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Spinal cord dynorphin may modulate nociception via a ℵ-opioid receptor in chronic arthritic rats
Brain Research, 3411( i 985 ) 156-159 Elsevier
156
BRE20969
Spinal cord d y n ~ i n
.ray m o d u ~ n ~ i ~ i o n in chronic arthritic rats
via a x-~ioid receptor
M. J. MILLANl, M. H. MILLANl, C. W. T. PILCHER1, A. CZLONKOWSKP, A. HERZ: and F. C. COLPAERT2-
1Department of Neuropharmacology, Max-Planck-lnstitutfar Psychiatrie, Am Klopferspitz 18a, D-8033 Planegg-Martinsried (F.R. G.) and 2Janssen Pharmaceutica Research Laboratories, B-2340 Beerse (Belgium) (Accepted March 12th, 1985)
Key words: dynorphin
Met-enkephalin - - opioid receptor - - spinal cord - - pare - - noclception - - arthritis
Inoculation of rats with Mycobacterium butyricum produced an arthritis of the limbs which revealed an enhanced sensitivity to noxious mechanical pressure (hyperalgesia). Arthritic rats displayed a pronounced rise in immtmoreactive dynorphin in lumbo,saeral spinal cord which correlated both with the intensity and time-course of this hyperalgesia. MR-2266, a relatively preferenfialantagonist at the x-opioid receptor (at which dynorphin is conside~;edto act) potentiated this hyperatgesia. In contrast, MR 2267 (its inactive stereoisomer) was ineffective. Further. naloxone (a weak'u-antagonist), and ICI 154,129 (a preferential &antagonist) were, in each case, inactive. The data demonstrate a pronounced response of spinal dynorphin to chronic arthritic pain in the rat. In addition, they raise the possibility of a function of spinal DYN, via a x-receptor, in the modulation of chronic arthritic pain.
Dynorphin A1-17 (DYN), a major product of the p r o - D Y N (pro-enkephalin B) gene, is present in high concentrations in the dorsal horn of the spinal cord in a position suggestive of its participation in the processing of afferent nociceptive informationl,9. D Y N displays a preference for the x-opioid receptor which is found in a high density in the dorsal spinal cord. and for which D Y N may be an endogenous ligand4,SA9. Both D Y N and synthetic x-agonists upon direct injection into the spinal cord of the rat produce antinociception, most notably against non-thermal stimuli7A4,15. Acute pain has been indicated to mobilize D Y N within the spinal cord and. under these conditions, a role of spinally-localized x-opioid receptors in the mediation of the concomitant antinociception has been proposed11,12, 20. Chronic pain has not, as yet, been examined as regards its possible influence upon the activity of spinal cord pools of DYN. It is, however, of particular interest in view of data acquired with chronic arthritic pain in the rat which is associated with an alteration in levels of immunoreactive (Jr) Met-enkephalin (ME) in the spinal cord 2,6.
In the light of the above observations, we have undertaken a parallel biochemical and behaviourat evaluation of the function response of spinal D Y N to chronic (arthritic) pain in the rat. Rats were intradermally inoculated with 0.05 ml Mycobacterium butyricum suspension (or vehicle as a control) at the taft-base to induce chronic arthritis3. They were housed individually with free access to water. Nociceptive thresholds were evaluated as follows: an automated gauge applied an incremental pressure via a wedge-shaped piston to the right hindpaw and the pressure required to elicit withdrawal, the paw pressure threshold (PPT) was determined. This was evaluated m rats subjected to biochemical analysis 1 day prior to sacrifice. In independent rats, for evaluation of the influence of drugs upon PPT. they (or saline) were injected s.c. in a volume of 4.0 ml/kg at the following doses: naloxone at 5 mg/kg; M R 2266 at 5 mg/kg; M R 2267 at 5 mg/kg and ICI-154.129 at 30 mg/kg. The PPT was measured 15 rain following drug application. The person monitoring the PPT was blind as to the drug injected. All animals were sacrificed by decapitation and hind-
Correspondence: M. J. Millan. Department of Neuropharmacology, Max-Planck-Institut fiir Psychiatrie. Am Klopferspitz 18a. D-8033 Pianegg-Martinsried. F. R. G. 0006-8993/85/$03.30 (~ 1985 Elsevier Science Publishers B.V. [Biomedical Division)
157
b.
Q°
150
~150 E _e
o
~
D
75
e~ i
o Q.
0
c
material and performance of the assays for ir-DYN and if-ME, in addition to properties of the antisera, were extensively described previouslyS,9.17. Briefly, the antiserum against DYN (generously provided by Dr. E. Weber) did not recognize DYN B, DYNI_ 8, a-neo-endorphin, leucine-or methionine-enkephalin, nor fl-endorphin. The detection limit was 5 fmol/tube. The antiserum against ME showed only 0.25% cross-reactivity to leucine-enkephalin and did not bind any of the above-specified opioid peptides. Ir-DYN was evaluated at one, 3 and 10 weeks postinoculation and if-ME at 3 weeks. At 3 weeks, rats showed a pronounced arthritic swelling and inflammation of the hind-limbs: control weight 2.21 _+ 0.11 g vs arthritis 4.06 +_ 0.15 g (P ~< 0.001; Student's two-tailed t-test). This was accompanied by a byperaigesia, that is a reduction in PPTs (Fig. la). These rats showed a pronounced increase in concentrations of ir-DYN in lumbo-sacral spinal cord to 212.8 + 12.9% of control values (= 100%) (Fig. lb). This rise was significantly and negatively correlated (Pearson Product-Moment Correlation Coefficient) with the intensity of hyperalgesia; i.e. rats showing the greatest hyperalgesia displayed the greatest rise in ir-DYN (Fig. lc). Further, a parallelism was seen in the time-course for the development and disappearance of the hyperalgesia and shifts in ir-DYN (Table I). In addition, in line with other authors 2,6, w e found a modest rise in ir-Met-enkephalin (ME) in the cord; (control, 436.2 + 38.2 vs arthritis 565.4 + 36.2, fmol/mg, P ~< 0.02, Student's twotailed t-test). This did not significantly correlate (r = _+ 0.07, Pearson Product-Moment Correlation Coefficient) with the intensity of the hyperalgesia. Further, we have found that neither ir-vasopressin nor ir-
o
E
8
0
A
C
A
C. "~ 200
E
r = - 0.69 p<- 0 0 2
o
E 150'
I-
-~ 100. i
._~
50.
80
160
paw-pressure threshold (g) Fig. 1. The influence of chronic arthritis upon (a) paw-pressure thresholds and (b) levels of immunoreactive (ir)-dynorphin in lumbo-sacral spinal cord. C = control (n = 10) and A = Arthritis (n - 11). Mean _+ S.E.M. depicted. Significance of A vs C values depicted. ***, P ~< 0.001 (Student's two-tailed t-test). In panel c, the correlation between levels of immunoreactivedynorphin and paw-pressure thresholds for individual arthritic rats is depicted, r - Pearson P r o d u c t - M o m e n t correlation coefficient.
paw weight determined as an index of the intensity of arthritic symptoms. In rats examined biochemically, the spinal cord was rapidly dissected out and, for preparation for radioimmunoassay of ir-DYN, and if-ME incubated in 5 vol. 0.1 HCI for 10 min at 97 °C, then frozen on dry ice. Procedures for preparation of TABLE I
Parallel appearance and disappearance of hyperalgesia and shifts in immunoreactive-dynorphin in lumbo-sacral spinal cord of arthritic rats Paw pressure thresholds are in g and immunoreactive dynorphin in fmol/mg tissue, wet weight. C signifies control and A arthritis. Data are given as m e a n + S.E.M. (n >/8 for each value presented). Significance of A vs C values indicated.
Weeks post-inoculation 1 Pawpressure C Thresholds A Immunoreactive C Dynorphin A
129.23 123.46 47.34 49.94
*** P ~ 0.001 (Student's two-tailed t-test).
_+ 10.62 _+ 6.21 + 4.28 + 6.20
3
10
125.16 _+ 8.94 75.31 _+ 7.67*** 59.51 _+ 4.82 126.59 _+ 4.41"**
116.62 109.22 51.16 56.72
_+ 7.51 _+ 8.11 _+ 2.32 + 4.90
158
2
o
ii io0
_2_
5oi
NLX PiR2266 MR2267 CONTROL
IC1154
SA-L"
NIX
M ~ ( g 5 MR2267 ICt154
ARTHRITIS
Fig. 2. The influence of acute treatment with various opioid receptor antagonists (see text) upon paw-pressure thresholds of control and arthritic rats. SAL = saline; NLX = naloxone. Mean + S.E.M. depicted. Significance of MR 2266 vs SAL values indicated. **, P<~0.0I (Student's two-tailedt-test). oxytocin are affected in the spinal cord of arthritic rats 10, suggestive of a certain neurochemical specificity in these effects. Fig. 2 shows that naloxone, a powerful #-receptor but a weak ~- (and d-) receptor antagonist TM, failed to signficantly affect PPTs in either control or arthritic rats. In contrast, MR 2266, which is a much more potent inhibitor of antinociception induced by intrathecal DYN and possesses greater n-receptor-blocking activity7,14, TM, significantly potentiated the hyperalgesia in arthritic rats. Its inactive stereoisomer, MR 2267, did not, in contrast, affect PPTs in arthritic rats indicating the stereospecificity of this action. Finally, a preferential 6-antagonist 16, ICI-154i129 failed to affect the PPT of arthritic rats. We found, in addition to this stereospecificity of action of MR 2266, that it was specific for non-thermal nociception since it did not affect the response of the paws to noxious heat (not shown). The present study shows chronic arthritic pain in the rat to be associated with a large and reversible rise in levels of ir-DYN in lumbo-sacral cord. That this rise reflects a response to the pain is supported by the following observations. Firstly, acute pain has been indicated to enhance the activity of DYN neurones in lumbo-sacral cordn, 20. Secondly, the chronic pain of recurrent foot-shock similarly elevates spinal cord levels of ir-DYN 13. Thirdly, the rise in ir-DYN in arthritic rats significantly correlated with the intensity of hyperalgesia (Fig. lc). Fourthly, inoculation
of rats in fore- or hind-paws leads to a rise in ir-DYN and ir-ME in, respectively, cervical or lumbo-sacral cord demonstrative that this is a response to a localized pain6,13. Thus, it is reasonable to infer that the rise in ir-DYN reflects a response to arthritic pain in our rats. Further, though recent studies of Jr-ME release in the spinal cord of arthritic rats failed to satisfactorily resolve the issue 2, the contention that the rise in ir-DYN reflects an enhanced activity appears the most reasonable hypothesis in the light of the above-mentioned points. Further, in current studies (Millan et al., in prep.) we have observed a downregulation in n-receptors in the spinal cord of arthritic rats; this is consistent with a facilitation rather than a suppression of the activity of DYN, since DYN is considered to act upon the x-receptor s:~. Indeed, in support of the above reasoning, MR 2266, an antagonist with greater activity than naloxone at x-receptors and much more potent in blocking antinociception induced by DYN injected into the cord 7,14,1s potentiated the hyperalgesia seen in arthritic rats. We suggest that this action on PPT, a response integrated in lumbo-sacral spinal cord, may reflect blockade of an action of endogenous DYN, the shift in levels of which correlated to the hyperalgesia both in its intensity and time-course. Support for a putative physiological role of spinal cord DYN in control of chronic pain is provided by the finding that both in arthritic and naive rats, exogenous DYN (or other x-agonists) intrathecally administered to the lumbo-sacral cord results in an antinociception 7,13-15. Interestingly, the present data with chronic pain offer a direct parallel to acute pain which has likewise been indicated to be accompanied by an augmented activity of DYN neurones and a modulation of nociception mediated by x-opioid receptors~.12.20. In conclusion, thus, the present and previous data suggest that acute or chronic pain activates spinal cord pools of DYN which may operate therein in the modulation of nociception. We thank Drs. V. H61tt and E. Weber f o r antisera against, respectively, DYN and ME. M 3 , M . was supported by the Deutsche Forschungsgemeinschaft. A.C. is a visiting scientist from the Department of Pharmacology, Institute of Physiological Sciences, Medical Academy of Warsaw, Poland.
159 1 Basbaum, A. I. and Fields, H. L., Endogenous pain control systems: brainstem spinal pathways and endorphin circuitry, Ann. Rev. Neurosci., 7 (1984) 309-338. 2 Cesselin, F., Bourgoin, S., Artaud, F. and Hamon, M., Basic and regulatory mechanisms of in vitro release of met-enkephalin from the dorsal horn of the rat spinal cord, J. Neurochem., 43 (1984) 763-773. 3 Colpaert, F. C. and van den Hoogen, R. M. V. M., Ventilatory response to adjuvant arthritis in the rat, Life Sci., 32 (1983) 957-963. 4 Cztonkowski, A., Costa, T., Przewlocki, R., Pasi, A. and Herz, A., Opiate receptor binding sites in human spinal cord, Brain Research, 267 (1983) 392-396. 5 Duka, Th., H611t, V., Przewlocki, R. and Wesche, D., Distribution of methionine and leucine-enkephalin within the rat as measured by a highly specific radioimmunoassay, Biochem. Biophys. Res. Commun., 85 (1978) 1119-1127. 6 Faccini, E., Uzumaki, M., Govoni, S., Missale, C., Spano, P. F., Covelli, V. and Trabucci, M., Afferent fibres mediate the increase of met-enkephalin elicited in rat spinal cord by localized pain, Pain, 18 (1984) 25-31. 7 Han, J.-S., Xie, G. X. and Goldstein, A., Analgesia induced by intrathecal injection of dynorphin B in the rat, Life Sci., 34 (1984) 1573-1579. 8 James, I. F., Fischli, W. and Goldstein, A., Opioid receptor selectivity of dynorphin gene products, J. Pharmacol. exp. Ther., 228 (1984) 88-93. 9 Millan, M. J., Millan, M. H., Czlonkowski, A. and Herz, A., Vasopressin and oxytocin in the rat spinal cord: localization and origin in comparison to dynorphin, Met-enkephalin and related opioid peptides and irresponsiveness to stimuli modifying neurohypophyseal secretion, Neuroscience, 13 (1984) 108-116. 10 Millan, M. J., Schmauss, C., Millan, M. H. and Herz, A., Vasopressin and oxytocin in the rat spinal cord: analysis of their role in control of nociception, Brain Research, 309 (1984) 384-388. 11 Millan, M. J., Tsang, Y. F., Przewlocki, R., HOllt, V. and
Herz, A., The influence of foot-shock stress upon brain, pituitary and spinal cord pools of immunoreactive dynorphin in the rat, Neurosci. Lett., 24 (1981) 75-79. 12 Panerai, A. E., Martini, A., Sacedote, P. and Mantegazza, P., x-receptor antagonist reverses 'non-opioid' stress-induced analgesia, Brain Research, 304 (1984) 153-156. 13 Przewlocki, R., Przewtocka, B., Larson, W., Garzon, J.,. Stala, L. and Herz, A., Opioid Peptides, Particularly Dynorphin, and Chronic Pain (Monograph Series), I.N.S.E.R.M., in press. 14 Przewtocki, R., Stala, L., Greczek, M., Shearman, G. T., Przewlocka, B. and Herz, A., Analgesic effects of/~-, b-and x-opiate agonists and, in particular, dynorphin at the spinal level, Life Sci., 33 Suppl. I (1983) 649-652. 15 Schmauss, C. and Yaksh, T. L., In vivo studies on spinal opiate receptors mediating antinociception II. Pharmacological profiles suggesting a differential association of mu, delta and kappa receptors with visceral, chemical and cutaneous thermal stimuli, J. Pharmacol. exp. Ther., 228 (1984) 7-14. 16 Shaw, J. S., Miller, L., Turnbull, M. J., Gormley, J. J. and Morley, J. S., Selective antagonists at the opioid delta receptor, Life Sci., 31 (1982) 1259-1262. 17 Weber, E., Evans, E. J. and Barchas, J. D., Predominance of the amino-terminal octapeptide fragment of dynorphin in rat brain regions, Nature (Lond.), 299 (1982) 77-79. 18 Vonvoigtlander, P. F., Lahti, R. A. and Lutens, J. H., U-50,488: A selective and structurally novel non-mu (kappa) opioid agonist, J. Pharmacol. exp. Ther., 224 (1983) 7-12. 19 W~ister, M., Rubini, P. and Schulz, R., The preference of putative proenkephalins for different types of opiate receptors, Life Sci., 29 (1981) 1219-1227. 20 Yaksh, T. D., Terenius, L., Nyberg, F., Jhamandas, K. and Wang, J. Y., Studies on the release by somatic stimulation from rat and cat spinal cord of active materials which displace dihydromorphine in an opiate-binding assay, Brain Research, 268 (1983) 119-128.
156
BRE20969
Spinal cord d y n ~ i n
.ray m o d u ~ n ~ i ~ i o n in chronic arthritic rats
via a x-~ioid receptor
M. J. MILLANl, M. H. MILLANl, C. W. T. PILCHER1, A. CZLONKOWSKP, A. HERZ: and F. C. COLPAERT2-
1Department of Neuropharmacology, Max-Planck-lnstitutfar Psychiatrie, Am Klopferspitz 18a, D-8033 Planegg-Martinsried (F.R. G.) and 2Janssen Pharmaceutica Research Laboratories, B-2340 Beerse (Belgium) (Accepted March 12th, 1985)
Key words: dynorphin
Met-enkephalin - - opioid receptor - - spinal cord - - pare - - noclception - - arthritis
Inoculation of rats with Mycobacterium butyricum produced an arthritis of the limbs which revealed an enhanced sensitivity to noxious mechanical pressure (hyperalgesia). Arthritic rats displayed a pronounced rise in immtmoreactive dynorphin in lumbo,saeral spinal cord which correlated both with the intensity and time-course of this hyperalgesia. MR-2266, a relatively preferenfialantagonist at the x-opioid receptor (at which dynorphin is conside~;edto act) potentiated this hyperatgesia. In contrast, MR 2267 (its inactive stereoisomer) was ineffective. Further. naloxone (a weak'u-antagonist), and ICI 154,129 (a preferential &antagonist) were, in each case, inactive. The data demonstrate a pronounced response of spinal dynorphin to chronic arthritic pain in the rat. In addition, they raise the possibility of a function of spinal DYN, via a x-receptor, in the modulation of chronic arthritic pain.
Dynorphin A1-17 (DYN), a major product of the p r o - D Y N (pro-enkephalin B) gene, is present in high concentrations in the dorsal horn of the spinal cord in a position suggestive of its participation in the processing of afferent nociceptive informationl,9. D Y N displays a preference for the x-opioid receptor which is found in a high density in the dorsal spinal cord. and for which D Y N may be an endogenous ligand4,SA9. Both D Y N and synthetic x-agonists upon direct injection into the spinal cord of the rat produce antinociception, most notably against non-thermal stimuli7A4,15. Acute pain has been indicated to mobilize D Y N within the spinal cord and. under these conditions, a role of spinally-localized x-opioid receptors in the mediation of the concomitant antinociception has been proposed11,12, 20. Chronic pain has not, as yet, been examined as regards its possible influence upon the activity of spinal cord pools of DYN. It is, however, of particular interest in view of data acquired with chronic arthritic pain in the rat which is associated with an alteration in levels of immunoreactive (Jr) Met-enkephalin (ME) in the spinal cord 2,6.
In the light of the above observations, we have undertaken a parallel biochemical and behaviourat evaluation of the function response of spinal D Y N to chronic (arthritic) pain in the rat. Rats were intradermally inoculated with 0.05 ml Mycobacterium butyricum suspension (or vehicle as a control) at the taft-base to induce chronic arthritis3. They were housed individually with free access to water. Nociceptive thresholds were evaluated as follows: an automated gauge applied an incremental pressure via a wedge-shaped piston to the right hindpaw and the pressure required to elicit withdrawal, the paw pressure threshold (PPT) was determined. This was evaluated m rats subjected to biochemical analysis 1 day prior to sacrifice. In independent rats, for evaluation of the influence of drugs upon PPT. they (or saline) were injected s.c. in a volume of 4.0 ml/kg at the following doses: naloxone at 5 mg/kg; M R 2266 at 5 mg/kg; M R 2267 at 5 mg/kg and ICI-154.129 at 30 mg/kg. The PPT was measured 15 rain following drug application. The person monitoring the PPT was blind as to the drug injected. All animals were sacrificed by decapitation and hind-
Correspondence: M. J. Millan. Department of Neuropharmacology, Max-Planck-Institut fiir Psychiatrie. Am Klopferspitz 18a. D-8033 Pianegg-Martinsried. F. R. G. 0006-8993/85/$03.30 (~ 1985 Elsevier Science Publishers B.V. [Biomedical Division)
157
b.
Q°
150
~150 E _e
o
~
D
75
e~ i
o Q.
0
c
material and performance of the assays for ir-DYN and if-ME, in addition to properties of the antisera, were extensively described previouslyS,9.17. Briefly, the antiserum against DYN (generously provided by Dr. E. Weber) did not recognize DYN B, DYNI_ 8, a-neo-endorphin, leucine-or methionine-enkephalin, nor fl-endorphin. The detection limit was 5 fmol/tube. The antiserum against ME showed only 0.25% cross-reactivity to leucine-enkephalin and did not bind any of the above-specified opioid peptides. Ir-DYN was evaluated at one, 3 and 10 weeks postinoculation and if-ME at 3 weeks. At 3 weeks, rats showed a pronounced arthritic swelling and inflammation of the hind-limbs: control weight 2.21 _+ 0.11 g vs arthritis 4.06 +_ 0.15 g (P ~< 0.001; Student's two-tailed t-test). This was accompanied by a byperaigesia, that is a reduction in PPTs (Fig. la). These rats showed a pronounced increase in concentrations of ir-DYN in lumbo-sacral spinal cord to 212.8 + 12.9% of control values (= 100%) (Fig. lb). This rise was significantly and negatively correlated (Pearson Product-Moment Correlation Coefficient) with the intensity of hyperalgesia; i.e. rats showing the greatest hyperalgesia displayed the greatest rise in ir-DYN (Fig. lc). Further, a parallelism was seen in the time-course for the development and disappearance of the hyperalgesia and shifts in ir-DYN (Table I). In addition, in line with other authors 2,6, w e found a modest rise in ir-Met-enkephalin (ME) in the cord; (control, 436.2 + 38.2 vs arthritis 565.4 + 36.2, fmol/mg, P ~< 0.02, Student's twotailed t-test). This did not significantly correlate (r = _+ 0.07, Pearson Product-Moment Correlation Coefficient) with the intensity of the hyperalgesia. Further, we have found that neither ir-vasopressin nor ir-
o
E
8
0
A
C
A
C. "~ 200
E
r = - 0.69 p<- 0 0 2
o
E 150'
I-
-~ 100. i
._~
50.
80
160
paw-pressure threshold (g) Fig. 1. The influence of chronic arthritis upon (a) paw-pressure thresholds and (b) levels of immunoreactive (ir)-dynorphin in lumbo-sacral spinal cord. C = control (n = 10) and A = Arthritis (n - 11). Mean _+ S.E.M. depicted. Significance of A vs C values depicted. ***, P ~< 0.001 (Student's two-tailed t-test). In panel c, the correlation between levels of immunoreactivedynorphin and paw-pressure thresholds for individual arthritic rats is depicted, r - Pearson P r o d u c t - M o m e n t correlation coefficient.
paw weight determined as an index of the intensity of arthritic symptoms. In rats examined biochemically, the spinal cord was rapidly dissected out and, for preparation for radioimmunoassay of ir-DYN, and if-ME incubated in 5 vol. 0.1 HCI for 10 min at 97 °C, then frozen on dry ice. Procedures for preparation of TABLE I
Parallel appearance and disappearance of hyperalgesia and shifts in immunoreactive-dynorphin in lumbo-sacral spinal cord of arthritic rats Paw pressure thresholds are in g and immunoreactive dynorphin in fmol/mg tissue, wet weight. C signifies control and A arthritis. Data are given as m e a n + S.E.M. (n >/8 for each value presented). Significance of A vs C values indicated.
Weeks post-inoculation 1 Pawpressure C Thresholds A Immunoreactive C Dynorphin A
129.23 123.46 47.34 49.94
*** P ~ 0.001 (Student's two-tailed t-test).
_+ 10.62 _+ 6.21 + 4.28 + 6.20
3
10
125.16 _+ 8.94 75.31 _+ 7.67*** 59.51 _+ 4.82 126.59 _+ 4.41"**
116.62 109.22 51.16 56.72
_+ 7.51 _+ 8.11 _+ 2.32 + 4.90
158
2
o
ii io0
_2_
5oi
NLX PiR2266 MR2267 CONTROL
IC1154
SA-L"
NIX
M ~ ( g 5 MR2267 ICt154
ARTHRITIS
Fig. 2. The influence of acute treatment with various opioid receptor antagonists (see text) upon paw-pressure thresholds of control and arthritic rats. SAL = saline; NLX = naloxone. Mean + S.E.M. depicted. Significance of MR 2266 vs SAL values indicated. **, P<~0.0I (Student's two-tailedt-test). oxytocin are affected in the spinal cord of arthritic rats 10, suggestive of a certain neurochemical specificity in these effects. Fig. 2 shows that naloxone, a powerful #-receptor but a weak ~- (and d-) receptor antagonist TM, failed to signficantly affect PPTs in either control or arthritic rats. In contrast, MR 2266, which is a much more potent inhibitor of antinociception induced by intrathecal DYN and possesses greater n-receptor-blocking activity7,14, TM, significantly potentiated the hyperalgesia in arthritic rats. Its inactive stereoisomer, MR 2267, did not, in contrast, affect PPTs in arthritic rats indicating the stereospecificity of this action. Finally, a preferential 6-antagonist 16, ICI-154i129 failed to affect the PPT of arthritic rats. We found, in addition to this stereospecificity of action of MR 2266, that it was specific for non-thermal nociception since it did not affect the response of the paws to noxious heat (not shown). The present study shows chronic arthritic pain in the rat to be associated with a large and reversible rise in levels of ir-DYN in lumbo-sacral cord. That this rise reflects a response to the pain is supported by the following observations. Firstly, acute pain has been indicated to enhance the activity of DYN neurones in lumbo-sacral cordn, 20. Secondly, the chronic pain of recurrent foot-shock similarly elevates spinal cord levels of ir-DYN 13. Thirdly, the rise in ir-DYN in arthritic rats significantly correlated with the intensity of hyperalgesia (Fig. lc). Fourthly, inoculation
of rats in fore- or hind-paws leads to a rise in ir-DYN and ir-ME in, respectively, cervical or lumbo-sacral cord demonstrative that this is a response to a localized pain6,13. Thus, it is reasonable to infer that the rise in ir-DYN reflects a response to arthritic pain in our rats. Further, though recent studies of Jr-ME release in the spinal cord of arthritic rats failed to satisfactorily resolve the issue 2, the contention that the rise in ir-DYN reflects an enhanced activity appears the most reasonable hypothesis in the light of the above-mentioned points. Further, in current studies (Millan et al., in prep.) we have observed a downregulation in n-receptors in the spinal cord of arthritic rats; this is consistent with a facilitation rather than a suppression of the activity of DYN, since DYN is considered to act upon the x-receptor s:~. Indeed, in support of the above reasoning, MR 2266, an antagonist with greater activity than naloxone at x-receptors and much more potent in blocking antinociception induced by DYN injected into the cord 7,14,1s potentiated the hyperalgesia seen in arthritic rats. We suggest that this action on PPT, a response integrated in lumbo-sacral spinal cord, may reflect blockade of an action of endogenous DYN, the shift in levels of which correlated to the hyperalgesia both in its intensity and time-course. Support for a putative physiological role of spinal cord DYN in control of chronic pain is provided by the finding that both in arthritic and naive rats, exogenous DYN (or other x-agonists) intrathecally administered to the lumbo-sacral cord results in an antinociception 7,13-15. Interestingly, the present data with chronic pain offer a direct parallel to acute pain which has likewise been indicated to be accompanied by an augmented activity of DYN neurones and a modulation of nociception mediated by x-opioid receptors~.12.20. In conclusion, thus, the present and previous data suggest that acute or chronic pain activates spinal cord pools of DYN which may operate therein in the modulation of nociception. We thank Drs. V. H61tt and E. Weber f o r antisera against, respectively, DYN and ME. M 3 , M . was supported by the Deutsche Forschungsgemeinschaft. A.C. is a visiting scientist from the Department of Pharmacology, Institute of Physiological Sciences, Medical Academy of Warsaw, Poland.
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