Naunyn-Schmiedeberg's Arch Pharmacol (1990) 342:67-71

Naunyn-Schmiedeberg's

Archivesof Pharmacology © Springer-Verlag1990

Different types of opioid receptors mediating analgesia induced by morphine, DAMGO, DPDPE, DADLE and/ -endorphin in mice Harold Hongwon Suh and Leon Fu Tseng Department of Pharmacology and Toxicology,Medical College of Wisconsin and Toxicological Research Laboratory, Clement J. Zablocki VA Medical Center, Milwaukee, WI, USA ReceivedJuly 7, 1989/AcceptedFebruary 19, 1990

Summary. The effects of intracerebroventricular (i.c.v.) administration of D-Phe-Cys-Tyr-D-Try-Orn-Thr-PenThr-NH2 (CTOP), a selective mu-opioid receptor antagonist, (Allyl)2-Tyr-Aib-Aib-Phe-Leu-OH (ICI 174864) and (N,N-Bisallyl-Tyr-Gly-Gly-~p-(CH2S)-Phe-Leu-OH (ICI 154129), selective delta-opioid receptor antagonists on blocking analgesia induced by fl-endorphin, morphine, D-Ala2-NMePhe4-Gly-ol-enkephalin (DAMGO), D-Ala2-D-LeuS-enkephalin (DADLE) and D-Pen 2enkephalin (DPDPE) administered i.c.v, were studied in male ICR mice. The analgesia was assessed by the tailflick and paw-licking (hot-plate) tests. The potencies of opioid agonists injected i.c.v, for producing analgesia were D A M G O > DADLE > /~-endorphin > morphine > DPDPE. Intracerebroventricular administration of CTOP (0.05 lag) selectively antagonized inhibition of the tail-flick and paw-licking response induced by morphine, D A M G O or DADLE but not/%endorphin or DPDPE. ICI 174864 (5 lag) and ICI 154129 (5 lag) injected i.c.v. selectively antagonized analgesia induced by DPDPE or DADLE but not fl-endorphin, morphine or D A M G O injected i.c.v. These results indicate that analgesia induced by morphine and D A M G O is mediated by the stimulation of mu-opioid receptors while analgesia induced by DPDPE is mediated by the stimulation of delta-opioid receptors. DADLE-induced analgesia is mediated by the stimulation of both mu- and delta-opioid receptors. Analgesia induced by/%endorphin is mediated by neither munor delta-opioid receptors. Send offprint requests to L. F. Tseng, Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA Abbreviations. i.c.v., intracerebroventricular; i.t. intrathecal; CTOP, D-Phe-Cys-Tyr-D-Try-Orn-Thr-Pen-Thr-NH2; DAMGO, D-Ala2NMePhe4-Gly-ol-enkephalin; DADLE, D-Ala2-D-LeuS-enke phalin; DPDPE, D-Pela2-D-PenS-enkephalin; ICI 174864 (Allyl)2Tyr-Aib-Aib-Phe-Leu-OH; ICI 154129, (N,N-Bisallyl-Tyr-Gly-Gly~v-(CH2S)-Phe-Leu-OH

Key words: Analgesia - Epsilon-, mu- and delta-receptor - /~-Endorphin - CTOP - ICI 174864 - ICI 154129

Introduction Opioid agonists produce their pharmacological effects by interaction with one or another of mu-, delta-, epsilon-, or kappa-opioid recetpor subtypes (Lord et al. 1977; Kosterlitz et al. 1980; Martin et al. 1976; Schulz et al. 1981 ; Wuster et al. 1980). Morphine and D A M G O have been known to produce analgesia by interacting with muopioid receptors (Tseng 1982, 1983; Suh and Tseng 1988; Suh et al. 1989; Martin et al. 1976; Porreca et al. 1984) while DPDPE produces analgesia via delta-opioid receptors (Mosberg et al. 1983; Corbett et al. 1983, 1984; Heyman et al. 1987; Porreca et al. 1987). Analgesic effects induced by DADLE are mediated by stimulation of mainly delta- and, to a lesser extent, mu-opioid receptors (Lord et al. 1977; Pfeiffer and Herz 1982; Barrett and Vaught 1983). We have previously proposed that/~-endorphin and morphine produce their analgesia by stimulating different types of opioid receptors (Suh and Tseng 1988, 1989; Suh et al. 1988). This hypothesis is based on the findings that ]~-endorphin(1-27) administered i.c.v, selectively antagonizes analgesia induced by /~-endorphin but not morphine, DAMGO, DPDPE and DADLE injected i.c.v. (Suh et al. 1988). fl-Fnnaltrexamine, a selective mu-opioid receptor antagonist, administered i.c.v, effectively antagonizes analgesia induced by morphine but not /~-endorphin given i.c.v. (Suh and Tseng 1988), suggesting that analgesia induced by/%endorphin administered i.c.v. is mediated by the stimulation of epsilon-opioid receptors

68 Table 1. Times for measuring the tail-flick and hot-plate paw-licking responses after intracerebroventricular administration of different opioids Drugs

Tail-flick test time (rain)

Hot-plate test time (min)

/~-Endorphin Morphine DAMGO DPDPE DADLE

30 30 20 20 15

15 15 5 10 10

9.5 s. The paw-licking and tail-flick latencies were measured before (To) and after (T1) the injection of opioid agonists. The inhibition of the tail-flick and paw-licking response was expressed as "percent analgesia" which was calculated as [(T1--T0)/(T2-To)] x 100, where the cutoff time (T2) was set at 10 and 30 s for the tail-flick and hot-plate tests, respectively. To establish the dose response curve, at least 3 drug doses were used and 8 - 10 mice were used for each dose. ED5o values and their 95% confidence intervals were determined by the method described by Litchfield and Wilcoxon (1949) with the aid of a computer program arranged by Tallarida and Murray (1981) using an Apple II computer.

Experimental protocol. A single i.c.v, injection of opioid agonist DAMGO = D-AlaZ-NMePhe#-Gly-ol-enkephalin DADLE = D-AlaZ-D-LeuS-enkephalin DPDPE = o-Pen2-D-PenS-enkephalin

w h e r e a s a n a l g e s i a i n d u c e d b y m o r p h i n e is m e d i a t e d b y the s t i m u l a t i o n o f m u - o p i o i d receptors. C T O P , a s o m a t o s t a t i n a n a l o g , is a h i g h l y selective ant a g o n i s t a g a i n s t m u - o p i o i d r e c e p t o r s ( G u l y a et al. 1986, 1988; H a w k i n s et al. 1989) a n d I C I 174864 a n d I C I 154129 are selective d e l t a - o p i o i d r e c e p t o r a n t a g o n i s t s ( C o r b e t t et al. 1984; S h a w et al. 1982; G o r m l e y et al. 1982; C o w a n a n d G m e r e k 1982; H e y m a n et al. 1987; Priestly et al. 1985). T h e p u r p o s e o f the p r e s e n t experi m e n t s was to use C T O P , I C I 174864 a n d I C I 154129 as selective m u - a n d d e l t a - o p i o i d r e c e p t o r b l o c k e r s , respectively, to d e m o n s t r a t e t h a t a n a l g e s i c effects i n d u c e d b y m o r p h i n e , D A M G O , D P D P E , D A D L E a n d /%endorp h i n a r e m e d i a t e d b y s t i m u l a t i o n o f different o p i o i d receptors. T h e effects o f a fixed d o s e o f C T O P , I C I 714864, I C I 154129 a d m i n i s t e r e d i.c.v, o n i n h i b i t i o n o f the tailflick a n d p a w - l i c k i n g r e s p o n s e i n d u c e d b y / ~ - e n d o r p h i n , morphine, DAMGO, DADLE or DPDPE administered i.c.v, were e v a l u a t e d .

alone or agonist in combination with antagonist was performed according to the method described by Haley and McCormick (1957). Injection volume for i.c.v, injection was 5 gl. The dose-response relationships were established by injecting i.c.v, with different doses of morphine sulfate, DAMGO, DPDPE, DADLE, or p-endorphin in the presence or absence of a fixed dose of CTOP (0.025 or 0.05 gg), ICI 174864 (5 ~tg), or ICI 154129 (5 gg). The agonists and antagonists were mixed, and given as a single i.c.v, injection. The times for measuring the tail-flick and paw-ticking responses after i.c.v, administration of the opioid agonist are shown in Table 1. These times chosen were based on the preliminary time course studies that inhibition of the paw-licking and tail-flick response reached a maximum after i.c.v, injection of opioid agonist. Injection sites were verified by injecting the same volume used in drug injection of 1% methylene blue.

Drugs. The drugs used in the present experiments were morphine sulfate (Mallinckrodt Chemical Works, St. Louis, MO, USA), fl-endorphin, D-Ala2-D-LeuS-enkephalin(DADLE) and D-Phe-CysTyr-D-Try-Orn-Thr-Pen-Thr-NH2 (CTOP) (Peninsula Laboratories Inc. Belmont, CA, USA), D-Pen2-D-PenS-enkephalin (DPDPE) and D-AlaZ-NMePhe4-Gly-ol-enkephalin (DAMGO) (Bachem Inc. Pasadena, CA, USA), (Allyl)2-Tyr-Aib-Aib-Phe-Leu-OH (ICI 174864) (Cambridge Research Biochemicals, Atlantic Beach, NY, USA), (N,N-Bisallyl-Tyr-Gly-Gly-lp-(CH2S)-Phe-Leu-OH (ICI 154129) (a gift from Dr. J. S. Shaw, Imperial Chemical Industry, Macclesfield Cheshire, England, UK). All the drugs used for injection were dissolved in sterile saline (0.9% NaC1 solution). The dose of all drug used are for the salt forms.

Methods

Results

Animals. Male ICR mice weighing 2 3 - 2 5 g (Harlan Sprague

Rank order potency

Dawley, Inc., Indianapolis, IN) were used for all experiments. Animals were housed 5 per cage in a room maintained at 22 + 0.5°C with an alternating 12 hour light-dark cycle. Food and water were availabie ad libitum. No pre-experimental training for the tail-flick and paw-ticking response was performed. Each animal was used only once.

Assessment of analgesia. Analgesia was assessed by measuring the spinally mediated tail-flick (D'Amour and Smith 1941) and the supraspinally mediated hot-plate paw-licking (Eddy and Leimbach 1953) responses. For the measurement of the latency of the tail-flick response, mice were gently held by hand with their tail positioned in apparatus (EMDIE Instrument Co, Maidens, VA, Model TF6) for radiant heat stimulation. The intensity of the radiant heat for the tail-flick test was adjusted so that the animal flicked its tail after 3 to 4.5 s. For measuring latency of the paw-licking response, each mouse was gently placed on a hot-plate (30 cmx 30 cm x 30 cm; iitc Inc, Woodland Hills, CA, Model 39 Hot Plate) and the time for the mouse to lick the front paws was measured. Control latency for the paw-licking response on the 55° C hot-plate was approximately

M o r p h i n e (0.75 to 6 . 0 n m o l ) , D A M G O (0.002 to 0.016 nmol), D P D P E (0.77 to 12.4 nmol), D A D L E (0.02 to 0 . 1 4 n m o l ) a n d ~ - e n d o r p h i n (0.04 to 0 . 2 9 n m o l ) a d m i n i s t e r e d i.c.v, a l o n e c a u s e d a dose d e p e n d e n t increase in i n h i b i t i n g the tail-flick a n d p a w - l i c k i n g response. A l l a g o n i s t s injected i.c.v, p r o d u c e d d o s e - d e p e n d e n t i n h i b i t i o n o f the tail-flick a n d p a w - l i c k i n g r e s p o n s e a n d p r o d u c e d full i n h i b i t i o n at high doses. T h e analgesic p o t e n c i e s ( E D s o ) for v a r i o u s o p i o i d a g o n i s t s for inhib i t i o n o f the tail-flick a n d p a w - l i c k i n g r e s p o n s e are s u m m a r i z e d in Table 2. T h e r a n k o r d e r p o t e n c y o f these o p i o i d s was D A M G O > D A D L E > f l - e n d o r p h i n > m o r p h i n e > D P D P E in b o t h in tail-flick a n d h o t - p l a t e tests. M o r p h i n e , D A M G O , D A D L E a n d D P D P E b u t n o t ~ - e n d o r p h i n at a n a l g e s i c d o s e i n c r e a s e d l o c o m o t o r activity. N o o t h e r a b n o r m a l m o t o r b e h a v i o r s were f o u n d .

69 Table 2. Effect of CTOP, ICI 174864 and ICI 154129 on the inhibition of the tail-flick and paw-licking responses induced by morphine, DAMGO, DADLE, fl-endorphin and DPDPE administered i.c.v. Drugs

Tail-flick test EDso (nmol/mouse) a

Paw-licking test EDso (nmol/mouse)

Morphine Morphine + CTOP (0.025 gg) Morphine + CTOP (0.05 gg) Morphine + ICI 174864 (5 gg) Morphine + ICI 154129 (5 gg) DAMGO DAMGO + CTOP (0.05 gg) DAMGO + ICI 174864 (5 gg) DAMGO + ICI 154129 (5 gg) DADLE DADLE + CTOP (0.05 gg) DADLE + ICI 174864 (5 gg) DADLE + ICI 154129 (5 gg) DPDPE DPDPE + CTOP (0.05 gg) DPDPE + ICI 174864 (5 gg) DPDPE + ICI 154129 (5 gg)

0.77 (0.42-- 1.40) 2.63 (1.05--6.61) 4.20 b ( 2 . 5 2 - - 1 1 . 4 9 ) 0.59 (0.27-- 1.32) 0.94 (0.38--2.72) 0.0063 (0.0041--0.0169) 0.0560 b (0.0230--0.1651) 0.0047 (0.0027-- 0.0089) 0.0061 (0.0032-- 0.0144) 0.030 (0.014--0.060) 0.305 b (0.096--0.971) 0.120 b (0.061-0.245) 0.116 b (0.064--0.254) 4.21 (1.99--8.11) 4.07 (2.60--9.19) 14.99b (8.30-27.07) 14.42b (8.17-27.13) 0.077 (0.038-0.153) 0.076 (0.017--0.312) 0.062 (0.038-0.106) 0.076 (0.040--0.141)

0.75 (0.33-- 1.73) 4.69 b (3.14--7.03) 5.43 b (2.52 - 15.79) 1.21 (0.78--2.63) 0.89 (0.36--2.12) 0.0031 (0.0018--0.0063) 0.0212 b (0.0151--0.0504) 0.0064 (0.0029-- 0.0133) 0.0052 (0.0019--0.0104) 0.027 (0.014--0.053) 0.140 b (0.066--0.295) 0.134b (0.072--0.297) 0.128 b (0.061 -- 0.277) 4.03 (2.32--5.50) 5.54 (2.77-- 11.12) 15.32b (9.89--32.17) 15.41 b (9.99-31.29) 0.072 (0.038-0.141) 0.084 (0.013--0.392) 0.068 (0.040-0.113) 0.094 (0.054-0.164)

fl-Endorphin fl-Endorphin + CTOP (0.05 ~g) fl-Endorphin + ICI 174864 (5 gg) fl-Endorphin + ICI 154129 (5 gg)

a EDso values were calculated according to the method described by Litchfield and Wilcoxon 0949) u Significantly different from control at P < 0.05 c Numbers in the parentheses indicate 95% confidence interval

Effects of i.c.v, administration of CTOP, ICI 174864 and ICI 154129 on blocking analgesia induced by morphine, DAMGO, DADLE, DPDPE and fl-endorphin given i.c.v. The CTOP, an mu-opioid receptor antagonist, administered i.c.v, at a dose of 0.025 or 0.05 gg in combination with different doses of morphine antagonized inhibition of the tail-flick and paw-licking responses induced by morphine given i.c.v, in a dose-dependent manner; the EDs0 values for morphine were increased by 3.4- and 5.5fold in the tail-flick test and by 6.3- and 7.2-fold in the paw-licking test by 0.025 and 0.5 gg of CTOP, respectively. Similarly, CTOP antagonized inhibition of the tailflick and paw-licking response induced by D A M G O and D A D L E administered i.c.v. However, CTOP did not antagonize inhibition of the tail-flick and paw-licking response induced by D P D P E and the ED5o values for D P D P E was not changed by i.c.v, administration of CTOP (Table 2). ICI 174864 and ICI 154129 at 5 gg injected i.c.v. blocked inhibition of the tail-flick and paw-licking response induced by D P D P E and D A D L E ; the EDso values for D P D P E and D A D L E were increased by 3.6and 3.4-fold in the tail-flick test and 3.81- and 3.82-fold in the paw-licking hot-plate test by 5 gg of ICI 174864 and ICI 154129, respectively (Table 2). CTOP (0.05 gg) and ICI compounds (5 gg) given i.c.v. did not affect inhibition of the tail-flick and paw-licking response induced by /~-endorphin administered i.c.v. (Table 2).

Discussion

The results of the present study indicate that analgesic effects induced by morphine and D A M G O are mediated by stimulation of mu-opioid receptors. This conclusion is based on the observation that CTOP, a selective mu-opioid receptor antagonist, administered i.c.v. antagonized inhibition of the tail-flick and paw-licking response induced by morphine. The result is consistent with the findings of previous studies that i.c.v, administration of CTOP antagonized inhibition of the tail-flick response induced by morphine given i.c.v, in mice (Gulya et al. 1988). Furthermore, we found that analgesic effect induced by D A M G O was also antagonized by i.c.v. administration of CTOP. However, ICI 174864 and ICI 154129, delta-receptor antagonists, did not antagonize inhibition of the tail-flick and paw-licking response induced by morphine and D A M G O given i.c.v. We have previously found that fl-endorphin (1-27), an epsilonopioid receptor antagonist, did not antagonize analgesia induced by morphine and D A M G O (Suh et al. 1988). These results indicate that analgesic effects induced by D A D L E administered i.c.v, is mediated by stimulation of both mu- and delta- but not epsilon-opioid receptors. Inhibition of the tail-flick and paw-licking response induced by D P D P E administered i.c.v, was effectively antagonized by ICI 174864 and ICI 154129 but not CTOP. The results are consistent with the finding by Heyman et al. (1987) that ICI 174864 injected i.c.v, effec-

70 tively antagonized D P D P E - i n d u c e d inhibiton of the tailflick response. However, inhibition of the tail-flick and paw-licking response induced by D P D P E administered i.c.v, was not antagonized by fl-endorphin(1-27) (Suh et al. 1988). Thus analgesia induced by D P D P E is mediated by the stimulation of delta- but not by mu- or epsilonopioid receptors. We found in the present study that CTOP, ICI 174864 or ICI 154129 injected i.c.v, effectively antagonized analgesia induced by D A D L E given i.c.v. However, fl-endorphin(1-27) injected i.c.v, was not effefctive in antagonizing analgesia induced by D A D L E administered i.c.v. (Suh et al. 1988). Thus analgesia induced by D A D L E administered i.c.v, is mediated by stimulation of both mu- and delta- but not epsilon-opioid receptors. Several lines of evidence seem to indicate that analgesia induced by fl-endorphin administered i.c.v, is mediated by the stimulation o f epsilon- but not mu- or deltaopioid receptors. This conclusion is based on the findings that fl-endorphin(1-27) administered i.c.v, selectively antagonized analgesia induced by fl-endorphin administered i.c.v, but not by morphine, D A M G O or D P D P E (Suh et al. 1988), We found in the present study that inhibition of the tail-flick and paw-licking responses induced by fl-endorphin administered i.c.v, was not antagonized by either C T O P or I C I c o m p o u n d s administered i.c.v. The lack o f m u - and delta-receptor activity of fl-endorphin for the production of analgesia was also supported by other pharmacological studies. fl-Funaltrexamine, a selective mu-opioid receptor antagonist (Takemori et al. 1981 ; Ward et al. 1982a, b) given i.c.v., did not antagonize analgesia induced by /% endorphin administered i.c.v. (Suh and Tseng 1988): Mice tolerant to fl-endorphin were not cross-tolerant to m o r phine and D P D P E and mice tolerant to morphine or D P D P E did not produce cross-tolerance to fl-endorphin (Sub and Tseng 1989). The results indicate that /3endorphin stimulates epsilon- but not mu- and deltaopioid receptors for the production o f analgesia. However, the finding that CTOP did not block the fl-endorphin-induced analgesia is not consistent with the findings by Shook et al. (1988). They found that C T P (D-Phe-Cys-Tyr-D-Try-Lys-Thr-Pen-Thr-NH2) antagonized fl-endorphin-induced analgesia in the hotplate test. Naloxone at 1 gg and ICI 174864 at 3 gg slightly antagonized fl-endorphin-induced analgesia. However, the selectivity of these receptor blockers is not absolute and the dose of C T P used was 1 gg which was too high. In our experiment, 50 ng of CTOP which blocked mu-opioid receptor activity did not block flendorphin-induced inhibition of the tail-flick and pawlicking response. We found that naloxone at 2 gg given i.c.v, blocked the morphine- but not fl-endorphin-induced analgesia (Sub et al. 1989). In summary, our results show that analgesia induced by morphine and D A M G O is mediated by the stimulation o f m u - o p i o i d receptor, D A D L E - i n d u c e d analgesia is mediated by b o t h delta- and mu-opioid receptor, D P D P E - i n d u c e d analgesia is mediated by delta-opioid receptor, and fl-endorphin-induced analgesia is mediated by epsilon- but not mu- or delta-opioid receptors.

Acknowledgements. This work was supported by US Public Health Grant, DA 03811. A preliminary report on some of these results was presented at the meeting of American Society of Pharmacology and Experimental Therapeutics, August 1989 (Pharmacologist, 31 : 126).

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