European Journal of Pharmacology, 175 (1990) 97-100
97
Elsevier EJP 20529
Short communication
Mediation of nitrous oxide analgesia in mice by spinal and supraspinal x-opioid receptors 1 R.M. Quock
2, J.A.
Best 2,6, D.C. Chen 3, L.K. Vaughn
2, P.S.
Portoghese 4 and A.E. Takemori s
2 Departments of Basic Sciences and 3 Pediatric Dentistry, Marquette University School of Dentistry, Milwaukee, WI 53233, U.S.A. and 4 Department of Medicinal Chemistry, College of Pharmacy, and 5 Department of Pharmacology, Medical School, University of Minnesota, Minneapolis, MN 55455, U.S.A. Received 1 November 1989, accepted 7 November 1989
Exposure to nitrous oxide produced concentration-dependent analgesia in the mouse abdominal constriction test. Intracerebroventricular or intrathecal pretreatment with naltrexone or nor-binaltorphimine significantly reduced nitrous oxide analgesia. However, similar pretreatment with fl-funaltrexamine had no appreciable effect. These findings suggest that nitrous oxide analgesia involves spinal and supraspinal x-opioid receptors. Nitrous oxide; Analgesia; Opioid receptors; Narcotic antagonists
1. Introduction Antagonism of nitrous oxide ( N 2 0 ) analgesia by systemically administered MR-2266 [(-)-5,9diethyl-a-5,9-dialkyl-2'-hydroxy-6,7-benzomorphan] suggests that r-opioid receptors might be involved in N20 analgesia (Quock and Graczak, 1988). But since MR-2266 has antagonist activity at /~ as well as ~¢-opioid receptors (Paterson et al., 1983), the present study was undertaken to assess the sensitivity of N20 analgesia to antagonism by a more selective x-opioid receptor blocker, nor-
1 This work was supported by U.S. Public Health Service Grants from the National Institute of Dental Research (NIDR) and National Institute on Drug Abuse (NIDA). 6 Present address: Department of Dental Research, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, U.S.A. Correspondence to: R.M. Quock, Department of Biomedical Sciences, University of Illinois College of Medicine at Rockford, 1601 Parkview Avenue, Rockford, IL 61107, U.S.A.
binaltorphimine (Takemori et al., 1988). We also investigated whether K-opioid receptors mediating N20 analgesia were located spinally or supraspinally.
2. Materials and methods
2.1. Analgesia testing Male ICR mice, weighing 20-25 g (Sasco/King Animal Laboratories, Oregon, WI), were tested for N20 analgesia, using the mouse abdominal constriction test. Mice were treated intraperitoneally (i.p.) with 10 m l / k g of a 0.6% acetic acid solution; exactly 5 min later, the number of abdominal constrictions in each animal was counted for 6-min period. Analgesic activity was expressed as a percentage reduction in the number of abdominal constrictions observed in NzO-exposed mice compared to the mean number of abdominal constrictions in a control group.
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
98
2.2. N20 exposure Cages of four to five mice were placed in an inflatable, polyethylene AtmosBag ® glovebag (Aldrich, Milwaukee, WI). N20 and oxygen (02) were delivered into the glovebag via a length of polyethylene tubing, using a standard N 2 0 / O 2 anesthesia machine (Adec, Newburg, OR). The proportions of N 2 0 and 02 were varied within a total inflow rate of 10 1/min to achieve the different test concentrations of N20 (25%, 50% and 75% in 02). A beaker of soda lime was enclosed inside the glovebag for absorption of carbon dioxide. Exhausted gas was vented from the glovebag to a hood. Control experiments were conducted in a glovebag open to room air. 2.3. Statistics Dose-response curves were constructed for N20 analgesia in different groups of animals pretreated with vehicle or various opioid receptor blockers. The ADs0 values with 95% confidence limits were determined by the method of Litchfield and Wilcoxon (1949). 2.4. Drugs The following drugs were used in this study: Nitrous oxide, U.S.P. and Oxygen, U.S.P. (Bentley Welding, Milwaukee, WI); naltrexone (DuPont Laboratories, Wilmington, DE); fl-funaltrexamine (Research Biochemicals, Wayland, MA); and nor-binaltorphimine (nor-BNI), which was synthesized by the method of Lipkowski et al. (1986). Solutions of these opioid receptor blockers were prepared in sterile distilled water. Different groups of mice were pretreated intracerebroventricularly (i.c.v.) or intrathecally (i.t.) with each of these opioid receptor blockers or distilled water under brief halothane anesthesia, using the microinjection methods of Haley and McCormick (1957) and Hylden and Wilcox (1980), respectively. Pretreatment doses and times were: 1.0/~g naltrexone (i.c.v. and i.t.) at 15 min prior to N 2 0 exposure, 5.0 i~g fl-funaltrexamine (i.c.v. and i.t.) at 24 h prior t o N 2 0 exposure, 50 nmol nor-BNI (i.c.v.) at 60 min prior t o N 2 0 exposure,
and 5 nmol nor-BNI (i.t.) at 15 min prior t o N 2 0 exposure. Central pretreatments were made in volumes of 4.0 /~1, using a modified Hamilton microsyringe.
3. Results
In experiments testing involvement of brain opioid receptors in N 2 0 analgesia, N20 exposure produced a concentration-dependent analgesic effect in control mice (pretreated i.c.v, with distilled water). Pretreatment with naltrexone or nor-BNI, but not fl-funaltrexamine, significantly reduced N20 analgesia (fig. 1). Naltrexone and nor-BNI pretreatments significantly elevated the ADs0 of N20 (P < 0.05, Litchfietd-Wilcoxon); ]3-funaltrexamine pretreatment was without appreciable effect. In experiments testing involvement of spinal opioid receptors in N20 analgesia, N20 exposure produced a concentration-dependent analgesic effect in control mice (pretreated i.t. with distilled water). Pretreatment with naltrexone or nor-BNI, but not /3-funaltrexamine, significantly reduced N20 analgesia (fig. 2). Naltrexone and nor-BNI pretreatments significantly increased the ADs0 of
100 %[ 80
ANALGESIA
.°4O I I ?
O:
10
100
% NITROUS OXIDE Fig. 1. Dose-response curves (ADs0 values and 95% confidence intervals) for N20 analgesia in different groups of mice pretreated i.c.v, with: A, distilled water (54.9%, 40.5-74.4%); Lx, l #g naltrexone (74.9%, 57.2-96.7%); ©, 50 nmol nor-BNI (ADso and 95% confidence intervals could not be calculated); and O, 5 ~g fl-funaltrexamine (55.8%, 39.9-78.0%). Values were determined from at least 45 mice per pretreatment group.
99
% ANALGESIA
loo I 8ol
A
4oi
oL lO
100 % NITROUS
OXIDE
Fig. 2. Dose-response curves for N20 analgesia in different groups of mice pretreated i.t. with: A, distilled water (76.6%, 52.9-111.0%); zx, 1 /zg naltrexone (233.0%, 140.0-387.9%); o, 5 nmol nor-BNI (281.1%, 202.0-391.1%); and O, 5 ~g /3funaltrexamine (84.6%, 71.1-100.6%). Values were determined from at least 45 mice per pretreatment group.
N20 (P < 0.05); /3-funaltrexamine pretreatment had no significant effect.
and duration of pretreatment with fl-funaltrexamine employed in this study, previous studies from one of our laboratories have demonstrated that /3-funaltrexamine administered either i.c.v, or i.t. to mice significantly and substantially attenuated the analgesic action of morphine that is administered subcutaneously, i.c.v, or i.t. (Ward et al., 1982; Takemori and Portoghese, 1987; Schwartz and Takemori, unpublished data). Morphine analgesia is thought to result from actions of morphine at both spinal and supraspinal sites (Yaksh and Rudy, 1978). The results of this investigation suggest that N20 may also have multiple sites of action in the central nervous system and that N20 analgesia in the mouse abdominal constriction test may be mediated by spinal and supraspinal x-opioid receptors.
Acknowledgement We thank DuPont Laboratories for its generous gift of naltrexone.
4. Discussion References The abdominal constriction test utilizes a chemical noxious stimulus that can detect analgesic drug effects mediated by/x- and x-opioid receptors (Tyers, 1980). We previously reported that N20 analgesia in the paradigm was sensitive to antagonism by systemically administered naltrexone (a non-selective opioid receptor blocker) and MR-2266 (a ~ >/x opioid receptor blocker) (Quock and Graczak, 1988). The results of the present study indicate that either i.c.v, or i.t. pretreatment with naltrexone significantly reduced N20 analgesia and elevated the ADs0 of N20. This effectiveness of naltrexone as an antagonist of N 2 0 w a s duplicated by i.c.v, or i.t. pretreatment with the highly selective K-opioid receptor blocker nor-BNI at doses previously reported to be effective at K-opioid receptors (Takemori et al., 1988). On the other hand, the highly selective #-opioid receptor blocker B-funaltrexamine administered either i.c.v. or i.t. failed to exert significant influence upon N 2 0 analgesia or the ADs0 of N20. At the dose
Haley, T.J. and W.G. McCormick 1957, Pharmacological effects produced by intracerebral injection of drugs in the conscious mouse, Br. J. Pharmacol. 12, 12. Hylden, J.L.K. and G.L. Wilcox, 1980, Intrathecal morphine in mice: A new technique, European J. Pharmacol. 67, 313. Lipkowski, A.W., H. Nagase and P.S. Portoghese, 1986, A novel pyrrole synthesis via reaction of ketones with Naminoamides, Tetrahedron Lett. 27. 4257. Litchfield, J.T. and F. Wilcoxon, 1949, A simplified method of evaluating dose-effect experiments, J. Pharmacol. Exp. Ther. 96, 99. Paterson, S.J., L.E. Robson and H.W. Kosterlitz, 1983, Classification of opioid receptors, Br. Med. Bull. 39, 31. Quock, R.M. and L.M. Graczak, 1988, Influence of narcotic antagonist drugs upon nitrous oxide analgesia in mice, Brain Res. 440, 35. Takemori, A.E., B.Y. Ho, J.S. Naeseth and P.S. Portoghese, 1988, Norbinaltorphimine, a highly selective r-opioid antagonist in analgesic and receptor binding assays, J. Pharmacol. Exp. Ther. 246, 255. Takemori, A.E. and P.S. Portoghese, 1987, Evidence for the interaction of morphine with kappa and delta opioid receptors to induce analgesia in fl-funaltrexamine-treated mice, J. Pharmacol. Exp. Ther. 243, 91.
100 Tyers, M.B., 1980, A classification of opiate receptors that mediate antinociception in animals, Br. J. Pharmacol. 69, 503. Ward, S.J., P.S. Portoghese and A.E. Takemori, 1982, Pharmacological characterization in vivo of the novel opiate fl-funaltrexamine, J. Pharmacol. Exp. Ther. 220, 494.
Yaksh, T.L. and T.A. Rudy, 1978, Narcotic analgetics: CNS sites and mechanisms of action as revealed by intracerebral injection techniques, Pain 4, 299.
97
Elsevier EJP 20529
Short communication
Mediation of nitrous oxide analgesia in mice by spinal and supraspinal x-opioid receptors 1 R.M. Quock
2, J.A.
Best 2,6, D.C. Chen 3, L.K. Vaughn
2, P.S.
Portoghese 4 and A.E. Takemori s
2 Departments of Basic Sciences and 3 Pediatric Dentistry, Marquette University School of Dentistry, Milwaukee, WI 53233, U.S.A. and 4 Department of Medicinal Chemistry, College of Pharmacy, and 5 Department of Pharmacology, Medical School, University of Minnesota, Minneapolis, MN 55455, U.S.A. Received 1 November 1989, accepted 7 November 1989
Exposure to nitrous oxide produced concentration-dependent analgesia in the mouse abdominal constriction test. Intracerebroventricular or intrathecal pretreatment with naltrexone or nor-binaltorphimine significantly reduced nitrous oxide analgesia. However, similar pretreatment with fl-funaltrexamine had no appreciable effect. These findings suggest that nitrous oxide analgesia involves spinal and supraspinal x-opioid receptors. Nitrous oxide; Analgesia; Opioid receptors; Narcotic antagonists
1. Introduction Antagonism of nitrous oxide ( N 2 0 ) analgesia by systemically administered MR-2266 [(-)-5,9diethyl-a-5,9-dialkyl-2'-hydroxy-6,7-benzomorphan] suggests that r-opioid receptors might be involved in N20 analgesia (Quock and Graczak, 1988). But since MR-2266 has antagonist activity at /~ as well as ~¢-opioid receptors (Paterson et al., 1983), the present study was undertaken to assess the sensitivity of N20 analgesia to antagonism by a more selective x-opioid receptor blocker, nor-
1 This work was supported by U.S. Public Health Service Grants from the National Institute of Dental Research (NIDR) and National Institute on Drug Abuse (NIDA). 6 Present address: Department of Dental Research, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, U.S.A. Correspondence to: R.M. Quock, Department of Biomedical Sciences, University of Illinois College of Medicine at Rockford, 1601 Parkview Avenue, Rockford, IL 61107, U.S.A.
binaltorphimine (Takemori et al., 1988). We also investigated whether K-opioid receptors mediating N20 analgesia were located spinally or supraspinally.
2. Materials and methods
2.1. Analgesia testing Male ICR mice, weighing 20-25 g (Sasco/King Animal Laboratories, Oregon, WI), were tested for N20 analgesia, using the mouse abdominal constriction test. Mice were treated intraperitoneally (i.p.) with 10 m l / k g of a 0.6% acetic acid solution; exactly 5 min later, the number of abdominal constrictions in each animal was counted for 6-min period. Analgesic activity was expressed as a percentage reduction in the number of abdominal constrictions observed in NzO-exposed mice compared to the mean number of abdominal constrictions in a control group.
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
98
2.2. N20 exposure Cages of four to five mice were placed in an inflatable, polyethylene AtmosBag ® glovebag (Aldrich, Milwaukee, WI). N20 and oxygen (02) were delivered into the glovebag via a length of polyethylene tubing, using a standard N 2 0 / O 2 anesthesia machine (Adec, Newburg, OR). The proportions of N 2 0 and 02 were varied within a total inflow rate of 10 1/min to achieve the different test concentrations of N20 (25%, 50% and 75% in 02). A beaker of soda lime was enclosed inside the glovebag for absorption of carbon dioxide. Exhausted gas was vented from the glovebag to a hood. Control experiments were conducted in a glovebag open to room air. 2.3. Statistics Dose-response curves were constructed for N20 analgesia in different groups of animals pretreated with vehicle or various opioid receptor blockers. The ADs0 values with 95% confidence limits were determined by the method of Litchfield and Wilcoxon (1949). 2.4. Drugs The following drugs were used in this study: Nitrous oxide, U.S.P. and Oxygen, U.S.P. (Bentley Welding, Milwaukee, WI); naltrexone (DuPont Laboratories, Wilmington, DE); fl-funaltrexamine (Research Biochemicals, Wayland, MA); and nor-binaltorphimine (nor-BNI), which was synthesized by the method of Lipkowski et al. (1986). Solutions of these opioid receptor blockers were prepared in sterile distilled water. Different groups of mice were pretreated intracerebroventricularly (i.c.v.) or intrathecally (i.t.) with each of these opioid receptor blockers or distilled water under brief halothane anesthesia, using the microinjection methods of Haley and McCormick (1957) and Hylden and Wilcox (1980), respectively. Pretreatment doses and times were: 1.0/~g naltrexone (i.c.v. and i.t.) at 15 min prior to N 2 0 exposure, 5.0 i~g fl-funaltrexamine (i.c.v. and i.t.) at 24 h prior t o N 2 0 exposure, 50 nmol nor-BNI (i.c.v.) at 60 min prior t o N 2 0 exposure,
and 5 nmol nor-BNI (i.t.) at 15 min prior t o N 2 0 exposure. Central pretreatments were made in volumes of 4.0 /~1, using a modified Hamilton microsyringe.
3. Results
In experiments testing involvement of brain opioid receptors in N 2 0 analgesia, N20 exposure produced a concentration-dependent analgesic effect in control mice (pretreated i.c.v, with distilled water). Pretreatment with naltrexone or nor-BNI, but not fl-funaltrexamine, significantly reduced N20 analgesia (fig. 1). Naltrexone and nor-BNI pretreatments significantly elevated the ADs0 of N20 (P < 0.05, Litchfietd-Wilcoxon); ]3-funaltrexamine pretreatment was without appreciable effect. In experiments testing involvement of spinal opioid receptors in N20 analgesia, N20 exposure produced a concentration-dependent analgesic effect in control mice (pretreated i.t. with distilled water). Pretreatment with naltrexone or nor-BNI, but not /3-funaltrexamine, significantly reduced N20 analgesia (fig. 2). Naltrexone and nor-BNI pretreatments significantly increased the ADs0 of
100 %[ 80
ANALGESIA
.°4O I I ?
O:
10
100
% NITROUS OXIDE Fig. 1. Dose-response curves (ADs0 values and 95% confidence intervals) for N20 analgesia in different groups of mice pretreated i.c.v, with: A, distilled water (54.9%, 40.5-74.4%); Lx, l #g naltrexone (74.9%, 57.2-96.7%); ©, 50 nmol nor-BNI (ADso and 95% confidence intervals could not be calculated); and O, 5 ~g fl-funaltrexamine (55.8%, 39.9-78.0%). Values were determined from at least 45 mice per pretreatment group.
99
% ANALGESIA
loo I 8ol
A
4oi
oL lO
100 % NITROUS
OXIDE
Fig. 2. Dose-response curves for N20 analgesia in different groups of mice pretreated i.t. with: A, distilled water (76.6%, 52.9-111.0%); zx, 1 /zg naltrexone (233.0%, 140.0-387.9%); o, 5 nmol nor-BNI (281.1%, 202.0-391.1%); and O, 5 ~g /3funaltrexamine (84.6%, 71.1-100.6%). Values were determined from at least 45 mice per pretreatment group.
N20 (P < 0.05); /3-funaltrexamine pretreatment had no significant effect.
and duration of pretreatment with fl-funaltrexamine employed in this study, previous studies from one of our laboratories have demonstrated that /3-funaltrexamine administered either i.c.v, or i.t. to mice significantly and substantially attenuated the analgesic action of morphine that is administered subcutaneously, i.c.v, or i.t. (Ward et al., 1982; Takemori and Portoghese, 1987; Schwartz and Takemori, unpublished data). Morphine analgesia is thought to result from actions of morphine at both spinal and supraspinal sites (Yaksh and Rudy, 1978). The results of this investigation suggest that N20 may also have multiple sites of action in the central nervous system and that N20 analgesia in the mouse abdominal constriction test may be mediated by spinal and supraspinal x-opioid receptors.
Acknowledgement We thank DuPont Laboratories for its generous gift of naltrexone.
4. Discussion References The abdominal constriction test utilizes a chemical noxious stimulus that can detect analgesic drug effects mediated by/x- and x-opioid receptors (Tyers, 1980). We previously reported that N20 analgesia in the paradigm was sensitive to antagonism by systemically administered naltrexone (a non-selective opioid receptor blocker) and MR-2266 (a ~ >/x opioid receptor blocker) (Quock and Graczak, 1988). The results of the present study indicate that either i.c.v, or i.t. pretreatment with naltrexone significantly reduced N20 analgesia and elevated the ADs0 of N20. This effectiveness of naltrexone as an antagonist of N 2 0 w a s duplicated by i.c.v, or i.t. pretreatment with the highly selective K-opioid receptor blocker nor-BNI at doses previously reported to be effective at K-opioid receptors (Takemori et al., 1988). On the other hand, the highly selective #-opioid receptor blocker B-funaltrexamine administered either i.c.v. or i.t. failed to exert significant influence upon N 2 0 analgesia or the ADs0 of N20. At the dose
Haley, T.J. and W.G. McCormick 1957, Pharmacological effects produced by intracerebral injection of drugs in the conscious mouse, Br. J. Pharmacol. 12, 12. Hylden, J.L.K. and G.L. Wilcox, 1980, Intrathecal morphine in mice: A new technique, European J. Pharmacol. 67, 313. Lipkowski, A.W., H. Nagase and P.S. Portoghese, 1986, A novel pyrrole synthesis via reaction of ketones with Naminoamides, Tetrahedron Lett. 27. 4257. Litchfield, J.T. and F. Wilcoxon, 1949, A simplified method of evaluating dose-effect experiments, J. Pharmacol. Exp. Ther. 96, 99. Paterson, S.J., L.E. Robson and H.W. Kosterlitz, 1983, Classification of opioid receptors, Br. Med. Bull. 39, 31. Quock, R.M. and L.M. Graczak, 1988, Influence of narcotic antagonist drugs upon nitrous oxide analgesia in mice, Brain Res. 440, 35. Takemori, A.E., B.Y. Ho, J.S. Naeseth and P.S. Portoghese, 1988, Norbinaltorphimine, a highly selective r-opioid antagonist in analgesic and receptor binding assays, J. Pharmacol. Exp. Ther. 246, 255. Takemori, A.E. and P.S. Portoghese, 1987, Evidence for the interaction of morphine with kappa and delta opioid receptors to induce analgesia in fl-funaltrexamine-treated mice, J. Pharmacol. Exp. Ther. 243, 91.
100 Tyers, M.B., 1980, A classification of opiate receptors that mediate antinociception in animals, Br. J. Pharmacol. 69, 503. Ward, S.J., P.S. Portoghese and A.E. Takemori, 1982, Pharmacological characterization in vivo of the novel opiate fl-funaltrexamine, J. Pharmacol. Exp. Ther. 220, 494.
Yaksh, T.L. and T.A. Rudy, 1978, Narcotic analgetics: CNS sites and mechanisms of action as revealed by intracerebral injection techniques, Pain 4, 299.
