Opioid receptor subtypes in the rat spinal cord: electrophysiological studies with μ- and δ-opioid receptor agonists in the control of nociception

36

Brain Research, 413 (1987) 36-44 Elsevier

BRE 12608

Opioid receptor subtypes in the rat spinal cord" electrophysiological studies with kt- and 6-opioid receptor agonists in the control of nociception A.H. Dickenson l, A.F. Sullivan 1, R. Knox l, J.M. Zajac 2 and B.P. Roques 2 1Department of Pharmacology, University College London, London (U. K.) and 21NSERM U206 and C.N.R.S. UA590, Paris (France) (Accepted 28 October 1986) Key words: Opioid receptor; Spinal cord;/~-Opioid; b-Opioid; Nociception; Analgesia; Intrathecal opioid; Rat dorsal horn; Enkephalin

We have compared the ability of selective ~- and b-opiate agonists to modulate nociceptive transmission at the level of the rat dorsal horn using electrophysiological approaches. Single-unit extraceUular recordings were made from neurones in the lumbar dorsal horn of the intact rat under halothane anaesthesia. Neurones could be activated by both A- and C-fibre electrical stimulation (and by natural innocuous and noxious stimuli). Agonists were applied directly onto the cord in a volume of 50/A. The intrathecal administration of 3 agonists, Tyr-D-Ala-Gly-MePhe-Gly-ol (DAGO) (~-selective) (2 x 10-3-10 nmol) Tyr-D-Thr-Gly-Phe-Leu-Thr (DTLET) (,u/b) (7 × 10-4-70 nmol), and cyclic Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE) (b) (2 × 10-2-100 nmol) produced dose-dependent inhibitions of Cfibre-evoked neuronal activity whilst A-fibre activity was relatively unchanged. DAGO produced near-maximal inhibitions which could be completely reversed by naloxone (1.5 nmol) whilst DPDPE causes less marked inhibitions which could only be partially reversed by naloxone (1.5-13.5 nmol). DTLET produced effects intermediate to those of DAGO and DPDPE, The results suggest that both ~-and 6-opioid receptors can modulate the transmission of nociceptive information in the rat spinal cord.

INTRODUCTION Clinical studies and behavioural approaches in animals 4° have demonstrated that the direct spinal application of opiates by the intrathecal (i.t.) or epidural route can produce a marked reduction in responses to noxious stimuli. This is in accordance with the high levels of opioid receptors 1'13'~8'25'28 and endogenous opioid peptides such as the enkephalins 17"2°in the spinal cord. However, the role of the different subtypes of opioid receptors in the control of pain and nociception at different levels of the central nervous system remains controversial 4'5"9"15A6"23"30'3t'35"37. A possible problem in the animal studies is the fact that motor, sedative or motivational effects may produce false positive results in some tests of analgesia.

We have developed an electrophysiological model for studying the actions of drugs applied directly onto the surface of the spinal cord on the electrically and naturally evoked activity of dorsal horn neurones. Results with morphine agree well with behavioural studies in animals and with experimental pain in man (see ref. 1t). Since substances interacting preferentially with #-sites such as morphine are used clinically there are obvious practical implications of comparing the potency of/~- and 6-agonists applied locally onto the spinal cord on the responses of dorsal horn neurones. Th compounds we have studied are Tyr-DAla-Gly-MePhe-Giy-ol ( D A G O ) 1~'24, Tyr-D-ThrGly-Phe-Leu-Thr ( D T L E T ) 4z and cyclic Tyr-D-PenGly-Phe-D-Pen ( D P D P E ) 26 a conformationally restricted enkephalin, which have/~-, 6//~- and 6-0pioid

Correspondence: A.H. Dickenson, Department of Pharmacology, University College London, London WCIE 6BT, U.K. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B .V. (Biomedical Division)

37 rent for the C-fibres and the Aft- and C-fibre-evoked

receptor selectivity respectively 6"s'22. Some preliminary findings have appeared in abstract form 7.

responses separated on latency measurements and quantified using gated counters. These current levels are highly suprathreshold (x50) for the Aft-fibres and so the Aft-responses may well be less amenable to opiate inhibition. D A G O and D T L E T were synthesized following the described methods 19"42. D P D P E was from Bachem (Switzerland). The peptides were applied at the indicated concentrations in a volume of 50ul onto the exposed surface of the spinal cord from a Hamilton syringe mimicking application of drugs via an i.t. cannula. Following two stable control sequences the responses were monitored for up to 3 h after drug application, lntrathecal naloxone (1.5-13.5 nmol) was used to attempt to reverse the effects of the peptides. The dose of naloxone administered was governed by the concentration of agonist already on the cord. An approximate 6:1 ratio in the dose (nmol) of agonist to antagonist was adhered to in order to examine the el-

MATERIALS AND METHODS The methods used have been described previously in detail n. Briefly, male S p r a g u e - D a w l e y rats were anaesthetized with halothane in 33% oxygen/66% nitrous oxide and the lumbar spinal cord exposed. Extracellular recordings were made (halothane at 0.7%) from single neurones, identified by their responses to cutaneous stimuli at various depths in the dorsal horn with the majority located approximately in laminae 1-V. All cells responded to touch, brush, pressure and pinch and to electrical stimulation of Aft- and C-fibres. The latter stimulus was a 2-ms wide pulse applied to the receptive fields by two fine needles at 0.5 Hz. Sixteen trials were presented at 3x threshold cur-

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38 fects of an equivalent dose of naloxone in reversing the different agonists. RESULTS Results are expessed as mean values (_+ S . E . M . ) and the Student's t-test was used for statistical comparison. A total of 75 single neurones (in 39 animals) were recorded in this study and for the whole population at a mean current of 2.5 +_ 0.4 m A (3 × threshold for C-fibre-evoked responses) a mean 18.3 _+ 1.7 Cfibre latency spikes and 8.6 _+ 0.3 A/J-spikes were e v o k e d per stimulus. All cells included in this study presented no obvious change in spike amplitude or characteristics over the course of the study and the overall form of the post-stimulus histograms following application of the agonists (in those cases of incomplete inhibition) and subsequently naloxone, were likewise not m a r k e d l y altered (Fig. l). Results are pooled for the whole population of cells since the magnitude of effects of the various drugs and indeed, the time courses, did not differ between the superficial and deep convergent cells. The analysis of the results includes the resting activity of the small proportion of cells (4%) exhibiting firing in the absence of overt stimulation. This resting activity was always sensitive to opioid inhibition and in none of the cases did it increase following administration of the 3 opi-

enced by locally applied D A G O (Figs. 1 and 2), The cells all received Aft- and (?-fibre inputs and responded to both innocuous and noxious stimuli. Twenty-two of the neurones were inhibited in a dosed e p e n d e n t m a n n e r by D A G O whilst the remaining two were excited by all doses. The inhibitory effects of D A G O were rapid in onset and the time course was similar notwithstanding the dose used with a mean maximal inhibitory effect 19 rain after the start of drug application (Fig. 3). The action of D A G O was selective for the C-fibre-evoked responses with an El)50 of 0.35 nmol and maximal inhibitions (94 _+_ 2 % ) were produced with 10 nmol. The Aft-fibreevoked responses were slightly reduced (30 _+.: 14%

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39 maximal inhibition) but the effect was not clearly dose-dependent and not significant except for the dose of 0.02 nmol (P < 0.05). It is quite possible that the Aft-responses recorded concurrently with the C-fibre responses will be less amenable to inhibition due to the suprathreshold nature of the stimulation for the former fibres. However, recording the Aft-responses at 3 × threshold following an effective dose of D A G O (10 nmol) which completely inhibited the C-fibre responses (n = 3) revealed maximum inhibitions of only 32% of the Aftresponses, comparable with the changes seen with suprathreshold stimulation. It may be that spatial spread of the transcutaneous stimulus provokes a response sufficiently asychronous to be amenable to inhibition despite the suprathreshold stimulus. By contrast the C-fibre responses were powerfully and dosedependently reduced with significant changes (P < 0.05) at doses higher than 0.1 nmol. However, it should be pointed out that the lowest doses tested (0.002 nmol n = 3, 0.02 nmol n = 5) tended to produce facilitatory effects on the C-fibre activity although these were not significant. Naloxone (1.5 nmol) rapidly reversed the effects of D A G O with the C-fibre responses regaining 91 _+ 7% of control values 20 rain after i.t. administration of the antagonist (n = 20 cells). The Aft-responses, by contrast, were not altered following naloxone. In a separate series of experiments we gauged the effects of the antagonist alone, applied locally onto the spinal cord on the response of 12 neurones to Aft- and C-fibreevoked activity. The dose of naloxone used above (1.5 nmol) had no effect on neuronal activity in the dorsal horn. A higher dose (40 nmol) than that used to reverse the effect of any of the agonists in the present study produced a slight facilitation (maximal effect 126 _+ 19%) but this was not statistically significant from controls. Thus the reversal of the effects of the agonists by naloxone are not likely to be due to excitatory effects of the antagonist.

about 0.014 nmol and an EDs0 of 4 nmol. However, the dose-response curve was not monotonic and could be arbitrarily divided into two components with the first one occurring in the range of 0 - 5 0 % inhibition. On this basis the EDs0 of D T L E T for the first and second part of the curve can be estimated to be 0.4 nmol and 8 nmol respectively (Fig. 4). The A-fibre responses were reduced by D T L E T but these were only significant changes with the highest doses of 7-50 nmol, reaching a maximum inhibition of 54%, a greater effect on these responses than the other two agonists. Naloxone (dose range 1.5-13.5 nmol) reversed the effects of D T L E T on both the Aft- and C-fibre evoked responses of all the neurones tested (n = 23) but the effects were incomplete with the C-fibre responses maximally attaining 75 + 8% of the control responses post naloxone. High doses of naloxone (40 nmoI) restored the responses to control values.

Effects of DPDPE D P D P E was tested on 22 neurones of which 18 were clearly inhibited dose-dependently whilst the remaining 4 were either slightly facilitated or unresponsive. The threshold was about 0.16 nmol and the EDs0 for D P D P E was 5 nmol. The apparent maximal effect of D P D P E was lower than the other two peptides, a 54% inhibition with 7 nmol. Interestingly, this value corresponds approximately to the change in slope observed with D T L E T (Fig. 4). It should be noted that higher doses of D P D P E were not investigated because of limited amounts of the agonist available, so the ED50 may be an underestimate. Howev100.

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er, other selective 6-opiate receptor agonists exhibit a plateau in the dose response curve in this range (unpublished observations). The Aft-fibre evoked responses were not significantly reduced by D P D P E except at the lowest dose tested (0.016 nmol) which produced a 16% inhibition (P < 0.05) (Fig. 5). The duration of the effects of D P D P E was similar to the other enkephalin analogues but the onset was slower with mean maximal effects for all doses occurring at 27 min after the i.t. injection (Fig. 3). Naloxone (1.5-13.5 nmol) produced an incomplete reversal of the C-fibre response with the maximal effect being 73 + 8% of the controls (n = 10). Again, as with D T L E T 40 nmol of naloxone completely reversed the inhibitions of the C-fibre-evoked activity but the lesser opioid-induced depression of the Aft-responses was not influenced by the antagonist. The results clearly demonstrate that the local application of D A G O , D T L E T and DPDPE onto the spinal cord produces a dose-dependent inhibition of C-fibre-evoked nociceptive responses of dorsal horn nociceptive neurones in the intact rat. All 3 peptides had relatively selective effects on neuronal responses to nociceptive electrical stimuli and only higher doses of D T L E T produced any marked changes in A-fibreevoked activity and even these were considerably less than the influences on C-fibre activity. However, since the Aft-responses concurrent with the C-fibre-evoked activity were obtained at highly

suprathreshold currents this apparent selectivity must be viewed with caution and experiments with controlled natural stimuli would be required in order to verify this point. Some of the inhibitions of Aft-responses may in fact be due to Ad-fibre effects since it is difficult to separate A/3- and Ad-responses in the rat. DISCUSSION

A previous study from our laboratory has shown that i.t. morphine under the same conditions produced marked inhibitory effects on nociceptive cells Ij (Table I). Several interesting points arise from the analysis of the concentration-dependent inhibitions elicited by the 3 enkephatin analogues. In all 3 cases the selectivity of the effects on C- vs A-fibre responses were similar to morphine. D A G O is about 20 times more potent than morphine but both compounds produced the same maximal inhibition,, about 95%, whereas the C-fibre-evoked responses were only reduced to an apparent maximum 54% inhibition with DPDPE. The dose-response curves of D A G O and D P D P E were monotonic while the curve produced by D T L E T seemed to be biphasic with the first and second components corresponding roughly to the shape of the curves of DPDPE and D A G O respectively (Fig. 6). Obviously the interpretation of these results relates to the potency and the selectivity of the various compounds for the ,~- and ?)-types of opioid receptors. The relative affinity for t~- and 6sites as well as cross-reactivity of D A G O , D T L E T and DPDPE have been deduced from competition experiments using [3H]DAGO as a selective it-ligand and [3H]DSLET or [3H]DTLET as highly prefer-

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Sullivan, 1986). Only the effects on C-fibre responses are shown here. • • : DAGO, ~ : DTLET, H : DPDPE, and C) O: morphine. Note the facilitations produced by low doses of morphine and DAGO. Each point represents the mean + S.E.M. *P < (I.05: **P < 0.01; ***P< 0.001. ential delta probes s'32. D A G O and D P D P E respectively, were found to be extremely selective for usites (K 1 = 3.9 riM) and d-sites (K 1 = 18 riM) in rat brain since the affinity of both ligands for their nonspecific site was higher than 700 nM. The linear hexapeptide D T L E T exhibited a low but significant residual cross-reactivity illustrated by its affinity for 6(K I = 1.35 nM) vs,u- (K I = 25 nM) receptors a2. Furthermore none of these enkephalin analogues recognizes the kappa receptor sub-type 32. Taken together these results account for the monophasic d o s e - r e sponse curve elicited by D A G O (EDs0 = 0.35 nmol) and D P D P E (ED50 = 5 nmol) (Table I). As compared to the/~-ligand, the overall 15-fold lower inhibitory potency of D P D P E contrasts with its 200 times weaker affinity for the u-receptor subtype. Moreover, the intrinsic activity of D A G O and D P D P E would seem to differ since the maximum inhibition induced by the cyclic peptide is about half that of D A G O . These features strongly suggest that the inhibitory effects elicited by each agonist are related to the stimulation of a single class of opioid receptors, i.e./~ for D A G O , 6 for D P D P E . Likewise the residual cross-reactivity of D T L E T would account for its biphasic dose-response curve with the first component attaining an approximately 5(Ff inhibition. The shape and the slope of this first component is ahnost identical to that induced by D P I ) P E and can be attributed to the interaction of D T I , E T with 6-opioid receptors. The 12-fold higher

potency of D T L E T (EDs0 = 0.4 nmol) as compared to D P D P E (EDs0 = 5 nmol) correlates well with the 18-fold greater affinity of the former peptide for the 6-sites 42. Furthermore the inhibition of nocJceptiveevoked responses induced by D T L E T in the second part of the dose-response curve is likely to reflect the binding of the peptide to u-receptors and occurs at doses (EDs0 = 9 nmol) considerably higher than D A G O in agreement with the weaker,u-affinity of DTLET. This interpretation is reinforced by the fact that only the effects of D A G O were completely reversed by low doses of naloxone in agreement with the highest affinity of this antagonist for u-receptors =2. It should be noted that we adjusted the dose of naloxone for each agonist on the basis of their differing potencies so that equivalent amounts of the antagonist were applied in each case although the effects of all 3 agonists could be reversed by high doses of naloxone which would be expected to be non-selective between opiate receptor subclasses. Our resuits would therefore indicate that activation of both u- and &opiate receptors in the rat dorsal horn can inhibit nociceptive activity evoked by cutaneous stimuli. These conclusions agree well with those of Yaksh and colleagues who have tested a variety of uand ()-ligands on behavioural nociceptive reactions in the rat following i.t. injection 3<~5. Although different ligands were used, peptides exhibiting some ()-selectivity such as D-AlaCD-LeuS-enkephalin ( D A D L ) and enkephalin dimer (DPE) had potencies about that of morphine whereas the peptide D-AIa 2 met-enkephalin amide ( M E A ) with predominant ,u-actions was 40 times as potent 34'35, in remarkable agreement with our present findings with more selective ligands. However, this concordance may be coincidental since the use of arbitrary cut-off time to gauge full analgesia in behavioural testing makes it difficult to compare effects directly. lntrathecal morphine, D A G O . I)TI.ET, and D P D P E have been studied using behavioural indices of nociception in the mouse 31. Our findings of a rank order of potency of D A G O = D T L E T > D P D P E > morphine is roughly in agreement with the [ ) T L E T > D A G O > morphine > D P D P E . The rank order of potency of these compounds injected intracerebroventricularly (i.c.v.) in rodents 4-~l~e is clearly different since D A G O is about 100 times more potent than D P D P E . More generally at the supraspinal level, the

42 antinociceptive potency of various agonists is linearly correlated with the affinity for/~-receptors 4"5"31. The relevance of our electrophysiological results to spinal analgesia is supported by the behavioural studies in animals discussed above but also by studies in experimental pain in humans. Since the neurones we recorded, so called convergent cells, are at the origins of some ascending somatosensory pathways > and also seem to be intercalated in flexion-reflex pathways 36 we would predict that similar reductions in reflex and sensory aspects of nociception would be produced by an inhibition of these cells. This is indeed the case in the human experiments with i.t. morphine 39 and in the animal studies since both the reflex tail flick and more integrated hot-plate tests show the same sensitivity to morphine and other opioids 35. These neurones may well be the critical neural element in the spinal action of opiates. In this regard. it is of interest to note that a similar structure-activity relationship for ~- and d-ligands has been reported in the primate using shock titration methods 41. Comparing the results with these I~- and 6-agonists to those we previously obtained with morphine the only major difference other than potency is the shape of the dose-response relationship: the curve is much steeper with morphine and D A G O . As both D A G O and morphine are j,-agonists, and we (Dickenson and Sullivan, unpublished observations) have found that methadone, another/~-agonist which is considerably more liposoluble than morphine, has a similar abrupt curve to these/~-ligands, pharmacokinetic factors cannot account for the differences. Similar curves for other peptides compared to morphine have recently been observed in behavioural studies in the rat 34. Possible reasons for these differences in curves could be spare receptors 3~, allosteric coupling 33 and/or metabolism of the opiates :~2. The superficial dorsal horn of the spinal cord is rich in /~-receptors and enkephalin-containing neurones (see Introduction). Although less abundant, d-sites are present in the substantia gelatinosa and also diffusely distributed in the central part of the spinal cord Is. By contrast with B-receptors, the selective stimulation of d-types leads to what appearsto be only a 50% inhibition of nociceptive inputs suggesting th~.t differ-

ent neuronal mechanisms could be involved in the 6or/~-induced effects. The limited action of h-agonists could be related to the release 3 of other peptides such as substance P which may enhance nociception 2~ or indirect antagonists of opioids such as cholecystokinin-8 (ref. 12). In our previous studies with morphine we noted a clear tendency for the lowest doses of the opiate to facilitate C-fibre-evoked activity whereas higher doses were always inhibitory ~l, This would seem to be a u-receptor-mediated response since similar effects were seen with D A G O but not with DTLET and DPDPE and could be related to various factors such as a decrease in the concentration of endogenous enkephalins by activation of autoreceptors or reuptake processes. Thus to conclude, both u- and d-receptor agonists with actions which seem confined to these receptor subclasses can attenuate the afferent transmission of nociceptive information in the rat dorsal horn by an action confined to the spinal cord. Since for the reasons discussed previously, the spinal cord seems a useful predictor of analgesic effects in man, both ~uand d-agonists may have useful clinical applications. at least following epidural or i.t. administration. Thus the mixed u/0-agonist D A D I . E ~'7'2'has been reported to be effective after epidural administration and metkephamid parenterally ff)r cancer and postoperative pain 2 and also in the prevention of parturition pain because these compounds induce less respiratory depression and do not cross the placental barrier la In more general terms, it thus seems that I~- and dopiate receptors can modulate cutaneous noxious inputs in the rat spinal cord indicating that at this level at least both the former receptor subclasses may have similar functional roles. ACKNOWLEDGEMENFS This work was supported by the Wellcome Trust, Medical Research Council and the Fondation pour la Recherche Medical Franqaise. R.K. has an MRC postgraduate studentship. We would like to thank Mrs. R. Fox and Mrs. J. Howe for their help in preparation of the manuscript.

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