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Lack of tolerance in peripheral opioid analgesia in mice
PII s0024-3205(98)00127-1
ELSEVIER
LACK OF TOLERANCE Shogo Tokuyama, Department
IN PERIPHERAL
Life Sciences, Vol. 62, Nos. 17/W, pp. 1677-1681, 1998 Copyright* 15% Elsevia Science Inc. Printed in the USA. All rights resewed 0024-3205/98 $19.00 + .oo
OPIOID ANALGESIA
IN MICE
Makoto Inoue, Takuya Fuchigami and Hiroshi Ueda*
of Molecular Pharmacology and Neuroscience, Nagasaki University School of Pharmaceutical Sciences, 1-14 Bunkyo-machi, Nagasaki 852, Japan
Summary We recently developed a sensitive peripheral analgesic test in mice. Bradykinin, a representative pain-producing substance, when given subcutaneously through a polyethylene tube into the plantar of the limb connected to a transducer, induced a flexor reflex response, in a dose dependent manner. When morphine, a u-opioid receptor agonist, was added to the plantar through another polyethylene tube, bradykinin-induced responses were completely abolished in a naloxone-reversible mamrer. These peripheral analgesic effects were also observed with DAMGO, another u-opioid receptor wnist, and U-69,593, a k-opioid receptor agonist, but not DSLET, a &opioid receptor agonist. When morphine was given subcutaneously to the back, a potent analgesia in the tail pinch test was observed. Repeated administrations of morphine once per day for 5 days showed a marked tolerance or reduction in morphine analgesia on the 6th day, while there was no significant reduction in the peripheral analgesia of morphine. These finding suggest that tolerance to morphine analgesia is mediated through synaptic plasticity in the central nervous system, but not through a receptor desensitization at the level of the single cell.
Key Worde
peripheral analgesia, bradykinin, opioid, morphine, tolerance
It is well accepted that opioids induce analgesic effects through opioid receptors located in the central nervous system (CNS), such as periaqueductal gray matter (1,2), nucleus reticularis para-glgantocellularis (3), nucleus raphe magnus (4) and spinal cord dorsal horn (5). However, there are reports that quaternary alkaloids that do not penetrate the blood-brain-barrier, such as could produce analgesia by systemic N-methyl morphine and N-methyl nalorphine, administration (6,7), and that the stress-induced analgesia in Freund’s adjuvant arthritis rats was antagwized by a local application of quaternary naloxone (8). The latter finding suggests that there exists an activation of a peripheral endogenous opioid system. *Corresponding
author: Dr. Hiroshi Ueda, at the above address. Tel: 8 l-95-843-0901, Fax: 81-95-844-4248, E-mail: ueda@,net.nagasaki-u.ac.jp
1678
Lack of Peripheral Opioid Tolerance
Vol. 62, Nos. U/18, 1998
The development of morphine tolerance by its repeated administration is one of the major limitations for its clinical use. Although it is accepted that morphine tolerance through CNS mechanisms is easily developed (9,10), little is known about tolerance-liability of peripherahnorphine analgesia Here we designed a new method to test peripheral analgesia using a specific pain-producing substance and examining the tolerance-liability of morphine analgesia through peripheral mechanisms. Methods Animals Male ddY-strain mice weighing 18 to 20 g (Otsubo Exp. Animals, Nagasaki, Japan) were purchased and housed in a group of 20 animals. They were kept in a room maintained at 21+ 2°C with free access to a standard laboratory diet (MF; Oriental Yeast, Tokyo, Japan) and tap water. These experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by The Declaration of Helsinki. Drugs Morphine (Takeda Pharm. Co., Osaka, Japan), naloxone (a u-opioid receptor antagonist, Sigma, St. Louis, MO, USA), DAMGO (Sigma, St. Louis, MO, USA), DSLET (Sigma, St. Louis, MO, USA), U-69,593 (Sigma, St. Louis, MO, USA), nor-binaltorphimine (nor-BNI, a h-opioid receptor antagonist, Sigma, St. Louis, MO, USA), bradykinin (Sigma, MO, St. Louis, USA) and Hoe 140 (B2 bradykinin receptor antagonist, Research Biochemical International, Natick, MA, USA) were dissolved in saline. They were administrated in a volume of 0.1 ml/l0 g of body weight for S.C.and in 2 ul for intraplantar injection. Evaluation ofperipheral analgesia Mice were suspended in a cloth sling with 4 limbs hanging free. The sling was suspended on a metal bar. All limbs were tied with strings, of which and 3 of the strings were fixed to the floor, while the fourth (right hind-limb) was connected to an isotonic transducer and recorder. For drug administration, two polyethylene cannulae (0.61 mm in outer diameter) tilled with drug solution were inserted into the plantar of the right hind-limb, and both cannulae were connected with microsyringes as shown in Fig. 1. Experiments were started when spontaneous reflex responses reached a baseline and an intraplantar injection of saline did not show significant responses. Bradykinin (BK)-induced pain-producing activity was evaluated by the ratio of BK-response to the maximal flexor reflexes observed immediately after cannulation. On the other hand, analgesic effects were evaluated by the ratio of BK-responses after drug treatments to the initial control responses (average of twice challenges).
Evaluation of morphine tolerance to central analgesia Analgesic effect was measured by a modified Haffner’s method (11) using an artery clip with a cut-off time of 15 set to avoid damage to the tail, at 15 min intervals for a period of 120 min. The effect was expressed as the area under the curve (AUC) obtained by plotting the increase in response time (set) on the ordinate and time interval (min) on the abscissa A significant decrease of AUC, compared with that of the first day, indicated the development of tolerance.
Vol. 62, Nos. 17/B, 1998
Lack of Peripheral Opioid Tolerance
1679
Microsyr inge
Fig. 1 Apparatus for the peripheral analgesic test in mice
Results BK, a representative pain-producing substance, in doses of 0.2 to 20 pmol induced flexor reflexes in a concentration dependent manner. This response induced by 2 pmol of bradykinin was completely antagonized by 0.4 pmol of Hoe 140, a B2 bradykinin receptor antagonist, indicating that the BK-responses are mediated by Bz bradykinin receptors. In addition, when 6 pmol of morphine was injected into the ipsilateral plantar, BK-responses were completely abolished in a naloxone-reversible manner. The EDso of morphine was 1.93 mnol. However, there was no sigpificant analpic effect after injection of morphine into the contralateral plantar, or of its subcutaneous injection to the back in doses up to 60 mnol. These results suggest that the analgesic effect of morphine as measured by this method is mediated by peripheral opioid receptors, and not throu& CNS receptors. Furthermore, peripheral analgesic effects were observed with 6 pmol of DAMGO or with 6 nmol of U-69,593, but not with 6 or 60 mnol of DSLET. These effects induced by DAMGO and U-69,593 were completely antagonized by 6 rnnol of naloxone and by 6 nmol of nor-BNI, respectively. When morphine in a dose of 10 mgkg was given subcutaneously to the back, a potent analgesia in the tail pinch test, which has been used for testing central analgesic activity of morphine (ll), was observed. Daily administration of morphine (once per day for 5 days) showed a marked tolerance or reduction in morphine analgesia on the 6th day. However, in mice treated with morphine for 5 days, there was no si&icant reduction in the peripheral analgesia of morphine on the 6th day. Similarly, in mice injected daily with 6 nmol morphine into the plantar for 5 days, tolerance development was not observed in this test.
1680
Lack of Peripheral
Opioid
Tolerance
Vol. 62, Nos. 17/l&
1998
Discussion The present analgesic test is considered to have advantages in that the pain-producing substances are specific, and thereby peripheral analgesic mechanisms become more simplified. This contrasts with the fact that pain stimuli in many analgesic tests include the involvement of various endogenous pain-producing substances, such as bradykinin, prostaglandin Ez and histamine. Furthermore, peripheral analgesic effects in this test were also observed with DAMGO and U-69,593 but not with DSLET. These findings suggest that both p and K opioid receptors are involved in peripheral analgesia, this being consistent with the fact that the expression of mRNA of p and K receptors are dominant over 6 receptors in the dorsai root gan#ia (12). Here we demonstrated the existence of peripheral morphine analgesia by this method. Of interest is the finding that this peripheral analgesia is resistant to the development of tolerance even when central analgesic effects are markedly reduced through repeated administration of morphine. Assuming that morphine-tolerance occurs at the single cell level throughout the body, peripheral morphine analgesia should also have shown tolerance after repeated challenges. The results of these experiments lead to the conclusion that morphine-tolerance is not attributable to single cell events throughout the body. Taking into consideration that the CNS consists of numerous synaptic networks, compared with nerve ending of peripheral pain&&rent neurons, the present study suggests that tolerance to morphine analgsia is mediated through synaptic plasticity in the CNS, but not through receptor desensitization at the single cell level. Thefollowing schematic serves as a working hypothesis of the pain inhibitory system in peripheral and CNS (Fig. 2).
Brain Stern
Descending pain intibitory pathway (NAB-HT neuons)
Fig. 2 Postulated diagrams of pain control circuitry in the spina cord and brain stem Abbreviations: B2R; B2 bradykinin receptor, P-OpR; p-opioid receptor, &End; p endorphin, CRH; corticotropin releasing hormone, Enk; enkephalin, NA; noradrenaline, 5-HT; serotonin.
Vol. 62, Nos. 17/l& 1998
Lack of Peripheral Opioid Tolerance
1681
References 1. J. BOWIE and B.A. MEYERSON, Pain 13 113-126 (1982). 2. Y. OSOBUCHI, J.E. ADAMS and R. LINCHITZ, Science 197 183-l 86 (1977). 3. J. AZAMI, M.D. LLEWELYN and M.H.T. ROBERTS, Pain 12 229-246 (1982). 4. A.H. DICKENSON, J.L. OLIVERAS and J.M. BESSON, Brain Res. 170 95-l 11 (1979). 5. J.C. YEUNG and R.A. RUDY, J. Pharmacol. Exp. Ther. 215 633-642 (1980). 6. T.W. SMITH, P. BUCHAN, D.N. PARSONS and S. WILKINSON, Life Sci. 31 1205-1208 (1982). 7. L. RIOS and J.J.C. JACOB, Eur. J. Pharmacol. 96 277-283 (1983). 8. C.G. PARSONS, A. CZLONKOWSKI, C. STEIN and A. HERZ, Pain 4181-93 (1990). 9. T.L. YAKSH, R.L. KOHL andT.A. RUDY, Eur. J. Phamacol. 42 275-284 (1977). 10. J.A. SIUCIAK, C. ADVOKAT, Brain Res. 424 3 1 l-3 19 (1987). 11. H. TAKAGI, T. INUKAI and M. NAKAMA, Jpn. J. Pharmcol. 16 287-294 (1966). 12. M. MINAMI, IS. MAEKAWA, K. YABUUCHI and M. SATOH, Mol. Brain Res. 30 203210 (1995).
ELSEVIER
LACK OF TOLERANCE Shogo Tokuyama, Department
IN PERIPHERAL
Life Sciences, Vol. 62, Nos. 17/W, pp. 1677-1681, 1998 Copyright* 15% Elsevia Science Inc. Printed in the USA. All rights resewed 0024-3205/98 $19.00 + .oo
OPIOID ANALGESIA
IN MICE
Makoto Inoue, Takuya Fuchigami and Hiroshi Ueda*
of Molecular Pharmacology and Neuroscience, Nagasaki University School of Pharmaceutical Sciences, 1-14 Bunkyo-machi, Nagasaki 852, Japan
Summary We recently developed a sensitive peripheral analgesic test in mice. Bradykinin, a representative pain-producing substance, when given subcutaneously through a polyethylene tube into the plantar of the limb connected to a transducer, induced a flexor reflex response, in a dose dependent manner. When morphine, a u-opioid receptor agonist, was added to the plantar through another polyethylene tube, bradykinin-induced responses were completely abolished in a naloxone-reversible mamrer. These peripheral analgesic effects were also observed with DAMGO, another u-opioid receptor wnist, and U-69,593, a k-opioid receptor agonist, but not DSLET, a &opioid receptor agonist. When morphine was given subcutaneously to the back, a potent analgesia in the tail pinch test was observed. Repeated administrations of morphine once per day for 5 days showed a marked tolerance or reduction in morphine analgesia on the 6th day, while there was no significant reduction in the peripheral analgesia of morphine. These finding suggest that tolerance to morphine analgesia is mediated through synaptic plasticity in the central nervous system, but not through a receptor desensitization at the level of the single cell.
Key Worde
peripheral analgesia, bradykinin, opioid, morphine, tolerance
It is well accepted that opioids induce analgesic effects through opioid receptors located in the central nervous system (CNS), such as periaqueductal gray matter (1,2), nucleus reticularis para-glgantocellularis (3), nucleus raphe magnus (4) and spinal cord dorsal horn (5). However, there are reports that quaternary alkaloids that do not penetrate the blood-brain-barrier, such as could produce analgesia by systemic N-methyl morphine and N-methyl nalorphine, administration (6,7), and that the stress-induced analgesia in Freund’s adjuvant arthritis rats was antagwized by a local application of quaternary naloxone (8). The latter finding suggests that there exists an activation of a peripheral endogenous opioid system. *Corresponding
author: Dr. Hiroshi Ueda, at the above address. Tel: 8 l-95-843-0901, Fax: 81-95-844-4248, E-mail: ueda@,net.nagasaki-u.ac.jp
1678
Lack of Peripheral Opioid Tolerance
Vol. 62, Nos. U/18, 1998
The development of morphine tolerance by its repeated administration is one of the major limitations for its clinical use. Although it is accepted that morphine tolerance through CNS mechanisms is easily developed (9,10), little is known about tolerance-liability of peripherahnorphine analgesia Here we designed a new method to test peripheral analgesia using a specific pain-producing substance and examining the tolerance-liability of morphine analgesia through peripheral mechanisms. Methods Animals Male ddY-strain mice weighing 18 to 20 g (Otsubo Exp. Animals, Nagasaki, Japan) were purchased and housed in a group of 20 animals. They were kept in a room maintained at 21+ 2°C with free access to a standard laboratory diet (MF; Oriental Yeast, Tokyo, Japan) and tap water. These experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by The Declaration of Helsinki. Drugs Morphine (Takeda Pharm. Co., Osaka, Japan), naloxone (a u-opioid receptor antagonist, Sigma, St. Louis, MO, USA), DAMGO (Sigma, St. Louis, MO, USA), DSLET (Sigma, St. Louis, MO, USA), U-69,593 (Sigma, St. Louis, MO, USA), nor-binaltorphimine (nor-BNI, a h-opioid receptor antagonist, Sigma, St. Louis, MO, USA), bradykinin (Sigma, MO, St. Louis, USA) and Hoe 140 (B2 bradykinin receptor antagonist, Research Biochemical International, Natick, MA, USA) were dissolved in saline. They were administrated in a volume of 0.1 ml/l0 g of body weight for S.C.and in 2 ul for intraplantar injection. Evaluation ofperipheral analgesia Mice were suspended in a cloth sling with 4 limbs hanging free. The sling was suspended on a metal bar. All limbs were tied with strings, of which and 3 of the strings were fixed to the floor, while the fourth (right hind-limb) was connected to an isotonic transducer and recorder. For drug administration, two polyethylene cannulae (0.61 mm in outer diameter) tilled with drug solution were inserted into the plantar of the right hind-limb, and both cannulae were connected with microsyringes as shown in Fig. 1. Experiments were started when spontaneous reflex responses reached a baseline and an intraplantar injection of saline did not show significant responses. Bradykinin (BK)-induced pain-producing activity was evaluated by the ratio of BK-response to the maximal flexor reflexes observed immediately after cannulation. On the other hand, analgesic effects were evaluated by the ratio of BK-responses after drug treatments to the initial control responses (average of twice challenges).
Evaluation of morphine tolerance to central analgesia Analgesic effect was measured by a modified Haffner’s method (11) using an artery clip with a cut-off time of 15 set to avoid damage to the tail, at 15 min intervals for a period of 120 min. The effect was expressed as the area under the curve (AUC) obtained by plotting the increase in response time (set) on the ordinate and time interval (min) on the abscissa A significant decrease of AUC, compared with that of the first day, indicated the development of tolerance.
Vol. 62, Nos. 17/B, 1998
Lack of Peripheral Opioid Tolerance
1679
Microsyr inge
Fig. 1 Apparatus for the peripheral analgesic test in mice
Results BK, a representative pain-producing substance, in doses of 0.2 to 20 pmol induced flexor reflexes in a concentration dependent manner. This response induced by 2 pmol of bradykinin was completely antagonized by 0.4 pmol of Hoe 140, a B2 bradykinin receptor antagonist, indicating that the BK-responses are mediated by Bz bradykinin receptors. In addition, when 6 pmol of morphine was injected into the ipsilateral plantar, BK-responses were completely abolished in a naloxone-reversible manner. The EDso of morphine was 1.93 mnol. However, there was no sigpificant analpic effect after injection of morphine into the contralateral plantar, or of its subcutaneous injection to the back in doses up to 60 mnol. These results suggest that the analgesic effect of morphine as measured by this method is mediated by peripheral opioid receptors, and not throu& CNS receptors. Furthermore, peripheral analgesic effects were observed with 6 pmol of DAMGO or with 6 nmol of U-69,593, but not with 6 or 60 mnol of DSLET. These effects induced by DAMGO and U-69,593 were completely antagonized by 6 rnnol of naloxone and by 6 nmol of nor-BNI, respectively. When morphine in a dose of 10 mgkg was given subcutaneously to the back, a potent analgesia in the tail pinch test, which has been used for testing central analgesic activity of morphine (ll), was observed. Daily administration of morphine (once per day for 5 days) showed a marked tolerance or reduction in morphine analgesia on the 6th day. However, in mice treated with morphine for 5 days, there was no si&icant reduction in the peripheral analgesia of morphine on the 6th day. Similarly, in mice injected daily with 6 nmol morphine into the plantar for 5 days, tolerance development was not observed in this test.
1680
Lack of Peripheral
Opioid
Tolerance
Vol. 62, Nos. 17/l&
1998
Discussion The present analgesic test is considered to have advantages in that the pain-producing substances are specific, and thereby peripheral analgesic mechanisms become more simplified. This contrasts with the fact that pain stimuli in many analgesic tests include the involvement of various endogenous pain-producing substances, such as bradykinin, prostaglandin Ez and histamine. Furthermore, peripheral analgesic effects in this test were also observed with DAMGO and U-69,593 but not with DSLET. These findings suggest that both p and K opioid receptors are involved in peripheral analgesia, this being consistent with the fact that the expression of mRNA of p and K receptors are dominant over 6 receptors in the dorsai root gan#ia (12). Here we demonstrated the existence of peripheral morphine analgesia by this method. Of interest is the finding that this peripheral analgesia is resistant to the development of tolerance even when central analgesic effects are markedly reduced through repeated administration of morphine. Assuming that morphine-tolerance occurs at the single cell level throughout the body, peripheral morphine analgesia should also have shown tolerance after repeated challenges. The results of these experiments lead to the conclusion that morphine-tolerance is not attributable to single cell events throughout the body. Taking into consideration that the CNS consists of numerous synaptic networks, compared with nerve ending of peripheral pain&&rent neurons, the present study suggests that tolerance to morphine analgsia is mediated through synaptic plasticity in the CNS, but not through receptor desensitization at the single cell level. Thefollowing schematic serves as a working hypothesis of the pain inhibitory system in peripheral and CNS (Fig. 2).
Brain Stern
Descending pain intibitory pathway (NAB-HT neuons)
Fig. 2 Postulated diagrams of pain control circuitry in the spina cord and brain stem Abbreviations: B2R; B2 bradykinin receptor, P-OpR; p-opioid receptor, &End; p endorphin, CRH; corticotropin releasing hormone, Enk; enkephalin, NA; noradrenaline, 5-HT; serotonin.
Vol. 62, Nos. 17/l& 1998
Lack of Peripheral Opioid Tolerance
1681
References 1. J. BOWIE and B.A. MEYERSON, Pain 13 113-126 (1982). 2. Y. OSOBUCHI, J.E. ADAMS and R. LINCHITZ, Science 197 183-l 86 (1977). 3. J. AZAMI, M.D. LLEWELYN and M.H.T. ROBERTS, Pain 12 229-246 (1982). 4. A.H. DICKENSON, J.L. OLIVERAS and J.M. BESSON, Brain Res. 170 95-l 11 (1979). 5. J.C. YEUNG and R.A. RUDY, J. Pharmacol. Exp. Ther. 215 633-642 (1980). 6. T.W. SMITH, P. BUCHAN, D.N. PARSONS and S. WILKINSON, Life Sci. 31 1205-1208 (1982). 7. L. RIOS and J.J.C. JACOB, Eur. J. Pharmacol. 96 277-283 (1983). 8. C.G. PARSONS, A. CZLONKOWSKI, C. STEIN and A. HERZ, Pain 4181-93 (1990). 9. T.L. YAKSH, R.L. KOHL andT.A. RUDY, Eur. J. Phamacol. 42 275-284 (1977). 10. J.A. SIUCIAK, C. ADVOKAT, Brain Res. 424 3 1 l-3 19 (1987). 11. H. TAKAGI, T. INUKAI and M. NAKAMA, Jpn. J. Pharmcol. 16 287-294 (1966). 12. M. MINAMI, IS. MAEKAWA, K. YABUUCHI and M. SATOH, Mol. Brain Res. 30 203210 (1995).