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Comparison of the antinociceptive effects of intracerebroventricular injection of kyotorphin, cyclo (N-methyl-tyr-arg) and met-enkephalin in mice
Neurupharmacolqv Vol. 23, No. 1, pp. 7-12, 1984 Printedin Great Britain.All rightsreserved
0028-3908/84 $3.00+ 0.00 Copyright 0 19&Q PergamonPressLtd
COMPARISON OF -THE ANT~NOCICEPT~VE EFFECTS OF INTRACEREBROVENTRICULAR INJECTION OF KYOTORPHIN, CYCLO (N-METHYL-TYR-ARG) AND MET-ENKEPHALIN IN MICE T. SAKURADA, S. SAKURADA, S. WATANABE, S. KAWAMURA, T. SATO,K. KISARA, Y. AKUTSU*, Y. SASAKI* and K. SUZUKI* Departments of Pharmacology and Biochemistry *, Tohoku College of Pharmacy, 4-4-l Komatsushima, Sendai 983, Japan (Accepted 17 May 1983)
Summary-Intracerebroventricular (i.c.v.) administration of kyotorphin (L-Tyrosine-L-Arginine) or Metenkephalin (Met-ENK) to conscious mice resulted in a dose-dependent antinociceptive effect as measured by three pain tests. Cyclo(N-methyl-r.-Tyrosine-L-Arginine) (cycle NMTA), an analogue of kyotorphin, increased the reaction time in the tail-pressure and tail-flick tests. Both dipeptides also decreased writhing induced by acetic acid. However, the antinoci~ptive activity of cycle NMTA was substantially greater than that of kyotorphin or Met-enkephalin. At the maximum effective dose of 62.7 nmol/mouse, this cyclic dipeptide produced a more long-lasting antinociceptive effect than did kyotorphin or Met-enkephalin. Antinociception induced by cycle NMTA or kyotorphin was significantly reversed by pretreatment with naloxone (2 or 8 mg/kg, i.p.), though naloxone was not as effective an antagonist of the antinociceptive action of these peptides as it was against Met-enkephalin. The results indicate that the antinociceptive effect induced by cycle NMTA may in part involve the endogenous opioid system in mice. Key words: kyotorphin, cycio(~-methyl-Tyr-Arg), cerebroventricular injection.
Met~nkephalin,
naloxone, antinociception,
intra-
METHODS
Kyotorphin is an endogenous dipeptide which produces morphine-like effects and which is widely distributed in the central nervous system of the rat brain (Ueda, Shiomi and Takagi, 1980). Previous studies have indicated that kyotorphin, after injection into the cisterna magna, can induce naloxone-reversible analgesic effects in experimental animals, and that kyotorphin does not inhibit the opiate-receptor binding in several ligand binding studies, unlike other opioid peptide analgesics (Takagi, Shiomi, Ueda and Amano, 1979b; Rackham, Wood and Hudgin, 1982). However, subsequent studies have suggested that kyotorphin produces release of Met-enkephalin from the striatum and spinal cord of the guinea pig (Shiomi, Kurahashi, Ueda, Harada, Amano and Takagi, 198 1). Takagi et al. (1979b) have also reported that L-Tyr-p-Arg, a linear analogue of kyotorphin, produced a more profound antinociceptive effect. In a brief communication, it was reported that cycle NMTA, an anlogue of kyotorphin and one of a series of diketopiper~ine derivatives, also produced a greater and longer-lasting antinociceptive effect in the mouse, compared to kyotorphin (Sakurada, Sakurada, Jin, Sato, Kisara, Sasaki and Suzuki, 1982). This action of cycle NMTA has now been extensively studied and compared with the antinociceptive properties of kyoto~hin or Met-enkeph~in.
Animals
Mice (ddY-male) weighing 20-23 g were used in a11 experiments. They were supplied with food and water ad libitum and kept on a 12-hr light-dark cycle. They were housed at least two days prior to their use and were used only once. Groups of 10 mice were used for each experiment. Antinociceptiue assay
Mice were injected intracerebroventricularly (i.c.v.) with Ringer solution or a peptide dissolved in Ringer, at various time intervals prior to being tested. The technique employed for the injection was described previously (Orikasa, Sakurada and Kisara, 1980). The mice were evaluated for responsiveness to noxious stimuli, using the three methods: the tailpressure test, the tail-flick test and the acetic acid induced-writhing test. The tail-pressure test was slightly modified from the original method (Green, Young and Godfrey, 1951); the base of the mouse’s tail was pressed mechanically and the level of pressure in mmHg (10 mmHg/sec) that evoked biting or struggling behaviour was noted. Only mice responding behaviourahy at a tail pressure of 40-50mmHg were selected for this test. The ED, in this mode1 was 7
8
T.
SAKURADA
arbitrarily defined as the dose of test drug capable of elevating the reaction latency to 1.5 times that of the pre-treated animals. The latency of the tail-flick response to a concentrated light beam stimulus was recorded at various time intervals after administration of drugs. Change in the tail-flick latency were expressed as a percentage maximum possible effect (% MPE), using a stimulus cut-off time of 20 set: Post-drug latency - pre-drug latency/maximum latency (20 set) - pre-drug latency x 100 = percentage maximum possible effect. The intensity of the light focussed on the tail was adjusted in preliminary experiments to give a reaction (flick of the tail) time of about 3-5 set in untreated animals. For writhing studies, mice were given intraperitoneal (i.p.) injections of 0.7% acetic acid (0.1 ml/10 g). The number of responses occurring between 5 and 10 min after administration of acetic acid was recorded. Acetic acid was given immediately after kyotorphin or cycle NMTA, and 8 min before Met-enkephalin. The response comprised a constriction of the abdominal muscles followed by an extension of the hind limbs. The ED,, value was defined as the dose of drug required to reduce the number of abdominal constrictions to 50% of the number observed in the vehicle-treated animals. The ED,, values and their 95% confidence limits were determined by the method of Litchfield and Wilcoxon (1949). Student t-test was used for comparison of several treatment groups with a control group. Drugs
Naloxone hydrochloride, supplied by Endo Laboratories Inc., was dissolved in sterile saline and administered 15 min before kyotorphin, cycle NMTA and Met-enkephalin. Cycle NMTA was synthetized by Sasaki, Akutsu, Matsui, Suzuki, Sakurada, Sato and Kisara (1982). Kyotorphin used in the present experiment moved as a single spot on TLC upon applying 50 pg to the silica gel plates: R, 0.13 [n -butanol-acetic acid-water (4: 1: 5, upper phase)], Rf 0.32 [n-butanol-pyridine-acetic acid-water (15: 10:3:12)]. RESULTS
Antinociception
measured by the tail-pressure
test
Kyotorphin, cycle NMTA and Met-enkephalin, administered intraventricularly, were found to have dose-related antinociceptive activity in the mouse tail-pressure test (Table 1). Cycle NMTA was much more potent than kyotorphin or Met-enkephalin. The effects of cycle NMTA or kyotorphin were longerlasting than that of Met-enkephalin. The effects of each dipeptide peaked at 5 min and lasted from 15 to 45 min for cycle NMTA and no more than 15 min for
et
al.
Administration
of kyotorphin, cycle NMTA and Met-ENK to mice
9
Table 2. Antinociceptiveeffects of cycle (IV-methyl-Tyr-Arg),kyotorphin and Met-enkephalinin the mouse ED, (95% confidence limits) (nmol/animal) Methods
Cyclo(N-methyl-Tyr-Arg)
Tail-pressure test
23.4 (15.5-35.5) 13.2 (10.9 - 16.1) 12.4 (7.3-21.1)
Tail-flick test Acetic acid-induced writhing syndrome test
kyotorphin. In contrast, antinociception induced by Met-enkephalin peaked at 2 min after the injection and was completely absent at 10min. In the tailpressure test, the ED,, values were 23.4nmol for cycle NMTA, 393.7 nmol for kyotorphin and 170.0 nmol for Met-enkephalin (Table 2). The antinociceptive effects of cycle NMTA or kyotorphin were attenuated by pretreatment with
8 2 NalOXO”e Rmger
Kyotorphin
Met-enkephalin
393.7 (3423452.7) 370.0 (308.3-444.0) 117.0 (108.3-126.4)
170.0 (146.61972) 140.0 (102.9-190.4) 88.0 (60.3-128.5)
2.0 mg/kg of naloxone. The degree of antagonism by naloxone of antinociception induced by both of the dipeptides was less than that seen with Metenkephalin (Table 1). Antinociception measured by the tail-jlick test Intraventricular injections of cycle NMTA, kyotorphin and Met-enkephalin also produced dose-
2 Img/kg) NalOXO”e +2414nmol/mouse) Met - e”keDholl”
+269 CVCIO c/V-methyl-Tyr-Arg)
100
80
LIIlL I
k r 6 #!
60
40
20
0
Ringer
100
28.9
Cycle IN-methyl-Tyr-Arg)
241.4
410 3 6926
Kyoforphin
vi-!
Ringer 63 5
nmol/mwse
Met-enkephalin
Fig. I. Upper panel: a comparison of the antinociceptive effect produced by intracerebroventricular injection of cyclo(N-methyl-Tyr-Arg), kyotorphin and Met-enkephalin as measured by the tail-flick test. All compounds used were evaluated at the time of the peak antinociceptive effects which was 2 min for Met-enkephalin, and 5 min for kyotorphin and cyclo(N-methyl-Tyr-Arg). Ten mice were used per group. The results are presented as mean and SE. Lower panel: the effect of naloxone on antinociception induced by cyclo(N-methyl-Tyr-Arg) and Met-enkephalin. Naloxone was given (i.p.) 15 min prior to the peptides. *P < 0.05 when compared with saline plus Met-enkephalin treatment.
10
T.
SAKURADA
dependent effects in the mouse tail-flick test (Fig. 1). Cycle NMTA exhibited the greatest antinociceptive activity and had a longer-lasting effect than Metenkephalin. In the tail-flick test, the ED, values were 13.2 nmol for cycle NMTA, 370.0 nmof for kyotorphin and 140.0nmol for Met-enkephalin (Table 2). This antinociceptive action of cycle NMTA was reduced by pretreatment with a relatively large dose of naloxone, 8.0 mg,lkg, but was not significant. Significant antagonistic effects of naloxone, 2 mg/kg on Met-enkephalin-induced antinociception were observed (Fig. 1). Antinociception measured writhing syndrome test
by
acetic
acid-induced
Intravent~cular administration of cycle NMTA, kyotorphin or Met-enkephalin caused dosedependent inhibition of the acetic acid-induced abdominal constrictions (Fig. 2). The EDSo values were 12.4 nmol for cycle NMTA, 117.0 nmol for kyotorphin and 88.0 nmol for Met-enkephalin (Table 2). The pretreatment with naloxone in a dose of 2 mg/kg (i.p.) did not produce significant antagonism of the effect of kyotorphin or Met-enkephalin. How-
et al.
ever, at 8 mg/kg of naloxone,
the effects of cycle NMTA or Met-enkephalin were significantly reversed, whereas the effects of kyotorphin were reduced slightly, although not to a statistically significant extent. DISCUSSION
It has been reported that in vivo, kyotorphin produces a naloxone-reversible effect in several antinociceptive assays (Takagi et al., 1979a; Vaught and Chipkin, 1982; Rackham et aZ., 1982). There is also electrophysiological evidence that kyotorphin has naloxone-sensitive actions on single neurones in the spinal dorsal horn of rabbits and the nucleus reticularis paragigantocellula~s in rats (Satoh, Kawajiri, Yamamoto, Akaike, Ukai and Takagi, 1980). The present study confirms that the antinociceptive actions of kyotorphin are mediated through opiate mechanisms. Takagi et al. (1979a) who first isolated and identified kyotorphin from bovine brain, reported the possibility that this dipeptide was regulating the release of endogenous opioids, especially Met-enkephalin (Takagi et al., 1979b). Moreover,
11h
I5 142 Or!mol/mouse
35 142.0
Cyclo(N-methyl-Tyr-Arg)
Met-enkepholm
Kyotorphtn
*
~ NCllO~O~~ NdOXOne -+ + 28 9 Ringer CYClO (N-methyl-Tyr-Arg)
Noloxone +4103 Kyotorphln
NC Fi
8 Saline 2 8 (q/kg) ,ne N0lOXOW +2414 lnmoL/moure) Met-enkephol~n !r
Fig. 2. Upper panel: a comparison of the antinoci~ptive effect produced by ~ntracerebroventricular injection of cyclo(N-methyl-Tyr-Arg), kyotorphin and Met-enkephahn as measured by the acetic acid-induced writhing test. Ten mice were used per group. The results are presented as mean and SE. Lower panel: the effect of naloxone on antinociception induced by cyclo(N-methyl-Tyr-Arg), kyotorphin and Met-enkephalin. Naloxone was given (i.p.) 15 min prior to the peptides. Details in Methods. Ten mice were used per group. *P c 0.01 when compared with saline plus cyclo(N-methyl-Tyr-Arg) or saline plus Met-enkephalin treatment.
Administration
of kyotorphin, cycle NMTA and Met-ENK to mice
there is a biochemical report supporting this concept that kyotorphin-induced antinociception is a result of an indirect action on opiate receptors and may possibly involve endogenous release of opioid peptides (Rackham et al., 1982). It seems obvious thal. kyotorphin is a unique endogenous dipeptide compared with any of the other previously identified opioid peptides. Depending on the situation, different authors have reported different results with kyotorphin. Vaught and Barrett (1980) could not demonstrate narcotic-like actions of kyotorphin in the mouse tail-flick test using a 55°C hot plate assay. However, they observed that kyotorphin exhibited a dose-dependent, lasting analgesia by lowering the temperature of the hot plate to 48°C. In the present experiments, the effective dose of kyotorphin, administered intracerebroventricularly was considerably large and nearly toxic. As judged from the ED,, of cycle NMTA, kyotorphin and Met-enkephalin, the intensity of nociceptive stimuli in the tail-flick test appears to be almost the same or slightly weaker than that of the tail pressed mechanically in the tailpressure test. It also seems evident that the intensity of chemical stimuli, induced by acetic acid, is much weaker than mechanical or heat nociceptive stimuli used in the present experiment. It is therefore probable that the ED,, dose of all peptides was the smallest in the acetic acid-induced writhing syndrome test. The ED,, value of kyotorphin in the acetic acidinduced writhing was approximately equal to the ED, of 105.6nmol in the hot plate test (weaker heat nociceptive stimuli; 48’C) obtained by Vaught and Chipkin (1982). It may be inferred from their and the present results that the antinociceptive activity of kyotorphin could be masked by the potent nociceptive heat or pressure stimuli. However, the effective dose of kyotorphin in the present study was
much larger than that obtained (1979a,b)
even if there
antinociceptive
by Takagi et al.
are some differences
in the
test system or routes of adminis-
tration of peptide. It may be of particular importance to consider the differences in the purity of kvotor-
phin. The data presented here we;e also almost in agreement with the result of Rackham et al. (1982), that the effective dose range of kyotorphin as measured by the tail-flick test in mice was lOO-2OOpg (armrox. 246492 nmol from calculation) after intracisternal administration. In the present experiment, it was not possible to demonstrate complete antagonistic effects of naloxone on kyotorphin-induced antinociceution measured bv the tail-oressure test. Thus, itseems unlikely thatihe antinoci’ceptive action of kyotorphin is duk only to an interaction with \&I
I
opiate receptors.
It has already been reported that cycle NMTA was found to be the most potent antinociceptive in a series of cyclic dipeptides (Sakaurada et al., 1982). Intraventricular administration of cycle NMTA to mice resulted in a dose-dependent, naloxone-reversible antinociception.
Despite the apparent
antagonism
of
11
naloxone on Met-enkephalin, the antinociceptive action of cycle NMTA was not reversed completely by naloxone (2 or 8 mg/kg, i.p.); in particular, a significant antagonistic effect was not seen in the tail-flick test. It has also been found in the tail-flick test in the rat that antinociception, induced by injection of cycle NMTA into the third ventricle, was not antagonized by naloxone and that cycle NMTA had no effect at the spinal cord level (Kawamura, Sakurada, Sakurada, Kisara, Akutsu, Sasaki and Suzuki, 1982). From the present three test systems, it seems that the mode of action of cycle NMTA may be only partly mediated through an opiate mechanism. Further experiments are required to settle more precisely the basis for the interaction with opiate receptors.
Acknowledgements-This work was supported in part by a research grant No. 57570081 from the Japanese Ministry of Education, Science and Culture.
REFERENCES Green A. F., Young P. A. and Godfrey E. I. (1951) A comparison of heat and pressure analgesiometric methods in rats. Br. J. Pharmac. 6: 572-585. Kawamura S., Sakurada S., Sakurada T., Kisara K., Akutsu Y., Sasaki Y. and Suzuki K. (1983) Antinociceptive effect of centrally administered cyclo(Nmethyl-L-Tyr-L-Arg) in the rat. Eur. J. Phurmac. 93: 1-8. Litchfield J. T. and Wilcoxon F. (1949) A simplified method of evaluating dose-effect experiments. J. Pharmuc. exp. Ther. 961 99-113.
Orikasa S., Sakurada S. and Kisara K. (1980) Head-twitch response induced by tyramine. Psychopharmacology 67: 53-59.
Rackham A., Wood P. L. and Hudgin R. L. (1982) Kyotorphin (tyrosine-arginine): further evidence for indirect opiate receptor activation. Life Sci. 30: 1337-1342. Sakurada S.. Sakurada T.. Jin H., Sato T.. Kisara K.. Sasaki Y. and Suzuki K. (1982) A&nocicebtive activities of synthetic cyclic dipeptides in mice. J. Pharm. Phurmac. 34: ___ -_. 7X&751. Sasaki Y., Akutsu Y., Matsui M., Suzuki K., Sakurada S., Sato T. and Kisara K. (1982) Studies on analgesic oligouentides. II. Structure-activitv relationshiu among
thiyt; analogs of a cyclic dipep;de, cyclo(-fyr-Arg-i Chem. Pharmuc. Bull. 30: 44354443.
Satoh M., Kawajiri S., Yamamoto M., Akaike A., Ukai Y. and Takagi H. (1980) Effect of tyrosyl-arginine (kyotorphin) a new opioid dipeptide, on single neurons in the sDina1dorsal horn of rabbits and the nucleus reticularis iaragigantocellularis of rats. Neurosci. Lett. 16: 3 19-322. Shiomi H., Kuraishi Y., Ueda H., Harada Y., Amano H. and Takagi H. (1981) Mechanism of kyotorphin-induced release of Met-enkephalin from guinea pig striatum and sninal cord. Bruin Res. 211: 161-169. Taiagi H., Shiomi H., Ueda H. and Amano H. (1979a) Morphine-like analgesia by a new dipeptide, t.-tyrosyl+arginine (Kyotorphin) and its analogue. Eur. J. Phurmuc. 55: 109-l 11. Takagi H., Shiomi H., Ueda H. and Amano H. (1979b) A novel analgesic dipeptide from bovine brain is a possible Met-enkephalin releaser. Nature 282: 41&412. Ueda H., Shiomi H. and Takagi H. (1980) Regional distribution of a novel analgesic dipeptide kyotorphin (TyrArg) in the rat brain and spinal cord. Bruin Res. 198:
460-464.
12
T. SAKURADAet al
Vaught J. L. and Barrett R. (1980) Inability of Kyotorphin (K) to produce analgesia and bind to the opioid receptor. Fedn Proc. Fedn Am. Sots exp. Biol. 39: 605.
Vaught J. L. and Chipkin R. E. (1982) A characterization of kyotorphin (Tyr-Arg)-induced antinociception. Eur. J. Pharmac. 19: 167-l 73.
0028-3908/84 $3.00+ 0.00 Copyright 0 19&Q PergamonPressLtd
COMPARISON OF -THE ANT~NOCICEPT~VE EFFECTS OF INTRACEREBROVENTRICULAR INJECTION OF KYOTORPHIN, CYCLO (N-METHYL-TYR-ARG) AND MET-ENKEPHALIN IN MICE T. SAKURADA, S. SAKURADA, S. WATANABE, S. KAWAMURA, T. SATO,K. KISARA, Y. AKUTSU*, Y. SASAKI* and K. SUZUKI* Departments of Pharmacology and Biochemistry *, Tohoku College of Pharmacy, 4-4-l Komatsushima, Sendai 983, Japan (Accepted 17 May 1983)
Summary-Intracerebroventricular (i.c.v.) administration of kyotorphin (L-Tyrosine-L-Arginine) or Metenkephalin (Met-ENK) to conscious mice resulted in a dose-dependent antinociceptive effect as measured by three pain tests. Cyclo(N-methyl-r.-Tyrosine-L-Arginine) (cycle NMTA), an analogue of kyotorphin, increased the reaction time in the tail-pressure and tail-flick tests. Both dipeptides also decreased writhing induced by acetic acid. However, the antinoci~ptive activity of cycle NMTA was substantially greater than that of kyotorphin or Met-enkephalin. At the maximum effective dose of 62.7 nmol/mouse, this cyclic dipeptide produced a more long-lasting antinociceptive effect than did kyotorphin or Met-enkephalin. Antinociception induced by cycle NMTA or kyotorphin was significantly reversed by pretreatment with naloxone (2 or 8 mg/kg, i.p.), though naloxone was not as effective an antagonist of the antinociceptive action of these peptides as it was against Met-enkephalin. The results indicate that the antinociceptive effect induced by cycle NMTA may in part involve the endogenous opioid system in mice. Key words: kyotorphin, cycio(~-methyl-Tyr-Arg), cerebroventricular injection.
Met~nkephalin,
naloxone, antinociception,
intra-
METHODS
Kyotorphin is an endogenous dipeptide which produces morphine-like effects and which is widely distributed in the central nervous system of the rat brain (Ueda, Shiomi and Takagi, 1980). Previous studies have indicated that kyotorphin, after injection into the cisterna magna, can induce naloxone-reversible analgesic effects in experimental animals, and that kyotorphin does not inhibit the opiate-receptor binding in several ligand binding studies, unlike other opioid peptide analgesics (Takagi, Shiomi, Ueda and Amano, 1979b; Rackham, Wood and Hudgin, 1982). However, subsequent studies have suggested that kyotorphin produces release of Met-enkephalin from the striatum and spinal cord of the guinea pig (Shiomi, Kurahashi, Ueda, Harada, Amano and Takagi, 198 1). Takagi et al. (1979b) have also reported that L-Tyr-p-Arg, a linear analogue of kyotorphin, produced a more profound antinociceptive effect. In a brief communication, it was reported that cycle NMTA, an anlogue of kyotorphin and one of a series of diketopiper~ine derivatives, also produced a greater and longer-lasting antinociceptive effect in the mouse, compared to kyotorphin (Sakurada, Sakurada, Jin, Sato, Kisara, Sasaki and Suzuki, 1982). This action of cycle NMTA has now been extensively studied and compared with the antinociceptive properties of kyoto~hin or Met-enkeph~in.
Animals
Mice (ddY-male) weighing 20-23 g were used in a11 experiments. They were supplied with food and water ad libitum and kept on a 12-hr light-dark cycle. They were housed at least two days prior to their use and were used only once. Groups of 10 mice were used for each experiment. Antinociceptiue assay
Mice were injected intracerebroventricularly (i.c.v.) with Ringer solution or a peptide dissolved in Ringer, at various time intervals prior to being tested. The technique employed for the injection was described previously (Orikasa, Sakurada and Kisara, 1980). The mice were evaluated for responsiveness to noxious stimuli, using the three methods: the tailpressure test, the tail-flick test and the acetic acid induced-writhing test. The tail-pressure test was slightly modified from the original method (Green, Young and Godfrey, 1951); the base of the mouse’s tail was pressed mechanically and the level of pressure in mmHg (10 mmHg/sec) that evoked biting or struggling behaviour was noted. Only mice responding behaviourahy at a tail pressure of 40-50mmHg were selected for this test. The ED, in this mode1 was 7
8
T.
SAKURADA
arbitrarily defined as the dose of test drug capable of elevating the reaction latency to 1.5 times that of the pre-treated animals. The latency of the tail-flick response to a concentrated light beam stimulus was recorded at various time intervals after administration of drugs. Change in the tail-flick latency were expressed as a percentage maximum possible effect (% MPE), using a stimulus cut-off time of 20 set: Post-drug latency - pre-drug latency/maximum latency (20 set) - pre-drug latency x 100 = percentage maximum possible effect. The intensity of the light focussed on the tail was adjusted in preliminary experiments to give a reaction (flick of the tail) time of about 3-5 set in untreated animals. For writhing studies, mice were given intraperitoneal (i.p.) injections of 0.7% acetic acid (0.1 ml/10 g). The number of responses occurring between 5 and 10 min after administration of acetic acid was recorded. Acetic acid was given immediately after kyotorphin or cycle NMTA, and 8 min before Met-enkephalin. The response comprised a constriction of the abdominal muscles followed by an extension of the hind limbs. The ED,, value was defined as the dose of drug required to reduce the number of abdominal constrictions to 50% of the number observed in the vehicle-treated animals. The ED,, values and their 95% confidence limits were determined by the method of Litchfield and Wilcoxon (1949). Student t-test was used for comparison of several treatment groups with a control group. Drugs
Naloxone hydrochloride, supplied by Endo Laboratories Inc., was dissolved in sterile saline and administered 15 min before kyotorphin, cycle NMTA and Met-enkephalin. Cycle NMTA was synthetized by Sasaki, Akutsu, Matsui, Suzuki, Sakurada, Sato and Kisara (1982). Kyotorphin used in the present experiment moved as a single spot on TLC upon applying 50 pg to the silica gel plates: R, 0.13 [n -butanol-acetic acid-water (4: 1: 5, upper phase)], Rf 0.32 [n-butanol-pyridine-acetic acid-water (15: 10:3:12)]. RESULTS
Antinociception
measured by the tail-pressure
test
Kyotorphin, cycle NMTA and Met-enkephalin, administered intraventricularly, were found to have dose-related antinociceptive activity in the mouse tail-pressure test (Table 1). Cycle NMTA was much more potent than kyotorphin or Met-enkephalin. The effects of cycle NMTA or kyotorphin were longerlasting than that of Met-enkephalin. The effects of each dipeptide peaked at 5 min and lasted from 15 to 45 min for cycle NMTA and no more than 15 min for
et
al.
Administration
of kyotorphin, cycle NMTA and Met-ENK to mice
9
Table 2. Antinociceptiveeffects of cycle (IV-methyl-Tyr-Arg),kyotorphin and Met-enkephalinin the mouse ED, (95% confidence limits) (nmol/animal) Methods
Cyclo(N-methyl-Tyr-Arg)
Tail-pressure test
23.4 (15.5-35.5) 13.2 (10.9 - 16.1) 12.4 (7.3-21.1)
Tail-flick test Acetic acid-induced writhing syndrome test
kyotorphin. In contrast, antinociception induced by Met-enkephalin peaked at 2 min after the injection and was completely absent at 10min. In the tailpressure test, the ED,, values were 23.4nmol for cycle NMTA, 393.7 nmol for kyotorphin and 170.0 nmol for Met-enkephalin (Table 2). The antinociceptive effects of cycle NMTA or kyotorphin were attenuated by pretreatment with
8 2 NalOXO”e Rmger
Kyotorphin
Met-enkephalin
393.7 (3423452.7) 370.0 (308.3-444.0) 117.0 (108.3-126.4)
170.0 (146.61972) 140.0 (102.9-190.4) 88.0 (60.3-128.5)
2.0 mg/kg of naloxone. The degree of antagonism by naloxone of antinociception induced by both of the dipeptides was less than that seen with Metenkephalin (Table 1). Antinociception measured by the tail-jlick test Intraventricular injections of cycle NMTA, kyotorphin and Met-enkephalin also produced dose-
2 Img/kg) NalOXO”e +2414nmol/mouse) Met - e”keDholl”
+269 CVCIO c/V-methyl-Tyr-Arg)
100
80
LIIlL I
k r 6 #!
60
40
20
0
Ringer
100
28.9
Cycle IN-methyl-Tyr-Arg)
241.4
410 3 6926
Kyoforphin
vi-!
Ringer 63 5
nmol/mwse
Met-enkephalin
Fig. I. Upper panel: a comparison of the antinociceptive effect produced by intracerebroventricular injection of cyclo(N-methyl-Tyr-Arg), kyotorphin and Met-enkephalin as measured by the tail-flick test. All compounds used were evaluated at the time of the peak antinociceptive effects which was 2 min for Met-enkephalin, and 5 min for kyotorphin and cyclo(N-methyl-Tyr-Arg). Ten mice were used per group. The results are presented as mean and SE. Lower panel: the effect of naloxone on antinociception induced by cyclo(N-methyl-Tyr-Arg) and Met-enkephalin. Naloxone was given (i.p.) 15 min prior to the peptides. *P < 0.05 when compared with saline plus Met-enkephalin treatment.
10
T.
SAKURADA
dependent effects in the mouse tail-flick test (Fig. 1). Cycle NMTA exhibited the greatest antinociceptive activity and had a longer-lasting effect than Metenkephalin. In the tail-flick test, the ED, values were 13.2 nmol for cycle NMTA, 370.0 nmof for kyotorphin and 140.0nmol for Met-enkephalin (Table 2). This antinociceptive action of cycle NMTA was reduced by pretreatment with a relatively large dose of naloxone, 8.0 mg,lkg, but was not significant. Significant antagonistic effects of naloxone, 2 mg/kg on Met-enkephalin-induced antinociception were observed (Fig. 1). Antinociception measured writhing syndrome test
by
acetic
acid-induced
Intravent~cular administration of cycle NMTA, kyotorphin or Met-enkephalin caused dosedependent inhibition of the acetic acid-induced abdominal constrictions (Fig. 2). The EDSo values were 12.4 nmol for cycle NMTA, 117.0 nmol for kyotorphin and 88.0 nmol for Met-enkephalin (Table 2). The pretreatment with naloxone in a dose of 2 mg/kg (i.p.) did not produce significant antagonism of the effect of kyotorphin or Met-enkephalin. How-
et al.
ever, at 8 mg/kg of naloxone,
the effects of cycle NMTA or Met-enkephalin were significantly reversed, whereas the effects of kyotorphin were reduced slightly, although not to a statistically significant extent. DISCUSSION
It has been reported that in vivo, kyotorphin produces a naloxone-reversible effect in several antinociceptive assays (Takagi et al., 1979a; Vaught and Chipkin, 1982; Rackham et aZ., 1982). There is also electrophysiological evidence that kyotorphin has naloxone-sensitive actions on single neurones in the spinal dorsal horn of rabbits and the nucleus reticularis paragigantocellula~s in rats (Satoh, Kawajiri, Yamamoto, Akaike, Ukai and Takagi, 1980). The present study confirms that the antinociceptive actions of kyotorphin are mediated through opiate mechanisms. Takagi et al. (1979a) who first isolated and identified kyotorphin from bovine brain, reported the possibility that this dipeptide was regulating the release of endogenous opioids, especially Met-enkephalin (Takagi et al., 1979b). Moreover,
11h
I5 142 Or!mol/mouse
35 142.0
Cyclo(N-methyl-Tyr-Arg)
Met-enkepholm
Kyotorphtn
*
~ NCllO~O~~ NdOXOne -+ + 28 9 Ringer CYClO (N-methyl-Tyr-Arg)
Noloxone +4103 Kyotorphln
NC Fi
8 Saline 2 8 (q/kg) ,ne N0lOXOW +2414 lnmoL/moure) Met-enkephol~n !r
Fig. 2. Upper panel: a comparison of the antinoci~ptive effect produced by ~ntracerebroventricular injection of cyclo(N-methyl-Tyr-Arg), kyotorphin and Met-enkephahn as measured by the acetic acid-induced writhing test. Ten mice were used per group. The results are presented as mean and SE. Lower panel: the effect of naloxone on antinociception induced by cyclo(N-methyl-Tyr-Arg), kyotorphin and Met-enkephalin. Naloxone was given (i.p.) 15 min prior to the peptides. Details in Methods. Ten mice were used per group. *P c 0.01 when compared with saline plus cyclo(N-methyl-Tyr-Arg) or saline plus Met-enkephalin treatment.
Administration
of kyotorphin, cycle NMTA and Met-ENK to mice
there is a biochemical report supporting this concept that kyotorphin-induced antinociception is a result of an indirect action on opiate receptors and may possibly involve endogenous release of opioid peptides (Rackham et al., 1982). It seems obvious thal. kyotorphin is a unique endogenous dipeptide compared with any of the other previously identified opioid peptides. Depending on the situation, different authors have reported different results with kyotorphin. Vaught and Barrett (1980) could not demonstrate narcotic-like actions of kyotorphin in the mouse tail-flick test using a 55°C hot plate assay. However, they observed that kyotorphin exhibited a dose-dependent, lasting analgesia by lowering the temperature of the hot plate to 48°C. In the present experiments, the effective dose of kyotorphin, administered intracerebroventricularly was considerably large and nearly toxic. As judged from the ED,, of cycle NMTA, kyotorphin and Met-enkephalin, the intensity of nociceptive stimuli in the tail-flick test appears to be almost the same or slightly weaker than that of the tail pressed mechanically in the tailpressure test. It also seems evident that the intensity of chemical stimuli, induced by acetic acid, is much weaker than mechanical or heat nociceptive stimuli used in the present experiment. It is therefore probable that the ED,, dose of all peptides was the smallest in the acetic acid-induced writhing syndrome test. The ED,, value of kyotorphin in the acetic acidinduced writhing was approximately equal to the ED, of 105.6nmol in the hot plate test (weaker heat nociceptive stimuli; 48’C) obtained by Vaught and Chipkin (1982). It may be inferred from their and the present results that the antinociceptive activity of kyotorphin could be masked by the potent nociceptive heat or pressure stimuli. However, the effective dose of kyotorphin in the present study was
much larger than that obtained (1979a,b)
even if there
antinociceptive
by Takagi et al.
are some differences
in the
test system or routes of adminis-
tration of peptide. It may be of particular importance to consider the differences in the purity of kvotor-
phin. The data presented here we;e also almost in agreement with the result of Rackham et al. (1982), that the effective dose range of kyotorphin as measured by the tail-flick test in mice was lOO-2OOpg (armrox. 246492 nmol from calculation) after intracisternal administration. In the present experiment, it was not possible to demonstrate complete antagonistic effects of naloxone on kyotorphin-induced antinociceution measured bv the tail-oressure test. Thus, itseems unlikely thatihe antinoci’ceptive action of kyotorphin is duk only to an interaction with \&I
I
opiate receptors.
It has already been reported that cycle NMTA was found to be the most potent antinociceptive in a series of cyclic dipeptides (Sakaurada et al., 1982). Intraventricular administration of cycle NMTA to mice resulted in a dose-dependent, naloxone-reversible antinociception.
Despite the apparent
antagonism
of
11
naloxone on Met-enkephalin, the antinociceptive action of cycle NMTA was not reversed completely by naloxone (2 or 8 mg/kg, i.p.); in particular, a significant antagonistic effect was not seen in the tail-flick test. It has also been found in the tail-flick test in the rat that antinociception, induced by injection of cycle NMTA into the third ventricle, was not antagonized by naloxone and that cycle NMTA had no effect at the spinal cord level (Kawamura, Sakurada, Sakurada, Kisara, Akutsu, Sasaki and Suzuki, 1982). From the present three test systems, it seems that the mode of action of cycle NMTA may be only partly mediated through an opiate mechanism. Further experiments are required to settle more precisely the basis for the interaction with opiate receptors.
Acknowledgements-This work was supported in part by a research grant No. 57570081 from the Japanese Ministry of Education, Science and Culture.
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Orikasa S., Sakurada S. and Kisara K. (1980) Head-twitch response induced by tyramine. Psychopharmacology 67: 53-59.
Rackham A., Wood P. L. and Hudgin R. L. (1982) Kyotorphin (tyrosine-arginine): further evidence for indirect opiate receptor activation. Life Sci. 30: 1337-1342. Sakurada S.. Sakurada T.. Jin H., Sato T.. Kisara K.. Sasaki Y. and Suzuki K. (1982) A&nocicebtive activities of synthetic cyclic dipeptides in mice. J. Pharm. Phurmac. 34: ___ -_. 7X&751. Sasaki Y., Akutsu Y., Matsui M., Suzuki K., Sakurada S., Sato T. and Kisara K. (1982) Studies on analgesic oligouentides. II. Structure-activitv relationshiu among
thiyt; analogs of a cyclic dipep;de, cyclo(-fyr-Arg-i Chem. Pharmuc. Bull. 30: 44354443.
Satoh M., Kawajiri S., Yamamoto M., Akaike A., Ukai Y. and Takagi H. (1980) Effect of tyrosyl-arginine (kyotorphin) a new opioid dipeptide, on single neurons in the sDina1dorsal horn of rabbits and the nucleus reticularis iaragigantocellularis of rats. Neurosci. Lett. 16: 3 19-322. Shiomi H., Kuraishi Y., Ueda H., Harada Y., Amano H. and Takagi H. (1981) Mechanism of kyotorphin-induced release of Met-enkephalin from guinea pig striatum and sninal cord. Bruin Res. 211: 161-169. Taiagi H., Shiomi H., Ueda H. and Amano H. (1979a) Morphine-like analgesia by a new dipeptide, t.-tyrosyl+arginine (Kyotorphin) and its analogue. Eur. J. Phurmuc. 55: 109-l 11. Takagi H., Shiomi H., Ueda H. and Amano H. (1979b) A novel analgesic dipeptide from bovine brain is a possible Met-enkephalin releaser. Nature 282: 41&412. Ueda H., Shiomi H. and Takagi H. (1980) Regional distribution of a novel analgesic dipeptide kyotorphin (TyrArg) in the rat brain and spinal cord. Bruin Res. 198:
460-464.
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T. SAKURADAet al
Vaught J. L. and Barrett R. (1980) Inability of Kyotorphin (K) to produce analgesia and bind to the opioid receptor. Fedn Proc. Fedn Am. Sots exp. Biol. 39: 605.
Vaught J. L. and Chipkin R. E. (1982) A characterization of kyotorphin (Tyr-Arg)-induced antinociception. Eur. J. Pharmac. 19: 167-l 73.