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Spinal antinociceptive action of three representative opioid peptides in frogs
Br~r~esearch, 402 (1987) 201-203 Elsevier
201
BRE 21997
Spinal antinociceptive action of three representative opioid peptides in frogs Craig W. Stevens 1, Paul D. Pezalla I and Tony L. Yaksh 2 1Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL 60680 (U.S.A.), and 2Department of Neurosurgical Research, Mayo Clinic, Rochester, MN 55905 (U.S.A.) (Accepted 9 September 1986) Key words: Amphibian; Rana pipiens; Antinociception; Dynorphin; fl-Endorphin; Met-enkephalin
Spinal administration of low doses of dynorphin, fl-endorphin or Met-enkephalin produces a potent, dose-dependent increase in the nociceptive threshold in the unanesthetized frog, Rana pipiens. Nociceptive thresholds were determined by using the acetic acid test, previously shown to be a sensitive indicator of antinociception in this amphibian species. Of particular interest, spinally administered dynorphin produces a potent antinociception in frogs without any signs of motor dysfunction seen after spinally administered dynorphin in mammalian species. Spinal administration of opiates and endogenous opioid peptides produces a behaviorally defined antinociception in a variety of mammalian models 24'25. Although endogenous opioid peptides and opiate binding sites are ubiquitous throughout phylogeny3'6'I1'20little attention has been focused on possible function(s) of endogenous opioid systems or development of behavioral models for antinociception in non-mammalian vertebrates. Recent work has shown that the amphibian, Rana pipiens, may serve as an appropriate model for assessment of antinociception following systemic 12'14 or spinal 21'22 administration of morphine and other opiate alkaloids. Furthermore, immobilization stress in frogs increases nociceptive threshold in frogs and this effect is blocked by naloxone 13. As immunocytochemical studies in amphibians demonstrate that high levels of dynorphin 4, fl-endorphin 7,8,15,26 and enkephalin 1'5'9'1° exist within the amphibian central nervous system and biochemical studies reveal high densities of opiate binding throughout the amphibian neuraxis2A6-18, the above results suggest endogenous opioid systems may likewise modulate central processing of noxious stimuli in amphibians as well as mammalians.
Given the powerful and well-defined antinociceptive effects of opioids in mammalian spinal cord, we sought in the present experiments to examine the effects of 3 endogenous representative opioid peptides, dynorphin, fl-endorphin and Met-enkephalin on nociception in the frog. We now report, for the first time, that spinal administration of the opioid peptides, dynorphin, fl-endorphin and Met-enkephalin produces a potent, dose-dependent increase in nociceptive thresholds in the frog, Rana pipiens. Leopard frogs, Rana pipiens (25-30 g; Nasco, Fort Atkinson, WI) were maintained as previously described 12 and two days before the start of experiments were randomly assigned to treatment groups and placed in individual pans with 0.5 cm tap water. Frogs were delivered in late August and experiments begun after two weeks acclimatization in our laboratory. Dynorphin Al_13, fl-endorphin and Met-enkephalin (Peninsula Labs, Palo Alto, CA) were dissolved in 0.7% NaC1 and injected intraspinally with a Hamilton microsyringe fitted with a tapered 26gauge needle (point no. 2). The site of injection was lateral to the neural spine at the articulation between the seventh and eighth vertebrae of the frog spinal column. Further details of the intraspinal injection
Correspondence: C.W. Stevens. Present address: Mayo Clinic, Rochester, MN 55905, U.S.A. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
202 32
lZ
24
c o3 r0
IE
~
s
BEP
DYN ENK
SAL
0~ 0
50 500
5
50 500
5
50 500
Dose (ng) Fig. 1. The effect of intraspinal Met-enkephalin (ENK), dynorphin (DYN) or fl-endorphin (BEP) on total change in nociceptive thresholds (NT). Data are plotted as the mean plus S.E.M. for 3-9 animals per treatment group. Asterisks denote significant difference from saline (SAL)-injected controls (P < 0.05, randomization test, one-tailed). method have been given zl. All doses were given in a volume of 1 pl/animal. Drugs and doses were coded and the experimenter was unaware of their identity until completion of the experiments. Estimation of the frog's nociceptive threshold (NT) were made with the acetic acid test as previously described 12. Briefly, 11 dilutions of acetic acid, equally spaced on a logarithmic scale from 0.26 to 15 M, were numbered in order of increasing concentration from 0 to 10. The NT is defined as the n u m b e r of the lowest concentration of acetic acid which causes a wiping response when applied to the dorsum of the frog's thigh. Data were plotted as the mean change in NT (post-injection NT minus pre-injection NT) summed over post-injection test times (5, 60,120 and 180 min). Non-parametric statistics were used 19 and significance set at P < 0.05. For each experiment presented there were no significant differences in pre-injection NT among treatment groups. The effect of intraspinal dynorphin, /3-endorphin
1 Adli, D.S., Yuen, G.L., Rosenthal, B.M., Ho, R.H. and Cruce, W.L.R., Enkephalin-like immunoreactive elements in Lissauer's tract and other regions of frog spinal cord, Soc. Neurosci. Abstr., 10 (1984) 488. 2 Audigier, Y., Duprat, A.M. and Cros, J., Comparative study of opiate and enkephalin receptors on lower vertebrates and higher vertebrates, Comp. Biochem. Physiol., 67C (1980) 191-193. 3 Buatti, M.C. and Pasternak, G.W., Multiple opiate receptors: phylogenetic differences, Brain Research, 218 (1981) 400-405. 4 Cone, R.I. and Goldstein, A., A dynorphin-like opioid in
or Met-enkephalin on nociceptive thresholds in summer frogs is shown in Fig. 1. Dynorphin (5, 50 or 500 ng) produced a significant dose-dependent increase in total change N T compared to saline controls. /3Endorphin and Met-enkephalin produced significant increases in N T at only the two highest doses (50 and 500 ng). The intraspinal injection of the 3 opioid peptides was not accompanied by any observable m o t o r deficits at any of the doses given, i.e. all frogs exhibited normal righting reflexes and wiping responses to the nociceptive stimulus with no abnormal tonus of hindlimbs. The present results demonstrate a potent, dose-dependent antinociceptive.action of spinally administered dynorphin,/3-endorphin and Met-enkephalin, and extend our earlier observations documenting the antinociceptive effects of spinal opiate alkaloids 14,21,22 in amphibians. To our knowledge, these results are the first demonstrating a behaviorally def;r.ed antinociceptive action of opioid peptides in a non-mammalian vertebrate. A post-hoc analysis of the potency of dynorphin,/3-endorphin and Met-enkephalin demonstrates an approximately 6-, 7- and 0.8-fold potency ratio, respectively, c o m p a r e d to spinal morphine. As spinally administered dynorphin in rodents produces severe motor dysfunction which is not easily dissociated from antinociceptive effects 23, the lack of any observable motor deficits after spinal dynorphin in frogs suggests that the acetic acid test in amphibians may be a suitable model for further studies of dynorphin antinociception.
We thank A n n Rockafellow for manuscript preparation. Supported by the Research Board of the University of Illinois at Chicago.
the central nervous system of an amphibian, Proc. Natl. Acad. Sci. U.S.A., 79 (1982)3345-3349.
5 Doerr-Schott, J., Dubois, M.P. and Lichte, C., Immunohistochemical localization of substances reactive to antisera against a- and fl-endorphin and Met-enkephalin in the brain of Rana temporaria, Cell Tissue Res., 217 (1981) 79-92. 6 Edley, S.M., Hall, L., Hevkenham, M. and Pert, C.B., Evolution of striatal opiate receptors, Brain Research, 249 (1982) 184-188. 7 Jackson, I.M.D., Bolaffi, J.L. and Guilleman, R., Presence of immunoreactive fl-endorphin and enkephalin-like material in the retina and other tissues of the frog, Rana pi-
203
piens, Gen. Comp. Endocrinol., 42 (1980) 505-508. 8 Jeqou, S., Tonon, M.C., Levoux, P., Leboulenger, F., Pelletier, G., Cote, J., Ling, N. and Vandry, N., Immunological characterization of endorphins, adrenocorticotropin and melanotropins in frog hypothalamus, Gen. Comp. Endocrinol., 51 (1983) 246-254. 9 Kilpatrick, D.L., Howells, R.D., Lahm, M.-W. and Udenfriend, S., Evidence for a proenkephalin-like precursor in amphibian brain, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 5772-5775. -10 Lorez, H.P. and Kemali, M., Substance P-, Met-enkephalin- and somatostatin-like immunoreactivity distribution in the frog spinal cord, Neurosci. Lett., 26 (1981) 119-124. 11 Pert, C.B., Aposhian, D. and Snyder, S.H., Phylogenetic distribution of opiate receptor binding, Brain Research, 75 (1974) 356-361. 12 Pezalla, P.D., Morphine-induced analgesia and explosive motor behavior in an amphibian, Brain Research, 273 (1983) 297-305. 13 Pezalla, P.D. and Dicig, M., Stress-induced analgesia in frogs: evidence for.the involvement of an opioid system, Brain Research, 296 (1984) 356-360. 14 Pezalla, P.D. and Stevens, C.W., Behavioral effects of morphine, levorphanol, dextrorphan and naloxone in the frog Rana pipiens, Pharmacol. Biochem. Behav., 21 (1984) 213-217. 15 Pezalla, P.D., Seidah, N.G., Benjannet, S., Crine, P., Lis, M. and Chretien, M., Biosynthesis of/3-endorphin,/3-1ipotropin and the putative ACTH-LPH precursor in the frog pars intermedia, Life Sci., 23 (1978) 2281-2292. 16 Puget, A., Jauzac, P. and Meunier, J.C., Distinct molecular forms of opiate binding in the frog brain, Life Sci., 33, Suppl. 1 (1983) 199-202.
17 Ruegg, V.T., Hiller, J.M. and Simon, E.J., Solubilization of an active opiate receptor from Bufo marinus, Eur. J. Pharmacol., 64 (1980) 367-368. 18 Ruegg, V.T., Cuenod, S., Hiller, J.M., Gioannini, T., Howells, R.D. and Simon, E.J., Characterization and partial purification of solubilized active opiate receptors from toad brain, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 4635-4638. 19 Siegel, S., Nonparametric Statistics for the Behavioral Sciences, McGraw-Hill, New York, 1956. 20 Simantov, R., Goodman, R., Aposhian, D. and Snyder, S.H., Phylogenetic distribution of a morphine-like peptide 'enkephalin', Brain Research, 111 (1976) 204-211. 21 Stevens, C.W. and Pezalla, P.D., A spinal site mediates opiate analgesia in frogs, Life Sci., 33 (1983) 2097-2103. 22 Stevens, C.W. and Pezalla, P.D., Naloxone blocks the analgesic action of levorphanol but not of dextrorphan in the leopard frog, Brain Research, 301 (1984) 171-174. 23 Stevens, C.W. and Yaksh, T.L., Dynorphin A and related peptides administered intrathecally in the rat: a search for putative kappa opiate receptor activity, J. Pharmacol. Exp. Ther., 238 (1986) 833-838. 24 Yaksh, T.L., Durant, P., Onofrio, B. and Stevens, C.W., The effect of spinally administered agents on pain transmission in man and animals. In: J.M. Besson and J. Lazorthes (Eds.), Spinal Opioids and the Relief of Pain, Inserm, 127 (1984) pp. 317-332. 25 Yaksh, T.L., The effects of intrathecally administered opioid and adrenergic agents on spinal function. In T.L. Yaksh (Ed.), Spinal Afferent Processing, Plenum Press, New York, 1986, pp. 505-531. 26 Yui, R., Immunohistochemical studies on peptide neurons in the hypothalamus of the bullfrog Rana catesbeiana, Gen. Comp. Endocrinol., 49 (1983) 195-209.
201
BRE 21997
Spinal antinociceptive action of three representative opioid peptides in frogs Craig W. Stevens 1, Paul D. Pezalla I and Tony L. Yaksh 2 1Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL 60680 (U.S.A.), and 2Department of Neurosurgical Research, Mayo Clinic, Rochester, MN 55905 (U.S.A.) (Accepted 9 September 1986) Key words: Amphibian; Rana pipiens; Antinociception; Dynorphin; fl-Endorphin; Met-enkephalin
Spinal administration of low doses of dynorphin, fl-endorphin or Met-enkephalin produces a potent, dose-dependent increase in the nociceptive threshold in the unanesthetized frog, Rana pipiens. Nociceptive thresholds were determined by using the acetic acid test, previously shown to be a sensitive indicator of antinociception in this amphibian species. Of particular interest, spinally administered dynorphin produces a potent antinociception in frogs without any signs of motor dysfunction seen after spinally administered dynorphin in mammalian species. Spinal administration of opiates and endogenous opioid peptides produces a behaviorally defined antinociception in a variety of mammalian models 24'25. Although endogenous opioid peptides and opiate binding sites are ubiquitous throughout phylogeny3'6'I1'20little attention has been focused on possible function(s) of endogenous opioid systems or development of behavioral models for antinociception in non-mammalian vertebrates. Recent work has shown that the amphibian, Rana pipiens, may serve as an appropriate model for assessment of antinociception following systemic 12'14 or spinal 21'22 administration of morphine and other opiate alkaloids. Furthermore, immobilization stress in frogs increases nociceptive threshold in frogs and this effect is blocked by naloxone 13. As immunocytochemical studies in amphibians demonstrate that high levels of dynorphin 4, fl-endorphin 7,8,15,26 and enkephalin 1'5'9'1° exist within the amphibian central nervous system and biochemical studies reveal high densities of opiate binding throughout the amphibian neuraxis2A6-18, the above results suggest endogenous opioid systems may likewise modulate central processing of noxious stimuli in amphibians as well as mammalians.
Given the powerful and well-defined antinociceptive effects of opioids in mammalian spinal cord, we sought in the present experiments to examine the effects of 3 endogenous representative opioid peptides, dynorphin, fl-endorphin and Met-enkephalin on nociception in the frog. We now report, for the first time, that spinal administration of the opioid peptides, dynorphin, fl-endorphin and Met-enkephalin produces a potent, dose-dependent increase in nociceptive thresholds in the frog, Rana pipiens. Leopard frogs, Rana pipiens (25-30 g; Nasco, Fort Atkinson, WI) were maintained as previously described 12 and two days before the start of experiments were randomly assigned to treatment groups and placed in individual pans with 0.5 cm tap water. Frogs were delivered in late August and experiments begun after two weeks acclimatization in our laboratory. Dynorphin Al_13, fl-endorphin and Met-enkephalin (Peninsula Labs, Palo Alto, CA) were dissolved in 0.7% NaC1 and injected intraspinally with a Hamilton microsyringe fitted with a tapered 26gauge needle (point no. 2). The site of injection was lateral to the neural spine at the articulation between the seventh and eighth vertebrae of the frog spinal column. Further details of the intraspinal injection
Correspondence: C.W. Stevens. Present address: Mayo Clinic, Rochester, MN 55905, U.S.A. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
202 32
lZ
24
c o3 r0
IE
~
s
BEP
DYN ENK
SAL
0~ 0
50 500
5
50 500
5
50 500
Dose (ng) Fig. 1. The effect of intraspinal Met-enkephalin (ENK), dynorphin (DYN) or fl-endorphin (BEP) on total change in nociceptive thresholds (NT). Data are plotted as the mean plus S.E.M. for 3-9 animals per treatment group. Asterisks denote significant difference from saline (SAL)-injected controls (P < 0.05, randomization test, one-tailed). method have been given zl. All doses were given in a volume of 1 pl/animal. Drugs and doses were coded and the experimenter was unaware of their identity until completion of the experiments. Estimation of the frog's nociceptive threshold (NT) were made with the acetic acid test as previously described 12. Briefly, 11 dilutions of acetic acid, equally spaced on a logarithmic scale from 0.26 to 15 M, were numbered in order of increasing concentration from 0 to 10. The NT is defined as the n u m b e r of the lowest concentration of acetic acid which causes a wiping response when applied to the dorsum of the frog's thigh. Data were plotted as the mean change in NT (post-injection NT minus pre-injection NT) summed over post-injection test times (5, 60,120 and 180 min). Non-parametric statistics were used 19 and significance set at P < 0.05. For each experiment presented there were no significant differences in pre-injection NT among treatment groups. The effect of intraspinal dynorphin, /3-endorphin
1 Adli, D.S., Yuen, G.L., Rosenthal, B.M., Ho, R.H. and Cruce, W.L.R., Enkephalin-like immunoreactive elements in Lissauer's tract and other regions of frog spinal cord, Soc. Neurosci. Abstr., 10 (1984) 488. 2 Audigier, Y., Duprat, A.M. and Cros, J., Comparative study of opiate and enkephalin receptors on lower vertebrates and higher vertebrates, Comp. Biochem. Physiol., 67C (1980) 191-193. 3 Buatti, M.C. and Pasternak, G.W., Multiple opiate receptors: phylogenetic differences, Brain Research, 218 (1981) 400-405. 4 Cone, R.I. and Goldstein, A., A dynorphin-like opioid in
or Met-enkephalin on nociceptive thresholds in summer frogs is shown in Fig. 1. Dynorphin (5, 50 or 500 ng) produced a significant dose-dependent increase in total change N T compared to saline controls. /3Endorphin and Met-enkephalin produced significant increases in N T at only the two highest doses (50 and 500 ng). The intraspinal injection of the 3 opioid peptides was not accompanied by any observable m o t o r deficits at any of the doses given, i.e. all frogs exhibited normal righting reflexes and wiping responses to the nociceptive stimulus with no abnormal tonus of hindlimbs. The present results demonstrate a potent, dose-dependent antinociceptive.action of spinally administered dynorphin,/3-endorphin and Met-enkephalin, and extend our earlier observations documenting the antinociceptive effects of spinal opiate alkaloids 14,21,22 in amphibians. To our knowledge, these results are the first demonstrating a behaviorally def;r.ed antinociceptive action of opioid peptides in a non-mammalian vertebrate. A post-hoc analysis of the potency of dynorphin,/3-endorphin and Met-enkephalin demonstrates an approximately 6-, 7- and 0.8-fold potency ratio, respectively, c o m p a r e d to spinal morphine. As spinally administered dynorphin in rodents produces severe motor dysfunction which is not easily dissociated from antinociceptive effects 23, the lack of any observable motor deficits after spinal dynorphin in frogs suggests that the acetic acid test in amphibians may be a suitable model for further studies of dynorphin antinociception.
We thank A n n Rockafellow for manuscript preparation. Supported by the Research Board of the University of Illinois at Chicago.
the central nervous system of an amphibian, Proc. Natl. Acad. Sci. U.S.A., 79 (1982)3345-3349.
5 Doerr-Schott, J., Dubois, M.P. and Lichte, C., Immunohistochemical localization of substances reactive to antisera against a- and fl-endorphin and Met-enkephalin in the brain of Rana temporaria, Cell Tissue Res., 217 (1981) 79-92. 6 Edley, S.M., Hall, L., Hevkenham, M. and Pert, C.B., Evolution of striatal opiate receptors, Brain Research, 249 (1982) 184-188. 7 Jackson, I.M.D., Bolaffi, J.L. and Guilleman, R., Presence of immunoreactive fl-endorphin and enkephalin-like material in the retina and other tissues of the frog, Rana pi-
203
piens, Gen. Comp. Endocrinol., 42 (1980) 505-508. 8 Jeqou, S., Tonon, M.C., Levoux, P., Leboulenger, F., Pelletier, G., Cote, J., Ling, N. and Vandry, N., Immunological characterization of endorphins, adrenocorticotropin and melanotropins in frog hypothalamus, Gen. Comp. Endocrinol., 51 (1983) 246-254. 9 Kilpatrick, D.L., Howells, R.D., Lahm, M.-W. and Udenfriend, S., Evidence for a proenkephalin-like precursor in amphibian brain, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 5772-5775. -10 Lorez, H.P. and Kemali, M., Substance P-, Met-enkephalin- and somatostatin-like immunoreactivity distribution in the frog spinal cord, Neurosci. Lett., 26 (1981) 119-124. 11 Pert, C.B., Aposhian, D. and Snyder, S.H., Phylogenetic distribution of opiate receptor binding, Brain Research, 75 (1974) 356-361. 12 Pezalla, P.D., Morphine-induced analgesia and explosive motor behavior in an amphibian, Brain Research, 273 (1983) 297-305. 13 Pezalla, P.D. and Dicig, M., Stress-induced analgesia in frogs: evidence for.the involvement of an opioid system, Brain Research, 296 (1984) 356-360. 14 Pezalla, P.D. and Stevens, C.W., Behavioral effects of morphine, levorphanol, dextrorphan and naloxone in the frog Rana pipiens, Pharmacol. Biochem. Behav., 21 (1984) 213-217. 15 Pezalla, P.D., Seidah, N.G., Benjannet, S., Crine, P., Lis, M. and Chretien, M., Biosynthesis of/3-endorphin,/3-1ipotropin and the putative ACTH-LPH precursor in the frog pars intermedia, Life Sci., 23 (1978) 2281-2292. 16 Puget, A., Jauzac, P. and Meunier, J.C., Distinct molecular forms of opiate binding in the frog brain, Life Sci., 33, Suppl. 1 (1983) 199-202.
17 Ruegg, V.T., Hiller, J.M. and Simon, E.J., Solubilization of an active opiate receptor from Bufo marinus, Eur. J. Pharmacol., 64 (1980) 367-368. 18 Ruegg, V.T., Cuenod, S., Hiller, J.M., Gioannini, T., Howells, R.D. and Simon, E.J., Characterization and partial purification of solubilized active opiate receptors from toad brain, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 4635-4638. 19 Siegel, S., Nonparametric Statistics for the Behavioral Sciences, McGraw-Hill, New York, 1956. 20 Simantov, R., Goodman, R., Aposhian, D. and Snyder, S.H., Phylogenetic distribution of a morphine-like peptide 'enkephalin', Brain Research, 111 (1976) 204-211. 21 Stevens, C.W. and Pezalla, P.D., A spinal site mediates opiate analgesia in frogs, Life Sci., 33 (1983) 2097-2103. 22 Stevens, C.W. and Pezalla, P.D., Naloxone blocks the analgesic action of levorphanol but not of dextrorphan in the leopard frog, Brain Research, 301 (1984) 171-174. 23 Stevens, C.W. and Yaksh, T.L., Dynorphin A and related peptides administered intrathecally in the rat: a search for putative kappa opiate receptor activity, J. Pharmacol. Exp. Ther., 238 (1986) 833-838. 24 Yaksh, T.L., Durant, P., Onofrio, B. and Stevens, C.W., The effect of spinally administered agents on pain transmission in man and animals. In: J.M. Besson and J. Lazorthes (Eds.), Spinal Opioids and the Relief of Pain, Inserm, 127 (1984) pp. 317-332. 25 Yaksh, T.L., The effects of intrathecally administered opioid and adrenergic agents on spinal function. In T.L. Yaksh (Ed.), Spinal Afferent Processing, Plenum Press, New York, 1986, pp. 505-531. 26 Yui, R., Immunohistochemical studies on peptide neurons in the hypothalamus of the bullfrog Rana catesbeiana, Gen. Comp. Endocrinol., 49 (1983) 195-209.