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Dynorphin-A-(1–13) antagonizes morphine analgesia in the brain and potentiates morphine analgesia in the spinal cord
Peptides. Vol. 6. pp. 1015-1020, 1985.' AnkhoInternationalInc. Printed in the U.S.A.
0196-9781/85 $3.00 + .00
Dynorphin-A-(1-13) Antagonizes Morphine Analgesia in the Brain and Potentiates Morphine Analgesia in the Spinal Cord M. F. REN,* C. H. LU A N D J. S. H A N Department o f Physiology, Beijing Medical University, Be{jing and *Department of Pharmacology Institute o f Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing, China R e c e i v e d 24 J u n e 1985 REN, M. F., C. H. LU AND J. S. HAN. Dynorphin-A-(l-13)antagonizes morphine analgesia in the brain and potentiates morphim, anal~,e.~ia in the spired cord. PEPTIDES 6(6) 1015-1020, 1985.--lntrathecal iqiection of subanalgesic doses of morphine (7.5 nmol) and dynorphin-A-(1-13) (I.25 nmol) in combination resulted in a marked analgesic effect as assessed by tail flick latency in the rat. The analgesic effect of the composite dynorphin/morphine was dose-dependent in serial dilutions so that a composition of I/8 of the analgesic dose of dynorphin and 1/3 that of morphine produced an analgesic effect equipotent to full dose of either drug applied separately. The analgesic effect induced by dynorphin/morphine mixture was not accompanied by motor dysfunction and was easily reversed by a small dose (0.5 mg/kg) of naloxone. Contrary to the augmentatory effect of dynorpbin on morphine analgesia in the spinal cord, intracerevroventricular (ICV) injection of 20 nmol of dynorphin-A-(I-13) exhibited a marked antagonistic effect on the analgesia produced by morphine (120 nmol, ICV). The theoretical considerations and practical implications of the differential interactions between dynorphin-A-(I-13) and morphine in the brain versus spinal cord are discussed. Dynorphin-A-(l-13)
Morphine
Analgesia
lntrathecal Injection of Drugs
THE role of dynorphin-A as an opioid peptide for antinociception in spinal cord has been extensively studied since 1982 [7-10, 17, 19, 20]. However, with large dose of intrathecal dynorphin-A there was usually a weakness or even motor paralysis in the hind limbs and the tail, thus interfering the assessment of nociception by tail flick test. One objective of this study was to observe whether the sensory and motor effects of dynorphin-A-(l-13) could be separated by changing the doses of dynorphin-A-(l-13) or by a combination of dynorphin-A-(1-13) with other narcotics so as to obtain a substantial analgesia without producing any motor dysfunction. Another issue was that dynorphin-A per se had little analgesic effect by ICV injection [5, 8, l l, 13, 21,24, 26]. Instead it was reported to antagonize morphine analgesia in the mice [5, 13, 21]. It seemed of interect to determine whether the same phenomenon exists in rats. The results indicate that dynorphin-A-(I-13) potentiates morphine analgesia in the spinal cord and antagonizes morphine analgesia in the brain. The underlying mechanisms of this bidirectional modulatory effect remain to be elucidated.
Intrathecal catheterization was performed according to Yaksh and Rudy. Rats were anesthetized with chlorohydrate (300 mg/kg, IP). After a midline incision over the skin at the lower border of the occipital crest and separation of the underlying muscles, the atlanto-occipital membrane and the dura were cut open and a PE l0 tubing was inserted down the spinal subarachnoid space for a length of about 7.5 cm, reaching the upper border of the lumbar enlargement. The experiment with intrathecal injection started 24-30 hours after the recovery of the animal from anesthesia. Drugs were dissolved in normal saline and injected via the catheter at a volume of 10/,I, followed by 5/,i of artificial CSF for flushing.
Intracerebroventricular h{/ection of Drugs Implantation of the cannula was performed stereotaxically under chlorohydrate anesthesia. Stainless steel guide cannulae of 0.8 mm outer diameter were implanted into the brain 1 mm posterior to the bregma and 2 mm lateral to the midline on both sides and were fixed on the skull with dental acrylic. Experiments with ICV injection were performed 5 days after the operation. A stainless steel injection tube of 0.4 mm outer diameter was inserted into the guide cannula and extended 1 mm beyond its lower end to reach the lateral ventricle. The injection volume was 8 t~l, to be finished in 30 sec.
METHOD
Animals Female albino rats weighing 200-250 g were supplied by the animal center of the Beijing Medical University. Each animal was used only for one experiment.
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Dose, nmol FIG. 1. The effect ofintrathecal injection of dynorphin-A-(I-13) and morphine on tail flick latency (TFL) in rats. The ordinate indicates percentage changes in TFL 30 min after the intrathecal injection. Numerals within the figure represent numbers of animals in each group. Vertical bar indicates S.E.
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Nociceptive Test Rats were kept in special holders for testing tail flick latency (TFL) with thermal irradiation given to the lower third of the tail [12]. Room temperature was kept within 2 0+- I°C. The results of the three successive measurements with 5 min apart obtained at the beginning of the experiment were averaged and taken as the basal threshold, usually in the range of 4-6 sec. The values of the subsequent measurements after drug administration were expressed as percent changes from the basal level. An elevation over 150% of the basal T F L was taken as the cutoff limit to avoid unnecessary skin damage.
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FIG. 3. Analgesic effect of dynorphin-A-(I-13) 1.25 nmol, morphine 7.5 nmol and both drugs in combination. ***Indicates p<0.001 compared to either dynorphin or morphine alone.
Chemicals Synthetic dynorphin-A-(1-13) was a gift from Dr. A. Goldstein (Addiction Research Foundation, Palo Alto). Naloxone HCI was obtained from Endo Laboratories. Morphine HCI was a product of Shenyang Drug Company, Shenyang, China. RESULTS
The Analgesic Effect of lntrathecal Injection of Dynorphin-A-( I-13) or Morphine HCI A total of 77 rats with intrathecal cannulae were divided into 6 groups of 6-13 in each. They were injected with normal saline, dynorphin-A-(1-13) 1.25, 2.5, 5 and 20 nmol or morphine HCI 7.5, 15 and 30 nmol, respectively, through the implanted cannula. Changes in T F L after the intrathecal injection were monitored for a total of 80 min. In the normal saline control group, the percentage changes in a period of 80 rain were within the limit of 6_+4% and - 1_+3%. Intrathecal injection of 1.25 nmol of dynorphin-A-(1-13) produced little change in T F L (maximal changes from 4_+2% to - 5 - 3 % ) , not exceeding the range of fluctuation seen in the normal saline control group. Intrathecal injection of 2.5 nmol of dynorphin-A-(1-13) or 7.5 nmol of morphine produced a slight increase (20-30%) in TFL. The difference
between these two groups and the saline control group was statistically not significant (p<0.05). Intrathecal injection of 5 and 20 nmol of dynorphin-A-(113) or 15 and 30 nmol of morphine produced significant increase in TFL. The effect emerged within 5 min and lasted for at least 60 min. Figure 1 shows the dose-response curves for dynorphin-A-(1-13) and morphine, where the percentage increase in TFL 30 min after the drug administration was taken for constructing the curves. Dynorphin-A-(1-13) is clearly more effective in raising the T F L than morphine on a molar basis. While morphine did not cause any motor disturbance in rats, high doses of dynorphia-A-(1-13) did. Four out of the 9 rats receiving 20 nmol of dynorphin-A-(1-13) and 3 out of 14 rats receiving 5 nmol of dynorphin-A-(1-13) exhibited a certain degree of muscle weakness or even paralysis in hind legs when they were allowed to move freely outside the holder one hour after the drug administration.
The Analgesic EfJbct of lntrathecal Injection of Dynorphin-A-( I-13) and Morphine in Combination A group of !1 rats was injected intrathecally with 1.25
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FIG. 4. The effect of naloxone on intrathecal dynorphin-A-(1-13) (A), morphine (B) and dynorphin/morphine composite (C). *p<0.05. **p<0.01, ***p<0.001 compared with NS control. nmol of dynorphin-A-(l-13) and 7.5 nmol of morphine in combination at a total volume of 10/xi. A marked increase in T F L over 100% of the basal level was seen 10 min after the injection, which maintained at a height between 104_+15% and 119---7% for the whole observation period of 80 min (Fig. 2). This effect was much stronger than the mathematical additive effect of the two components (Fig. 3). None of the rats showed any signs of motor disfunction when they were allowed to move freely out of the holders. To test the potential synergistic effect existing between dynorphin-A-(l-13) and morphine, the dynorphin/morphine mixture was serially diluted and injected intrathecally to groups of rats. These results are also shown in Fig. 2. A mixture of dynorphin-A-(1-13) 0.625 nmol and morphine 3.75 nmol ( n = l l ) produced an increase in T F L over 60% within 1 hour (p<0.05 as compared to the corresponding NS control). A mixture of dynorphin-A-(l-13) 0.313 nmol and morphine 1.875 nmol produced an increase in T F L of over 30% 30 rain after the drug administration, showing a clear dose-response relationship.
The Effect of Nah)xone on Analgesia Induced by Dynorphin-A-( I-13). Morphine and Dynorphhl/Morphine Mixture In an attempt to evaluate the naloxone reversibility of the opioid analgesia, equipotent doses of dynorphin-A-(l-13) (20 nmol), morphine (30 nmol) and dynorphin/morphine mixture (D 1.25 nmol/M7.5 nmol) were injected intrathecally to groups of rats to produce an increase in T F L 100% above the base line level. Thirty min after intrathecal injection, the rats were given a subcutaneous injection of naloxone (0.5 mg/kg) or normal saline (1 ml/rat). T F L was measured at 10 min intervals for a period of 100 min. The results are shown in Fig. 4.
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FIG. 5. The effect of ICV injection of various doses of dynorphin-A-I 1-13) and morphine in combination. In contrast to dynorphin-A-(l-13) induced analgesia which was resistant to naloxone blockade (Fig. 4A), morphine analgesia was sensitive to naloxone as was indicated by the 67% reduction of the analgesic effect 20 min after the naloxone administration (Fig. 4B). Figure 4C shows that the analgesic effect produced by dynorphin/morphine mixture was almost totally abolished by naloxone. The antagonistic effect of naloxone lasted for at least 1 hour.
The E fJ~,ct of lCV Injection of Dynorphin-A-(I-13) or Morphine Five groups of rats with 6--11 in each group were given ICV injection of normal saline 8/zl, dynorphin-A-(l-13) 20 nmol or morphine 30, 60, 120 nmol, respectively. T F L was measured at 10 min intervals for a total of 90 rain.
1018
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DISCUSSION
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The changes in T F L in the 90 min period after NS injection were in the range of 3-_4% to -7_+5%. Dynorphin-A(1-13) 20 nmol produced a slight increase in T F L by 24___13% 10 min after ICV injection which was not significantly different from the NS control group. ICV injection of morphine produced a dose-dependent increase in TFL. Peak values were reached 30-40 min after the ICV injection, being 19-+ 12%, 74+18% and 139+7% in 30, 60 and 120 nmol group, respectively.
The Effect on TFL of ICV Injection of Dynorphin-A-( I-13) and Morphine in Combination Two groups of 8 rats were injected ICV with a dynorphin/morphine mixture followed by an observation period of 90 min. A mixture of 10 nmol of dynorphin-A-(l-13) and 120 nmol of morphine produced an increase in T F L of 109_+ 15% which was only slightly lower than that produced by 120 nmol of morphine alone (139_7%, p<0.05). However, addition of 20 nmol of dynorphin-A-(1-13) to 120 nmol of morphine resulted in a dramatical suppression of morphine analgesia. The increase in T F L 30 min after ICV injection was only 16_+8% which was 12% of that seen in 120 nmol morphine group (p<0.001) (see Fig. 5). The suppressive effect of dynorphin-A-(1-13) on morphine analgesia was most prominent within 40 min of ICV injection and subsided gradually in a period of 80 min.
Antagonistic Effect of lCV Dynorphin-A(l-13) on Morphine Analgesia Two groups of rats with 6 and 9 in each were given ICV injection of 120 nmol of morphine which produced an increase in T F L of 126_+14% and 121-+12%, respectively, 30 min after the injection. One group (n=6) was then given ICV injection of normal saline and the other group (n=9) 20 nmol of dynorphin-A-(l-13). The results are shown in Fig. 6. Dynorphin-A-(I-13) produced a 68% reversal (p<0.001) of the morphine analgesia 20 min after its ICV administration as compared to the NS control group. This antagonistic effect was quick in onset (a reversal of 56%, p<0.01, within 10 min) and lasted about 50 min.
Our data clearly show that intrathecal dynorphin-A-(1-13) produced a dose-dependent prolongation of T F L , an effect one fold stronger than morphine on a molar basis. While high dose of dynorphin could result in motor dysfunction, such disfunction does not explain the prolongation of T F L in lower doses. This becomes evident since: (1) 5 nmol of dynorphin-A-(1-13) produced an increase of T F L by 69_+ 13% (p<0.001 compared to NS control group) yet only 3/14 of the rats exhibited signs of motor disturbance, (2) a subanalgesic dose of dynorphin-A-(1-13) (1.25 and 0.625 nmol) produced a marked potentiation on morphine analgesia whereas none of the animals had any sign of motor dysfunction. Thus different effects of dynorphin-A on sensory and motor function may occur depending on the dose: a small amount of exogenously applied [7, 9, 10, 17, 19, 20, 27] or endogenously released [8] dynorphin-A primarily affects nociception. Motor activities are impaired only when a relatively large dose is administered or a huge amount of dynorphin-A is released in such cases as cord injury [4]. The supposition that dynorphin-A in spinal cord may be more involved in sensory rather than in motor function seems to be in line with the findings that the dynorphin-A content in the dorsal spinal cord is much higher than in the ventral cord [1]. It is worthwhile to mention that the paralytic effect of dynorphin-A may well be a non-opioid effect. The commonly used criterion to differentiate opioid with non-opioid nature of a physiological or pharmacological effect is its susceptibility to naloxone blockade. However, the conclusion can be different depending on the dose of naloxone being used. Goldstein has pointed out that dynorphin-A-(l-13) is 13-fold more resistant to naloxone blockade than [LeuS]enkephalin in the guinea pig ileum assay [6]. We have found in the in vivo study that the dose of naloxone (10 mg/kg, SC) required to produce 50% reversal of the analgesic effect of dynorphin-A was 14 times as high as that needed for morphine (0.7 mg/kg, SC), when an equipotent dose of dynorphin-A or morphine was injected intrathecally [9]. These and some other evidence from receptor-protection studies and cross tolerance studies point to the conclusion that dynorphin-A is an endogenous ligand of the kappa receptors [3] and that dynorphin-A induced analgesia is mediated by kappa receptors in the spinal cord [26]. Based on the data mentioned above, a naloxone dose of 10-20 mg/kg should be tried (in rats) and prove to be ineffective before a "non-opioid" nature can be assigned to an effect. To this context it is interesting to note that the paralytic effect of intrathecally administered dynorphin-A is not affected by 20 mg/kg of naloxone (J. B. Long, presented in the International Narcotic Research Conference, June, 1985), therefore is most probably nonopioid. The augmentation of morphine analgesia by a small amount of dynorphin-A-(l-13) merits special attention. The joint effect of dynorphin-A-(l-13) (1.25 nmol) and morphine (7.5 nmol) was not simply additive. It was equivalent to 12 nmol of dynorphin-A-(1-13) or 20 nmol of morphine given separately, as could be estimated from the dose-response curves shown in Fig. 1. The analgesic effect was still prominent even when the mixture was diluted 1-2 fold, where single dose of dynorphin-A-(1-13) or morphine was already several times smaller than the minimal effective doses (see Figs. 1 and 2). These findings strongly suggest that dynorphin-A-(1-13) and morphine are acting on different receptor populations, resulting in a synergistic effect.
DYNORPHIN-A A N T A G O N I Z E S MORPHINE A N A L G E S I A The result of naloxone blockade seems to throw some light on the type of receptors implicated in analgesia produced by the dynorphin/morphine mixture. Since dynorphin-A is believed to act through kappa receptors and requires a large dose of naloxone (10 mg/kg) for partial antagonism [6, 9, 27], an almost complete abolishment of the analgesia produced by dynorphin/morphine mixture by a small dose of naloxone (0.5 mg/kg) seem to suggest that the composite probably acts mainly on mu receptors. However, if a mutual interaction between kappa and mu receptors is required for the synergistic effect to appear, then selective blockade of the kappa receptors, leaving mu receptors intact, would also have resulted in a disruption of the synergism. This issue deserves further investigation. Modulatory effect on morphine analgesia of opioid peptides other than dynorphin have been reported [2, 14, 15, 23] and various models have been proposed to explain the possible interaction between delta and mu receptors [22] or between enkephalin sites and alkaloid sites of a unique opioid receptor [16]. In the spinal cord, a potentiation of morphine analgesia by 100/xg of [leu]enkephalin has been reported [14]. The amount of [leu]enkephalin needed (around 200 nmol) for potentiation of morphine analgesia is about 200 times that of the dynorphin-A-(l-13) used in the present study (0.625-1.25 nmol), implying that the interaction between dynorphin-A-(l-13) and morphine (or kappa and mu receptors) in the spinal cord is much more intimate than that between enkephalin and morphine (or delta and mu receptors). Whether there is an allosterically coupled kappa/mu receptor combination remains to be determined. While the theoretical explanation is not clear, the synergism between dynorphin-A-(l-13) and morphine may have some practical implications. Since only a minimal activation of kappa and mu receptors is required to produce a substantial analgesia, one would anticipate less development of
1019
tolerance when a mixture of dynorphin/morphine or kappa/mu agonists is developed for clinical trial. Another interesting finding in our study is the sharp contrast between brain and spinal cord for dynorphin/morphine interaction. While dynorphin-A-(l-13) potentiated morphine analgesia in the spinal cord, it antagonized morphine analgesia in the brain. This phenomenon was first reported in mice [5, 13, 21] and is here confirmed in the rat. Controversial results have been obtained previously as to whether dynorphin-A produces an analgesic effect after ICV injection [5, I 1, 17, 18, 21, 24, 26]. Most of the authors using tail flick for nociceptive test obtained negative results [5, 24, 26]. We have also found that ICV injection of dynorphin-A(1-13) alone produced no significant effect on T F L at a wide dose range of 1-20 nmol (only the 20 nmol data are shown in Fig. 5). However when administered in combination with morphine (Fig. 5) or superimposed on morphine analgesia (Fig. 6), a marked suppression of the morphine effect was observed. The phenomenon is straight forward and clearcut. However, the amount of dynorphin-A-(1-13) (20 nmol) required for this negative modulation is large, and half this dose (10 nmol) proved ineffective (Fig. 5). The physiological relevance of this phenomenon awaits for clarification. It is techniquely difficult to use naloxone for testing the nature (opioid or non-opioid) of the antagonistic effect of dynorphin-A-(i-13) on morphine analgesia. However, it was reported that des-tyrosine dynorphin-A was also effective in antagonizing morphine analgesia [25], indicating that this effect could be non-opioid, as was the paralytic effect of this peptide. ACKNOWLEDGEMENTS We thank Dr. Avram Goldstein for the gift of dynorphin-A-(113), and Endo Laboratories for naloxone. This work was supported by the Ministry of Health, People's Republic of China.
REFERENCES
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20. Przewlocki, R., G. T. Shearman and A. Herz. Mixed opioid/nonopioid effects of dynorphin and dynorphin relayed peptides after their intrathecal injection in rats. Neuropeptides 3: 233-240, 1983. 21. Tulunay, F. C., M. F. Jen, J. K. Chang, H. H. Loh and N. M. Lee. Possible regulatory role of dynorphin on morphine- and fl-endorphin-induced analgesia..I Pharmacol Exp Ther 219: 296-298, 1981. 22. Vaught, J. L., R. B. Rothman and T. C. Westfall. Mu and delta receptors: Their role in analgesia and in the differential effects of opioid peptides on analgesia. Lift, Sci 30: 1443-1455, 1982. 23. Vaught, J. L. and A. E. Takemori. Differential effects of leucine- and methionine-enkephalin on morphine-induced analgesia, acute tolerance and dependence. J Pharmacol Exp Ther 208: 86-90, 1978.
REN, LU AND HAN
24. Walker, J. M., R. J. Katz and H. Akil. Behavioral effects of dynorphin 1-13 in the mouse and rat: Initial observations. Peptides 1: 341-345, 1980. 25. Walker, J. M., D. E. Tucker, D. H. Coy, B. B. Walker and H. Akil. Des-tyrosine-dynorphin antagonizes morphine analgesia. Eur J Pharmacol 85: 121-122, 1982. 26. Wuster, M., R. Schulz and A. Herz. Opiate activity and receptor selectivity of dynorphin 11-13) in the mouse vas deferens. Eur J Pharmacol 62: 235-236, 1980. 27. Xie, G. X. and J. S. Han. Dynorphin: Analgesic effect via kappa receptors in spinal cord of rats. ('hung Kuo Yao Li Hsyeh Pao 5: 231-234, 1984.
0196-9781/85 $3.00 + .00
Dynorphin-A-(1-13) Antagonizes Morphine Analgesia in the Brain and Potentiates Morphine Analgesia in the Spinal Cord M. F. REN,* C. H. LU A N D J. S. H A N Department o f Physiology, Beijing Medical University, Be{jing and *Department of Pharmacology Institute o f Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing, China R e c e i v e d 24 J u n e 1985 REN, M. F., C. H. LU AND J. S. HAN. Dynorphin-A-(l-13)antagonizes morphine analgesia in the brain and potentiates morphim, anal~,e.~ia in the spired cord. PEPTIDES 6(6) 1015-1020, 1985.--lntrathecal iqiection of subanalgesic doses of morphine (7.5 nmol) and dynorphin-A-(1-13) (I.25 nmol) in combination resulted in a marked analgesic effect as assessed by tail flick latency in the rat. The analgesic effect of the composite dynorphin/morphine was dose-dependent in serial dilutions so that a composition of I/8 of the analgesic dose of dynorphin and 1/3 that of morphine produced an analgesic effect equipotent to full dose of either drug applied separately. The analgesic effect induced by dynorphin/morphine mixture was not accompanied by motor dysfunction and was easily reversed by a small dose (0.5 mg/kg) of naloxone. Contrary to the augmentatory effect of dynorpbin on morphine analgesia in the spinal cord, intracerevroventricular (ICV) injection of 20 nmol of dynorphin-A-(I-13) exhibited a marked antagonistic effect on the analgesia produced by morphine (120 nmol, ICV). The theoretical considerations and practical implications of the differential interactions between dynorphin-A-(I-13) and morphine in the brain versus spinal cord are discussed. Dynorphin-A-(l-13)
Morphine
Analgesia
lntrathecal Injection of Drugs
THE role of dynorphin-A as an opioid peptide for antinociception in spinal cord has been extensively studied since 1982 [7-10, 17, 19, 20]. However, with large dose of intrathecal dynorphin-A there was usually a weakness or even motor paralysis in the hind limbs and the tail, thus interfering the assessment of nociception by tail flick test. One objective of this study was to observe whether the sensory and motor effects of dynorphin-A-(l-13) could be separated by changing the doses of dynorphin-A-(l-13) or by a combination of dynorphin-A-(1-13) with other narcotics so as to obtain a substantial analgesia without producing any motor dysfunction. Another issue was that dynorphin-A per se had little analgesic effect by ICV injection [5, 8, l l, 13, 21,24, 26]. Instead it was reported to antagonize morphine analgesia in the mice [5, 13, 21]. It seemed of interect to determine whether the same phenomenon exists in rats. The results indicate that dynorphin-A-(I-13) potentiates morphine analgesia in the spinal cord and antagonizes morphine analgesia in the brain. The underlying mechanisms of this bidirectional modulatory effect remain to be elucidated.
Intrathecal catheterization was performed according to Yaksh and Rudy. Rats were anesthetized with chlorohydrate (300 mg/kg, IP). After a midline incision over the skin at the lower border of the occipital crest and separation of the underlying muscles, the atlanto-occipital membrane and the dura were cut open and a PE l0 tubing was inserted down the spinal subarachnoid space for a length of about 7.5 cm, reaching the upper border of the lumbar enlargement. The experiment with intrathecal injection started 24-30 hours after the recovery of the animal from anesthesia. Drugs were dissolved in normal saline and injected via the catheter at a volume of 10/,I, followed by 5/,i of artificial CSF for flushing.
Intracerebroventricular h{/ection of Drugs Implantation of the cannula was performed stereotaxically under chlorohydrate anesthesia. Stainless steel guide cannulae of 0.8 mm outer diameter were implanted into the brain 1 mm posterior to the bregma and 2 mm lateral to the midline on both sides and were fixed on the skull with dental acrylic. Experiments with ICV injection were performed 5 days after the operation. A stainless steel injection tube of 0.4 mm outer diameter was inserted into the guide cannula and extended 1 mm beyond its lower end to reach the lateral ventricle. The injection volume was 8 t~l, to be finished in 30 sec.
METHOD
Animals Female albino rats weighing 200-250 g were supplied by the animal center of the Beijing Medical University. Each animal was used only for one experiment.
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Dose, nmol FIG. 1. The effect ofintrathecal injection of dynorphin-A-(I-13) and morphine on tail flick latency (TFL) in rats. The ordinate indicates percentage changes in TFL 30 min after the intrathecal injection. Numerals within the figure represent numbers of animals in each group. Vertical bar indicates S.E.
i
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Experimental Data Analysis Data are expressed as mean+S.E, and the significance of difference between two groups was examined by two tailed t test.
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FIG. 2. Dose-response effect of serial dilutions ofintrathecal dynorphinlmorphine composite. Numerals in parentheses indicate number of animals.
150
Nociceptive Test Rats were kept in special holders for testing tail flick latency (TFL) with thermal irradiation given to the lower third of the tail [12]. Room temperature was kept within 2 0+- I°C. The results of the three successive measurements with 5 min apart obtained at the beginning of the experiment were averaged and taken as the basal threshold, usually in the range of 4-6 sec. The values of the subsequent measurements after drug administration were expressed as percent changes from the basal level. An elevation over 150% of the basal T F L was taken as the cutoff limit to avoid unnecessary skin damage.
i / D 0~3 nmol uo) A-2~. . . . _~_--4>--~--~-_ ~~'--6~M 1.875nmol .o--<>--_~_-~) . . . . . . ~-- - ~ - - - 6 NS fOul(e)
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FIG. 3. Analgesic effect of dynorphin-A-(I-13) 1.25 nmol, morphine 7.5 nmol and both drugs in combination. ***Indicates p<0.001 compared to either dynorphin or morphine alone.
Chemicals Synthetic dynorphin-A-(1-13) was a gift from Dr. A. Goldstein (Addiction Research Foundation, Palo Alto). Naloxone HCI was obtained from Endo Laboratories. Morphine HCI was a product of Shenyang Drug Company, Shenyang, China. RESULTS
The Analgesic Effect of lntrathecal Injection of Dynorphin-A-( I-13) or Morphine HCI A total of 77 rats with intrathecal cannulae were divided into 6 groups of 6-13 in each. They were injected with normal saline, dynorphin-A-(1-13) 1.25, 2.5, 5 and 20 nmol or morphine HCI 7.5, 15 and 30 nmol, respectively, through the implanted cannula. Changes in T F L after the intrathecal injection were monitored for a total of 80 min. In the normal saline control group, the percentage changes in a period of 80 rain were within the limit of 6_+4% and - 1_+3%. Intrathecal injection of 1.25 nmol of dynorphin-A-(1-13) produced little change in T F L (maximal changes from 4_+2% to - 5 - 3 % ) , not exceeding the range of fluctuation seen in the normal saline control group. Intrathecal injection of 2.5 nmol of dynorphin-A-(1-13) or 7.5 nmol of morphine produced a slight increase (20-30%) in TFL. The difference
between these two groups and the saline control group was statistically not significant (p<0.05). Intrathecal injection of 5 and 20 nmol of dynorphin-A-(113) or 15 and 30 nmol of morphine produced significant increase in TFL. The effect emerged within 5 min and lasted for at least 60 min. Figure 1 shows the dose-response curves for dynorphin-A-(1-13) and morphine, where the percentage increase in TFL 30 min after the drug administration was taken for constructing the curves. Dynorphin-A-(1-13) is clearly more effective in raising the T F L than morphine on a molar basis. While morphine did not cause any motor disturbance in rats, high doses of dynorphia-A-(1-13) did. Four out of the 9 rats receiving 20 nmol of dynorphin-A-(1-13) and 3 out of 14 rats receiving 5 nmol of dynorphin-A-(1-13) exhibited a certain degree of muscle weakness or even paralysis in hind legs when they were allowed to move freely outside the holder one hour after the drug administration.
The Analgesic EfJbct of lntrathecal Injection of Dynorphin-A-( I-13) and Morphine in Combination A group of !1 rats was injected intrathecally with 1.25
DYNORPHIN-A ANTAGONIZES MORPHINE ANALGESIA Dynorphin ,<7,
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• Naloxone 0.5 mg/kg
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f Dynorphin1.25 nmol C, Morphine 7.5 nmol
N = 6--'7 rats
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FIG. 4. The effect of naloxone on intrathecal dynorphin-A-(1-13) (A), morphine (B) and dynorphin/morphine composite (C). *p<0.05. **p<0.01, ***p<0.001 compared with NS control. nmol of dynorphin-A-(l-13) and 7.5 nmol of morphine in combination at a total volume of 10/xi. A marked increase in T F L over 100% of the basal level was seen 10 min after the injection, which maintained at a height between 104_+15% and 119---7% for the whole observation period of 80 min (Fig. 2). This effect was much stronger than the mathematical additive effect of the two components (Fig. 3). None of the rats showed any signs of motor disfunction when they were allowed to move freely out of the holders. To test the potential synergistic effect existing between dynorphin-A-(l-13) and morphine, the dynorphin/morphine mixture was serially diluted and injected intrathecally to groups of rats. These results are also shown in Fig. 2. A mixture of dynorphin-A-(1-13) 0.625 nmol and morphine 3.75 nmol ( n = l l ) produced an increase in T F L over 60% within 1 hour (p<0.05 as compared to the corresponding NS control). A mixture of dynorphin-A-(l-13) 0.313 nmol and morphine 1.875 nmol produced an increase in T F L of over 30% 30 rain after the drug administration, showing a clear dose-response relationship.
The Effect of Nah)xone on Analgesia Induced by Dynorphin-A-( I-13). Morphine and Dynorphhl/Morphine Mixture In an attempt to evaluate the naloxone reversibility of the opioid analgesia, equipotent doses of dynorphin-A-(l-13) (20 nmol), morphine (30 nmol) and dynorphin/morphine mixture (D 1.25 nmol/M7.5 nmol) were injected intrathecally to groups of rats to produce an increase in T F L 100% above the base line level. Thirty min after intrathecal injection, the rats were given a subcutaneous injection of naloxone (0.5 mg/kg) or normal saline (1 ml/rat). T F L was measured at 10 min intervals for a period of 100 min. The results are shown in Fig. 4.
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FIG. 5. The effect of ICV injection of various doses of dynorphin-A-I 1-13) and morphine in combination. In contrast to dynorphin-A-(l-13) induced analgesia which was resistant to naloxone blockade (Fig. 4A), morphine analgesia was sensitive to naloxone as was indicated by the 67% reduction of the analgesic effect 20 min after the naloxone administration (Fig. 4B). Figure 4C shows that the analgesic effect produced by dynorphin/morphine mixture was almost totally abolished by naloxone. The antagonistic effect of naloxone lasted for at least 1 hour.
The E fJ~,ct of lCV Injection of Dynorphin-A-(I-13) or Morphine Five groups of rats with 6--11 in each group were given ICV injection of normal saline 8/zl, dynorphin-A-(l-13) 20 nmol or morphine 30, 60, 120 nmol, respectively. T F L was measured at 10 min intervals for a total of 90 rain.
1018
REN, L U A N D H A N
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Dynorphim~-ts) 20 nmol,icv
DISCUSSION
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90 rain Time ]FIG. 6. The effect of ]CV injection of dynorphin-A-(l-13) on morphine analgesia. M, morphine 120 nmol; D, dynorphin-A-(]-]3) 20 nmol; NS, normal saline.
The changes in T F L in the 90 min period after NS injection were in the range of 3-_4% to -7_+5%. Dynorphin-A(1-13) 20 nmol produced a slight increase in T F L by 24___13% 10 min after ICV injection which was not significantly different from the NS control group. ICV injection of morphine produced a dose-dependent increase in TFL. Peak values were reached 30-40 min after the ICV injection, being 19-+ 12%, 74+18% and 139+7% in 30, 60 and 120 nmol group, respectively.
The Effect on TFL of ICV Injection of Dynorphin-A-( I-13) and Morphine in Combination Two groups of 8 rats were injected ICV with a dynorphin/morphine mixture followed by an observation period of 90 min. A mixture of 10 nmol of dynorphin-A-(l-13) and 120 nmol of morphine produced an increase in T F L of 109_+ 15% which was only slightly lower than that produced by 120 nmol of morphine alone (139_7%, p<0.05). However, addition of 20 nmol of dynorphin-A-(1-13) to 120 nmol of morphine resulted in a dramatical suppression of morphine analgesia. The increase in T F L 30 min after ICV injection was only 16_+8% which was 12% of that seen in 120 nmol morphine group (p<0.001) (see Fig. 5). The suppressive effect of dynorphin-A-(1-13) on morphine analgesia was most prominent within 40 min of ICV injection and subsided gradually in a period of 80 min.
Antagonistic Effect of lCV Dynorphin-A(l-13) on Morphine Analgesia Two groups of rats with 6 and 9 in each were given ICV injection of 120 nmol of morphine which produced an increase in T F L of 126_+14% and 121-+12%, respectively, 30 min after the injection. One group (n=6) was then given ICV injection of normal saline and the other group (n=9) 20 nmol of dynorphin-A-(l-13). The results are shown in Fig. 6. Dynorphin-A-(I-13) produced a 68% reversal (p<0.001) of the morphine analgesia 20 min after its ICV administration as compared to the NS control group. This antagonistic effect was quick in onset (a reversal of 56%, p<0.01, within 10 min) and lasted about 50 min.
Our data clearly show that intrathecal dynorphin-A-(1-13) produced a dose-dependent prolongation of T F L , an effect one fold stronger than morphine on a molar basis. While high dose of dynorphin could result in motor dysfunction, such disfunction does not explain the prolongation of T F L in lower doses. This becomes evident since: (1) 5 nmol of dynorphin-A-(1-13) produced an increase of T F L by 69_+ 13% (p<0.001 compared to NS control group) yet only 3/14 of the rats exhibited signs of motor disturbance, (2) a subanalgesic dose of dynorphin-A-(1-13) (1.25 and 0.625 nmol) produced a marked potentiation on morphine analgesia whereas none of the animals had any sign of motor dysfunction. Thus different effects of dynorphin-A on sensory and motor function may occur depending on the dose: a small amount of exogenously applied [7, 9, 10, 17, 19, 20, 27] or endogenously released [8] dynorphin-A primarily affects nociception. Motor activities are impaired only when a relatively large dose is administered or a huge amount of dynorphin-A is released in such cases as cord injury [4]. The supposition that dynorphin-A in spinal cord may be more involved in sensory rather than in motor function seems to be in line with the findings that the dynorphin-A content in the dorsal spinal cord is much higher than in the ventral cord [1]. It is worthwhile to mention that the paralytic effect of dynorphin-A may well be a non-opioid effect. The commonly used criterion to differentiate opioid with non-opioid nature of a physiological or pharmacological effect is its susceptibility to naloxone blockade. However, the conclusion can be different depending on the dose of naloxone being used. Goldstein has pointed out that dynorphin-A-(l-13) is 13-fold more resistant to naloxone blockade than [LeuS]enkephalin in the guinea pig ileum assay [6]. We have found in the in vivo study that the dose of naloxone (10 mg/kg, SC) required to produce 50% reversal of the analgesic effect of dynorphin-A was 14 times as high as that needed for morphine (0.7 mg/kg, SC), when an equipotent dose of dynorphin-A or morphine was injected intrathecally [9]. These and some other evidence from receptor-protection studies and cross tolerance studies point to the conclusion that dynorphin-A is an endogenous ligand of the kappa receptors [3] and that dynorphin-A induced analgesia is mediated by kappa receptors in the spinal cord [26]. Based on the data mentioned above, a naloxone dose of 10-20 mg/kg should be tried (in rats) and prove to be ineffective before a "non-opioid" nature can be assigned to an effect. To this context it is interesting to note that the paralytic effect of intrathecally administered dynorphin-A is not affected by 20 mg/kg of naloxone (J. B. Long, presented in the International Narcotic Research Conference, June, 1985), therefore is most probably nonopioid. The augmentation of morphine analgesia by a small amount of dynorphin-A-(l-13) merits special attention. The joint effect of dynorphin-A-(l-13) (1.25 nmol) and morphine (7.5 nmol) was not simply additive. It was equivalent to 12 nmol of dynorphin-A-(1-13) or 20 nmol of morphine given separately, as could be estimated from the dose-response curves shown in Fig. 1. The analgesic effect was still prominent even when the mixture was diluted 1-2 fold, where single dose of dynorphin-A-(1-13) or morphine was already several times smaller than the minimal effective doses (see Figs. 1 and 2). These findings strongly suggest that dynorphin-A-(1-13) and morphine are acting on different receptor populations, resulting in a synergistic effect.
DYNORPHIN-A A N T A G O N I Z E S MORPHINE A N A L G E S I A The result of naloxone blockade seems to throw some light on the type of receptors implicated in analgesia produced by the dynorphin/morphine mixture. Since dynorphin-A is believed to act through kappa receptors and requires a large dose of naloxone (10 mg/kg) for partial antagonism [6, 9, 27], an almost complete abolishment of the analgesia produced by dynorphin/morphine mixture by a small dose of naloxone (0.5 mg/kg) seem to suggest that the composite probably acts mainly on mu receptors. However, if a mutual interaction between kappa and mu receptors is required for the synergistic effect to appear, then selective blockade of the kappa receptors, leaving mu receptors intact, would also have resulted in a disruption of the synergism. This issue deserves further investigation. Modulatory effect on morphine analgesia of opioid peptides other than dynorphin have been reported [2, 14, 15, 23] and various models have been proposed to explain the possible interaction between delta and mu receptors [22] or between enkephalin sites and alkaloid sites of a unique opioid receptor [16]. In the spinal cord, a potentiation of morphine analgesia by 100/xg of [leu]enkephalin has been reported [14]. The amount of [leu]enkephalin needed (around 200 nmol) for potentiation of morphine analgesia is about 200 times that of the dynorphin-A-(l-13) used in the present study (0.625-1.25 nmol), implying that the interaction between dynorphin-A-(l-13) and morphine (or kappa and mu receptors) in the spinal cord is much more intimate than that between enkephalin and morphine (or delta and mu receptors). Whether there is an allosterically coupled kappa/mu receptor combination remains to be determined. While the theoretical explanation is not clear, the synergism between dynorphin-A-(l-13) and morphine may have some practical implications. Since only a minimal activation of kappa and mu receptors is required to produce a substantial analgesia, one would anticipate less development of
1019
tolerance when a mixture of dynorphin/morphine or kappa/mu agonists is developed for clinical trial. Another interesting finding in our study is the sharp contrast between brain and spinal cord for dynorphin/morphine interaction. While dynorphin-A-(l-13) potentiated morphine analgesia in the spinal cord, it antagonized morphine analgesia in the brain. This phenomenon was first reported in mice [5, 13, 21] and is here confirmed in the rat. Controversial results have been obtained previously as to whether dynorphin-A produces an analgesic effect after ICV injection [5, I 1, 17, 18, 21, 24, 26]. Most of the authors using tail flick for nociceptive test obtained negative results [5, 24, 26]. We have also found that ICV injection of dynorphin-A(1-13) alone produced no significant effect on T F L at a wide dose range of 1-20 nmol (only the 20 nmol data are shown in Fig. 5). However when administered in combination with morphine (Fig. 5) or superimposed on morphine analgesia (Fig. 6), a marked suppression of the morphine effect was observed. The phenomenon is straight forward and clearcut. However, the amount of dynorphin-A-(1-13) (20 nmol) required for this negative modulation is large, and half this dose (10 nmol) proved ineffective (Fig. 5). The physiological relevance of this phenomenon awaits for clarification. It is techniquely difficult to use naloxone for testing the nature (opioid or non-opioid) of the antagonistic effect of dynorphin-A-(i-13) on morphine analgesia. However, it was reported that des-tyrosine dynorphin-A was also effective in antagonizing morphine analgesia [25], indicating that this effect could be non-opioid, as was the paralytic effect of this peptide. ACKNOWLEDGEMENTS We thank Dr. Avram Goldstein for the gift of dynorphin-A-(113), and Endo Laboratories for naloxone. This work was supported by the Ministry of Health, People's Republic of China.
REFERENCES
I. Botticelli, L. I., B. M. Cox and A. Goldstein. Immunoreactive dynorphin in mammalian spinal cord and dorsal root ganglia. Proc Natl A c a d Sci USA 78: 7783-7786, 1981. 2. Chapman, D. B., J. Hu and E. L. Way. Methionine-enkephalin antagonism and endorphin potentiation of narcotic-induced analgesia, Eur .I Pharmacol 65: 36%377, 1980. 3. Chavin, C., I. F. James and A. Goldstein. Dynorphin is a specific ligand of the Kopioid receptor. Science 215: 413-415, 1982. 4. Faden, A. I., T. P. Jacobs, G. P. Smith, B. Green, J. A. Zivin. Neuropeptides in spinal cord injury: Comparative experimental models. Peptides 4: 631-634, 1983. 5. Friedman, H. J., M. F. Jen, J. K. Chang, N. M. Lee and H. H. Loh. Dynorphin: A possible modulatory peptide on morphine or fl-endorphin analgesia in mouse. Eur J Pharmacol 69: 357-360, 1981. 6. Goldstein, A., S. Tachibana, L. I. Lowney, M. Hunkapiller and L. Hood. Dynorphin-(1-13), an extraordinarily potent opioid peptide. Proc Natl A c a d Sci USA 76: 6666-6670, 1979. 7. Han, J. S. and C. W. Xie. Dynorphin: Potent analgesic effect in spinal cord of the rat. Lift" Sci 31: 1781-1783, 1982. 8. Han, J. S. and G. X. Xie. Dynorphin: Important mediator for electroacupuncture analgesia in the spinal cord of the rabbit. Pain 18: 367-376, 1984. 9. Han, J. S., G. X. Xie and A. Goldstein. Analgesia induced by intrathecal injection of dynorphin B in the rat. L(/b Sci 34: 1573-1579, 1984. 10. Herman, B. H. lntrathecal dynorphin induces antinociception and paralysis. Soc N e u r o s c i Abstr 8: 91, 1982.
I I. Herman, B. H., F. Leslie and A. Goldstein. Behavioral effects and in vivo degradation of intraventricularly administered dynorphin-(l-13) and D-Ala-dynorphin-(l-ll) in rats. Lift, Sci 27: 883-892, 1980. 12. Jen, M. F. and J. S. Han. Rat tail flick acupuncture analgesia model. Chin M e d J 92: 576-582, 1979. 13. Jen, M. F., F. C. Tulunay, E. Y. Way and N. M. Lee. Modulatory role of dynorphin in morphine tolerant and nontolerant mice. IUPHAR 8th Intern Congr Pharmacol Abstr p. 285, 1981. 14. Larson, A. A., J. L. Vaught and E. Tachemori. The potentiation of spinal analgesia by leucine enkephalin. Eur J Pharmacol 61: 381-383, 1098. 15. Lee, N. M., H. J. Friedman, L. Leybin, T. M. Cho, H. H. Loh and C. H. Li. Peptide inhibitor of morphine- and /3endorphin-induced analgesia. Proc Nat! A c a d Sci USA 77: 5525-5526, 1980. 16. Lee, N. M. and A. P. Smith. A protein-lipid model of the opiate receptor. LiJb Sci 26: 145%1464, 1980. 17. Nakazawa, T., M. Ikeda, T. Kaneko and K. Yamatzu. Analgesic effect of dynorphin-A and morphine in mice. Peptides 6: 75-78, 1985. 18. Petrie, E. C., S. T. Tiffany, T. B. Baker and J. L. Dahl. Dynorphin (1-13): Analgesia, hypothermia, cross tolerance with morphine and fl-endorphin. Peptides 3: 41-47, 1982. 19. Piercey, M. F., K. Varner and L. A. Schroeder. Analgesic activity of intraspinally administered dynorphin and ethylketocyclazocine. Eur J P h a r m a c o l 80: 283-284, 1982.
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20. Przewlocki, R., G. T. Shearman and A. Herz. Mixed opioid/nonopioid effects of dynorphin and dynorphin relayed peptides after their intrathecal injection in rats. Neuropeptides 3: 233-240, 1983. 21. Tulunay, F. C., M. F. Jen, J. K. Chang, H. H. Loh and N. M. Lee. Possible regulatory role of dynorphin on morphine- and fl-endorphin-induced analgesia..I Pharmacol Exp Ther 219: 296-298, 1981. 22. Vaught, J. L., R. B. Rothman and T. C. Westfall. Mu and delta receptors: Their role in analgesia and in the differential effects of opioid peptides on analgesia. Lift, Sci 30: 1443-1455, 1982. 23. Vaught, J. L. and A. E. Takemori. Differential effects of leucine- and methionine-enkephalin on morphine-induced analgesia, acute tolerance and dependence. J Pharmacol Exp Ther 208: 86-90, 1978.
REN, LU AND HAN
24. Walker, J. M., R. J. Katz and H. Akil. Behavioral effects of dynorphin 1-13 in the mouse and rat: Initial observations. Peptides 1: 341-345, 1980. 25. Walker, J. M., D. E. Tucker, D. H. Coy, B. B. Walker and H. Akil. Des-tyrosine-dynorphin antagonizes morphine analgesia. Eur J Pharmacol 85: 121-122, 1982. 26. Wuster, M., R. Schulz and A. Herz. Opiate activity and receptor selectivity of dynorphin 11-13) in the mouse vas deferens. Eur J Pharmacol 62: 235-236, 1980. 27. Xie, G. X. and J. S. Han. Dynorphin: Analgesic effect via kappa receptors in spinal cord of rats. ('hung Kuo Yao Li Hsyeh Pao 5: 231-234, 1984.