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Differential mechanisms mediating β-endorphin- and morphine-induced analgesia in mice
European Journal of Pharmacology, 168 (1989) 61-70
61
Elsevier EJP 50939
Differential mechanisms mediating fl-endorphinand morphine-induced analgesia in mice Harold Hongwon Suh, James Masao Fujimoto and Leon Liang-Fu Tseng
*
Department of Pharmacology and Toxicology, Medical College of Wisconsin and Toxicological Research Laboratory, CJ Zabloeki, VA Medical Center, Milwaukee, 141153226, U.S.A.
Received 16 March 1989, revised MS received 30 May 1989, accepted 13 June 1989
Effects of yohimbine, methysergide and naloxone given intrathecally (i.t.) and naloxone given intracerebroventriculady (i.c.v.) on inhibition of the tail-flick and hot-plate response induced by fl-endorphin and morphine given i.c.v. were studied in male ICR mice. Yohimbine (1.5 and 15/~g) and methysergide (1.5 and 15/~g) injected i.t. antagonized inhibition of the tail-flick response induced by morphine but not fl-endorphin administered i.c.v. On the other hand, naloxone (20 ng) injected i.t. antagonized inhibition of the tail-flick response induced by i.c.v, administered fl-endorphin but not morphine. Yohimbine and methysergide given i.t. did not antagonize inhibition of the hot-plate response induced by morphine nor did naloxone given i.t. antagonized i.c.v, fl-endorphin-induced inhibition of the hot-plate response. Naloxone given i.c.v, was more effective in antagonizing morphine-induced inhibition of the tail-flick and hot-plate response than inhibition induced by fl-endorphin given i.c.v. Naloxone at doses (0.1 and 1 ~g) which effectively reversed inhibition of the tail-flick response to i.c.v, morphine was not effective in reversing the i.c.v. fl-endorphin-induced inhibition of the tail-flick response. Our results indicate that fl-endorphin and morphine produce analgesia by stimulating separate types of opioid receptors, c- for fl-endorphin and /~- for morphine, and activate separate descending pain modulatory control systems. The supraspinal c system stimulated by fl-endorphin is mediated by activation of spinal opioid receptors whereas the supraspinal /~ system stimulated by morphine is mediated by activation of spinal aE-adrenoceptors and serotonin receptors for the production of analgesia. fl-Endorphin; Morphine; Analgesia; Pain control systems (descending)
1. Introduction fl-Endorphin, an endogenous opioid peptide found in the brain produces an antinociceptive response when injected into the brain in different species of animals and human (Feldberg and Smith, 1977; Loh et al., 1976; Hosobuchi and Li, 1978; Meglio et al., 1977; Tseng et al., 1979).
* To whom all correspondence should be addressed: Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Millwaukee, WI 53226, U.S.A.
Early publications seem to suggest that the pharmacological actions of fl-endorphin closely resemble those of morphine. Both analgesia produced by fl-endorphin and morphine are antagonized by systemic injection of naloxone (Lob et al., 1976). Like morphine, multiple injection or chronic administration of fl-endorphin into the brain produces tolerance and dependence (Tseng et al., 1977; Wei and Loh; 1976). fl-Endorphin also substitutes for morphine in animals tolerant to and dependent on morphine (Tseng et al., 1976; 1977). In in vitro opioid receptor binding studies, fl-endorphin like morphine binds to both /~- and 8-opioid receptors (Hazum et al., 1975; Law et al.,
0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
62 1979). However, the contention that fl-endorphin has the same or similar mechanism of action as that of morphine has been questioned. We have previously demonstrated that fl-endorphin and morphine produce analgesia by activating different descending pain modulatory systems (Tseng and Fujimoto, 1984; 1985; Suh and Tseng, 1988). Unlike morphine which produces analgesia in part by activation of the descending noradrenergic and serotonergic systems, the analgesia induced by fl-endorphin applied supraspinally appears to be mediated by the release of [MetS]enkephalin in the spinal cord (Camarata and Yaksh, 1985; Tseng and Fujimoto, 1984; 1985; Tseng et al., 1985; 1986; Yaksh, 1979). These concepts are supported by the pharmacological studies which show that blockade of a 2adrenoceptors or serotonin receptors in the spinal cord by i.t. injection of yohimbine or methysergide antagonize the analgesia induced by morphine administered supraspinally and blockade of spinal opioid receptors by i.t. injection of naloxone or fl-funaltrexamine antagonizes supraspinally administered fl-endorphin-induced analgesia (Jensen and Yaksh, 1986; Kuraishi et al., 1983; Shiomi et al., 1978; Wigdor and Wilcox, 1987; Yaksh and Tyce, 1979; Tseng and Fujimoto, 1984; 1985; Suh and Tseng, 1988). Biochemical studies have shown that i.c.v, injection of fl-endorphin but not morphine releases [MetS]enkephalin from the spinal cord (Tseng et al., 1985; 1986). The types of opioid receptors stimulated by fl-endorphin and morphine for the production of analgesia appear to be different. Previous studies in mice have demonstrated that i.c.v, pretreatment with fl-funaltrexamine antagonizes analgesia induced by morphine but not fl-endorphin given i.c.v. On the other hand, fl-endorphin-(1-27) given i.c.v, antagonizes i.c.v, fl-endorphin- but not morphine-induced analgesia (Suh et al., 1988). The purpose of the present experiments was to systematically examine the effects of naloxone given i.c.v, and naloxone, yohimbine or methysergide given i.t. on analgesia induced by i.c.v. administered fl-endorphin and morphine. The studies are designed to further delineate the differential mechanisms of analgesic actions produced by fl-endorphin and morphine given i.c.v.
2. M a t e r i a l s
and methods
2.1. Animals Male ICR mice weighing 23-25 g (Harlan Sprague Dawley, Inc., Indianapolis, IN) were used for all the experiments. Animals were housed five per cage in a room maintained at 22 _+0.5 °C with an alternating 12 h light-dark cycle. Food and water were available ad libitum. Animals were used only once in all the experiments.
2.2. Assessment of analgesia Analgesia was determined by the tail-flick (D'Amour and Smith, 1941) and 55°C hot-plate (Eddy and Leimbach, 1953) assays. In most experiments, tail-flick assay was followed by hotplate assay. The intensity of the heat stimulus in the tail-flick test was adjusted so that the animal flicked its tail in 3-4.5 s. Control latencies for the hot-plate test were approximately 9 s. The hotplate and tail-flick latencies were measured before (To) and after (T1) i.c.v, injection of fl-endorphin and morphine. The inhibition of the tail-flick and hot-plate response was expressed as 'percent analgesia' which was calculated as [(T1 - T 0 ) / ( T 2 - To)] × 100, where the cutoff time (T2) was set at 10 and 30 s for the tail-flick and hot-plate tests, respectively. To calculate EDs0 values, at least three different doses were used and eight to nine mice were used for each dose. EDs0 values and their 95% confidence intervals were determined by the method of Litchfield and Wilcoxon (1949) with the aid of a computer program described by Tallarida and Murray (1981) using an Apple II computer.
2.3. Experimental protocol I.t. injection was made according to the procedure of Hylden and Wilcox (1980) using a Hamilton syringe with 30 gauge needle. The i.c.v, administration was performed following the method of Haley and McCormick (1957). Injection volumes were 4 and 5/~1 for i.t. and i.c.v, injection, respectively. Injection sites were verified using 1%
63
2.4. Drugs
methylene blue in testing animals until a 90% success rate was achieved. Yohimbine (1.5 or 15/Lg) and methysergide (1.5 or 15 /~g) were injected i.t. 20 min prior to the i.c.v, injection of various doses of/3-endorphin or morphine, while naloxone (20 ng) was injected i.t. immediately before the i.c.v, injection of /3-endorphin or morphine. In another study, naloxone (2 /~g) was coadministered i.c.v, with different doses of /3-endorphin or morphine. These doses were chosen based on the preliminary studies that these doses of antagonists effectively block the effects of the respective agonists injected i.t. or i.c.v (Wigdor and Wilcox, 1987; Gulya et al., 1988). Hot-plate and tail-flick responses were tested 15 and 30 min, respectively, after the i.c.v. injection. These times used were determined based on the preliminary time course studies which showed the hot-plate and tail-flick inhibition reached a maximum at 15 and 30 min, respectively, after i.c.v, injection of 2 ttg (6 nmol) of morphine or 1 /~g (0.3 nmol) of/3-endorphin. In another experiment, different doses of naloxone were given i.c.v. 15 and 30 min after i.c.v, injection of a fixed dose (4 /~g) of /3-endorphin and morphine, respectively and the tail-flick response was tested 2.5, 5, 10, 15, 20, 30 and 45 min after i.c.v, injection of naloxone.
Morphine sulfate was purchased from Mallinckrodt Chemical Works (St. Louis, MO). /3-Endorphin was obtained from Bachem Inc. (Torrance, CA). Yohimbine hydrochloride was purchased from Sigma Chemical Company (St. Louis, MO). Methysergide maleate was a gift from Sandoz Pharmaceuticals (Hanover, N J). Naloxone hydrochloride was a gift from Endo Laboratories (Wilmingtonl DE). Morphine sulfate, /3-endorphin and naloxone hydrochloride were dissolved in a sterile saline. Yohimbine hydrochloride and methysergide maleate were dissolved in a vehicle consisting of 15% dimethyl sulfoxide and 85% saline (0.9% NaC1). Doses refer to the salt form.
3. Results
3.1. Effects of yohimbine and methysergide injected i.t. on the analgesia induced by morphine and ~-endorphin administered i.c.v. Mice were injected i.t. with vehicle (4 #1), yohimbine (1.5 or 15 t~g), methysergide (1.5 or 15 /~g), or a combination of two drugs (1.5 + 1.5, 15 + 15 #g) 20 rain prior to i.c.v, injection of
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Fig. 1. The effects of intrathecal (i.t.) pretreatment with 15/.tg yohimbine (Yoh), methysergide (Methy) or the combination of the two drugs (Yoh + Methy) on intracerebroventricularly (i.c.v.) fl-endorphin (fl-EP)- and morphine (MS)-induced tail-flick (a) and hot-plate (b) inhibition. Mice were pretreated i.t. with either vehicle, yohimbine, methysergide or combination of yohimbine and methysergide for 20 min. Then fl-endorphin or morphine was injected i,c.v, and hot-plate and tail-flick response were tested at 15 and 30 min, respectively, later. The vertical bars are the S.E.M. The number of animals used for each dose was 8.
64 TABLE 1 Effect of i.t. yohimbine, methysergide and a combination of both on EDs0 values for/3-endorphin and morphine for the inhibition of the tail-flick response. EDs0 (nmol/mouse)
i.c.v. MS
a
Saline
Yohimbine (1.5 #g)
Methysergide (1.5/tg)
Yohimbine + methysergide (1.5/zg) (1.5/~g)
1.90 (1.30-3.20) c
7.90 b (4.30-14.5)
7.10 b (3.50-14.6)
14.1 b (6.40-17.7)
Yohirnbine (15/~g)
Methysergide (15/~g)
Yohimbine + methysergide (15/Lg) (15 #g)
10.1 b (4.70-21.8) 0.35 (0.14-0.87)
8.80 b (5.10-15.5) 0.20 (0.10-0.30)
19.7 b (9.90-39.2) 0.20 (0.10-0.30)
i.c.v. MS i.C.V. /3-EP
0.25 (0.13-0.49)
" The EDs0 values were calculated according to the method described by Litchfield and Wilcoxon (1949). b Significantly different from vehicle control, P < 0.05. ¢ Numbers in parentheses indicate the 95% confidence interval.
various doses of morphine or /3-endorphin. The hot-plate and tail-flick responses were measured at 15 and 30 min, respectively, after i.c.v, injection of morphine or/3-endorphin. As shown in fig. la and b, /3-endorphin administered i.c.v, at doses 0.078-0.58 nmol (0.25-2/~g) caused a dose-dependent increase of inhibition of the tail-flick and hot-plate response in mice pretreated i.t. with vehicle. I.t. injection with yohimbine (15 ~g), methysergide (15 /~g) or combination of two antagonists (15 #g + 15 ~g) did not affect inhibition of the tail-flick and hot-plate response produced by/3-endorphin injected i.c.v. The EDs0
values and the slopes of dose-response lines for /3-endorphin injected i.c.v, for inhibition of the tail-flick and hot-plate response in mice pretreated with yohimbine, methysergide or combination of ,two antagonists were not significantly different from those in mice treated with vehicle i.t. (tables 1 and 2). Morphine at doses 0.7-6.0 nmol (0.25-2 ~g) caused a dose-dependent increase of inhibition of the tail-flick and hot-plate response in vehicle pretreated mice. In contrast to the lack of effects of i.t. yohimbine and methysergide on /3-endorphin-induced analgesia, i.t. injection of yohim-
TABLE 2 Effect of i.t. yohimbine, methysergide and a combination of both on EDs0 values for/3-endorphin and morphine for the inhibition of the hot-plate response. EDs0 (nmol/mouse)
i.c.v. MS
Saline
Yohimbine (1.5/~g)
Methysergide (1.5/Lg)
Yohimbine + methysergide (1.5/~g) (1.5 ~tg)
1.49 (0.60-3.73) b
1.30 (0.60-3.0)
2.20 (1.40-3.40)
1.20 (0.60-2.30)
Yohimbine (15 tLg)
Methysergide (15 ~,g)
Yobimbine + methysergide (15 t'g) (15 tLg)
2.18 (1.25-3.83) 0.11 (0.07-0.20)
1.10 (0.30-.3.50) 0.11 (0.05-0.30)
1.60 (0.60-2.30) 0.20 (0.10-0.30)
i.c.v. MS i.c.v. /3-EP
a
0.11 (0.08-0.16)
a The EDs0 values were caluculated according to the method described by Litchfield and Wilcoxon (1949). b Numbers in parentheses indicate the 95% confidence interval.
65 affail- f l i c k Test
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Fig. 2. The effects of intrathecal (i.t.) injection of naloxone on intracerebroventricular (i.c.v.) fl-endorphin (fl-EP)- and morphine (MS)-induced tail-flick (a) and hot-plate (b) inhibition. Mice were injected i.t. with either saline or 20 ng of naloxone, at the same time they were injected i.c.v, with various doses of fl-endorphin and morphine. Tail-flick response and hot-plate response were tested as above (fig. 1). The vertical bars are the S.E.M. The n u m b e r of animals used for each dose was 8.
bine or methysergide at 1.5 or 15 ~g effectively antagonized inhibition of the tail-flick response induced by morphine injected i.c.v. A combination of yohimbine and methysergide (1.5 + 1.5 or 15 + 15 ~g) given i.t. further increased antagonism of inhibition of the tail-flick response induced by morphine administered i.c.v, and shifted the dose-response curve further to the right (fig. la and table 1). The EDs0 values for morphine for inhibition of the tail-flick response were increased 3.7-, 4.2-, 7.4-fold and 4.6-, 5.3-, 10.4-fold by 1.5 and 15 /~g of methysergide, yohimbine and the combination of methysergide and yohimbine, respectively, injected i.t. without any significant changes of the slopes of the dose-response lines (table 1). However, i.t. administration of yohimbine, methysergide and a combination of the two did not affect the inhibition of the hot-plate response induced by morphine given i.c.v. (fig. lb and table 2). In control groups, i.t. injection with yohimbine, methysergide or a combination of two drugs did not alter the tail-flick and hot-plate latencies of mice injected i.c.v, with vehicle, yohimbine, methysergide and the combination of yohimbine and methysergide, respectively, in the tail-flick test (N = 40), mean __+S.E.: 3.68 + 0.08, 3.76 + 0.09, 3.79 + 0.07 and 3.75 _ 0.08 s; in the hot-plate test (N = 40): 9.4 + 0.29, 9.6 + 0.30, 9.2 __+0.27 and 9.4 _ 0.40 s.
3.2. Effects of naloxone injected i.t. on the analgesia induced by fl-endorphin and morphine administered i.c.v. As shown in fig. 2a, blockade of spinal opioid receptors by i.t. injection of naloxone antagonized inhibition of the tail-flick response induced by TABLE 3 Effect of naloxone on ED5o values for fl-endorphin and morphine administered i.c.v, for the inhibition of the tail-flick and hot-plate response. EDso ( n m o l / m o u s e ) " i.t. Saline
i.c.v. Naloxone (20 ng)
Saline
Naloxone (2 ~tg)
A. Tail-flick test i.c.v, fl-EP 0.12 0.62 b 0.06 0.07 (0.03-0.30) c (0.30-1.30) (0.03-0.20) (0.04-0.15) i.c.v. MS 1.90 2.30 1.83 10.1 b (1.10-3.20) (1.30-4.00) (1.30-3.10) (5.50-18.7) B. Hot-plate test i.c.v, fl-EP 0.08 0.08 0.07 0.22 (0.04-0.14) (0.03-0.16) (0.04-0.12) (0.09-0.56) i.c.v. MS 1.1 1.20 1.10 13.1 b (0.60-2.00) (0.70-2.00) (0.60-2.10) (8.00-21.6) a EDso values were calculated according to the method described by Litchfield and Wilcoxon (1949). b Significantly different from saline control, P < 0.05. ¢ N u m b e r s in the parentheses indicate the 95% confidence interval.
66 a}Tail-flick Test
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Fig. 3. The effects of intracerebroventricular (i.c.v.) injection with naloxone on i.c.v, fl-endorphin (fl-EP)- and morphine (MS)-induced tail-flick (a) and hot-plate (b) inhibition. Mice were injected i.c.v, with various doses of fl-endorphin or morphine in the presence or absence of 2 / x g of naloxone. Tail-flick and hot-plate resonse was tested as above. The n u m b e r of animals used for each dose was 8.
fl-endorphin but not morphine administered i.c.v. EDs0 value for fl-endorphin for inhibition of the tail-flick response in mice injected with i.t. naloxone was increased 5-fold above that for saline controls (table 3) while i.t. naloxone did not affect the EDs0 for morphine for the inhibition. 1.t. injection of naloxone did not antagonize inhibition of the hot-plate response induced by fl-endorphin or morphine administered i.c.v. (fig. 2b and table 3).
3.3. Effects of naloxone injected i.c.v, on the analgesia induced by fl-endorphin and morphine administered i. c. v.
Naloxone at a dose of 2/.tg given i.c.v, did not antagonize inhibition of the tail-flick and hot-plate response induced by fl-endorphin administered i.c.v, while the same dose of naloxone markedly attenuated the i.c.v, morphine-induced inhibition of the tail-flick and hot-plate response (fig. 3a and
a,Tail-flick test
b. Hot- plate test
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Fig. 4. The effects of intracerebroventricular (i.c.v.) injection of various doses of naloxone on i.c.v, injected fixed dose of fl-endorphin (fl-EP)- and morphine (MS)-induced tail-flick (a) and hot-plate (b) inhibition. In one study, mice were injected i.c.v, with 2 /~g of fl-endorphin the presence of 2, 4, 8 or 16 ~g of naloxone. In another study, mice were injected i.c.v, with 2 /~g of morphine in the presence of 0.1, 0.2, 0.4 or 2 /~g of naloxone, Tail-flick response and hot-plate response were tested as above. The n u m b e r of animals used for each dose was 8.
67
b). The EDs0 values for fl-endorphin injected i.c.v. for inhibition of the tail-flick and hot-plate response were not significantly changed by naloxone given i.c.v, whereas EDs0 values for morphine injected i.c.v, were increased 7.8- and 11.9-fold, respectively, above saline controls, in mice coinjected with i.c.v, naloxone (table 3). The effects of various doses of naloxone in combination with a fixed dose of fl-endorphin (2 /~g) or morphine (2 /~g) given i.c.v, on inhibition of the tail-flick and hot-plate response were studied. Based on the calculation of the potency ratio of these two compounds, this dose (2 #g) of fl-endorphin and morphine produced approximately the same intensity of analgesic response. Naloxone was found to be about 27-fold more potent in antagonizing the analgesia induced by morphine than fl-endorphin in both tail-flick and hot-plate test (fig. 4). The differential actions of naloxone on fl-endorphinand morphine-induced analgesia can also be shown clearly in studies in which naloxone injected i.c.v. reversed the analgesia already developed by
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"rinm(min) Fig. 5. The effects of intracerebroventricular (i.c.v.) injection of naloxone on reversing the inhibition of the tail-flick response induced by fl-endorphin or morphine injected i.c.v. Mice were pretreated i.c.v, with 4 ~ g of fl-endorphin or morphine for 15 and 30 rain, respectively. Tail-flick responses, tested 2.5, 5, 10, 15, 20, 30 and 45 rain after i.c.v, injection of saline or naloxone. The vertical bars are the S.E.M. * Different from the saline control group (P < 0.05). The n u m b e r of the animals used for each dose was 8.
morphine or fl-endorphin. Naloxone at doses 0.2 and 1 #g caused a dose-dependent reversal of the inhibition of the tail-flick response induced by morphine (4 #g) given i.c.v, while 1 /xg of naloxone did not significantly affect the inhibition of the tail-flick response induced by fl-endorphin (4 #g) given i.c.v. (fig. 5).
4. Discussion
The results of the present studies showed that blockade of the spinal a2-adrenoceptors and serotonin receptors by i.t. injection of yohimbine and methysergide antagonized inhibition of the tail-flick response induced by morphine but not fl-endorphin. The findings indicate that spinopetal noradrenergic and serotonergic systems are involved in inhibition of the tail-flick response induced by i.c.v, administered morphine whereas the descending monoaminergic systems are not involved in inhibition of the tail-flick response induced by i.c.v, administered fl-endorphin. The results of experiments with morphine in the present study are in agreement with previous studies by others which demonstrated that spinal noradrenergic or serotonergic antagonists attenuated the analgesia induced by morphine injected supraspinally in rats (Yaksh and Tyce, 1979; Kuraishi et al., 1978; 1979; 1983; Jensen and Yaksh, 1986; Yaksh, 1985) and mice (Wigdor and Wilcox, 1987). A combination of yohimbine and methysergide producing more antagonism of inhibition than each antagonist injected alone seems to suggest that adrenergic and serotonergic systems are separately involved in inhibition of the tail-flick response induced by supraspinal morphine. The aadrenoceptors involved in the antinociception induced by morphine has been identified to be a 2but not al-adrenoceptors (Yaksh, 1979; Howe et al., 1982). Further evidence that the analgesia induced by morphine but not fl-endorphin is mediated by activation of spinopetal noradrenergic and serotonergic systems can be obtained from the studies with biogenic amine uptake inhibitors. I.t. injection of desipramine or fluoxetine which inhibits the reuptake of norepinephrine and serotonin, respectively, potentiates the analgesia induced
68 by morphine (Larsen and Arnt, 1984) but not fl-endorphin given i.c.v. (unpublished observation). Blockade of spinal opioid receptors by i.t. injection of naloxone antagonized inhibition of the tail-flick response induced by i.c.v, administered fl-endorphin but not morphine. The results are consistent with that of previous studies in rats and mice that naloxone, fl-funaltrexamine, ICI 174864 or ICI 154129 given i.t. attenuates the analgesia induced by i.c.v, administered fl-endorphin but not morphine (Tseng and Fujimoto, 1984; 1985; Suh and Tseng, 1988; 1989a). A single i.c.v, injection of fl-endorphin but not morphine produces an acute tolerance to opioid receptors in the spinal cord (Suh and Tseng, 1989a). This pharmacological evidence that fl-endorphin but not morphine induces the release of endogenous opioids is further supported by biochemical studies, fl-Endorphin but not morphine administered i.c.v, releases [MetS]enkephalin from the spinal cord in anesthetized rats (Tseng et al., 1985), mice and other species (unpublished observations) of laboratory animals. Thus inhibition of the tail-flick response induced by supraspinally administered fl-endorphin is mediated by activation of spinal opioid receptors whereas inhibition induced by morphine does not involve spinal opioid systems. I.t. administration of yohimbine and methysergide did not antagonize inhibition of the hotplate response induced by morphine nor did i.t. injection of naloxone antagonize inhibition of the hot-plate response induced by fl-endorphin. These results are not surprising because the hot-plate response is believed to be a supraspinally integrated nociceptive response and the blockade of the spinal a-adrenoceptors and serotonin and opioid receptors should not affect the supraspinally integrated hot-plate responses (Jensen and Yaksh, 1986; Schmauss and Yaksh, 1984). At supraspinal sites, naloxone given i.c.v, was found to be more effective in antagonizing i.c.v. administered morphine-induced analgesia than flendorphin-induced analgesia in the tail-flick and hot-plate test. Naloxone at doses which effectively reversed the analgesia induced by morphine was found to be not effective in reversing the analgesia induced by fl-endorphin. The results indicate that the types of opioid receptors acted on by morphine
and fl-endorphin are different. This conclusion is further supported by other studies. (1) fl-Endorphin-(1-27) injected i.c.v, blocks i.c.v, administered fl-endorphin- but not morphine-induced analgesia (Sub et al., 1988). (2) fl-Funaltrexalnine or CTOP (D-Phe-Cys-Tyr-D-Try-Orn-Thr-PenThr-NH2), /~-receptor antagonists, injected i.c.v. blocks i.c.v, administered morphine- but not fl-endorphin-induced analgesia (Suh and Tseng, 1988; 1989b). (3) In cross tolerance study, we found that a single i.c.v, injection of fl-endorphin produced an acute tolerance to fl-endorphin but not morphine administered i.c.v. (Suh and Tseng, 1989b). (4) A single i.c.v, pretreatment with morphine produced an acute tolerance to morphine but not fl-endorphin administered i.c.v. (Suh and Tseng, 1989b). The specific opioid receptor for fl-endorphin has been designated as c-receptors (Wuster et al., 1980; Johnson et al., 1982; Goodman et al., 1983; Houghton et al., 1984). Our results support the proposal for separate opioid receptors supraspinally, c-opioid receptors for fl-endorphin and #-opioid receptors for morphine for the production of analgesia as represented by inhibition of the tail-flick response. High dose of naloxone (27 times higher than the dose which blocks morphine-induced analgesia) injected i.c.v, was found to antagonize fl-endorphin-induced analgesia. Inasmuch as naloxone is a highly lipid soluble compound, the possibility exists that small amount of naloxone injected i.c.v. might diffuse into the spinal cord by redistribution and antagonizes i.c.v, injected fl-endorphininduced analgesia by blocking the opioid receptors at the spinal site. We l~ave previously demonstrated that 2.5 #g of fl-funaltrexamine injected i.c.v, for 24 h does not block analgesia induced by fl-endorphin given i.c.v~ (Suh and Tseng, 1988). The findings that naloxone in combination with fl-endorphin given i.c.v, did not antagonize the analgesia induced by fl-endorphin while i.t. or systemic injection of naloxone is effective in antagonizing the fl-endorphin-induced inhibition of the tail-flick response (Tseng and Fujimoto, 1984; 1985; Tseng et al., 1983) indicate that naloxone given systemically blocks the fl-endorphin-induced analgesia by blocking the effects of released [MetS]enkephalin at the postsynaptic opioid re-
69
ceptor sites rather than blocking the opioid receptors acted on directly by injected fl-endorphin. It is concluded that fl-endorphin and morphine stimulate separate types of opioid receptor supraspinally, c-receptors for/3-endorphin and it-receptors for morphine. The supraspinal ~ system activated by fl-endorphin involves spinal opioid receptor whereas supraspinal /t system activated by morphine involves spinal a2-adrenoceptors and serotonin receptors for the production of the tailflick inhibition.
Acknowledgements This work was supported by US Public Health Grant, DA 03811, (L.F.T.) and VA Medical Research Fund (J.M.F.). A preliminary report on some of these results was presented at the meeting of Federation of American Societies for Experimental Biology, May, 1988 (FASEB Journal 2, A368, 1988).
References Camarata, P. and T.L. Yaksh, 1985, Characterization of the spinal adrenergic receptors mediating the spinal effect producing by the microinjection of morphine into the periaqueductal gray, Brain Res. 336, 133. D'Amour, F.E. and D.L. Smith, 1941, A method for determining loss pain sensation, J. Pharmacol. Exp. Ther. 72, 74. Eddy, N.B. and D. Leimbach, 1953, Synthesis analgesics. II. Dithienylbutenyl and dithienylbutylamides, J. Pharmacol. Exp. Ther. 107, 385. Feldberg, W. and D.G. Smyth, 1977, The C fragment of lipoprotein - An endogenous potent analgesic peptide, Br. J. Pharmacol. 60, 445. Goodman, R.G., R.A. Houghton and G.W. Pasternak, 1983, Autoradiography of [3H]fl-endorphin binding in brain, Brain Res. 288, 334. Gulya, K., M. Krivian, N. Nyolczas, Z. Sarnyai and G.L. Kovfics, 1988, Central effects of the potent and highly selective tt opioid antagonist D-Phe-Cys-Tyr-D-Try-OrnThr-Pen-Thr-NH2 (CTOP) in mice, European J. Pharmacol. 150, 355. Haley, T.J. and W.G. McCormick, 1957, Pharmacological effects produced by intracerebral injections of drugs in the conscious mouse, Br. J. Pharmacol. 12, 12. Hazum, E., K. Chang and P. Cuatrecasas, 1979, Interaction of iodinated human [D-Ala2]fl-endorphin with opiate receptors, J. Biol. Chem. 254, 1765. Hosobuchi, Y. and C.H. Li, 1978, The analgesia of human fl-endorphin in man, Commun. Psychopharmacol. 2, 33.
Houghton, R.A., N. Johnson and G.W. Pasternak, 1984, [3H]fl-Endorphin binding in rat brain, J. Neurosci. 4, 2460. Howe, J.R., J.Y. Wang and T.L. Yaksh, 1983, Selective antagonism of the antinociceptive effect of intrathecally applied alpha adrenergic agonists by intrathecal prazosin and intrathecal yohimbine, J. Pharmacol. Exp. Ther. 224, 552. Johnson, N., R. Houghton and G.W. Pasternak, 1982, Binding of [3H]-fl-endorphin in rat brain, Life Sci. 31, 1381. Hylden, J.L.K. and G.L. Wilcox, 1980, Intrathecal morphine in mice: A new technique, European J. Pharmacol. 67, 313. Jensen, T.S. and T.L Yaksh, 1986, II. Examination of spinal monoamine receptors through which brainstem opiate-sensitive systems act in the rat brain, Brain Res. 363, 114. Kuraishi, Y., K. Fukui, H. Shiomi, A. Akaike and H. Takagi, 1978, Microinjection of opioids into the nucleus reticularis gigantocellularis of the rat: Analgesia and increase in the normetanephrine level in the spinal cord, Biochem. Pharmacol. 272, 756. Kuraishi, Y., Y. Harada, S. Aratani, M. Satoh and H. Takagi, 1983, Separate involvement of the spinal noradrenergic and serotonergic systems in morphine analgesia: the difference in mechanical and thermal analgesic tests, Brain Res. 273, 245. Kuraishi, Y., Y. Harada and H. Takagi, 1979, Noradrenaline regulation of pain-transmission in the spinal cord mediated by alpha-adrenoceptors, Brain Res. 174, 333. Larsen, J.J. and J. Arnt, 1984, Spinal 5-HT or NA uptake inhibition potentiates supraspinal morphine antinociception in rats, Acta Pharmacol. Toxicol. 54, 72. Law, P.Y., H.H. Loh and C.H. Li, 1979, Properties and localization of fl-endorphin receptor in rat brain, Proc. Natl. Acad. Sci. U.S.A. 76, 5455. Litchfield, J.T. and F. Wilcoxon, 1949, A simplified method of evaluating dose effect experiments, J. Pharmacol. Exp. Ther. 96, 99. Loh, H.H., L.F. Tseng, E. Wei and C.H. Li, 1976, fl-Endorphin is a potent analgesic agent, Proc. Acad. Sci. U.S.A. 73, 2895. Meglio, M., Y. Hosobuchi, H.H. Loh, J.E. Adams and C.H. Li, 1977, /3-Endorphin: Behavioral and analgesic activity in cats, Proc. Natl. Acad. Sci. U.S.A. 74, 774. Schmauss, C. and T.L Yaksh, 1984, In vivo studies on spinal opiate receptor systems mediating antinociception. I1. Pharmacological profiles suggesting a differential association of mu, delta and kappa receptors with visceral chemical and cutaneous thermal stimuli in the rat, J. Pharmacol. Exp. Ther. 228, 1. Shiomi, H., H. Murakami and H. Takagi, 1978, Morphine analgesia and bulbospinal serotonergic system: Increase in concentration of 5-hydroxyindolacetic acid in the rat spinal cord with analgesia, European J. Pharmacol. 52, 335. Sub, H.H. and L.F. Tseng, 1988, Intrathecal fl-funaltrexamine antagonizes intracerebroventricular fl-endorphin- but not morphine-induced analgesia in mice, J. Pharmacol. Exp. Ther. 245, 587.
70 Suh, H.H. and L.F. Tseng, 1989a, Spinal delta- but not muopioid receptors are involved in tail-flick inhibition induced by fl-endorphin given intracerebroventricularly (i.c.v.) in mice, FASEB J. 3, A279. Suh, H.H. and L.F. Tseng, 1989b, fl-EP, morphine and DPDPE stimulate different opioid receptors for analgesia, Pharmacologist 31, 126. Suh, H.H., L.F. Tseng and C.H. Li, 1988, fl-Endorphin-(1-27) antagonizes fl-endorphin- but not morphine-, D-Pen2-D PenS-enkephalin- and U50,488H-induced analgesia in mice, Neuropharmacology 279, 57. Tallarida, R.I. and R.B. Murray, 1981, Manual of Pharmacologic Calculations with Computer Programs (SpringerVerlag, New York). Tseng, L.F., S.S. Cheng and J.M. Fujimoto, 1983, Inhibition of tail-flick and shaking responses by intrathecal and intraventricular D-Ala2-D-LeuS-enkephalin and fl-endorphin in anesthetized rats, J. Pharmacol. Exp. Ther. 224, 51. Tseng, L.F. and J.M. Fujimoto, 1984, Evidence that spinal endorphin mediates intraventricular fl-endorphin-induced tail-flick inhibition and catalepsy, Brain Res. 302, 231. Tseng, L.F. and J.M. Fujimoto, 1985, Differential actions of intrathecal naloxone on blocking the tail-flick inhibition induced by intraventricular fl-endorphin and morphine, J. Pharmacol. Exp. Ther. 232, 74. Tseng, L.F., M.J. Higgins, J.S. Hong, P.M. Hudson and J.M. Fujimoto, 1985, Release of immunoreactive Met-enkephalin from the spinal cord by intraventricular fl-endorphin but not morphine in anesthetized rats, Brain Res. 343, 60. Tseng, L.F., R.C. King and J.M. Fujimoto, 1986, Release of immunoreactive Met-enkephalin by intraventricular fl-endorphin in anesthetized rats, Reg. Pept. 14, 181.
Tseng, L.F., H.H. Loh and C.H. Li, 1976, fl-Endorphin: Cross tolerance to and cross physical dependence on morphine, Proc. Natl. Acad. Sci. U.S.A. 73, 4187. Tseng, L.F., H.H. Loh and C.H. Li, 1977, Development of tolerance and behavioral activity in rats, Biochem. Biophys. Res. Commun. 74, 390. Tseng, L.F., T.J. Oswald, H.H. Loh and C.H. Li, 1979, Behavioral activities of opioid peptides and morphine sulfate in golden hamsters and rats, Psychopharmacology 64, 215. Wei, E. and H.H. Loh, 1976, Chronic intracerebral infusion of peptides with osmotic minipump and the development of physical dependence, in: Opiates and Endogenous Opioid Peptides, ed. H.W. Kosterlitz (Elsevier/North-Holland Biochemical Press, Amsterdam) p. 303. Wigdor, S. and G.L. Wilcox, 1987, Central and systemic morphine-induced antinociception in mice: Contribution of descending serotonergic and noradrenergic pathways, J. Pharmacol. Exp. Ther. 242, 90. Wuster, M., R. Schulz and R. Herz, 1980, The direction of opioid agonists towards mu-, delta- and epsilon-receptors in the vas deferens of the mouse and the rat, Life Sci. 27, 163. Yaksh, T.L., 1979, Direct evidence that spinal serotonin and noradrenaline terminals mediate the spinal antinociceptive effect of morphine in the periaqueductal gray, Brain Res. 160, 180. Yaksh, Y.L., 1985, Pharmacology of spinal adrenergic systems which modulate spinal nociceptive processing, Pharmacol. Biochem. Behav. 22, 845. Yaksh, T.L. and G.M. Tyce, 1979, Microinjection of morphine into the periaqueductal gray evokes the release of serotonin from the spinal cord, Brain Res. 171, 176.
61
Elsevier EJP 50939
Differential mechanisms mediating fl-endorphinand morphine-induced analgesia in mice Harold Hongwon Suh, James Masao Fujimoto and Leon Liang-Fu Tseng
*
Department of Pharmacology and Toxicology, Medical College of Wisconsin and Toxicological Research Laboratory, CJ Zabloeki, VA Medical Center, Milwaukee, 141153226, U.S.A.
Received 16 March 1989, revised MS received 30 May 1989, accepted 13 June 1989
Effects of yohimbine, methysergide and naloxone given intrathecally (i.t.) and naloxone given intracerebroventriculady (i.c.v.) on inhibition of the tail-flick and hot-plate response induced by fl-endorphin and morphine given i.c.v. were studied in male ICR mice. Yohimbine (1.5 and 15/~g) and methysergide (1.5 and 15/~g) injected i.t. antagonized inhibition of the tail-flick response induced by morphine but not fl-endorphin administered i.c.v. On the other hand, naloxone (20 ng) injected i.t. antagonized inhibition of the tail-flick response induced by i.c.v, administered fl-endorphin but not morphine. Yohimbine and methysergide given i.t. did not antagonize inhibition of the hot-plate response induced by morphine nor did naloxone given i.t. antagonized i.c.v, fl-endorphin-induced inhibition of the hot-plate response. Naloxone given i.c.v, was more effective in antagonizing morphine-induced inhibition of the tail-flick and hot-plate response than inhibition induced by fl-endorphin given i.c.v. Naloxone at doses (0.1 and 1 ~g) which effectively reversed inhibition of the tail-flick response to i.c.v, morphine was not effective in reversing the i.c.v. fl-endorphin-induced inhibition of the tail-flick response. Our results indicate that fl-endorphin and morphine produce analgesia by stimulating separate types of opioid receptors, c- for fl-endorphin and /~- for morphine, and activate separate descending pain modulatory control systems. The supraspinal c system stimulated by fl-endorphin is mediated by activation of spinal opioid receptors whereas the supraspinal /~ system stimulated by morphine is mediated by activation of spinal aE-adrenoceptors and serotonin receptors for the production of analgesia. fl-Endorphin; Morphine; Analgesia; Pain control systems (descending)
1. Introduction fl-Endorphin, an endogenous opioid peptide found in the brain produces an antinociceptive response when injected into the brain in different species of animals and human (Feldberg and Smith, 1977; Loh et al., 1976; Hosobuchi and Li, 1978; Meglio et al., 1977; Tseng et al., 1979).
* To whom all correspondence should be addressed: Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Millwaukee, WI 53226, U.S.A.
Early publications seem to suggest that the pharmacological actions of fl-endorphin closely resemble those of morphine. Both analgesia produced by fl-endorphin and morphine are antagonized by systemic injection of naloxone (Lob et al., 1976). Like morphine, multiple injection or chronic administration of fl-endorphin into the brain produces tolerance and dependence (Tseng et al., 1977; Wei and Loh; 1976). fl-Endorphin also substitutes for morphine in animals tolerant to and dependent on morphine (Tseng et al., 1976; 1977). In in vitro opioid receptor binding studies, fl-endorphin like morphine binds to both /~- and 8-opioid receptors (Hazum et al., 1975; Law et al.,
0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
62 1979). However, the contention that fl-endorphin has the same or similar mechanism of action as that of morphine has been questioned. We have previously demonstrated that fl-endorphin and morphine produce analgesia by activating different descending pain modulatory systems (Tseng and Fujimoto, 1984; 1985; Suh and Tseng, 1988). Unlike morphine which produces analgesia in part by activation of the descending noradrenergic and serotonergic systems, the analgesia induced by fl-endorphin applied supraspinally appears to be mediated by the release of [MetS]enkephalin in the spinal cord (Camarata and Yaksh, 1985; Tseng and Fujimoto, 1984; 1985; Tseng et al., 1985; 1986; Yaksh, 1979). These concepts are supported by the pharmacological studies which show that blockade of a 2adrenoceptors or serotonin receptors in the spinal cord by i.t. injection of yohimbine or methysergide antagonize the analgesia induced by morphine administered supraspinally and blockade of spinal opioid receptors by i.t. injection of naloxone or fl-funaltrexamine antagonizes supraspinally administered fl-endorphin-induced analgesia (Jensen and Yaksh, 1986; Kuraishi et al., 1983; Shiomi et al., 1978; Wigdor and Wilcox, 1987; Yaksh and Tyce, 1979; Tseng and Fujimoto, 1984; 1985; Suh and Tseng, 1988). Biochemical studies have shown that i.c.v, injection of fl-endorphin but not morphine releases [MetS]enkephalin from the spinal cord (Tseng et al., 1985; 1986). The types of opioid receptors stimulated by fl-endorphin and morphine for the production of analgesia appear to be different. Previous studies in mice have demonstrated that i.c.v, pretreatment with fl-funaltrexamine antagonizes analgesia induced by morphine but not fl-endorphin given i.c.v. On the other hand, fl-endorphin-(1-27) given i.c.v, antagonizes i.c.v, fl-endorphin- but not morphine-induced analgesia (Suh et al., 1988). The purpose of the present experiments was to systematically examine the effects of naloxone given i.c.v, and naloxone, yohimbine or methysergide given i.t. on analgesia induced by i.c.v. administered fl-endorphin and morphine. The studies are designed to further delineate the differential mechanisms of analgesic actions produced by fl-endorphin and morphine given i.c.v.
2. M a t e r i a l s
and methods
2.1. Animals Male ICR mice weighing 23-25 g (Harlan Sprague Dawley, Inc., Indianapolis, IN) were used for all the experiments. Animals were housed five per cage in a room maintained at 22 _+0.5 °C with an alternating 12 h light-dark cycle. Food and water were available ad libitum. Animals were used only once in all the experiments.
2.2. Assessment of analgesia Analgesia was determined by the tail-flick (D'Amour and Smith, 1941) and 55°C hot-plate (Eddy and Leimbach, 1953) assays. In most experiments, tail-flick assay was followed by hotplate assay. The intensity of the heat stimulus in the tail-flick test was adjusted so that the animal flicked its tail in 3-4.5 s. Control latencies for the hot-plate test were approximately 9 s. The hotplate and tail-flick latencies were measured before (To) and after (T1) i.c.v, injection of fl-endorphin and morphine. The inhibition of the tail-flick and hot-plate response was expressed as 'percent analgesia' which was calculated as [(T1 - T 0 ) / ( T 2 - To)] × 100, where the cutoff time (T2) was set at 10 and 30 s for the tail-flick and hot-plate tests, respectively. To calculate EDs0 values, at least three different doses were used and eight to nine mice were used for each dose. EDs0 values and their 95% confidence intervals were determined by the method of Litchfield and Wilcoxon (1949) with the aid of a computer program described by Tallarida and Murray (1981) using an Apple II computer.
2.3. Experimental protocol I.t. injection was made according to the procedure of Hylden and Wilcox (1980) using a Hamilton syringe with 30 gauge needle. The i.c.v, administration was performed following the method of Haley and McCormick (1957). Injection volumes were 4 and 5/~1 for i.t. and i.c.v, injection, respectively. Injection sites were verified using 1%
63
2.4. Drugs
methylene blue in testing animals until a 90% success rate was achieved. Yohimbine (1.5 or 15/Lg) and methysergide (1.5 or 15 /~g) were injected i.t. 20 min prior to the i.c.v, injection of various doses of/3-endorphin or morphine, while naloxone (20 ng) was injected i.t. immediately before the i.c.v, injection of /3-endorphin or morphine. In another study, naloxone (2 /~g) was coadministered i.c.v, with different doses of /3-endorphin or morphine. These doses were chosen based on the preliminary studies that these doses of antagonists effectively block the effects of the respective agonists injected i.t. or i.c.v (Wigdor and Wilcox, 1987; Gulya et al., 1988). Hot-plate and tail-flick responses were tested 15 and 30 min, respectively, after the i.c.v. injection. These times used were determined based on the preliminary time course studies which showed the hot-plate and tail-flick inhibition reached a maximum at 15 and 30 min, respectively, after i.c.v, injection of 2 ttg (6 nmol) of morphine or 1 /~g (0.3 nmol) of/3-endorphin. In another experiment, different doses of naloxone were given i.c.v. 15 and 30 min after i.c.v, injection of a fixed dose (4 /~g) of /3-endorphin and morphine, respectively and the tail-flick response was tested 2.5, 5, 10, 15, 20, 30 and 45 min after i.c.v, injection of naloxone.
Morphine sulfate was purchased from Mallinckrodt Chemical Works (St. Louis, MO). /3-Endorphin was obtained from Bachem Inc. (Torrance, CA). Yohimbine hydrochloride was purchased from Sigma Chemical Company (St. Louis, MO). Methysergide maleate was a gift from Sandoz Pharmaceuticals (Hanover, N J). Naloxone hydrochloride was a gift from Endo Laboratories (Wilmingtonl DE). Morphine sulfate, /3-endorphin and naloxone hydrochloride were dissolved in a sterile saline. Yohimbine hydrochloride and methysergide maleate were dissolved in a vehicle consisting of 15% dimethyl sulfoxide and 85% saline (0.9% NaC1). Doses refer to the salt form.
3. Results
3.1. Effects of yohimbine and methysergide injected i.t. on the analgesia induced by morphine and ~-endorphin administered i.c.v. Mice were injected i.t. with vehicle (4 #1), yohimbine (1.5 or 15 t~g), methysergide (1.5 or 15 /~g), or a combination of two drugs (1.5 + 1.5, 15 + 15 #g) 20 rain prior to i.c.v, injection of
b)Hot- Plate test
a~Tail- f l i c k t e s t
c.~e EP. * ;' v t , ~ c l *
t uc.v. e-EP* . . . . . . . . . . . . . . . .Yoh i.t. (Yoh + Methy ) / \ 4• ia.Methy
tc.v.MS * o
CP C~ m m
A *~v MS÷
T
1 ~.c.v. e - E p * i.t (Yoh • Methy)
-
I
I
I
IIII/rto
/
•
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i
i
®
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i 1
,
,
,
,,JJ,i
lO
,
,
,
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.
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Fig. 1. The effects of intrathecal (i.t.) pretreatment with 15/.tg yohimbine (Yoh), methysergide (Methy) or the combination of the two drugs (Yoh + Methy) on intracerebroventricularly (i.c.v.) fl-endorphin (fl-EP)- and morphine (MS)-induced tail-flick (a) and hot-plate (b) inhibition. Mice were pretreated i.t. with either vehicle, yohimbine, methysergide or combination of yohimbine and methysergide for 20 min. Then fl-endorphin or morphine was injected i,c.v, and hot-plate and tail-flick response were tested at 15 and 30 min, respectively, later. The vertical bars are the S.E.M. The number of animals used for each dose was 8.
64 TABLE 1 Effect of i.t. yohimbine, methysergide and a combination of both on EDs0 values for/3-endorphin and morphine for the inhibition of the tail-flick response. EDs0 (nmol/mouse)
i.c.v. MS
a
Saline
Yohimbine (1.5 #g)
Methysergide (1.5/tg)
Yohimbine + methysergide (1.5/zg) (1.5/~g)
1.90 (1.30-3.20) c
7.90 b (4.30-14.5)
7.10 b (3.50-14.6)
14.1 b (6.40-17.7)
Yohirnbine (15/~g)
Methysergide (15/~g)
Yohimbine + methysergide (15/Lg) (15 #g)
10.1 b (4.70-21.8) 0.35 (0.14-0.87)
8.80 b (5.10-15.5) 0.20 (0.10-0.30)
19.7 b (9.90-39.2) 0.20 (0.10-0.30)
i.c.v. MS i.C.V. /3-EP
0.25 (0.13-0.49)
" The EDs0 values were calculated according to the method described by Litchfield and Wilcoxon (1949). b Significantly different from vehicle control, P < 0.05. ¢ Numbers in parentheses indicate the 95% confidence interval.
various doses of morphine or /3-endorphin. The hot-plate and tail-flick responses were measured at 15 and 30 min, respectively, after i.c.v, injection of morphine or/3-endorphin. As shown in fig. la and b, /3-endorphin administered i.c.v, at doses 0.078-0.58 nmol (0.25-2/~g) caused a dose-dependent increase of inhibition of the tail-flick and hot-plate response in mice pretreated i.t. with vehicle. I.t. injection with yohimbine (15 ~g), methysergide (15 /~g) or combination of two antagonists (15 #g + 15 ~g) did not affect inhibition of the tail-flick and hot-plate response produced by/3-endorphin injected i.c.v. The EDs0
values and the slopes of dose-response lines for /3-endorphin injected i.c.v, for inhibition of the tail-flick and hot-plate response in mice pretreated with yohimbine, methysergide or combination of ,two antagonists were not significantly different from those in mice treated with vehicle i.t. (tables 1 and 2). Morphine at doses 0.7-6.0 nmol (0.25-2 ~g) caused a dose-dependent increase of inhibition of the tail-flick and hot-plate response in vehicle pretreated mice. In contrast to the lack of effects of i.t. yohimbine and methysergide on /3-endorphin-induced analgesia, i.t. injection of yohim-
TABLE 2 Effect of i.t. yohimbine, methysergide and a combination of both on EDs0 values for/3-endorphin and morphine for the inhibition of the hot-plate response. EDs0 (nmol/mouse)
i.c.v. MS
Saline
Yohimbine (1.5/~g)
Methysergide (1.5/Lg)
Yohimbine + methysergide (1.5/~g) (1.5 ~tg)
1.49 (0.60-3.73) b
1.30 (0.60-3.0)
2.20 (1.40-3.40)
1.20 (0.60-2.30)
Yohimbine (15 tLg)
Methysergide (15 ~,g)
Yobimbine + methysergide (15 t'g) (15 tLg)
2.18 (1.25-3.83) 0.11 (0.07-0.20)
1.10 (0.30-.3.50) 0.11 (0.05-0.30)
1.60 (0.60-2.30) 0.20 (0.10-0.30)
i.c.v. MS i.c.v. /3-EP
a
0.11 (0.08-0.16)
a The EDs0 values were caluculated according to the method described by Litchfield and Wilcoxon (1949). b Numbers in parentheses indicate the 95% confidence interval.
65 affail- f l i c k Test
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- PlateTeet
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,
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100
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10
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Dose(nmoles)
Fig. 2. The effects of intrathecal (i.t.) injection of naloxone on intracerebroventricular (i.c.v.) fl-endorphin (fl-EP)- and morphine (MS)-induced tail-flick (a) and hot-plate (b) inhibition. Mice were injected i.t. with either saline or 20 ng of naloxone, at the same time they were injected i.c.v, with various doses of fl-endorphin and morphine. Tail-flick response and hot-plate response were tested as above (fig. 1). The vertical bars are the S.E.M. The n u m b e r of animals used for each dose was 8.
bine or methysergide at 1.5 or 15 ~g effectively antagonized inhibition of the tail-flick response induced by morphine injected i.c.v. A combination of yohimbine and methysergide (1.5 + 1.5 or 15 + 15 ~g) given i.t. further increased antagonism of inhibition of the tail-flick response induced by morphine administered i.c.v, and shifted the dose-response curve further to the right (fig. la and table 1). The EDs0 values for morphine for inhibition of the tail-flick response were increased 3.7-, 4.2-, 7.4-fold and 4.6-, 5.3-, 10.4-fold by 1.5 and 15 /~g of methysergide, yohimbine and the combination of methysergide and yohimbine, respectively, injected i.t. without any significant changes of the slopes of the dose-response lines (table 1). However, i.t. administration of yohimbine, methysergide and a combination of the two did not affect the inhibition of the hot-plate response induced by morphine given i.c.v. (fig. lb and table 2). In control groups, i.t. injection with yohimbine, methysergide or a combination of two drugs did not alter the tail-flick and hot-plate latencies of mice injected i.c.v, with vehicle, yohimbine, methysergide and the combination of yohimbine and methysergide, respectively, in the tail-flick test (N = 40), mean __+S.E.: 3.68 + 0.08, 3.76 + 0.09, 3.79 + 0.07 and 3.75 _ 0.08 s; in the hot-plate test (N = 40): 9.4 + 0.29, 9.6 + 0.30, 9.2 __+0.27 and 9.4 _ 0.40 s.
3.2. Effects of naloxone injected i.t. on the analgesia induced by fl-endorphin and morphine administered i.c.v. As shown in fig. 2a, blockade of spinal opioid receptors by i.t. injection of naloxone antagonized inhibition of the tail-flick response induced by TABLE 3 Effect of naloxone on ED5o values for fl-endorphin and morphine administered i.c.v, for the inhibition of the tail-flick and hot-plate response. EDso ( n m o l / m o u s e ) " i.t. Saline
i.c.v. Naloxone (20 ng)
Saline
Naloxone (2 ~tg)
A. Tail-flick test i.c.v, fl-EP 0.12 0.62 b 0.06 0.07 (0.03-0.30) c (0.30-1.30) (0.03-0.20) (0.04-0.15) i.c.v. MS 1.90 2.30 1.83 10.1 b (1.10-3.20) (1.30-4.00) (1.30-3.10) (5.50-18.7) B. Hot-plate test i.c.v, fl-EP 0.08 0.08 0.07 0.22 (0.04-0.14) (0.03-0.16) (0.04-0.12) (0.09-0.56) i.c.v. MS 1.1 1.20 1.10 13.1 b (0.60-2.00) (0.70-2.00) (0.60-2.10) (8.00-21.6) a EDso values were calculated according to the method described by Litchfield and Wilcoxon (1949). b Significantly different from saline control, P < 0.05. ¢ N u m b e r s in the parentheses indicate the 95% confidence interval.
66 a}Tail-flick Test
b~Hot- Platetest
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at
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.,
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Fig. 3. The effects of intracerebroventricular (i.c.v.) injection with naloxone on i.c.v, fl-endorphin (fl-EP)- and morphine (MS)-induced tail-flick (a) and hot-plate (b) inhibition. Mice were injected i.c.v, with various doses of fl-endorphin or morphine in the presence or absence of 2 / x g of naloxone. Tail-flick and hot-plate resonse was tested as above. The n u m b e r of animals used for each dose was 8.
fl-endorphin but not morphine administered i.c.v. EDs0 value for fl-endorphin for inhibition of the tail-flick response in mice injected with i.t. naloxone was increased 5-fold above that for saline controls (table 3) while i.t. naloxone did not affect the EDs0 for morphine for the inhibition. 1.t. injection of naloxone did not antagonize inhibition of the hot-plate response induced by fl-endorphin or morphine administered i.c.v. (fig. 2b and table 3).
3.3. Effects of naloxone injected i.c.v, on the analgesia induced by fl-endorphin and morphine administered i. c. v.
Naloxone at a dose of 2/.tg given i.c.v, did not antagonize inhibition of the tail-flick and hot-plate response induced by fl-endorphin administered i.c.v, while the same dose of naloxone markedly attenuated the i.c.v, morphine-induced inhibition of the tail-flick and hot-plate response (fig. 3a and
a,Tail-flick test
b. Hot- plate test
99
~o
'i
C 9C
/#,E,
80
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' 0.1
1' Naloxone
11) Dose(#g)
o
8c
"-- 6C •~ s~ m 4(: 10
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Dose(#g)
Fig. 4. The effects of intracerebroventricular (i.c.v.) injection of various doses of naloxone on i.c.v, injected fixed dose of fl-endorphin (fl-EP)- and morphine (MS)-induced tail-flick (a) and hot-plate (b) inhibition. In one study, mice were injected i.c.v, with 2 /~g of fl-endorphin the presence of 2, 4, 8 or 16 ~g of naloxone. In another study, mice were injected i.c.v, with 2 /~g of morphine in the presence of 0.1, 0.2, 0.4 or 2 /~g of naloxone, Tail-flick response and hot-plate response were tested as above. The n u m b e r of animals used for each dose was 8.
67
b). The EDs0 values for fl-endorphin injected i.c.v. for inhibition of the tail-flick and hot-plate response were not significantly changed by naloxone given i.c.v, whereas EDs0 values for morphine injected i.c.v, were increased 7.8- and 11.9-fold, respectively, above saline controls, in mice coinjected with i.c.v, naloxone (table 3). The effects of various doses of naloxone in combination with a fixed dose of fl-endorphin (2 /~g) or morphine (2 /~g) given i.c.v, on inhibition of the tail-flick and hot-plate response were studied. Based on the calculation of the potency ratio of these two compounds, this dose (2 #g) of fl-endorphin and morphine produced approximately the same intensity of analgesic response. Naloxone was found to be about 27-fold more potent in antagonizing the analgesia induced by morphine than fl-endorphin in both tail-flick and hot-plate test (fig. 4). The differential actions of naloxone on fl-endorphinand morphine-induced analgesia can also be shown clearly in studies in which naloxone injected i.c.v. reversed the analgesia already developed by
Tug-flick t ~ l t ~v.~
100
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u
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"rinm(min) Fig. 5. The effects of intracerebroventricular (i.c.v.) injection of naloxone on reversing the inhibition of the tail-flick response induced by fl-endorphin or morphine injected i.c.v. Mice were pretreated i.c.v, with 4 ~ g of fl-endorphin or morphine for 15 and 30 rain, respectively. Tail-flick responses, tested 2.5, 5, 10, 15, 20, 30 and 45 rain after i.c.v, injection of saline or naloxone. The vertical bars are the S.E.M. * Different from the saline control group (P < 0.05). The n u m b e r of the animals used for each dose was 8.
morphine or fl-endorphin. Naloxone at doses 0.2 and 1 #g caused a dose-dependent reversal of the inhibition of the tail-flick response induced by morphine (4 #g) given i.c.v, while 1 /xg of naloxone did not significantly affect the inhibition of the tail-flick response induced by fl-endorphin (4 #g) given i.c.v. (fig. 5).
4. Discussion
The results of the present studies showed that blockade of the spinal a2-adrenoceptors and serotonin receptors by i.t. injection of yohimbine and methysergide antagonized inhibition of the tail-flick response induced by morphine but not fl-endorphin. The findings indicate that spinopetal noradrenergic and serotonergic systems are involved in inhibition of the tail-flick response induced by i.c.v, administered morphine whereas the descending monoaminergic systems are not involved in inhibition of the tail-flick response induced by i.c.v, administered fl-endorphin. The results of experiments with morphine in the present study are in agreement with previous studies by others which demonstrated that spinal noradrenergic or serotonergic antagonists attenuated the analgesia induced by morphine injected supraspinally in rats (Yaksh and Tyce, 1979; Kuraishi et al., 1978; 1979; 1983; Jensen and Yaksh, 1986; Yaksh, 1985) and mice (Wigdor and Wilcox, 1987). A combination of yohimbine and methysergide producing more antagonism of inhibition than each antagonist injected alone seems to suggest that adrenergic and serotonergic systems are separately involved in inhibition of the tail-flick response induced by supraspinal morphine. The aadrenoceptors involved in the antinociception induced by morphine has been identified to be a 2but not al-adrenoceptors (Yaksh, 1979; Howe et al., 1982). Further evidence that the analgesia induced by morphine but not fl-endorphin is mediated by activation of spinopetal noradrenergic and serotonergic systems can be obtained from the studies with biogenic amine uptake inhibitors. I.t. injection of desipramine or fluoxetine which inhibits the reuptake of norepinephrine and serotonin, respectively, potentiates the analgesia induced
68 by morphine (Larsen and Arnt, 1984) but not fl-endorphin given i.c.v. (unpublished observation). Blockade of spinal opioid receptors by i.t. injection of naloxone antagonized inhibition of the tail-flick response induced by i.c.v, administered fl-endorphin but not morphine. The results are consistent with that of previous studies in rats and mice that naloxone, fl-funaltrexamine, ICI 174864 or ICI 154129 given i.t. attenuates the analgesia induced by i.c.v, administered fl-endorphin but not morphine (Tseng and Fujimoto, 1984; 1985; Suh and Tseng, 1988; 1989a). A single i.c.v, injection of fl-endorphin but not morphine produces an acute tolerance to opioid receptors in the spinal cord (Suh and Tseng, 1989a). This pharmacological evidence that fl-endorphin but not morphine induces the release of endogenous opioids is further supported by biochemical studies, fl-Endorphin but not morphine administered i.c.v, releases [MetS]enkephalin from the spinal cord in anesthetized rats (Tseng et al., 1985), mice and other species (unpublished observations) of laboratory animals. Thus inhibition of the tail-flick response induced by supraspinally administered fl-endorphin is mediated by activation of spinal opioid receptors whereas inhibition induced by morphine does not involve spinal opioid systems. I.t. administration of yohimbine and methysergide did not antagonize inhibition of the hotplate response induced by morphine nor did i.t. injection of naloxone antagonize inhibition of the hot-plate response induced by fl-endorphin. These results are not surprising because the hot-plate response is believed to be a supraspinally integrated nociceptive response and the blockade of the spinal a-adrenoceptors and serotonin and opioid receptors should not affect the supraspinally integrated hot-plate responses (Jensen and Yaksh, 1986; Schmauss and Yaksh, 1984). At supraspinal sites, naloxone given i.c.v, was found to be more effective in antagonizing i.c.v. administered morphine-induced analgesia than flendorphin-induced analgesia in the tail-flick and hot-plate test. Naloxone at doses which effectively reversed the analgesia induced by morphine was found to be not effective in reversing the analgesia induced by fl-endorphin. The results indicate that the types of opioid receptors acted on by morphine
and fl-endorphin are different. This conclusion is further supported by other studies. (1) fl-Endorphin-(1-27) injected i.c.v, blocks i.c.v, administered fl-endorphin- but not morphine-induced analgesia (Sub et al., 1988). (2) fl-Funaltrexalnine or CTOP (D-Phe-Cys-Tyr-D-Try-Orn-Thr-PenThr-NH2), /~-receptor antagonists, injected i.c.v. blocks i.c.v, administered morphine- but not fl-endorphin-induced analgesia (Suh and Tseng, 1988; 1989b). (3) In cross tolerance study, we found that a single i.c.v, injection of fl-endorphin produced an acute tolerance to fl-endorphin but not morphine administered i.c.v. (Suh and Tseng, 1989b). (4) A single i.c.v, pretreatment with morphine produced an acute tolerance to morphine but not fl-endorphin administered i.c.v. (Suh and Tseng, 1989b). The specific opioid receptor for fl-endorphin has been designated as c-receptors (Wuster et al., 1980; Johnson et al., 1982; Goodman et al., 1983; Houghton et al., 1984). Our results support the proposal for separate opioid receptors supraspinally, c-opioid receptors for fl-endorphin and #-opioid receptors for morphine for the production of analgesia as represented by inhibition of the tail-flick response. High dose of naloxone (27 times higher than the dose which blocks morphine-induced analgesia) injected i.c.v, was found to antagonize fl-endorphin-induced analgesia. Inasmuch as naloxone is a highly lipid soluble compound, the possibility exists that small amount of naloxone injected i.c.v. might diffuse into the spinal cord by redistribution and antagonizes i.c.v, injected fl-endorphininduced analgesia by blocking the opioid receptors at the spinal site. We l~ave previously demonstrated that 2.5 #g of fl-funaltrexamine injected i.c.v, for 24 h does not block analgesia induced by fl-endorphin given i.c.v~ (Suh and Tseng, 1988). The findings that naloxone in combination with fl-endorphin given i.c.v, did not antagonize the analgesia induced by fl-endorphin while i.t. or systemic injection of naloxone is effective in antagonizing the fl-endorphin-induced inhibition of the tail-flick response (Tseng and Fujimoto, 1984; 1985; Tseng et al., 1983) indicate that naloxone given systemically blocks the fl-endorphin-induced analgesia by blocking the effects of released [MetS]enkephalin at the postsynaptic opioid re-
69
ceptor sites rather than blocking the opioid receptors acted on directly by injected fl-endorphin. It is concluded that fl-endorphin and morphine stimulate separate types of opioid receptor supraspinally, c-receptors for/3-endorphin and it-receptors for morphine. The supraspinal ~ system activated by fl-endorphin involves spinal opioid receptor whereas supraspinal /t system activated by morphine involves spinal a2-adrenoceptors and serotonin receptors for the production of the tailflick inhibition.
Acknowledgements This work was supported by US Public Health Grant, DA 03811, (L.F.T.) and VA Medical Research Fund (J.M.F.). A preliminary report on some of these results was presented at the meeting of Federation of American Societies for Experimental Biology, May, 1988 (FASEB Journal 2, A368, 1988).
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Tseng, L.F., H.H. Loh and C.H. Li, 1976, fl-Endorphin: Cross tolerance to and cross physical dependence on morphine, Proc. Natl. Acad. Sci. U.S.A. 73, 4187. Tseng, L.F., H.H. Loh and C.H. Li, 1977, Development of tolerance and behavioral activity in rats, Biochem. Biophys. Res. Commun. 74, 390. Tseng, L.F., T.J. Oswald, H.H. Loh and C.H. Li, 1979, Behavioral activities of opioid peptides and morphine sulfate in golden hamsters and rats, Psychopharmacology 64, 215. Wei, E. and H.H. Loh, 1976, Chronic intracerebral infusion of peptides with osmotic minipump and the development of physical dependence, in: Opiates and Endogenous Opioid Peptides, ed. H.W. Kosterlitz (Elsevier/North-Holland Biochemical Press, Amsterdam) p. 303. Wigdor, S. and G.L. Wilcox, 1987, Central and systemic morphine-induced antinociception in mice: Contribution of descending serotonergic and noradrenergic pathways, J. Pharmacol. Exp. Ther. 242, 90. Wuster, M., R. Schulz and R. Herz, 1980, The direction of opioid agonists towards mu-, delta- and epsilon-receptors in the vas deferens of the mouse and the rat, Life Sci. 27, 163. Yaksh, T.L., 1979, Direct evidence that spinal serotonin and noradrenaline terminals mediate the spinal antinociceptive effect of morphine in the periaqueductal gray, Brain Res. 160, 180. Yaksh, Y.L., 1985, Pharmacology of spinal adrenergic systems which modulate spinal nociceptive processing, Pharmacol. Biochem. Behav. 22, 845. Yaksh, T.L. and G.M. Tyce, 1979, Microinjection of morphine into the periaqueductal gray evokes the release of serotonin from the spinal cord, Brain Res. 171, 176.