Brain Research, 548 (1991) 287-291 Elsevier ADONIS 000689939116587L
287
BRES 16587
Autoradiographic distribution of/ , 6 and r opioid binding sites in the superficial dorsal hom, over the rostrocaudal axis of the rat spinal cord D. Besse, M.C. Lombard, and J.M. Besson Unit# de Recherche de Neurophysiologie Pharmacologique (INSERM, Unit~ 161) and Laboratoire de Physiopharmacologie de la Douleur (EPHE), Paris (France)
(Accepted 4 December 1990) Key words: Opioid receptor; Rat spinal cord; Lamina I; Lamina II; Quantitative autoradiography
The purpose of this study was to use [3H]DAMGO, [3H]DTLET and [3H]EKC in the presence of 100 nM DAMGO and 100 nM DTLET, combined with a quantitative autoradiography to analyse the different proportions and the rostrocaudal distribution of/~, t5 and r opioid binding sites in the superficial layers (laminae I and II) of the cervical (Ct-Cs), thoracic (Ts-TT), lumbar (La-L5) and sacral ($2-$3) dorsal horn of the rat. The proportions of the three main types of opioid binding sites, assessed by autoradiography in laminae I and II, were found homogeneous at each segmental level considered: 70.4-74.3%, 18.4-20.3% and 7.3-9.5% for/~, 6, r sites, respectively. The physiological relevance of these data is discussed.
INTRODUCTION Opioid binding sites are classically known to be principally localized in the superficial layers (laminae I and II) of the dorsal horn 9'14'18. In a recent autoradiographic study 1, we have reported the proportions of the three main types of opioid binding sites/~, 6 and r, in laminae I and II of the cervical enlargement. These binding sites have a physiological role since iontophoretic application of opioid substances in the superficial dorsal horn reduces the responses of dorsal horn neurons to nociceptive stimulation (see references in ref. 6). This observation is relevant to the analgesic effects induced by intrathecal or systemic (in spinalized animals) administration of opioids (see ref. 17). There is a great interest in the relationship between physiological effects of opioids and anatomical data (localization, density) on opioid receptors. Thus, some binding studies have described rostrocaudal differences in spinal cord opioid receptors s'9 while, in others, homogeneous distribution o f / , , 6 and r opioid binding sites was reported 15 (see also ref. 3). Thus, the purpose of the present study was to use highly selective ligands and quantitative autoradiography to gauge the rostrocaudal distribution and the different proportions of/~, 6 and r opioid binding sites in the
superficial layers of the cervical, thoracic, lumbar and sacral dorsal horn of the rat. Autoradiography offers the great advantage of being able to focus analysis on laminae I and II and to allow a quantification of autoradiograms. Preliminary binding assays on slices were c a r d e d out to assess affinities and binding capacities for/~, 6 and r opioid sites in the spinal cord of the rat. MATERIALS AND METHODS Experiments were performed on Sprague-Dawley albino rats, weighing 200-225 g. Rats were killed by decapitation and cervical (Ct-C s or C4-T2), thoracic (Ts-TT) , lumbar (La-L5) and sacral ($2-$3) parts of spinal cord were rapidly removed, frozen in isopentane at -40 °C and preserved at -80 °C. For preliminary saturation studies, cervico-thoracic extents C4-T2 of 3 rats were cut (transversal sections 15/zm thick) in a cryostat at -20 °C. Crossections were thaw-mounted onto gelatinized slides (30 spinal cord sections per slide) and kept at -80 *(2 up to incubation. Sections were distributed so that every spinal segment was present on each slide. Sections were then brought to room temperature and placed in incubation chambers maintained at 25 °C and 60-80% of relative humidity. Seven different concentrations were used for each ligand. Sets of two slides per rat were incubated for 60 rain in 500 /~l of 50 mM Tris-HCl buffer (pH 7.4) containing 0.5-25 nM [3H]Tyr-D-Ala-Gly-NMe-Phe-Gly-ol ([3H]DAMGO, 1.85 TBq/ mmol, CEN Saelay) to label /~ sites, 0.5-20 nM [3H]Tyr-DThr-Gly-Phe-Leu-Thr ([3H]DTLET, 2.22 TBq/mmol, CEN Saclay) to label 6 sites and 0.5-18 nM [3H]ethylketocyclazocine ([3H]EKC, 0.55 TBq/mmol, NEN) in the presence of DAMGO 100 nM and DTLET 100 nM, to label r sites. The non-specific binding was
Correspondence: Unit6 de Recherche de Neurophysiologie Pharmacologique (INSERM, Unit6 161) and Laboratoire de Physiopharmacologie de la Douleur (EPHE), 2 rue d'Altsia 75014 Paris, France.
288 evaluated in the presence of l0 -~ M levorphanol for the 3 ligands. At the end of the incubation, the sections were washed twice in 400 ml of ice-cold 50 mM Tris-HCl buffer (pH 7.4) for l0 min each, rinsed in ice-cold bidistilled water and air-dried. The 30 total crossections were scraped with a blade and placed inside a scintillation vial (5 ml of NEN Biofluor emulsifier cocktail) to count radioactivity. Each of the 7 saturation curves points was an average of two measurements (one measurement corresponding to one slide) for each of the 3 rats. The non-specific binding was measured from one slide per concentration, for each rat. Binding parameters at equilibrium were determined from the linear regression of Scatchard. For autoradiography, spinal cord sections of each segmental level were incubated for 60 min at 25 °C in 50 mM Tris-HCl buffer (pH 7.4) containing [3H]DAMGO 3 nM for/~ sites, [3H]DTLET 3 nM for 6 sites and [3H]EKC 10 nM, in the presence of DAMGO 100 nM and DTLET 100 nM, for r sites. The non specific binding was evaluated in presence of 10 -6 M levorphanol for the 3 ligands. At the end of the incubation, the sections were washed twice in 400 ml of ice-cold 50 mM Tris-HCI buffer (pH 7.4) for 10 min each, rinsed in ice-cold bidistilled water and air-dried. The labeled sections were brought into close contact with tritium-sensitive film (Amersham) and exposed for 15 weeks at 4 °C. Autoradiograms were developed about 2 min in Kodak D19. Quantitative measurements of autoradiograms were performed every 120/~m with a Biocom analyser system (about 15 sections measured for each spinal cord level). The Biocom system allows us to convert grey values to binding site density values by reference to calibrated tritium standards (Amersham). The area of measurements corresponded to the superficial layers of the dorsal horns (laminae I and II, see ref. 1). The specific binding was calculated by subtracting the amount of ligand bound from total binding in sections incubated for non-specific labeling. The quantity of sites was calculated as:
Rostrocaudal distribution of/~, 6 and ~ opioid binding sites in the superficial layers of the dorsal horn For the three ligands, as previously mentioned by various authors, autoradiograms reveal a p r e d o m i n a n t binding in the superficial layers of the dorsal horn (Fig. 2). Yet, the total [3H]EKC b o u n d observed in the present study seems very weak. The non-specific binding corresponds to about 13-18%, 2 0 - 3 0 % and 5 5 - 7 5 % of the total b o u n d for [ 3 H ] D A M G O , [3H]DTLET and [3H]EKC, respectively in the superficial layers of the dorsal horn. For the 3 ligands, the binding capacities have been calculated by taking into account our measurements
10 z B / F
15
40 lO
DAMGO
o
10
10
20
30 F(nM)
20
B(fmot/section)
107 B/F
Bin.x = B (1 + Kd/F) with B corresponding to the amount of bound ligand for a concentration F of free ligand and Kd its constant of affinity. Statistical analysis was made using an ANOVA-test followed by the multiple range test LSD (Least Significant Difference).
15
60
10
40
DTLET
RESULTS
o
O
10
20
20
B (fmell section)
10
20
20
Characterization of binding properties at equilibrium, on total crossections of the spinal cord Saturation analysis of the specific binding of [ 3 H ] D A M G O , [3H]DTLET and [3H]EKC (in the presence of 100 n M D A M G O and 100 n M D T L E T ) has revealed saturable binding. Linear Scatchard curves (Fig. 1) indicated binding to one type of receptor for each ligand. Data shows that [ 3 H ] D A M G O binds to approximately 50% (13.5 + 0.5 fmoi/section), [3H]DTLET 30% (9.0 __+ 0.2 fmol/section) and [3H]EKC 20% (5.3 + 0.2 fmol/ section) of the total a m o u n t of opioid binding sites in the cervico-thoracic (C4-T2) spinal cord. The K d values were found as follows: 4.2 + 0.3, 1.6 + 0.2 and 1.4 + 0.1 nM (expressed as mean + S . E . M . , n = 3) for [ 3 H ] D A M G O , [3H]DTLET and [3H]EKC, respectively. The constants of dissociation which were obtained from this preliminary study, have been used to calculate the maximal binding capacities in the autoradiographical study.
30 FlnM)
L 0
10
106 B/F 6 40 3
EKC
0 0
20
30 F(nM)
=
0
5
10
B (fmol/section)
Fig. 1. Scatchard analysis of the specific binding of [3H]DAMGO, [3H]DTLET and [3H]EKC (in the presence of 100 nM DAMGO and 100 nM DTLET), to rat spinal cord slices. The corresponding saturation curves are inserted in the top right of each diagram. Each experimental point corresponds to the mean value of 3 determinations (n = 3) performed in duplicate.
289
Fig. 2. Photographs of the autoradiographic distribution of the binding of the 3 ligands at various levels of the rat spinal cord: cervical (eer.), thoracic (th.), lumbar (lum.) and sacral (sac.). A: bindingof [3H]DAMGO ~ sites). B: binding of [aH]DTLET (6 sites). C: binding of [3H]EKC in the presence of 100 nM DAMGO and 100 nM DTLET (r sites). of specific bound and the above values of K d. The binding capacities clearly indicate a majority of/~ opioid binding sites, while 6 binding sites are found in moderate levels with only very few r binding sites (Table I). This arrangement is found whatever the spinal cord level (i.e. cervical, thoracic, lumbar and sacral levels). In addition, binding capacities for [3H]DAMGO (55.4-59.9 fmol/mg of tissue equivalent), [3H]DTLET (14.8-16.2) and
[3H]EKC (5.8-7.5) are similar at each considered spinal cord level. Consequently, the whole specific opioid binding appears homogeneous for all the 4 levels. Fig. 3 illustrates at each level, the relative proportions of/a, and r opioid binding sites expressed as percentages of total opioid binding sites present in the superficial layers of the dorsal horn.
290 CERVICAL
THORACIC
74%
72%
8%
SACRAL
LUMBAR
70%
74%
10%
7"/o
zu~o
p sites
~
~"sites
I
X sites
Fig. 3. Respective proportions of/~, ~ and ~ opioid binding sites in the superficial layers of the rat dorsal horn, at the cervical (C6-C8), thoracic (Ts-T7), lumbar (La-L5) and sacral ($2-$3) levels.
DISCUSSION The present study confirms our previous data 1 obtained at the cervical enlargement demonstrating, at the level of the superficial dorsal horn, high percentages of/~ opioid binding sites, with intermediate 6 and low binding sites, and extends them to the thoracic, lumbar
TABLE I Specific binding capacities of [3H]DAMGO, [3H]DTLET and [3H]EKC (in the presence of lO0 nM DAMGO and 10(9nM DTLET) at various rostrocaudal levels of the superficial dorsal horn, as determined by computerized densitometry
Values are expressed in fmol/mg of tissue equivalent and represent means _+S.E.M. (n = 3). The total opioid binding sites correspond to the sum oI/~, dtand r binding sites. For each ligand, statistical analysis has revealed homogeneous groups whatever the spinal cord level (/73,8 = 0.082 for [3H]DAMGO, F~,~ = 0.283 for [3H]DTLET and F3.8 = 0.834 for [3H]EKC). Spinal cord level
Specific binding capacity DAMGO
DTLET
EKC
Total opioid binding sites
Cervical(C6-C8) Thoracic(Ts-TT) Lumbar(L3-Ls) Sacral(S2-S3)
59.4 + 2.9 57,2 + 4.9 55.4 + 5.7 59.9_+ 2.9
14.8+ 0.6 16.2+ 0.7 15.8+ 1.1 14.8+ 0.4
6.3 + 0.7 6.3 + 0.6 7.5 + 0.2 5.8 + 0.2
80.5 79.6 78.6 80.5
and sacral levels. Moreover, for each ligand, similar values of specific binding were measured along the axis of the spinal cord (see Table I). Accordingly, the relative proportions of/~ (70.4-74.4%), di (18.4-20.3%) and ~¢ (7.3-9.5%) sites remain stable in the various studied levels. To our knowledge, this is the first quantitative study demonstrating a homogeneous distribution of the 3 main types of opioid binding sites all along the superficial dorsal horn of the rat spinal cord. Our data strongly differ from those of previous binding 3's']s or autoradiographical 9 studies showing different proportions for the 3 types of opioid receptors 3' 8,9.15 and/or a rostrocaudai gradient of/~, 6 and • opioid binding sites in the rat and guinea pig spinal cord s'9. Unfortunately, our study is difficult to compare with those previously mentioned, since, due to methodological reasons, in these studies, the proportion of ~cbinding sites was overestimated. In fact, in these studies, the labeling of ~c sites was performed by using non-specific tritiated ligands (etorphine, bremazocine, diprenorphine) in the presence of high concentration of more or less selective /~ and 6 blockers. In addition, it has been shown that the quantity of ~¢ sites measured with [3H]etorphine was greater than with [3H]EKC4, under the same experimental conditions. This could be related to the fact that [3H]etorphine still binds to a number of sites defined as /~ and 6 sites in the presence of high concentrations of/~ and ~ ligands inducing an overestimation of the number of ~¢ sites. The technical approach can also explain, at least in part, the divergent proportions o f ~ , b and K sites observed in literature. Effectively, as illustrated in the present study, different values for the respective proportions of the 3 main types of opioid binding sites were obtained accordingly, as only the total cross-section of the spinal cord, or the superficial layers of the dorsal horn were measured. In this respect, for each of the 3 ligands, we have observed different values for the ratios 'superficial layers (laminae I and II) versus deeper layers (laminae I, 11, IV and V)' (results not published). The ratio calculated with [ 3 H ] D A M G O was found to be greater than with [3H]DTLET or [3H]EKC. This could explain why, in our study, the results are quite different between autoradiography and the experiment using impulse counting. The quantification, as we performed it, takes into account the means of 15 values measured systematically every 120/~m. The data presented in this study reflect means obtained in about 2 spinal segments at each level. The only autoradiographical study reported in the literature 9 relative to the rostrocaudai distribution of bt, 6 and 1¢ binding sites in the spinal cord presents only qualitative data. Now, such data are well known to support a non-negligible part of subjective interpretation.
291 From the present autoradiographical study, using highly selective ligands and quantitative measurements, several conclusions can be drawn: (a) Since, in the superficial layers of the dorsal horn, opioid binding sites mainly reflect presynaptic binding sites 1 located on thin primary afferent fibers 7'13, our data suggest that there is an identical proportion of the three main types of opioid receptors, whatever the peripheral location and/or origin of nociceptors. (b) The high levels o f / z , intermediate 6 and low r binding sites are in good agreement with several electrophysiological 5'1° and pharmacological H (see also ref. 16) studies based on intrathecal administration of opioid substances, showing that the analgesic effects of/z agonists were greater than that produced by r agonists, with 6 ligands eliciting intermediate effects.
(c) Although speculative, the similar densities o f / z opioid receptors in the superficial layers of the dorsal horn along the length of the spinal cord support the idea that morphine could be equally effective in the relief of pain due to an excess of nociception arising from any part of the body. Despite the lack of systematic study in animals in order to assess the efficacy of intrathecal administration of morphine at different levels of the neuraxis, clinical reports (see refs. in ref. 2) claim that a high percentage of cancer pain originating from various parts of the body can be adequately managed by intrathecal or epidural administration of morphine. Acknowledgements. We thank M.J. Besson and her group for their friendly aid to perform binding studies on spinal cord slices, D.A. Dickenson for critical reading of the manuscript and E. Dehausse for drawings and photographies.
REFERENCES 1 Besse, D., Lombard, M.C., Zajac, J.M., Roques, B.P. and Besson, J.M., Pre- and postsynaptic distribution of/~, 6 and r opioid receptors in the superficial layers of the cervical dorsal horn of the rat spinal cord, Brain Research, 521 (1990) 15-22. 2 Besson, J.M. and Lazorthes, Y. (Eds.), Spinal opioids and the relief of pain; basic mechanisms and clinical applications, Les Editions INSERM, (Colloq. INSERM), Paris, Vol. 127, 1985, pp. 1-518. 3 Czlonkowski, A., Costa, T., Przewlocki, R., Pasi, A. and Herz, A., Opiate receptor binding sites in human spinal cord, Brain Research, 267 (1983) 392-396. 4 Delay-Goyet, E, Roques, B.P. and Zajac, J.M., Differences of binding characteristics of non-selective opiates towards/z and receptors types, Life Sci., 41 (1987) 723-731. 5 Dickenson, A.H., Sullivan, A.F., Knox, R.J., Zajac, J.M. and Roques, B.P., Opioid receptor subtypes in the rat spinal cord: electrophysiological studies with/~ and t5opioid receptor agonists in the control of nociception, Brain Research, 413 (1987) 36-44. 6 Duggan, A.W. and North, R.A., Electrophysiology of opioids, PharmacoL Rev., 35 (1984) 219-281. 7 Gamse, R., Holzer, P. and Lembeck, F., Indirect evidence for presynaptic location of opiate receptors on chemosensitive primary sensory neurones, Naunyn-Schmiedeberg's Arch. Pharmacol., 308 (1979) 281-285. 8 Gouard~,res, C. and Cros, J., Opioid binding sites in different levels of rat spinal cord, Neuropeptides, 5 (1984) 113-116. 9 Gouard~.res, C., Cros, J. and Quirion, R., Autoradiographic localization of mu, delta and kappa opioid receptor binding sites in rat and guinea pig spinal cord, Neuropeptides, 6 (1985) 331-342. 10 Knox, R.J. and Dickenson, A.H., Effects of selective and
11
12 13
14 15 16
17 18
non-selective r opioid receptor agonists on cutaneous Cfibre-evoked responses of rat dorsal horn neurones, Brain Research, 415 (1987) 21-29. Leighton, G.E., Rodriguez, R.E., Hill, R.G. and Hughes, J., K-Opioid agonists produce antinociception after i.v. and i.c.v. but not intrathecal administration in the rat, Br. J. PharmacoL, 93 (1988) 553-560. Morris, B.J. and Herz, A., Distinct distribution of opioid receptor types in rat lumbar spinal cord, Naunyn-Schmiedeberg's Arch. Pharmacol., 336 (1987) 240-243. Nagy, J.I., Vincent, S.R., Staines, W.M.A., Fibiger, H.C., Reisine, T.D. and Yamamura, H.I., Neurotoxic' action of capsaicin on spinal substance P neurons, Brain Research, 186 (1980) 435-444. Pert, C.B., Kuhar, M.J. and Snyder, S.H., Autoradiographic localization of the opiate receptor in rat brain, Life Sci., 16 (1975) 1849-1854. Traynor, J.R. and Wood, M.S., Distribution of opioid binding sites in spinal cord, Neuropeptides, 10 (1987) 313-320. Yaksh, T.L., Durant, E, Onofrio, B. and Stevens, C.W., The effect of spinally administered agents on pain transmission in man and animals. In J.M. Besson and Y. Lazorthes (Eds.), Substances opioides medullaires et analg~sie (Spinal opioids and the relief of pain), Les Editions INSERM, Paris (Colloq. INSERM), Paris, Vol. 127, 1985, pp. 267-306. Yaksh, T.L. and Noueihed, R., The physiology and pharmacology of spinal opiates, Annu. Rev. Pharmacol. Toxicol., 25 (1985) 433-462. Zajac, J.M., Peschanski, M., Besson, J.M. and Roques, B.P., High-resolution autoradiography of opioid receptors and enkephalinase in the rat spinal cord. In Proceedings of the Vth World Congress on Pain, Pain Research and Clinical Management, Vol. 3, 1988, pp. 436-441.
287
BRES 16587
Autoradiographic distribution of/ , 6 and r opioid binding sites in the superficial dorsal hom, over the rostrocaudal axis of the rat spinal cord D. Besse, M.C. Lombard, and J.M. Besson Unit# de Recherche de Neurophysiologie Pharmacologique (INSERM, Unit~ 161) and Laboratoire de Physiopharmacologie de la Douleur (EPHE), Paris (France)
(Accepted 4 December 1990) Key words: Opioid receptor; Rat spinal cord; Lamina I; Lamina II; Quantitative autoradiography
The purpose of this study was to use [3H]DAMGO, [3H]DTLET and [3H]EKC in the presence of 100 nM DAMGO and 100 nM DTLET, combined with a quantitative autoradiography to analyse the different proportions and the rostrocaudal distribution of/~, t5 and r opioid binding sites in the superficial layers (laminae I and II) of the cervical (Ct-Cs), thoracic (Ts-TT), lumbar (La-L5) and sacral ($2-$3) dorsal horn of the rat. The proportions of the three main types of opioid binding sites, assessed by autoradiography in laminae I and II, were found homogeneous at each segmental level considered: 70.4-74.3%, 18.4-20.3% and 7.3-9.5% for/~, 6, r sites, respectively. The physiological relevance of these data is discussed.
INTRODUCTION Opioid binding sites are classically known to be principally localized in the superficial layers (laminae I and II) of the dorsal horn 9'14'18. In a recent autoradiographic study 1, we have reported the proportions of the three main types of opioid binding sites/~, 6 and r, in laminae I and II of the cervical enlargement. These binding sites have a physiological role since iontophoretic application of opioid substances in the superficial dorsal horn reduces the responses of dorsal horn neurons to nociceptive stimulation (see references in ref. 6). This observation is relevant to the analgesic effects induced by intrathecal or systemic (in spinalized animals) administration of opioids (see ref. 17). There is a great interest in the relationship between physiological effects of opioids and anatomical data (localization, density) on opioid receptors. Thus, some binding studies have described rostrocaudal differences in spinal cord opioid receptors s'9 while, in others, homogeneous distribution o f / , , 6 and r opioid binding sites was reported 15 (see also ref. 3). Thus, the purpose of the present study was to use highly selective ligands and quantitative autoradiography to gauge the rostrocaudal distribution and the different proportions of/~, 6 and r opioid binding sites in the
superficial layers of the cervical, thoracic, lumbar and sacral dorsal horn of the rat. Autoradiography offers the great advantage of being able to focus analysis on laminae I and II and to allow a quantification of autoradiograms. Preliminary binding assays on slices were c a r d e d out to assess affinities and binding capacities for/~, 6 and r opioid sites in the spinal cord of the rat. MATERIALS AND METHODS Experiments were performed on Sprague-Dawley albino rats, weighing 200-225 g. Rats were killed by decapitation and cervical (Ct-C s or C4-T2), thoracic (Ts-TT) , lumbar (La-L5) and sacral ($2-$3) parts of spinal cord were rapidly removed, frozen in isopentane at -40 °C and preserved at -80 °C. For preliminary saturation studies, cervico-thoracic extents C4-T2 of 3 rats were cut (transversal sections 15/zm thick) in a cryostat at -20 °C. Crossections were thaw-mounted onto gelatinized slides (30 spinal cord sections per slide) and kept at -80 *(2 up to incubation. Sections were distributed so that every spinal segment was present on each slide. Sections were then brought to room temperature and placed in incubation chambers maintained at 25 °C and 60-80% of relative humidity. Seven different concentrations were used for each ligand. Sets of two slides per rat were incubated for 60 rain in 500 /~l of 50 mM Tris-HCl buffer (pH 7.4) containing 0.5-25 nM [3H]Tyr-D-Ala-Gly-NMe-Phe-Gly-ol ([3H]DAMGO, 1.85 TBq/ mmol, CEN Saelay) to label /~ sites, 0.5-20 nM [3H]Tyr-DThr-Gly-Phe-Leu-Thr ([3H]DTLET, 2.22 TBq/mmol, CEN Saclay) to label 6 sites and 0.5-18 nM [3H]ethylketocyclazocine ([3H]EKC, 0.55 TBq/mmol, NEN) in the presence of DAMGO 100 nM and DTLET 100 nM, to label r sites. The non-specific binding was
Correspondence: Unit6 de Recherche de Neurophysiologie Pharmacologique (INSERM, Unit6 161) and Laboratoire de Physiopharmacologie de la Douleur (EPHE), 2 rue d'Altsia 75014 Paris, France.
288 evaluated in the presence of l0 -~ M levorphanol for the 3 ligands. At the end of the incubation, the sections were washed twice in 400 ml of ice-cold 50 mM Tris-HCl buffer (pH 7.4) for l0 min each, rinsed in ice-cold bidistilled water and air-dried. The 30 total crossections were scraped with a blade and placed inside a scintillation vial (5 ml of NEN Biofluor emulsifier cocktail) to count radioactivity. Each of the 7 saturation curves points was an average of two measurements (one measurement corresponding to one slide) for each of the 3 rats. The non-specific binding was measured from one slide per concentration, for each rat. Binding parameters at equilibrium were determined from the linear regression of Scatchard. For autoradiography, spinal cord sections of each segmental level were incubated for 60 min at 25 °C in 50 mM Tris-HCl buffer (pH 7.4) containing [3H]DAMGO 3 nM for/~ sites, [3H]DTLET 3 nM for 6 sites and [3H]EKC 10 nM, in the presence of DAMGO 100 nM and DTLET 100 nM, for r sites. The non specific binding was evaluated in presence of 10 -6 M levorphanol for the 3 ligands. At the end of the incubation, the sections were washed twice in 400 ml of ice-cold 50 mM Tris-HCI buffer (pH 7.4) for 10 min each, rinsed in ice-cold bidistilled water and air-dried. The labeled sections were brought into close contact with tritium-sensitive film (Amersham) and exposed for 15 weeks at 4 °C. Autoradiograms were developed about 2 min in Kodak D19. Quantitative measurements of autoradiograms were performed every 120/~m with a Biocom analyser system (about 15 sections measured for each spinal cord level). The Biocom system allows us to convert grey values to binding site density values by reference to calibrated tritium standards (Amersham). The area of measurements corresponded to the superficial layers of the dorsal horns (laminae I and II, see ref. 1). The specific binding was calculated by subtracting the amount of ligand bound from total binding in sections incubated for non-specific labeling. The quantity of sites was calculated as:
Rostrocaudal distribution of/~, 6 and ~ opioid binding sites in the superficial layers of the dorsal horn For the three ligands, as previously mentioned by various authors, autoradiograms reveal a p r e d o m i n a n t binding in the superficial layers of the dorsal horn (Fig. 2). Yet, the total [3H]EKC b o u n d observed in the present study seems very weak. The non-specific binding corresponds to about 13-18%, 2 0 - 3 0 % and 5 5 - 7 5 % of the total b o u n d for [ 3 H ] D A M G O , [3H]DTLET and [3H]EKC, respectively in the superficial layers of the dorsal horn. For the 3 ligands, the binding capacities have been calculated by taking into account our measurements
10 z B / F
15
40 lO
DAMGO
o
10
10
20
30 F(nM)
20
B(fmot/section)
107 B/F
Bin.x = B (1 + Kd/F) with B corresponding to the amount of bound ligand for a concentration F of free ligand and Kd its constant of affinity. Statistical analysis was made using an ANOVA-test followed by the multiple range test LSD (Least Significant Difference).
15
60
10
40
DTLET
RESULTS
o
O
10
20
20
B (fmell section)
10
20
20
Characterization of binding properties at equilibrium, on total crossections of the spinal cord Saturation analysis of the specific binding of [ 3 H ] D A M G O , [3H]DTLET and [3H]EKC (in the presence of 100 n M D A M G O and 100 n M D T L E T ) has revealed saturable binding. Linear Scatchard curves (Fig. 1) indicated binding to one type of receptor for each ligand. Data shows that [ 3 H ] D A M G O binds to approximately 50% (13.5 + 0.5 fmoi/section), [3H]DTLET 30% (9.0 __+ 0.2 fmol/section) and [3H]EKC 20% (5.3 + 0.2 fmol/ section) of the total a m o u n t of opioid binding sites in the cervico-thoracic (C4-T2) spinal cord. The K d values were found as follows: 4.2 + 0.3, 1.6 + 0.2 and 1.4 + 0.1 nM (expressed as mean + S . E . M . , n = 3) for [ 3 H ] D A M G O , [3H]DTLET and [3H]EKC, respectively. The constants of dissociation which were obtained from this preliminary study, have been used to calculate the maximal binding capacities in the autoradiographical study.
30 FlnM)
L 0
10
106 B/F 6 40 3
EKC
0 0
20
30 F(nM)
=
0
5
10
B (fmol/section)
Fig. 1. Scatchard analysis of the specific binding of [3H]DAMGO, [3H]DTLET and [3H]EKC (in the presence of 100 nM DAMGO and 100 nM DTLET), to rat spinal cord slices. The corresponding saturation curves are inserted in the top right of each diagram. Each experimental point corresponds to the mean value of 3 determinations (n = 3) performed in duplicate.
289
Fig. 2. Photographs of the autoradiographic distribution of the binding of the 3 ligands at various levels of the rat spinal cord: cervical (eer.), thoracic (th.), lumbar (lum.) and sacral (sac.). A: bindingof [3H]DAMGO ~ sites). B: binding of [aH]DTLET (6 sites). C: binding of [3H]EKC in the presence of 100 nM DAMGO and 100 nM DTLET (r sites). of specific bound and the above values of K d. The binding capacities clearly indicate a majority of/~ opioid binding sites, while 6 binding sites are found in moderate levels with only very few r binding sites (Table I). This arrangement is found whatever the spinal cord level (i.e. cervical, thoracic, lumbar and sacral levels). In addition, binding capacities for [3H]DAMGO (55.4-59.9 fmol/mg of tissue equivalent), [3H]DTLET (14.8-16.2) and
[3H]EKC (5.8-7.5) are similar at each considered spinal cord level. Consequently, the whole specific opioid binding appears homogeneous for all the 4 levels. Fig. 3 illustrates at each level, the relative proportions of/a, and r opioid binding sites expressed as percentages of total opioid binding sites present in the superficial layers of the dorsal horn.
290 CERVICAL
THORACIC
74%
72%
8%
SACRAL
LUMBAR
70%
74%
10%
7"/o
zu~o
p sites
~
~"sites
I
X sites
Fig. 3. Respective proportions of/~, ~ and ~ opioid binding sites in the superficial layers of the rat dorsal horn, at the cervical (C6-C8), thoracic (Ts-T7), lumbar (La-L5) and sacral ($2-$3) levels.
DISCUSSION The present study confirms our previous data 1 obtained at the cervical enlargement demonstrating, at the level of the superficial dorsal horn, high percentages of/~ opioid binding sites, with intermediate 6 and low binding sites, and extends them to the thoracic, lumbar
TABLE I Specific binding capacities of [3H]DAMGO, [3H]DTLET and [3H]EKC (in the presence of lO0 nM DAMGO and 10(9nM DTLET) at various rostrocaudal levels of the superficial dorsal horn, as determined by computerized densitometry
Values are expressed in fmol/mg of tissue equivalent and represent means _+S.E.M. (n = 3). The total opioid binding sites correspond to the sum oI/~, dtand r binding sites. For each ligand, statistical analysis has revealed homogeneous groups whatever the spinal cord level (/73,8 = 0.082 for [3H]DAMGO, F~,~ = 0.283 for [3H]DTLET and F3.8 = 0.834 for [3H]EKC). Spinal cord level
Specific binding capacity DAMGO
DTLET
EKC
Total opioid binding sites
Cervical(C6-C8) Thoracic(Ts-TT) Lumbar(L3-Ls) Sacral(S2-S3)
59.4 + 2.9 57,2 + 4.9 55.4 + 5.7 59.9_+ 2.9
14.8+ 0.6 16.2+ 0.7 15.8+ 1.1 14.8+ 0.4
6.3 + 0.7 6.3 + 0.6 7.5 + 0.2 5.8 + 0.2
80.5 79.6 78.6 80.5
and sacral levels. Moreover, for each ligand, similar values of specific binding were measured along the axis of the spinal cord (see Table I). Accordingly, the relative proportions of/~ (70.4-74.4%), di (18.4-20.3%) and ~¢ (7.3-9.5%) sites remain stable in the various studied levels. To our knowledge, this is the first quantitative study demonstrating a homogeneous distribution of the 3 main types of opioid binding sites all along the superficial dorsal horn of the rat spinal cord. Our data strongly differ from those of previous binding 3's']s or autoradiographical 9 studies showing different proportions for the 3 types of opioid receptors 3' 8,9.15 and/or a rostrocaudai gradient of/~, 6 and • opioid binding sites in the rat and guinea pig spinal cord s'9. Unfortunately, our study is difficult to compare with those previously mentioned, since, due to methodological reasons, in these studies, the proportion of ~cbinding sites was overestimated. In fact, in these studies, the labeling of ~c sites was performed by using non-specific tritiated ligands (etorphine, bremazocine, diprenorphine) in the presence of high concentration of more or less selective /~ and 6 blockers. In addition, it has been shown that the quantity of ~¢ sites measured with [3H]etorphine was greater than with [3H]EKC4, under the same experimental conditions. This could be related to the fact that [3H]etorphine still binds to a number of sites defined as /~ and 6 sites in the presence of high concentrations of/~ and ~ ligands inducing an overestimation of the number of ~¢ sites. The technical approach can also explain, at least in part, the divergent proportions o f ~ , b and K sites observed in literature. Effectively, as illustrated in the present study, different values for the respective proportions of the 3 main types of opioid binding sites were obtained accordingly, as only the total cross-section of the spinal cord, or the superficial layers of the dorsal horn were measured. In this respect, for each of the 3 ligands, we have observed different values for the ratios 'superficial layers (laminae I and II) versus deeper layers (laminae I, 11, IV and V)' (results not published). The ratio calculated with [ 3 H ] D A M G O was found to be greater than with [3H]DTLET or [3H]EKC. This could explain why, in our study, the results are quite different between autoradiography and the experiment using impulse counting. The quantification, as we performed it, takes into account the means of 15 values measured systematically every 120/~m. The data presented in this study reflect means obtained in about 2 spinal segments at each level. The only autoradiographical study reported in the literature 9 relative to the rostrocaudai distribution of bt, 6 and 1¢ binding sites in the spinal cord presents only qualitative data. Now, such data are well known to support a non-negligible part of subjective interpretation.
291 From the present autoradiographical study, using highly selective ligands and quantitative measurements, several conclusions can be drawn: (a) Since, in the superficial layers of the dorsal horn, opioid binding sites mainly reflect presynaptic binding sites 1 located on thin primary afferent fibers 7'13, our data suggest that there is an identical proportion of the three main types of opioid receptors, whatever the peripheral location and/or origin of nociceptors. (b) The high levels o f / z , intermediate 6 and low r binding sites are in good agreement with several electrophysiological 5'1° and pharmacological H (see also ref. 16) studies based on intrathecal administration of opioid substances, showing that the analgesic effects of/z agonists were greater than that produced by r agonists, with 6 ligands eliciting intermediate effects.
(c) Although speculative, the similar densities o f / z opioid receptors in the superficial layers of the dorsal horn along the length of the spinal cord support the idea that morphine could be equally effective in the relief of pain due to an excess of nociception arising from any part of the body. Despite the lack of systematic study in animals in order to assess the efficacy of intrathecal administration of morphine at different levels of the neuraxis, clinical reports (see refs. in ref. 2) claim that a high percentage of cancer pain originating from various parts of the body can be adequately managed by intrathecal or epidural administration of morphine. Acknowledgements. We thank M.J. Besson and her group for their friendly aid to perform binding studies on spinal cord slices, D.A. Dickenson for critical reading of the manuscript and E. Dehausse for drawings and photographies.
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