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Age related changes of enkephalin in rat spinal cord
160
Brain Research, 262 (1983) 160-162 Elsevier Biomedical Press
Age related changes of enkephalin in rat spinal cord C. MISSALE, S. GOVONI, L. CASTELLETTI, P. F. SPANO and M. TRABUCCHI*
Department of Pharmacology, University of Brescia, Via Valsabbina 19, Brescia (Italy) (Accepted November 2nd, 1982)
Key words: a g i n g - Met-enkephalin - opiate receptors
Met-enkephalin immunoreactive material content was found to be decreased in the cervical and thoracic segments of the spinal cord from rats aged 25 months as compared to young, 3-month-old, rats. No age-related variations were detectable at the lumbar level. Bio-Gel P 30 column chromatography of thoracic segment extracts indicates that the composition of the immunoreactive material is similar in the two age-groups investigated. At the thoracic level opiate receptor binding was also measured. Opiate receptor number is increased in the thoracic segments of the spinal cord from older rats. These age-related changes in immunoreactive Met-enkephalin content and opiate receptor number at spinal levels may contribute to determine an altered pain sensitivity during aging.
Clinical reports indicate an increased effectiveness of morphine-like analgesics in geriatric patients. This p h e n o m e n o n does not appear to be linked to pharmacokinetic variations in morphine absorption or disposition but to agerelated changes in pain regulatory mechanisms 3. Convincing evidence has been accumulated indicating Met-enkephalin as an important modulator of pain transmission in the dorsal horns of the spinal cord where it acts by inhibiting substance P release from primary afferent neurons 14A6. In fact, a population of opiate receptors in the dorsal horn is located on primary afferent terminals 1.~° which release substance p7.~5; in addition, Met-enkephalin immunoreacrive interneurons are present in the substantia gelatinosa 5.8. On the basis of this evidence it was interesting to study whether age-related alterations in the content of Met-enkephalin immunoreactive material (ME-IR) take place at spinal level. Young (3 months old) and aged (25 months old) male Sprague-Dawley rats were used. The animals were exposed to a light cycle of 12 h (from 06.00 to 18.00 h); water and food were administered ad libitum. Senescent rats with * To whom correspondence should be addressed.
0006-8993/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
pathological affections (such as gross tumors, pituitary tumors, lung infections) were excluded from the study. The rats were decapitated, the spinal cords dissected and frozen on dry-ice. The cervical, thoracical and lumbar segments were cut in about 2 m m thick slices and the dorsal and ventral halves were then dissected. ME-IR was measured according to Yang et al. 17 by radioimmunoassay. Aliquots of neutralized 0.1 M acetic acid extracts of dorsal and ventral halves were incubated at 4°C for 16 h with an antiserum directed toward Met-enkephalin in 0.5 ml of 0.2 M Tris-HC1 buffer (pH 7.4) containing 0.1% bovine serum albumin and 0.15% dextran. The reaction was stopped by adding 0.2 ml of 1.5% charcoal slurry containing 0.15% dextran and 0.9% NaC1. For gel chromatography aliquots of the extracts from young and aged rats were pooled separately, lyophilized, resuspended in 1 M acetic acid and applied to a Bio-Gel P 30 column (0.9 × 60 cm). One molar acetic acid was used as eluant and 1 ml fractions were collected every 30 min. Eluate fractions were assayed for ME-IR. Opiate receptors were analyzed by using [3H]DAla2-Met-enkephalinamide (20 Ci/mmol, Radiochemical Centre, Amersham) as ligand. Tis-
161 TABLE I
Met-enkephalin immunoreactive material (ME-IR) content in the spinal cord ofyoung (3 months) and aged (25 months) rats Each v a l u e is the m e a n ± S.D. o f at least 10 a n i m a l s . A ge
ME-IR (ng/mgprotein) Cervical
3 months 25 m o n t h s
Thoracic
Lumbar
Ventral
Dorsal
Ventral
Dorsal
Ventral
Dorsal
1.33 _+ 0.20 1.21 _ 0 . 1 4
3.94 ± 0.40 1.78±0.18"
1.12 _ 0.16 1.26±0.17
3.48 _ 0.71 1.93±0.19"
1.21 _ 0.13 1.17+0.16
2.92 ± 0.31 2.85±0.32
* P < 0.001 in respect to 3 - m o n t h - o l d rats, two-tailed S t u d e n t ' s t-test.
sues were homogenized in 30 vol. (w/v) of 0.05 M Tris-HC1 buffer (pH 7.7) and centrifuged at 49,000 g for 15 min. Pellets, resuspended in the original volume of buffer, were incubated at 37 °C for 30 min, centrifuged again and finally resuspended in 45 vols. of the same buffer. A1iquots of the homogenate were incubated at 25 °C for 30 min with the tracer in the presence or absence of 1/~M D-Ala2-Met-enkephalinamide. The reaction was stopped by filtration. The specific binding was defined as the difference between the total binding and the binding in the presence of 1 /~M D-Ala2-Met-enkephalinamide. Protein content was measured according to Lowry et al. ~ . Table I shows that ME-IR content is decreased in the cervical and thoracic portions of the spinal cord from 25-month-old rats, while no age-related variations are detectable at the lumbar level. The age-related decrease of ME-IRVo
Met-enk.
50_ 4,0.
}~ 3.0_
e---e
=m
0 - - 0 2 5 months
3 months
content at spinal level is confined to the dorsal halves while no changes are observed in the ventral ones. Bio-Gel P 30 column chromatography of the thoracic segment extracts reveals that the immunoreactive material is heterogeneous with a prevalence of material eluting in the elution volume of synthetic MetLenkephalin. The immunoreactive material composition was similar in the two age groups investigated (3 and 25 months, Fig. 1). These data, although describing a rather selective decrease of ME-IR content in the dorsal halves of the thoracic spinal cord of aged rats, are not sufficient to establish whether or not the decreased peptide content is associated with a slower Met-enkephalin turnover. An indirect method to obtain information on the functional activity of enkephalinergic neurons was provided by the measure of the opiate receptor activity. For this purpose, the binding of [3H]D-Alaz-Met-enkephalinamide was studied in the thoracic segment of the spinal cord of young and aged rats. The kinetic analysis of the binding data indicates that the number of opiate receptors is increased in the thoracic segment of the spinal cord of aged rats (Table II). T A B L E II
Kinetic characteristics of [3H]D-A LA 2-Met-enkephalinamide binding in the thoracical spinal cord of young (3 months) and aged (25 months) rats
2o~
10i
E a c h v a l u e is the m e a n +_ S.D. o f 3 - 4 i n d e p e n d e n t determinations.
~10
120
130
140 Fraction number
Fig. 1. B i o - G e l P 30 c o l u m n c h r o m a t o g r a p h y o f thoracic spinal cord extracts o f y o u n g (3 m o n t h s ) a n d aged (25 m o n t h s ) rats.
A ge
K d (rim)
Bmax 6fmol/mgprotein)
3 months 25months
10.3 _ 1.1 12.4 _ 1.5
88 _+ 7 123 ± 11"
* P < 0.05 in respect to 3 - m o n t h - o l d rats.
162 The opiate receptor increase observed at thoracic level may indicate that the reduced ME-IR content in this spinal segment reflects a decreased turnover of the peptide or a decreased number of enkephalinergic neurons. The fact that the peptide content changes are observed only in the dorsal half of the spinal cord favors the concept of an altered function of the neuronal pathways carrying nociceptive signals. At this level, enkephalinergic neurons and opiate receptors are strategically arranged to modulate noxious stimuli. An increased receptor availability at the dorsal horn level may be of relevance for the greater pharmacological response to morphine observed during aging. At present we are unable to explain why the lumbar ME-IR content is not affected by aging. It should be remembered that in aged rats selective changes of the endogenous opioid content and receptor density take place also in other sites in the CNS as well as in the periphery. In particular, Met-enkephalin and fi-
endorphin content seems to be depressed in some brain areas such as the hypothalam u s 2,4,6,9,13 and the tuberculum olfactorium (Missale et al., unpublished observations); opiate receptor number is diminished in striatum, hypothalamus, and cerebral cortex ~2. On the contrary, increased Met-enkephalin and fi-endorphin concentrations were observed in the pituitary~.9.J3; this increase in the case offi-endorphin is also associated with an increase of circulating fl-endorphin levels6. Obviously, not all of these changes should be connected with changes in pain sensitivity. However, pain perception is an event mediated at different neuronal levels and each one of these modifications has to be considered as a possible contribution to determine an altered pain sensitivity in aged organisms.
1 Atweh, S. F. and Kuhar, M. H., Autoradiographic localization of opiate receptors in rat brain, Brain Research, 124 (1977) 53-67. 2 Barden, N., Dupont, A., Labrie, F., M6raud, Y,, Rouleau, D., Vaudry, H. and Boissier, J. R., Age dependent changes in the fl-endorphin content of discrete brain nuclei, Brain Research, 208 ( 1981) 209-212. 3 Belville, J. W., Forrest, W. H., Miller, E. and Brown, B. W., Influence of age on pain relief from analgesics, JAMA, 217(1971)1835 1841. 4 Dupont, A., Savard, P, M6raud, Y., Labrie, F. and Boissier, J. R., Age-related changes in central nervous system enkephalins and substance P, Life Sci., 29 (1981) 23172322. 5 Fields, M. L., Emson, P. C., Leigh, B. K., Gilber, R. F. T. and Iversen, L. L., Multiple opiate receptors sites on primary afferent fibers, Nature (Lond.), 284 (1980) 351-353. 6 Forman, L. J., Sonntag, W. E., Van Vugt, D. A. and Meites, J., Immunoreactive fl-endorphin in the plasma, pituitary and hypothalamus of young and old male rats, Neurobiol. Ageing, 2 ( 1981) 280-284. 7 H6kfelt, T., Kelleth, J. O., Nilsson, G. and Pernow, B., Experimental immunohistochemicalstudies on the localization and distribution of substance P in cat primary sensory neurons, Brain Research, 100 (1975) 232-252. 8 HOkfelt, T., Ljungdahl, A., Terenius, L., Elde, R. and Nilsson, G., Immunohistochemical analysis of peptide pathways possibly related to pain and analgesia: enkephalin and substance P, Proc. nat. Acad. Sci. U.S.A., 74(1977) 3081-3085. 9 Kumar, M. S, A, Chen, C. L. and Huang, H. H., Pituitary and hypothalamic concentrations of Met-enkepha-
lin in young and old rats, Neurobiol. Ageing, 1 (1980) 153 155. 10 Lamotte, C., Pert, C. B. and Snyder, S. H., Opiate receptor binding in primate spinal cord: distribution and changes after dorsal root section, Brain Research, 112 (1976)407 412. 11 Lowry, O. M., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 ( 1951) 265-275. 12 Messing, R. B., Vasquez, B. J., Samaniego, B.. Jensen, R. A., Martinez, J. L. and McGaugh, J. L., Alterations in dihydromorphine binding in cerebral hemispheres of aged male rats, J. Neurochem,, 36 ( 1981) 784 787. 13 Missale, C., Govoni, S., Croce, L., Bosio, A., Spano. P. F. and Trabucchi, M., Changes of endorphin and Met-enkephalin content in the hypothalamus pituitary axis induced by aging, J. Neurochem., (1982) in press. 14 Mudge, A. W., Leeman, S. E. and Fishbach. G. D.. Enkephalin inhibits the release of substance P from sensory' neurons in culture and decreases action potential duration, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 526-530. 15 Yaksh, T. L., Farb, D. H., Leeman, S. E. and Jessel. T. M., Intrathecal capsaicin depletes substance P in the rat spinal cord and produces prolonged thermal analgesia. Science, 206 (1979) 481-483. 16 Yaksh, T. L., Jessel, T. M., Gamsc, R.. Mudge, A. W. and Leeman, S. E., Intrathecal morphine inhibits substance P release from mammalian spinal cord in vivo, Nature (Lond.), 286 (1980) 155- 157. 17 Yang, H. Y. T.. Hong, J. S. and Costa, E., Regional distribution of Leu- and Met-enkephalin in rat brain, Neuropharmacology, 16 (1977) 303-307.
The animals used in this study were kindly supplied by the Italian Study Group on Brain Aging.
Brain Research, 262 (1983) 160-162 Elsevier Biomedical Press
Age related changes of enkephalin in rat spinal cord C. MISSALE, S. GOVONI, L. CASTELLETTI, P. F. SPANO and M. TRABUCCHI*
Department of Pharmacology, University of Brescia, Via Valsabbina 19, Brescia (Italy) (Accepted November 2nd, 1982)
Key words: a g i n g - Met-enkephalin - opiate receptors
Met-enkephalin immunoreactive material content was found to be decreased in the cervical and thoracic segments of the spinal cord from rats aged 25 months as compared to young, 3-month-old, rats. No age-related variations were detectable at the lumbar level. Bio-Gel P 30 column chromatography of thoracic segment extracts indicates that the composition of the immunoreactive material is similar in the two age-groups investigated. At the thoracic level opiate receptor binding was also measured. Opiate receptor number is increased in the thoracic segments of the spinal cord from older rats. These age-related changes in immunoreactive Met-enkephalin content and opiate receptor number at spinal levels may contribute to determine an altered pain sensitivity during aging.
Clinical reports indicate an increased effectiveness of morphine-like analgesics in geriatric patients. This p h e n o m e n o n does not appear to be linked to pharmacokinetic variations in morphine absorption or disposition but to agerelated changes in pain regulatory mechanisms 3. Convincing evidence has been accumulated indicating Met-enkephalin as an important modulator of pain transmission in the dorsal horns of the spinal cord where it acts by inhibiting substance P release from primary afferent neurons 14A6. In fact, a population of opiate receptors in the dorsal horn is located on primary afferent terminals 1.~° which release substance p7.~5; in addition, Met-enkephalin immunoreacrive interneurons are present in the substantia gelatinosa 5.8. On the basis of this evidence it was interesting to study whether age-related alterations in the content of Met-enkephalin immunoreactive material (ME-IR) take place at spinal level. Young (3 months old) and aged (25 months old) male Sprague-Dawley rats were used. The animals were exposed to a light cycle of 12 h (from 06.00 to 18.00 h); water and food were administered ad libitum. Senescent rats with * To whom correspondence should be addressed.
0006-8993/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
pathological affections (such as gross tumors, pituitary tumors, lung infections) were excluded from the study. The rats were decapitated, the spinal cords dissected and frozen on dry-ice. The cervical, thoracical and lumbar segments were cut in about 2 m m thick slices and the dorsal and ventral halves were then dissected. ME-IR was measured according to Yang et al. 17 by radioimmunoassay. Aliquots of neutralized 0.1 M acetic acid extracts of dorsal and ventral halves were incubated at 4°C for 16 h with an antiserum directed toward Met-enkephalin in 0.5 ml of 0.2 M Tris-HC1 buffer (pH 7.4) containing 0.1% bovine serum albumin and 0.15% dextran. The reaction was stopped by adding 0.2 ml of 1.5% charcoal slurry containing 0.15% dextran and 0.9% NaC1. For gel chromatography aliquots of the extracts from young and aged rats were pooled separately, lyophilized, resuspended in 1 M acetic acid and applied to a Bio-Gel P 30 column (0.9 × 60 cm). One molar acetic acid was used as eluant and 1 ml fractions were collected every 30 min. Eluate fractions were assayed for ME-IR. Opiate receptors were analyzed by using [3H]DAla2-Met-enkephalinamide (20 Ci/mmol, Radiochemical Centre, Amersham) as ligand. Tis-
161 TABLE I
Met-enkephalin immunoreactive material (ME-IR) content in the spinal cord ofyoung (3 months) and aged (25 months) rats Each v a l u e is the m e a n ± S.D. o f at least 10 a n i m a l s . A ge
ME-IR (ng/mgprotein) Cervical
3 months 25 m o n t h s
Thoracic
Lumbar
Ventral
Dorsal
Ventral
Dorsal
Ventral
Dorsal
1.33 _+ 0.20 1.21 _ 0 . 1 4
3.94 ± 0.40 1.78±0.18"
1.12 _ 0.16 1.26±0.17
3.48 _ 0.71 1.93±0.19"
1.21 _ 0.13 1.17+0.16
2.92 ± 0.31 2.85±0.32
* P < 0.001 in respect to 3 - m o n t h - o l d rats, two-tailed S t u d e n t ' s t-test.
sues were homogenized in 30 vol. (w/v) of 0.05 M Tris-HC1 buffer (pH 7.7) and centrifuged at 49,000 g for 15 min. Pellets, resuspended in the original volume of buffer, were incubated at 37 °C for 30 min, centrifuged again and finally resuspended in 45 vols. of the same buffer. A1iquots of the homogenate were incubated at 25 °C for 30 min with the tracer in the presence or absence of 1/~M D-Ala2-Met-enkephalinamide. The reaction was stopped by filtration. The specific binding was defined as the difference between the total binding and the binding in the presence of 1 /~M D-Ala2-Met-enkephalinamide. Protein content was measured according to Lowry et al. ~ . Table I shows that ME-IR content is decreased in the cervical and thoracic portions of the spinal cord from 25-month-old rats, while no age-related variations are detectable at the lumbar level. The age-related decrease of ME-IRVo
Met-enk.
50_ 4,0.
}~ 3.0_
e---e
=m
0 - - 0 2 5 months
3 months
content at spinal level is confined to the dorsal halves while no changes are observed in the ventral ones. Bio-Gel P 30 column chromatography of the thoracic segment extracts reveals that the immunoreactive material is heterogeneous with a prevalence of material eluting in the elution volume of synthetic MetLenkephalin. The immunoreactive material composition was similar in the two age groups investigated (3 and 25 months, Fig. 1). These data, although describing a rather selective decrease of ME-IR content in the dorsal halves of the thoracic spinal cord of aged rats, are not sufficient to establish whether or not the decreased peptide content is associated with a slower Met-enkephalin turnover. An indirect method to obtain information on the functional activity of enkephalinergic neurons was provided by the measure of the opiate receptor activity. For this purpose, the binding of [3H]D-Alaz-Met-enkephalinamide was studied in the thoracic segment of the spinal cord of young and aged rats. The kinetic analysis of the binding data indicates that the number of opiate receptors is increased in the thoracic segment of the spinal cord of aged rats (Table II). T A B L E II
Kinetic characteristics of [3H]D-A LA 2-Met-enkephalinamide binding in the thoracical spinal cord of young (3 months) and aged (25 months) rats
2o~
10i
E a c h v a l u e is the m e a n +_ S.D. o f 3 - 4 i n d e p e n d e n t determinations.
~10
120
130
140 Fraction number
Fig. 1. B i o - G e l P 30 c o l u m n c h r o m a t o g r a p h y o f thoracic spinal cord extracts o f y o u n g (3 m o n t h s ) a n d aged (25 m o n t h s ) rats.
A ge
K d (rim)
Bmax 6fmol/mgprotein)
3 months 25months
10.3 _ 1.1 12.4 _ 1.5
88 _+ 7 123 ± 11"
* P < 0.05 in respect to 3 - m o n t h - o l d rats.
162 The opiate receptor increase observed at thoracic level may indicate that the reduced ME-IR content in this spinal segment reflects a decreased turnover of the peptide or a decreased number of enkephalinergic neurons. The fact that the peptide content changes are observed only in the dorsal half of the spinal cord favors the concept of an altered function of the neuronal pathways carrying nociceptive signals. At this level, enkephalinergic neurons and opiate receptors are strategically arranged to modulate noxious stimuli. An increased receptor availability at the dorsal horn level may be of relevance for the greater pharmacological response to morphine observed during aging. At present we are unable to explain why the lumbar ME-IR content is not affected by aging. It should be remembered that in aged rats selective changes of the endogenous opioid content and receptor density take place also in other sites in the CNS as well as in the periphery. In particular, Met-enkephalin and fi-
endorphin content seems to be depressed in some brain areas such as the hypothalam u s 2,4,6,9,13 and the tuberculum olfactorium (Missale et al., unpublished observations); opiate receptor number is diminished in striatum, hypothalamus, and cerebral cortex ~2. On the contrary, increased Met-enkephalin and fi-endorphin concentrations were observed in the pituitary~.9.J3; this increase in the case offi-endorphin is also associated with an increase of circulating fl-endorphin levels6. Obviously, not all of these changes should be connected with changes in pain sensitivity. However, pain perception is an event mediated at different neuronal levels and each one of these modifications has to be considered as a possible contribution to determine an altered pain sensitivity in aged organisms.
1 Atweh, S. F. and Kuhar, M. H., Autoradiographic localization of opiate receptors in rat brain, Brain Research, 124 (1977) 53-67. 2 Barden, N., Dupont, A., Labrie, F., M6raud, Y,, Rouleau, D., Vaudry, H. and Boissier, J. R., Age dependent changes in the fl-endorphin content of discrete brain nuclei, Brain Research, 208 ( 1981) 209-212. 3 Belville, J. W., Forrest, W. H., Miller, E. and Brown, B. W., Influence of age on pain relief from analgesics, JAMA, 217(1971)1835 1841. 4 Dupont, A., Savard, P, M6raud, Y., Labrie, F. and Boissier, J. R., Age-related changes in central nervous system enkephalins and substance P, Life Sci., 29 (1981) 23172322. 5 Fields, M. L., Emson, P. C., Leigh, B. K., Gilber, R. F. T. and Iversen, L. L., Multiple opiate receptors sites on primary afferent fibers, Nature (Lond.), 284 (1980) 351-353. 6 Forman, L. J., Sonntag, W. E., Van Vugt, D. A. and Meites, J., Immunoreactive fl-endorphin in the plasma, pituitary and hypothalamus of young and old male rats, Neurobiol. Ageing, 2 ( 1981) 280-284. 7 H6kfelt, T., Kelleth, J. O., Nilsson, G. and Pernow, B., Experimental immunohistochemicalstudies on the localization and distribution of substance P in cat primary sensory neurons, Brain Research, 100 (1975) 232-252. 8 HOkfelt, T., Ljungdahl, A., Terenius, L., Elde, R. and Nilsson, G., Immunohistochemical analysis of peptide pathways possibly related to pain and analgesia: enkephalin and substance P, Proc. nat. Acad. Sci. U.S.A., 74(1977) 3081-3085. 9 Kumar, M. S, A, Chen, C. L. and Huang, H. H., Pituitary and hypothalamic concentrations of Met-enkepha-
lin in young and old rats, Neurobiol. Ageing, 1 (1980) 153 155. 10 Lamotte, C., Pert, C. B. and Snyder, S. H., Opiate receptor binding in primate spinal cord: distribution and changes after dorsal root section, Brain Research, 112 (1976)407 412. 11 Lowry, O. M., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 ( 1951) 265-275. 12 Messing, R. B., Vasquez, B. J., Samaniego, B.. Jensen, R. A., Martinez, J. L. and McGaugh, J. L., Alterations in dihydromorphine binding in cerebral hemispheres of aged male rats, J. Neurochem,, 36 ( 1981) 784 787. 13 Missale, C., Govoni, S., Croce, L., Bosio, A., Spano. P. F. and Trabucchi, M., Changes of endorphin and Met-enkephalin content in the hypothalamus pituitary axis induced by aging, J. Neurochem., (1982) in press. 14 Mudge, A. W., Leeman, S. E. and Fishbach. G. D.. Enkephalin inhibits the release of substance P from sensory' neurons in culture and decreases action potential duration, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 526-530. 15 Yaksh, T. L., Farb, D. H., Leeman, S. E. and Jessel. T. M., Intrathecal capsaicin depletes substance P in the rat spinal cord and produces prolonged thermal analgesia. Science, 206 (1979) 481-483. 16 Yaksh, T. L., Jessel, T. M., Gamsc, R.. Mudge, A. W. and Leeman, S. E., Intrathecal morphine inhibits substance P release from mammalian spinal cord in vivo, Nature (Lond.), 286 (1980) 155- 157. 17 Yang, H. Y. T.. Hong, J. S. and Costa, E., Regional distribution of Leu- and Met-enkephalin in rat brain, Neuropharmacology, 16 (1977) 303-307.
The animals used in this study were kindly supplied by the Italian Study Group on Brain Aging.