6 animals
lmipramine dose
;20
Imipramine content (nmol/g tissue) Spinal cord
Intraperitoneal a 20 mg/kg Intrathecal b 15.8. nmol 31.6 nmol
38.5 + 4.1
48.3 + 4.7
55.2 + 6.0 104.4 + 9.8
-* -*
o e~
,1 10
aMice were decapitated 30 min after administration of imipramine. bMice were decapitated 5 min after administration of imipramine. *Imipramine was not detected.
tion of imipramine with the dose of 20 mg/kg. The dose of 20 mg/kg was selected because the EDs0 value of the tail-pinch test 30 min after i.p. imipramine injection was 19.5 mg/kg. The contents of imipramine in the whole brain and the spinal cord were 48.3 + 4.7 nmol/g tissue and 38.5 + 4.1 nmol/g tissue, respectively (Table I). The EDs0 values of the tail-pinch, tail-flick test and SP-induced behavior 5 min after i.t. injection were between A
I
0 o
I
i
1.0
2.0
0 10 20 30 B (fmol/mg protein)
1
3.0
i 4.0
3 H - S P (nM)
Fig. 6. [3H]SP binding to homogenate of the mouse spinal cord. A: specific and non-specific binding of [3H]SP to the homogenate of the mouse spinal cord as function of radiolabeled peptide concentration. The abscissa shows [3H]SP concentration (nM) and ordinate shows [3H]SP bound (fmol/mg protein). Homogenate was prepared as described in Materials and Methods and incubated at 20°C for 1 h with increasing concentrations of [3H]SP. Specific binding (O) and non-specific binding (&) were calculated as described in Materials and Methods. B: Scatchard analysis of specific binding from the experiment in (A) is presented. The abscissa shows bound [3H]SP (fmol/mg protein) and the ordinate shows bound/free (fmol/mg protein/nM). Each point is the mean of duplicates.
B
(%)
(%) tt
150
150 tO e~ g~
i
B
,o
Whole brain
+
100
100
.E
(%) 125
Jo
cO
o
100
o 0) t-i
=o
cO~
so
50
IX
a.
o Morphine
o +
+
75
I "1" o)
Imiprarnine
--~ -
+
ig
50
¢J
+
25 c
NaJoxone
+
+
Naloxone
+
+
Fig. 5. Antinociceptive effect of morphine (A) or imipramine (B) on SP-induced behavior, and antagonism of this effect by opioid receptor antagonist naloxone. Each column represents the mean _+ S.E.M. (n = 8) of the percentage of control response. Control response means the same as described in Fig. 3. A: control response was 37.5 _+ 6.5 counts/min. Morphine (0.125 nmol) was i.t. coadministered with SP. Naloxone (4 nmol) was also i.t. co-administered with SP. B: control response was 75.6 ___ 15.8 counts/rain. Imipramine (31.6 nmol) was i.t. administered 5 min before SP administration. Naloxone (4 nmol) was i.t. co-administered with SP. *'**Indicate significant differences from the control response, P < 0.05 or P < 0.01, respectively. +*Indicates significant difference between morphine group and morphine + naloxone group, P < 0.01.
8 el
0 -1(
a -9
t
I
I
I
I
-8
-7
-6
-5
-4
I
-3
IOglo (concentration, M) Fig. 7. Inhibition of [3H]SP binding to the homogenate of the mouse spinal cord by unlabeled SP or imipramine. The 0.84 nM [3H]SP and increasing concentrations of unlabeled SP (11) or imipramine (O) were mixed and incubated with the homogenate of the mouse spinal cord. Incubating conditions are defined as described in Materials and Methods. The abscissa shows lOgl0(concentration; M) of unlabeled drugs and the ordinate shows the percentage of the binding of [3H]SP alone. The data are the mean _+ S.E.M. of duplicate determinations from 5 separate experiments.
64 TABLE II Inhibition of [~H]SP binding to the homogenate of the mouse spinal cord by unlabeled SP and imipramine
The concentrations of the drugs required to inhibit the specific binding of 0.84 nM [3H]SP to the homogenate of the mouse spinal cord by 50% (IC5o) and Hill coefficients (nn) were estimated from the Hill plots of the displacement curves represented in Fig. 7. All values are expressed as the mean + S.E.M. of data obtained in n experiments.
Unlabeled SP Imipramine
1Cso (m)*
nl4
n
(2.46+0.70)× 10-9 (2.34+0.39)× 10 -4
1.01 + 0.15 0.77 + 0.07
4 5
*Indicates significant difference between unlabeled SP and imipramine, P < 0.05.
15.8 nmol and 31.6 nmol. The imipramine contents 5 min after the i.t. injection of this agent were measured for these doses of imipramine. In the spinal cord, the contents of imipramine after i.t. injection of 15.8 nmol and 31.6 nmol of this agent were 55.2 + 6.0 nmol/g tissue and 104.4 + 9.8 nmol/g tissue, respectively (Table I). In the whole brain, imipramine could not be detected 5 min after i.t. injection of imipramine. [3H]SP binding assay
In the homogenate of the spinal cord, [3H]SP binding was saturable and Scatchard analysis of saturation data yielded a straight line from which the density of binding sites (Bmax) w a s 29.5 + 1.5 fmol/mg protein and the dissociation constant (Kd) was 0.45 + 0.04 nM (n = 5) (Fig. 6), Unlabeled SP or imipramine dose-dependently inhibited [3H]SP binding (Fig. 7). The Hill plot yielded the IC50 values for unlabeled SP and imipramine against [3H]SP binding of 2.46 (+_ 0.70) x 10-9 M and 2.37 (_+ 0.39) × 10 -4 M , respectively. The Hill plot yielded Hill coefficients for unlabeled SP and imipramine of 1.01 _+ 0.15 and 0.77 _+ 0.07, respectively (Table II). DISCUSSION Many investigators have shown that antidepressants have antinociceptive effect in r a t s 1'3'9'20'30'35'41, mice 7,s' 14,39 and humans TM. The antinocieeptive effects of antidepressants were shown in animals by the systemic administration 1'3'7-9A8'2°'3°'35'39'41, by the intracerebroventricular administration 39 and by the i.t. administration TM. In the present study we confirmed antinocieeptive effect of imipramine by both the tail-pinch test and the tail-flick test. In the tail-pinch test, imipramine produced an antinociceptive effect in both treatment of i.p. and i.t. with EDso values of 19.5 mg/kg and 27.5 nmol, respectively. If imipramine produced an antinociceptive effect
at the spinal level, the contents of imipramine in the spinal cord would be the same when mice were administered imipramine at the dose to produce the same degree of antinociception by i.p. and i.t. When 20 mg/kg imipramine was injected i.p., the content of imipramine in the spinal cord 30 min after administration was 38.5 nmol/g tissue. On the other hand, when 15.8 nmol of imipramine (this dose was smaller than the EDs0 dose of i.t. imipramine) was injected i.t.. the content of imipramine in the spinal cord 5 min after injection was 55.2 nmol/g tissue. When the EDso dose of imipramine was injected i.t., imipramine did not reach the supraspinal site, and this fact indicates that imipramine can produce antinociception at the spinal level. But the supraspinal mechanism cannot be excluded because the content of imipramine in the spinal cord after i.p. injection of the EDso dose imipramine was lower than the content of imipramine in the spinal cord after i.t. injection of the ED50 dose imipramine. To study the spinal mechanism of the antinociceptive effect of imipramine in detail, the effect of imipramine on SP-induced behavior was investigated with several receptor antagonists that are related to nociception. SP is thought to be one of the neurotransmitter candidates of somatosensory primary afferent fibers especially for nociception 36. It is reported in mice that SP administered i.t. produces caudally directed biting, licking and scratching 18'28 which is similar to the behavior induced by peripheral irritation 15. This SP-induced behavior is reported to be reduced by i.t. co-administered opioids, NE 19 and 5-HT iv, and in the present study NE, 5-HT and morphine reduced SP-induced behavior. This inhibitory effect was reversed by the a-adrenergic receptor antagonist phentolamine, the 5-HT receptor antagonist methysergide and the opioid receptor antagonist naloxone, respectively. We observed in the present study that 6.5 nmol of 5-HT was necessary to inhibit SP-induced behavior but other investigators have shown that a smaller dose of 5-HT (6.5-12.9 pmol) is sufficient to inhibit SP-induced behavior and a larger dose of 5-HT (25.8 pmol-10.3 nmol) by itself produces scratching behavior 1°'11'17'42. In our observation 6.5 nmol of 5-HT did not produce the scratching behavior by itself but 65.0 nmol of 5-HT produced approximately 40 counts/min of scratching behavior. The discrepancy between previous studies and the present data of 5-HT doses to inhibit SP-induced behavior and to produce scratching behavior by itself cannot be clearly explained but the difference of the mouse strain may be related. The mechanism of the antinociceptive effect of antidepressants is not clearly understood but two possible mechanisms are suggested. One possibility is that antidepressants produce the antinociceptive effect through
65 the serotonergic mechanism 7'9'35'4~. The other possibility is that antidepressants interact with opioid receptors and produce antinociception 1'3'~-9'2°'3°'35. Because antidepressants are known to inhibit NE uptake at nerve endings 34 and NE is recognized to have an antinociceptive effect at the spinal level 29, NE involvement in the antinociceptive effect of imipramine could not be ruled out. But in the present study, imipramine reduced SP-induced behavior as previously reported 12'14 and this effect was not antagonized by an a-adrenergic receptor antagonist, a serotonergic receptor antagonist or an opioid receptor antagonist. This fact suggests that imipramine inhibits SP-induced behavior by a mechanism other than those previously suggested. Antidepressants interact with muscarinic receptors 38 or N-methyl-D-aspartate receptors 31 and these mechanisms may contribute to the antinociceptive effect of antidepressants but one of the possible mechanisms by which imipramine inhibits SPinduced behavior may be by direct action on SP receptors. To determine the direct effect of imipramine on SP receptors, we performed in vitro studies of [3H]SP binding to the receptors. Imipramine inhibited [3H]SP binding with IC50 value of 2.37 × 10-4 M. The tissue preparation for SP binding contained protein of 0.4 mg/ml and in this procedure 14.45 + 0.17 mg protein was obtained from 1 g spinal cord. Therefore, 2.37 × 10-4 M of imipramine corresponds to 8.6 #mol/g tissue concentration. The EDs0 value of imipramide for inhibition of SP-induced behavior was 20.2 nmol, the imipramine content in the spinal cord 5 min after i.t. injection of this dose was between 55.2 nmol/g tissue and 104.4 nmol/g tissue. Therefore IC50 of imipramine for [3H]SP binding is at least 80 times higher than EDso for SP-induced behavior. This dissociation cannot be clearly explained but in vivo studies on imipramine may exist at the site of action in higher concentrations than calculated. Imipramine has also been reported to inhibit [3H]naloxone binding 1'3, [3H]naltrexone binding 2° and [3H]MK-801 bind-
ing3t. IC50 values of imipramine for these effects were approximately 10 times lower than that for inhibition of SP binding. In addition, there are several nociceptive neurotransmitter candidates and Kuraishi et al. have reported that in rabbits thermal and mechanical noxious stimuli are transmitted with different neurotransmitters and SP is related only to mechanical noxious stimuli 22. In the present study, i.t. imipramine produces antinociception for both thermal and mechanical noxious stimuli in mice. From these facts the antinociceptive effect of imipramine cannot be explained only by SP receptor blockade but noxious stimuli transmitted with SP may be partially blocked at the SP receptor level. In the [3H]SP binding assay, the Hill plot revealed that the Hill coefficient for imipramine was smaller than unity. More detailed study of the interaction between imipramine and SP receptors is necessary. Recently some studies have shown that chronic antidepressant treatment increases the effect of SP at the spinal level a3 and at the supraspinal level 21. These facts may indicate the up-regulation of SP receptors by the SP binding inhibition of antidepressant. In conclusion, imipramine produced antinociceptive effect at the spinal level on the thermal or mechanical noxious stimuli and SP-induced behavior. Imipramine inhibited SP-induced behavior through the non-adrenergic, non-serotonergic and non-opioidergic mechanisms. In vitro, imipramine inhibited [3H]SP binding with IC5o value of 2.37 × 10-4 M but this dose corresponded to about 100 times higher tissue concentration of imipramine than the antinociceptive effect.
REFERENCES
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Acknowledgements.We express our thanks to Professor Takeo Fukuda and Dr. Masahiro Nomoto from the Department of Pharmacology, School of Medicine, Kagoshima University for their valuable suggestions and to Professor Nozomu Yoshimura from the Department of Anesthesiology, School of Medicine, Kagoshima University for his encouragement and to Mr. Yoshiteru Toriire for his technical assistance. And we thank Mr. Robert Hodierne for reading the manuscript.
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26 Messing, R.B. and Lytle, L.D., Serotonm-contaming neurons: their possible role in pain and analgesizt, Pain, 4 (1977) 1-2~. 27 Perrone, M.H., Diehl, R.E. and Haubrich, D.R.. Binding of [3H]substance P to putative substance P receptors in rat brain membranes, Eur. J. Pharmacol., 95 (19831 131-- [33. 28 Piercey, M,F., Dobry, P.J.K., Schroedcr. L.A. and Einspahr, EJ., Behavioral evidence that substance P may be a spinal cord sensory neurotransmitter, Brain Res., 210 ( i981 ) 407-412. 29 Reddy, S.V.R., Maderdrut. J.L. and Yaksh, T.L.. Spinal cord pharmacology of adrenergic agonist-mediated antinociception, J. Pharmacol. Exp. Ther.. 213 (1980) 525--533. 30 Reichenberg, K., Gaillard-Plaza, G. and Montastruc, J.L., Influence of naloxone on the antinociceptive effects of some antidepressant drugs, Arch. Int. Pharmacodvn. Ther., 275 (1985) 78-85. 31 Reynolds, I.J. and Miller, R.J., Tricyctic antidepressants block N-methyl-o-aspartate receptors: similarities to the action of zinc, Br. J. Pharmacol., 95 (1988) 95-102. 32 Roberst, M.H.T., 5-Hydroxytryptamine and antinociception. Neuropharmacology, 23 (1984) 1529-1536. 33 Ross, S.B. and Renyi, A.L., Accumulation of tritiated 5-hydroxytryptamine in brain slices, Life Sci., 6 (1967) 1407-1415. 34 Ross, S.B. and Renyi, A.L., Inhibition of the uptake of tritiated catecholamines by antidepressant and related agents, Eur. J. Pharmacol., 2 (1967) 181-186. 35 Sacerdote, P., Brini, A., Mantegazza, P. and Panerai, A.E., A role for serotonin and/3-endorphin in the analgesia induced by some tricyclic antidepressant drugs, Pharmacol. Biochem. Behay., 26 (1987) 153-158. 36 Salt, T.E. and Hill, R.G.. Neurotransmitter candidates of somatosensory primary afferent fibres, Neuroscience, 10 (1983) 1083-1103. 37 Segawa, T. and Kuruma, 1., The influences of drugs on the uptake of 5-hydroxytryptamine by nerve-ending particles of rabbit brain stem, J. Pharm. Pharmacol., 20 (1968) 320-322. 38 Snyder, S.H. and Yamamura, H.I., Antidepressants and the muscarinic acetylcholine receptor, Arch. Gen. Psychiatry,, 34 (1977) 236-239. 39 Spiegel, K., Kalb, R. and Pasternak, G.W., Analgesic activity of tricyclic antidepressants, Ann. Neurol.. 13 (1983) 462-465. 40 Takagi, H., Inukai, T. and Nakama, M., A modification of Haffner's method for testing analgesics, Jpn. J. Pharmacol., 16 (1966) 287-294. 41 Tura, B. and Tura, S.M., The analgesic effect of tricyctic antidepressants, Brain Res., 518 (1990) 19-22. 42 Vaught, J.L. and Scott, R., Interactions of substance P antagonists with serotonin in the mouse spinal cord, Peptides, 9 (1988) 909-913. 43 Watson, C.P., Evans, R.J., Reed, K., Merskey, H., Goldsmith, L. and Warsh, J., Amitriptyline versus placebo in postherpetic neuralgia, Neurology, 32 (1982) 671-673
59
BRES 17747
Imipramine inhibits intrathecal substance P-induced behavior and blocks spinal cord substance P receptors in mice Tatsuro Iwashita and Takao Shimizu Department of Pharmacology, School of Medicine, Kagoshima University, Kagoshima (Japan) (Accepted 31 December 1991) Key words: Antinociception; Imipramine; Substance P; Intrathecal administration; Behavioral response; [3H]SP binding; Spinal cord
The mechanism of the antinociceptive effect of the tricyclic antidepressant imipramine was investigated in mice. Intrathecal (i.t.) administration of imipramine produced dose-dependent antinociception in the tail-pinch and tail-flick tests with EDso values (95% confidence limit) of 27.5 (17.0-43.9) and 20.2 (12.6-32.2) nmol, respectively. In substance P (SP)-induced nociceptive behavior, imipramine (i.t.) also produced dose-dependent antinociception with EDs0 value of 20.2 (16.1-25.2) nmol. Tissue concentration of imipramine was between 55.2 and 104.4 nmol/g tissue when these EDs0 values of imipramine were i.t. administered. In the SP-induced behavior, the antinociceptive effect of 31.6 nmol of imipramine was not antagonized by the a-adrenergic receptor antagonist phentolamine, the serotonergic receptor antagonist methysergide, or the opioid receptor antagonist naloxone. In vitro study, imipramine dose-dependently inhibited specific [3H]SP binding in the spinal cord homogenate with IC50 value of 2.37 × 10-4 M and this value corresponds to 8.6/~mol/g tissue concentration. These data indicate that imipramine produces antinociceptive effect at about 100 times lower dose than SP receptor blockade. INTRODUCTION
depressants is antagonized by the opioid antagonist naloxone 3,7-9,20,30,35.
Antidepressants applied for the treatment of chronic pain in humans and prospective studies with amitriptyline suggest that this effect is independent of an antidepressant effect 5'25'43. In experimental animals, many investigators also have reported that the antidepressants amitryptyline v,35,39,41, clomipramine 9,3°,35, desipramine 3, 14,30,39 and imipramine L7'2°'39'4~ have an antinociceptive
Substance P (SP) is generally thought to be a sensory neurotransmitter of pain 36. Intrathecally (i.t.) administered SP produces caudally directed biting, licking and scratching behavior in mice ~8"28 and this behavior resembles the behavior produced by cutaneous irritants tS. Previously, SP-induced behavior has been shown to be reversed by i.t. opioids, norepinephrine (NE) 19, and 5-HT 17 like other noxious stimuli. Therefore, this SP-induced behavior is thought to be a nociceptive response. Antidepressants are also reported to reduce the SP-induced behavior 12'14.
effect on various nociceptive stimuli. Imipramine is also reported to have an antinociceptive effect in experimental pain with human volunteers 4. Although the antinociceptive mechanism of antidepressants is not still clearly understood, two possible mechanisms are suggested. The first possible mechanism is the influence of antidepressants on the serotonergic (5-HT) system. Several bodies of evidence exist showing that the bulbospinal serotonergic system inhibits nociceptive transmission 2'26'32. Antidepressants are known to inhibit the monoamine uptake mechanism to the nerve endings 33'34'37, and the antinociceptive effect of some antidepressants is reported to be reversed by the 5-HT antagonist methysergide 7'9 or metergoline 35. A n o t h e r possible mechanism is the interaction with opioid receptors. Antidepressants are known to bind to the opioid receptors x'3'2° and many studies demonstrate that antinociceptive effect of anti-
In the present study, we have observed the antinociceptive effect of tricyclic antidepressant imipramine on thermal, mechanical noxious stimuli and SP-induced behavior in mice. We have examined the site of antinociceptive effect of imipramine by comparing the tissue concentration of imipramine between i.t. administration and systemic administration. The possible mechanisms of the antinociceptive effect of imipramine were investigated, whether the effect of imipramine on SP-induced behavior is reversed by the administration of various neurotransmitter antagonists, or whether imipramine also affects SP receptors.
Correspondence: T. Iwashita, Department of Pharmacology, School of Medicine, Kagoshima University, Kagoshima 890, Japan. Fax: (81) (992) 65-8567.
6O MATERIALS AND METHODS
Animals Male adult ddY mice weighing 26-40 g (6-9 weeks of age) were used. Mice were fed at the Institute of Laboratory Animal Sciences, Kagoshima University, at a room temperature of 22-24°C and humidity of 60-70%. They were housed in colony cages with free access to food and water in 12/12 light-dark cycle.
(LC-4B, Bioanalytical SystemsJ set at a potential oi +11.8 V versa~ an Ag/AgC1 reference electrode and a computing integrator-primer (Waters 740, Waters Associates). The analytical column temperature was controlled at 34°(;. The mobile phase consisted of 40% acetonitrile containing 0.05 M KzHPO 4 adjusted to pH 3.50 with 10% phosphoric acid, and was pumped through the column at a rate of l ml/min.
[~H]SP binding assay Drug administration Intrathecal injections were made in conscious mice by the Hylden and Wilcox 16 method. The lumbar puncture was made approximately at the fifth and sixth lumbar intervertebral space using a 26-gauge needle on a 10 pl Hamilton syringe. Puncture of the dura was reliably indicated by a flick of the tail, 5 ktl of the drug was injected.
Antinociceptive assay Tail-flick test. The latency for a mouse to flick its tail away from a source of radiant heat 6 was measured with a thermal analgesimeter (Natsume, KN-205E). Mouse was restrained in a plastic holder and its tail blackened and placed beneath a focussed spotlight lamp (heat source). A foot-switch simultaneously activated the heat source and a digital timer. When the tail flicked away from the heat source, the timer was stopped and tall flick latency was recorded. A baseline tail-flick latency (pre-value) was between 2.0 and 7.0 s. A 25 s cutoff time was used as a maximal effect for protection from tissue injury. Tail-pinch test. The quantitative tall-pinch test by Takagi et al. 4° was used with a small modification. The base of the mouse tail was pinched with artery clips. The pressure ranged from 100 to 600 g in 50 g increments. Pain sensitivity was assessed when a mouse turned its head to its tail and bit the artery clip within 5 s. The decreased sensitivity to pain was scored from 1 to 7. A pre-value of the sensitivity to pain was between 2.0 and 3.5. A 7 as cutoff value was used when mouse did not bite the 600 g pressure artery clip. Following drug administration, the alternative rate of the nociceptive response was calculated as a maximal possible effect (MPE).
M P E (%) =
post-value - pre-value
x 100
cutoff value - pre-value
SP-induced behavior. Mice were i.t. injected with 6 pmol of SP and caudally directed biting, licking and scratching behavior was counted for 1 min after injection. Values for 50% effective dose (EDs0) were determined by the Litchfield and Wilcoxon 23 method. In this test, an antinociceptive effect was defined as 50% MPE in the tall-flick and tall-pinch test, and 50% of the control response in the SP-induced behavior.
Measurement of imipramine content in the spinal cord and the whole brain Mice were injected intraperitoneally (i.p.) with imipramine, 20 mg/kg or 15.8 nmol, 31.6 nmol i.t., and decapitated 30 rain after i.p. injection or 5 min after i.t. injection. The whole brain and the spinal cord were removed and weighed, then frozen on dry-ice and stored at -4&C until the assay. The tissue sample was homogenized in 10 vol. (w/v) of methanol, using an ultrasonic cell disruptor (40% pulsed power for 30 s; Model 185, Branson), and centrifuged at 26,000 x g for 20 rain at 4°(2 (Kubota, KR-2000(Yr). The supernatant was filtered through a 0.45/~m membrane filter (LC3A, Gelman Sciences) and a 100/A aliquot of the filtered solution was injected into a high-performance liquid chromatography (HPLC) system. The HPLC system consisted of a delivery pump (Waters 510, Waters Associates), a sample injector (WISP 710B, Waters Associates), a reverse-phase column (250 mm length × 4.6 mm i.d.; Eicompak 5-ODS, Eicom Co.), an electrochemical detector
[3H]SP binding assay was performed by the Perrone et al.-': method with a small modification. [3H]SP (42.6 Ci/mmol specific activity) was obtained from New England Nuclear (Boston). Mice were decapitated and the spinal cord was rapidly removed and weighed. The tissue samples were homogenized in 10 vol. (w/v) of ice-cold 50 mM Tris buffer (Trizma; Sigma Chemical Co.) (pH 7.7 at 20°C) using a Polytron homogenizer (setting 6 for 30 s; Kinematical. The homogenate was centrifuged twice at 12,000 × g for 20 min at 0°C (Kubota, KR-20000T) with intermediate rehomogenization of the pellet in fresh buffer following the first centrifugation. The resultant pellet was resuspended in 5 vol. (w/v) of assay buffer consisting of 50 mM Tris buffer (pH 7.7 at 20°C), 2 mM CaC12, 2 mM MgCI 2, 4 ktg/ml leupeptin (Peptide Institute, Osaka, Japan), 2 #g/ml chymostatin (Peptide Institute), 40 pg/ml bacitracin (Sigma Chemical Co., St. Louis, MO) and 0.02% bovine serum albumin (Sigma Chemical Co.), then frozen and stored at -40°C until binding assay. For the binding assay, the tissue preparation was diluted with an assay buffer to 2 mg/ml protein concentration and a 100 kd aliquot of this tissue suspension was incubated at 20°C in a final volume of 500 #1 of the assay buffer with [3H]SP for 60 rain. Specific binding was defined as that displaced by 1/~M unlabeled SP. Incubation was terminated by filtration under reduced pressure over GF/F filter (Whatman Int. UK) that had been presoaked with 0.1% polyethyleneimine (Nacalai Tesque, Kyoto, Japan) for 3 h. The filters were rinsed with 3 × 3 ml ice-cold Tris buffer (pH 7.7 at 20°C) and emulsified by shaking with 10 ml Biofluor (New England Nuclear, Boston) for 30 min. Samples were counted by liquid scintillation spectrometry (Packard, TRI-CARB 4430). Protein was determined by the Lowry et al. 24 method. To investigate the effect of unlabeled SP or imipramine on [3H]SP specific binding, various concentrations of unlabeled SP or imipramine were co-incubated with 0.84 nM [3H]SP and the displacement curves were drawn. Values for 50% inhibitory concentration (IC50) of drugs against [3H]SP binding were determined from linear regression analysis of Hill plots.
Drugs Imipramine hydrochloride, norepinephrine hydrochloride, 5-hydroxytryptamine creatinine sulfate and naloxone hydrochloride were purchased from Sigma Chemical Co. (St. Louis, MO); morphine hydrochloride was from Takeda Chemical Industries (Osaka, Japan); phentolamine mesylate was from Ciba-Geigy Japan Co. (Takarazuka, Japan); methysergide maleate was from Sandoz (Basel, Switzerland); substance P was from Peptide Institute (Osaka, Japan). All drugs administered i.t. were dissolved in sterile artificial cerebrospinal fluid (NaCI 138 mM, KCI 3 mM, CaCI 2 1.25 mM, MgCI 2 1 mM and D-glucose 1 mM) and made up to a volume of 5 pl. Imipramine administered i.p. was dissolved in sterile 0.9% NaC1 and made in a volume of 10 ml/kg.
Statistical analysis The results were analysed by the Kruskal-Wallis test followed by the Wilcoxon U-test. All values are expressed as the mean +__ S.E.M.
RESULTS
Antinociceptiye effect o f imipramine Tail-pinch test. I m i p r a m i n e a d m i n i s t e r e d i.t. o r i.p.
61 (%)
p r o d u c e d a d o s e - d e p e n d e n t antinociceptive effect (Fig. 1). M a x i m u m effects were o b t a i n e d 5 min after i.t. injection (Fig. 1A) and 15-30 min after i.p. injection (Fig.
120
c 0 (z ¢0
100
==
MPE
(%) c 0 o
100
c o 50
50
0 I
-20
0 5
I
roll
15 30 Time a f t e r injection
I
60
7.9
15.8
23.7
31.6
i 47.3
Imipramine (nmol) min
MPE
(%)
**
lOO
lii I
G.
Fig. 2. Antinociceptive effect of imipramine on intrathecally administered SP-induced behavior. Each column represents the mean + S.E.M. (n = 8) of the percentage of control response. Imipramine was i.t. administered 5 min before SP administration. Control response indicates the number of the SP-induced behavior 5 min after i.t. administration of 5 pl artificial CSF (vehicle). *'**Indicate significant difference from the control response, P < 0.05 or P < 0,01, respectively.
50
0 -20
,, 0
i i i 15 30 45 Time a f t e r injection
I-" 6 0 min
MPE
(%)
C
100 ~ * *
**
50
0 -20
I I 0 5
l i 15 30 T i m e a f t e r injection
I 60
min
Fig. 1. Time-course of antinociceptive effect of imipramine on tailpinch test (A,B) and tail-flick test (C) in mice. The ordinate shows the antinociceptive effect expressed as MPE (%) indicated in Materials and Methods, and the abscissa shows time after drug administration. Each vertical bar is the mean + S.E.M. from at least 8 animals. A,C: mice were intrathecally administered 5/A volume of artificial cerebrospinal fluid (CSF) (o) or imipramine (15.8 nmol, O; 31.6 nmol, A; 63.1 nmol, II). B: mice were intraperitoneally administered 10 ml/kg volume of saline ((3) or imipramine (12.5 mg/kg, O; 25.0 mg/kg; a ; 50.0 mg/kg, II). *'** Indicate significant difference from the vehicle control group, P < 0.05 or P < 0.01, respectively.
1B). The EDs0 value (95% confidence limit) for 5 min after i.t. injection was 27.5 (17.0-43.9) nmol, and the EDs0 for 30 min after i.p. injection was 19.5 (9.8-39.0) mg/kg. Tail-flick test. I m i p r a m i n e injected i.t. p r o d u c e d a d o s e - d e p e n d e n t antinociceptive effect (Fig. 1C) and the m a x i m u m effect was observed 5-15 min after i.t. injection. The EDs0 value for 5 min after i.t. injection was 20.2 (12.6-32.2) nmol. SP-induced behavior. In control animals, i.t. injection of SP p r o d u c e d the nociceptive b e h a v i o r of biting, licking and scratching within 1 min of injection. A v e r a g e total count of the behavior was 64.4 + 7.8 counts/min (mean + S . E . M . , n = 8). Since i.t. administered imip r a m i n e p r o d u c e d its m a x i m u m antinociceptive effect 5 min after injection in both the tail-pinch and tail-flick tests, imipramine was injected 5 min before SP administration. I m i p r a m i n e p r o d u c e d a d o s e - d e p e n d e n t inhibition of SP-induced behavior (Fig. 2), with an EDs0 value of 20.2 (16.1-25.2) nmol.
Influences of several neurotransmitter antagonists on the effect of imipramine in SP-treated mice a-Adrenergic receptor antagonist. N E (40 pmol) i.t. coadministered with SP r e d u c e d SP-induced behavior to 29.6 + 13.4% of the control response. P h e n t o l a m i n e (10 nmol) i.t. co-administered with SP did not significantly increase SP-induced b e h a v i o r (114.8 + 28.9% of the con-
62
A
B
(%)
(%)
2oo
"
O
2OO
150
150
tt
¢o
==
-6 i. ,-
100
O o
+
100
c
~
50 0.
o NE Phentolamine
-
÷
-
÷
-
-
~
+
Imipramine Phentolamine
-
*
-
+
-
-
*
+
Fig. 3. Antinociceptive effect of NE (A) or imipramine (B) on SPinduced behavior, and antagonism of this effect by a-adrenergic receptor antagonist phentolamine. Each column represents the mean + S.E.M. (n = 8) of the percentage of control response. Control response indicates the number of the SP-induced behavior i.t. coadministered with vehicle for NE, phentolamine or given the vehicle for imipramine 5 min before SP administration. A: control response was 41.9 + 2.6 counts/rain. NE (40 pmol) was i.t. co-administered with SP. Phentolamine (10 nmol) was also i.t. coadministered with SP. B: control response was 45.0 + 13.2 counts/min. Imipramine (31.6 nmol) was i.t. administered 5, min before SP administration. Phentolamine (10 nmol) was i.t. coadministered with SP. *'**Indicate significant difference from the control response, P < 0.05 or P < 0.01, respectively. **Indicates significant difference between NE group and NE + Phentolamine group, P < 0.01.
trol response), but reversed the effect of N E on SP-induced b e h a v i o r (123.9 + 11.5% of the control response) (Fig. 3A). I m i p r a m i n e (31.6 nmol) injected i.t. 5 min before SP administration also r e d u c e d SP-induced behavior to 16.2 + 6.4% of the control response, but phentolamine (10 nmol) i.t. co-administered with SP did not significantly reverse the effect of i m i p r a m i n e on SP-induced b e h a v i o r (41.3 + 16.4% of the control response)
(Fig. 3B). Serotonergic receptor antagonist. 5-HT (6.5 nmol) i.t. co-administered with SP r e d u c e d the SP-induced behavior to 14.0 _ 12.3% of the control response. A l t h o u g h i.t. injection of 65.0 nmol 5-HT p r o d u c e d scratching behavior, the administration of 6.5 nmol 5 - H T did not produce scratching b e h a v i o r by itself. Methysergide (40 nmol) i.t. co-administered with SP did not significantly increase SP-induced b e h a v i o r (130.8 + 20.5% of the control response) but reversed the effect of 5-HT on SP-induced b e h a v i o r (165.3 _+ 33.3% of the control response) (Fig. 4A). I m i p r a m i n e (31.6 nmol) injected i.t. 5 min before SP administration also r e d u c e d SP-induced be-
havior to 13.2 + 3.8% of the control response, but methysergide (40 nmol) i.t. co-administered with SP did not reverse the effect of imipramine on SP-induced behavior (26.9 _+ 10.5% of the control response) (Fig. 4B). Opioid receptor antagonist. M o r p h i n e (0.125 nmol) i.t. co-administered with SP reduced SP-induced behavior to 38.4 + 13.9% of the control response. Naloxone (4 nmol) i.t. co-administered with SP did not significantly increase SP-induced behavior (98.9 + 22.7% of the control response), but reversed the effect of morphine on SP-induced behavior (125.1 _+ 18.4% of the control response) (Fig. 5A). I m i p r a m i n e (31.6 nmol) injected i.t. 5 min before SP administration reduced SPinduced behavior to 4.6 + 3.2% of the control response, but naloxone (4 nmol) i.t. co-administered with SP did not reverse the effect of imipramine on SP-induced behavior (6.3 + 3.6% of the control response) (Fig. 5B).
Measurement of imipramine in the spinal cord and the whole brain The imipramine contents in the spinal cord and the whole brain were m e a s u r e d 30 min after the i.p. injec-
A
B (%) 200
t t
(%)
I
200
]too
+
,oo f c
g.
g.
l i
50
o_;i 5-HT
Methysergide
-
+
-
+
Imipramlne
-
+
-
-
+
+
Methyserglde
-
-
+
+
+
Fig. 4. Antinociceptive effect of 5-HT (A) or imipramine (B) on SP-induced behavior, and antagonism of this effect by serotonergic receptor antagonist methysergide. Each column represents the mean + S.E.M. (n = 8) of the percentage of control response. Control response means the same as described in Fig. 3. A: control response was 41.5 + 9.0 counts/min, 5-HT (6,5 nmol) was i.t. co-administered with SP. Methysergide (40 nmol) was also i.t. coadministered with SP. B: control response was 53.1 + 8.0 counts/ min. Imipramine (31.6 nmol) was i.t. administered 5 min before SP administration. Methysergide (40 nmol) was i.t. coadministered with SP. **Indicates significant difference from the control response, P < 0,01. ttIndicates significant difference between 5-HT group and 5-HT + methysergide group, P < 0,01.
63 A
TABLE I
Contents of imipramine in the spinal cord and the whole brain after intraperitoneal or intrathecal administration of imipramine
30
All values are expressed as the mean + S.E.M. of data obtained in 6 animals
lmipramine dose
;20
Imipramine content (nmol/g tissue) Spinal cord
Intraperitoneal a 20 mg/kg Intrathecal b 15.8. nmol 31.6 nmol
38.5 + 4.1
48.3 + 4.7
55.2 + 6.0 104.4 + 9.8
-* -*
o e~
,1 10
aMice were decapitated 30 min after administration of imipramine. bMice were decapitated 5 min after administration of imipramine. *Imipramine was not detected.
tion of imipramine with the dose of 20 mg/kg. The dose of 20 mg/kg was selected because the EDs0 value of the tail-pinch test 30 min after i.p. imipramine injection was 19.5 mg/kg. The contents of imipramine in the whole brain and the spinal cord were 48.3 + 4.7 nmol/g tissue and 38.5 + 4.1 nmol/g tissue, respectively (Table I). The EDs0 values of the tail-pinch, tail-flick test and SP-induced behavior 5 min after i.t. injection were between A
I
0 o
I
i
1.0
2.0
0 10 20 30 B (fmol/mg protein)
1
3.0
i 4.0
3 H - S P (nM)
Fig. 6. [3H]SP binding to homogenate of the mouse spinal cord. A: specific and non-specific binding of [3H]SP to the homogenate of the mouse spinal cord as function of radiolabeled peptide concentration. The abscissa shows [3H]SP concentration (nM) and ordinate shows [3H]SP bound (fmol/mg protein). Homogenate was prepared as described in Materials and Methods and incubated at 20°C for 1 h with increasing concentrations of [3H]SP. Specific binding (O) and non-specific binding (&) were calculated as described in Materials and Methods. B: Scatchard analysis of specific binding from the experiment in (A) is presented. The abscissa shows bound [3H]SP (fmol/mg protein) and the ordinate shows bound/free (fmol/mg protein/nM). Each point is the mean of duplicates.
B
(%)
(%) tt
150
150 tO e~ g~
i
B
,o
Whole brain
+
100
100
.E
(%) 125
Jo
cO
o
100
o 0) t-i
=o
cO~
so
50
IX
a.
o Morphine
o +
+
75
I "1" o)
Imiprarnine
--~ -
+
ig
50
¢J
+
25 c
NaJoxone
+
+
Naloxone
+
+
Fig. 5. Antinociceptive effect of morphine (A) or imipramine (B) on SP-induced behavior, and antagonism of this effect by opioid receptor antagonist naloxone. Each column represents the mean _+ S.E.M. (n = 8) of the percentage of control response. Control response means the same as described in Fig. 3. A: control response was 37.5 _+ 6.5 counts/min. Morphine (0.125 nmol) was i.t. coadministered with SP. Naloxone (4 nmol) was also i.t. co-administered with SP. B: control response was 75.6 ___ 15.8 counts/rain. Imipramine (31.6 nmol) was i.t. administered 5 min before SP administration. Naloxone (4 nmol) was i.t. co-administered with SP. *'**Indicate significant differences from the control response, P < 0.05 or P < 0.01, respectively. +*Indicates significant difference between morphine group and morphine + naloxone group, P < 0.01.
8 el
0 -1(
a -9
t
I
I
I
I
-8
-7
-6
-5
-4
I
-3
IOglo (concentration, M) Fig. 7. Inhibition of [3H]SP binding to the homogenate of the mouse spinal cord by unlabeled SP or imipramine. The 0.84 nM [3H]SP and increasing concentrations of unlabeled SP (11) or imipramine (O) were mixed and incubated with the homogenate of the mouse spinal cord. Incubating conditions are defined as described in Materials and Methods. The abscissa shows lOgl0(concentration; M) of unlabeled drugs and the ordinate shows the percentage of the binding of [3H]SP alone. The data are the mean _+ S.E.M. of duplicate determinations from 5 separate experiments.
64 TABLE II Inhibition of [~H]SP binding to the homogenate of the mouse spinal cord by unlabeled SP and imipramine
The concentrations of the drugs required to inhibit the specific binding of 0.84 nM [3H]SP to the homogenate of the mouse spinal cord by 50% (IC5o) and Hill coefficients (nn) were estimated from the Hill plots of the displacement curves represented in Fig. 7. All values are expressed as the mean + S.E.M. of data obtained in n experiments.
Unlabeled SP Imipramine
1Cso (m)*
nl4
n
(2.46+0.70)× 10-9 (2.34+0.39)× 10 -4
1.01 + 0.15 0.77 + 0.07
4 5
*Indicates significant difference between unlabeled SP and imipramine, P < 0.05.
15.8 nmol and 31.6 nmol. The imipramine contents 5 min after the i.t. injection of this agent were measured for these doses of imipramine. In the spinal cord, the contents of imipramine after i.t. injection of 15.8 nmol and 31.6 nmol of this agent were 55.2 + 6.0 nmol/g tissue and 104.4 + 9.8 nmol/g tissue, respectively (Table I). In the whole brain, imipramine could not be detected 5 min after i.t. injection of imipramine. [3H]SP binding assay
In the homogenate of the spinal cord, [3H]SP binding was saturable and Scatchard analysis of saturation data yielded a straight line from which the density of binding sites (Bmax) w a s 29.5 + 1.5 fmol/mg protein and the dissociation constant (Kd) was 0.45 + 0.04 nM (n = 5) (Fig. 6), Unlabeled SP or imipramine dose-dependently inhibited [3H]SP binding (Fig. 7). The Hill plot yielded the IC50 values for unlabeled SP and imipramine against [3H]SP binding of 2.46 (+_ 0.70) x 10-9 M and 2.37 (_+ 0.39) × 10 -4 M , respectively. The Hill plot yielded Hill coefficients for unlabeled SP and imipramine of 1.01 _+ 0.15 and 0.77 _+ 0.07, respectively (Table II). DISCUSSION Many investigators have shown that antidepressants have antinociceptive effect in r a t s 1'3'9'20'30'35'41, mice 7,s' 14,39 and humans TM. The antinocieeptive effects of antidepressants were shown in animals by the systemic administration 1'3'7-9A8'2°'3°'35'39'41, by the intracerebroventricular administration 39 and by the i.t. administration TM. In the present study we confirmed antinocieeptive effect of imipramine by both the tail-pinch test and the tail-flick test. In the tail-pinch test, imipramine produced an antinociceptive effect in both treatment of i.p. and i.t. with EDso values of 19.5 mg/kg and 27.5 nmol, respectively. If imipramine produced an antinociceptive effect
at the spinal level, the contents of imipramine in the spinal cord would be the same when mice were administered imipramine at the dose to produce the same degree of antinociception by i.p. and i.t. When 20 mg/kg imipramine was injected i.p., the content of imipramine in the spinal cord 30 min after administration was 38.5 nmol/g tissue. On the other hand, when 15.8 nmol of imipramine (this dose was smaller than the EDs0 dose of i.t. imipramine) was injected i.t.. the content of imipramine in the spinal cord 5 min after injection was 55.2 nmol/g tissue. When the EDso dose of imipramine was injected i.t., imipramine did not reach the supraspinal site, and this fact indicates that imipramine can produce antinociception at the spinal level. But the supraspinal mechanism cannot be excluded because the content of imipramine in the spinal cord after i.p. injection of the EDso dose imipramine was lower than the content of imipramine in the spinal cord after i.t. injection of the ED50 dose imipramine. To study the spinal mechanism of the antinociceptive effect of imipramine in detail, the effect of imipramine on SP-induced behavior was investigated with several receptor antagonists that are related to nociception. SP is thought to be one of the neurotransmitter candidates of somatosensory primary afferent fibers especially for nociception 36. It is reported in mice that SP administered i.t. produces caudally directed biting, licking and scratching 18'28 which is similar to the behavior induced by peripheral irritation 15. This SP-induced behavior is reported to be reduced by i.t. co-administered opioids, NE 19 and 5-HT iv, and in the present study NE, 5-HT and morphine reduced SP-induced behavior. This inhibitory effect was reversed by the a-adrenergic receptor antagonist phentolamine, the 5-HT receptor antagonist methysergide and the opioid receptor antagonist naloxone, respectively. We observed in the present study that 6.5 nmol of 5-HT was necessary to inhibit SP-induced behavior but other investigators have shown that a smaller dose of 5-HT (6.5-12.9 pmol) is sufficient to inhibit SP-induced behavior and a larger dose of 5-HT (25.8 pmol-10.3 nmol) by itself produces scratching behavior 1°'11'17'42. In our observation 6.5 nmol of 5-HT did not produce the scratching behavior by itself but 65.0 nmol of 5-HT produced approximately 40 counts/min of scratching behavior. The discrepancy between previous studies and the present data of 5-HT doses to inhibit SP-induced behavior and to produce scratching behavior by itself cannot be clearly explained but the difference of the mouse strain may be related. The mechanism of the antinociceptive effect of antidepressants is not clearly understood but two possible mechanisms are suggested. One possibility is that antidepressants produce the antinociceptive effect through
65 the serotonergic mechanism 7'9'35'4~. The other possibility is that antidepressants interact with opioid receptors and produce antinociception 1'3'~-9'2°'3°'35. Because antidepressants are known to inhibit NE uptake at nerve endings 34 and NE is recognized to have an antinociceptive effect at the spinal level 29, NE involvement in the antinociceptive effect of imipramine could not be ruled out. But in the present study, imipramine reduced SP-induced behavior as previously reported 12'14 and this effect was not antagonized by an a-adrenergic receptor antagonist, a serotonergic receptor antagonist or an opioid receptor antagonist. This fact suggests that imipramine inhibits SP-induced behavior by a mechanism other than those previously suggested. Antidepressants interact with muscarinic receptors 38 or N-methyl-D-aspartate receptors 31 and these mechanisms may contribute to the antinociceptive effect of antidepressants but one of the possible mechanisms by which imipramine inhibits SPinduced behavior may be by direct action on SP receptors. To determine the direct effect of imipramine on SP receptors, we performed in vitro studies of [3H]SP binding to the receptors. Imipramine inhibited [3H]SP binding with IC50 value of 2.37 × 10-4 M. The tissue preparation for SP binding contained protein of 0.4 mg/ml and in this procedure 14.45 + 0.17 mg protein was obtained from 1 g spinal cord. Therefore, 2.37 × 10-4 M of imipramine corresponds to 8.6 #mol/g tissue concentration. The EDs0 value of imipramide for inhibition of SP-induced behavior was 20.2 nmol, the imipramine content in the spinal cord 5 min after i.t. injection of this dose was between 55.2 nmol/g tissue and 104.4 nmol/g tissue. Therefore IC50 of imipramine for [3H]SP binding is at least 80 times higher than EDso for SP-induced behavior. This dissociation cannot be clearly explained but in vivo studies on imipramine may exist at the site of action in higher concentrations than calculated. Imipramine has also been reported to inhibit [3H]naloxone binding 1'3, [3H]naltrexone binding 2° and [3H]MK-801 bind-
ing3t. IC50 values of imipramine for these effects were approximately 10 times lower than that for inhibition of SP binding. In addition, there are several nociceptive neurotransmitter candidates and Kuraishi et al. have reported that in rabbits thermal and mechanical noxious stimuli are transmitted with different neurotransmitters and SP is related only to mechanical noxious stimuli 22. In the present study, i.t. imipramine produces antinociception for both thermal and mechanical noxious stimuli in mice. From these facts the antinociceptive effect of imipramine cannot be explained only by SP receptor blockade but noxious stimuli transmitted with SP may be partially blocked at the SP receptor level. In the [3H]SP binding assay, the Hill plot revealed that the Hill coefficient for imipramine was smaller than unity. More detailed study of the interaction between imipramine and SP receptors is necessary. Recently some studies have shown that chronic antidepressant treatment increases the effect of SP at the spinal level a3 and at the supraspinal level 21. These facts may indicate the up-regulation of SP receptors by the SP binding inhibition of antidepressant. In conclusion, imipramine produced antinociceptive effect at the spinal level on the thermal or mechanical noxious stimuli and SP-induced behavior. Imipramine inhibited SP-induced behavior through the non-adrenergic, non-serotonergic and non-opioidergic mechanisms. In vitro, imipramine inhibited [3H]SP binding with IC5o value of 2.37 × 10-4 M but this dose corresponded to about 100 times higher tissue concentration of imipramine than the antinociceptive effect.
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Acknowledgements.We express our thanks to Professor Takeo Fukuda and Dr. Masahiro Nomoto from the Department of Pharmacology, School of Medicine, Kagoshima University for their valuable suggestions and to Professor Nozomu Yoshimura from the Department of Anesthesiology, School of Medicine, Kagoshima University for his encouragement and to Mr. Yoshiteru Toriire for his technical assistance. And we thank Mr. Robert Hodierne for reading the manuscript.
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