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Intrinsic mechanisms of antinociception in inflammation: local opioid receptors and beta-endorphin

This study examined antinociception induced through the activation of local opioid receptors in inflammation by endogenous opioids. Rats developed a unilateral localized inflammation upon injection of Freund's adjuvant into one hindpaw. Four to 6 d later they were subjected to cold water swim (CWS), an environmental stimulus known to activate intrinsic opioid systems. Following CWS (1 min) the animals' withdrawal threshold to noxious pressure applied onto the paws increased significantly more on the inflamed paw than on the noninflamed paw. This unilateral antinociceptive effect in inflamed paws was dose-dependently and stereospecifically reversible by intraplantar (i.pl.) but not systemic (i.v. or s.c.) administration of the opioid antagonist naloxone (18 micrograms). This suggested that CWS- induced antinociception in inflamed tissue was brought about by the activation of local opioid receptors. Antiinflammatory or vasoconstrictive events, as measured by paw volume and temperature, did not contribute to this unilateral antinociception. Receptor-selective antagonists indicated the involvement of mu- and delta- but not kappa- receptors. Intravenous application of a universal antibody to endogenous opioid peptides (3-E7) and a specific antibody to beta- endorphin, but not antisera against metenkephalin or dynorphin, abolished the CWS effect. Finally, the i.pl. injection of synthetic beta-endorphin (1–31) produced an antinociceptive effect in inflamed paws which was reversible by i.pl. naloxone and selective mu- and delta- receptor antagonists. These findings suggest that antinociception in inflamed tissue can be induced through the activation of local opioid receptors by endogenous beta-endorphin released during CWS.

Receptorselective antagonists indicated the involvement of P-and 6but not K-receptors. Intravenous application of a universal antibody to endogenous opioid peptides (3-E7) and a specific antibody to @-endorphin, but not antisera against metenkephalin or dynorphin, abolished the CWS effect. Finally, the i.pl. injection of synthetic @-endorphin (l-3 1) produced an antinociceptive effect in inflamed paws which was reversible by i.pl. naloxone and selective CC-and s-receptor antagonists.
These findings suggest that antinociception in inflamed tissue can be induced through the activation of local opioid receptors by endogenous 8-endorphin released during CWS.
Traditionally, antinociception produced by exogenous as well as endogenous opioids has been associated with activation of opioid receptors in the central nervous system. Recently, however, we and others have demonstrated that exogenous opioid agonists can exert antinociceptive effects by interacting with local opioid receptors in inflamed tissue of the rat (Ferreira and Nakamura, 1979;Joris et al., 1987;Stein et al., 1988bStein et al., , 1989a. Furthermore, we have been able to differentiate the types of opioid receptors involved (Stein et al., 1989a). The physiological significance of these receptors, however, is yet to be elucidated. In particular, the question arises as to which are the endogenous ligands for these receptors and what stimuli call them into play.
Three families of endogenous opioid peptides derived from 3 precursor peptides are known to date: the pro-opiomelanocortin (POMC), the pro-enkephalin, and the pro-dynorphin system. These precursors undergo differential processing in the various regions of the central and peripheral nervous systems (HBllt, 1986;Lewis et al., 1987) and the major cleavage products have differing affinities to the 3 opioid receptor types p, 6, and K. Intrinsic opioid antinociceptive systems can be activated by certain environmental stimuli (Terman et al., 1984;Millan and Herz, 1985;Bodnar, 1986). Out of a wide variety of models we chose cold water swim (CWS) and examined its ability to elicit antinociception through local opioid receptor-specific mechanisms in inflammation.
Specifically, this study investigated (1) the antinociceptive effect of different durations of CWS in unilateral hindpaw inflammation, (2) whether this effect is reversible by locally, compared to systemically, applied naloxone and its stereoisomer, (3) whether antiinflammatory and/or vasoconstrictive events contribute to this antinociception, (4) which types of opioid receptors are involved, (5) whether this effect can be abolished by antibodies against endogenous opioid peptides, and (6) whether this effect can be mimicked by local administration of the opioid peptide @-endorphin.
Stereospecific and dose-dependent reversibility by local, but not systemic, application of naloxone provides evidence for an involvement of peripherally located opioid receptors. Differential abolition of an effect by antagonists that are highly selective for particular receptor types and by specific antibodies will point to the identity of the endogenous ligand(s) involved. Imitation of this effect by exogenous administration of the putative ligand will confirm its functional significance. Using these criteria we have concluded that antinociception in inflamed tissue can be induced through activation of local p-and &opioid receptors by endogenous P-endorphin released during CWS.

Materials and Methods
Subjects. Experiments were carried out in male Wistar rats (Ivanovas, Kisslegg, ERG) (180-200 gm) housed individually in cages lined with sawdust. Standard laboratory rodent chow and water were available ad libitum. Room temperature and relative humidity were maintained at 22 * 0.5"C and 60%, respectively. A 12 hr/12 hr (8 A.M./~ P.M.) ligbtdark cycle was used. All testing was conducted in the light phase. The guidelines on ethical standards for investigations of experimental pain in animals (Zimmermann, 1983) were followed.

Induction of inflammation.
The inflammatory agent used was modified Freund's complete adjuvant (FCA), containing 0.1% heat-killed and dried Mycobacterium butyricum in 85% Marco1 52 and 15% Aracel A mannide monooleate emulsifier (Calbiochem, La Jolla, CA). Rats received an intraplantar injection of 0.15 ml of this suspension into the right hindfoot under brief ether anesthesia. Control animals were anesthetized but not injected. Inflammation of the injected paws was apparent within 12 hr following treatment with FCA. A detailed description of the time course and magnitude of the inflammatory reaction is given elsewhere (Stein et al., 1988a). In all rats studied, the inflammation remained confined to the inoculated paw. All testing was conducted between 4 and 6 d postinoculation.
Parameters of inflammation. Paw volume was measured by submerging the hindpaw to the tibiotarsal joint into the water-filled Perspex cell of a plethysmometer (Ugo Basile, Comerio, Italy). The volume of displacement, which is equal to the paw volume, was indicated on a digital display. The surface temperature ofthe plantar skin was measured with an Infrared radiation thermometer (Ultrakust, Ruhmannsfelden, FRG).
Algesiometric testing. Antinociceptive effects were evaluated using the paw pressure test. The animal was gently restrained under paper wadding and incremental pressure applied via a wedge-shaped, blunt piston onto an area of 1.75 mm2 of the dorsal surface of the hindpaw by means of an automated gauge (Ugo Basile, Comerio, Italy). The pressure required to elicit paw withdrawal, the paw pressure threshold (PPT), was determined. A cutoff of 250 gm was employed. Three consecutive trials, separated by intervals of 10 set, were conducted and the average determined. The same procedure was then carried out on the contralateral side; the sequence of sides was alternated between subjects to preclude "order" effects. Separate groups of animals were used for each treatment, with the observer blind to the experimental condition employed. Drugs and their administration.
The following drugs were used: naloxone-HCl (DuPont. Geneva. Switzerland). Antagonists were given concomitantly with agonists in a total volume of 200 ~1. All substances were injected under brief halothane anesthesia. Antisera. A monoclonal antibody (3-E7) against virtually all opioid peptides  as well as polyclonal antibodies from rabbit against B-EP. methionine-enkephalin (ME). and dvnorohin A ( l-17) (Dfi) were generated in our labor&ory. All a&bodies were purifikd by chromatography on staphylococcal protein A-Sepharose (Pharmacia, Freiburg, FRG) and tested for binding activity and specificity in standard radioimmunoassays.
Cross-reactivities are shown in Table 1. The IgG fraction of antisera was dissolved in saline and doses were calculated in rg IgG. Control experiments for nonspecific binding were carried out with purified normal rabbit IgG. Experiment 1. Antinociceptive effects of different durations of CWS in controls and in animals with unilateral hindpaw inflammation were examined.
Baseline PPTs were taken and rats were returned to their home cages. Ten minutes later, rats were placed into a tub (20 cm deep) containing water at l-2°C. Separate groups (n = 6) of animals were exposed to CWS for 0.5, 1, and 2 min, respectively. PPTs were redetermined at 1, 5, and 15 min following CWS.
The data were analyzed as follows. The increase in PPT was expressed as percentage of maximum possible effect (O/a MPE) according to the following formula: % MPE = (oost CWS PPT -basal PPTk(250 basal PFT) x 100. At the timcof peak effect (1 min post Civ'S), elevations of PPT in right paws were compared to those in left paws using Wilcoxon's matched-pairs test. PPT elevations in control animals were compared to those in FCA-treated animals using the Mann Whitney U-Test (Kirk, 1982). Experiment 2. This experiment examined whether the antinociceptive effect following CWS in inflamed paws was brought about by a local opioid receptor-specific mechanism.
To test this hypothesis, various doses of i.pl. (-)-naloxone were administered prior to CWS. Equivalent doses were also given S.C. and i.v. to exclude a central site of action. Stereospecificity of antagonism was assessed using the inactive dextrorotatory isomer of naloxone. A CWS duration of 1 min was chosen since it produced a consistent elevation of PPT in inflamed paws while not significantly affecting PPT in noninflamed paws. Following baseline PP? recordings, 5 groups (n = 6) ofrats were given the following injections under brief halothane anesthesia: 3 groups received either saline (0.1 ml), (-)-, or (+)-naloxone (18 pg) into the right hindpaw; the fourth and fifth groups received (-)-naloxone (18 fig) i.v. and s.c., respectively. Ten minutes later, the animals were subjected to CWS for 1 min and PPT were reevaluated at 1, 5, and 15 min post cws.
A separate experiment examined the dose-response relationship of i.pl. naloxone. After determination of baseline PPT, 4 groups of animals received saline and 6, 12, or 18 Mug of (-)-naloxone i.pl. 10 min before CWS. PPT were recorded 1 min thereafter.
In previous control experiments we have shown that i.pl. injections of saline, (+)-, and (-)-naloxone alone are without effect in both FCAtreated and normal rais (Stein et al., 1989a).
The data were analvzed as follows: elevation of PPT (in % MPE1 in the inflamed paw was compared to that in the contralatkral paw using Wilcoxon's test, the (1 -tailed) hypothesis being that the former is greater than the latter (Kirk, 1982). This analysis was performed for each measurement (1, 5, and 15 min post CWS) and the Bonferroni correction was applied as appropriate.
In the group given (-)-naloxone (18 pg i.pl.), average PPT obtained at the 4 time points were analyzed separately for each side by Friedman's analysis of variance. The null hypothesis was that there is no change in PPT over time.
To construct a dose-response curve, PPT elevation (in % MPE) was plotted against the dose of (-)-naloxone.
A linear regression analysis of variance (ANOVA) was performed to test the zero slope hypothesis (Kirk, 1982). Experiment 3. This experiment examined the effect of 1 min CWS upon hindpaw volume and temperature. Two groups of rats (n = 6) received either saline (0.1 ml) or (-)-naloxone (18 pg) into the inflamed paw; baseline values were then taken and 10 min later the animals were subiected to 1 min CWS. Both parameters were redetermined immediaiely thereafter.
Changes in paw volume and temperature were expressed as percentage of baseline values (100%). Left and right paws were then compared using the Wilcoxon test grid between-&o& comparisons were made using the Mann-Whitney test. Experiment 4. In this experiment we sought to clarify which types of opioid receptors are involved in the mediation of the antinociceptive effect of CWS in inflammation.
were subjected to linear regression ANOVA. Data obtained in control Increases in PPT (in % MPE) were plotted against the dose and a experiments were analyzed by the Friedman test. linear regression ANOVA was used to test the zero slope hypothesis. Experiment 6. This experiment examined whether the antinociceptive Data obtained in control experiments without CWS were analyzed by effect of CWS could be mimicked by i.pl. administration of p-EP. After the Friedman test, the hypothesis being that there is no change in PPT. determination of baseline PPT, 3 groups of rats (n = 5-6) received Experiment 5. This experiment examined whether the antinocicep-P-EP into both hindpaws (0.25, 0.5, and 1 fig per paw) under brief tion induced by CWS in inflamed paws could be abolished by antibodies halothane anesthesia. PPT were reevaluated at 5, 10, and 20 min postagainst endogenous opioid peptides. Analogous to the above protocols, injection. Doses and testing intervals were chosen based on pilot exseparate groups of rats (n = 5-6) received i.v. injections of a pan-opioid periments. antibody (3-E7) (0.05, 0.15, 0.25, 0.5, and 1.0 Mg) as well as specific Analogously to the previous experiments, PPT elevation (in % MPE) antibodies to @-EP (0.25, 0.5, and 1.0 pg) to ME (1, 4, and 8 pg) and in the right paw was compared to that in the left paw using Wilcoxon's to DYN (1, 4, and 8 pg) 5 min before CWS. In control experiments for test. Dose-response curves were constructed by plotting the increase of nonspecific binding, normal rabbit IgG (5 pg) was administered.
PPT PPT (% MPE) at the time of peak effect (5 min) against the dose and were measured 1 min post CWS. All doses and time intervals for testing were then subjected to a linear regression ANOVA.  Whether the antinociceptive effect of i.pl. p-EP could be reversed by the same antagonists as used in the foregoing experiments was finally examined. To assess this question, the same doses of (-)-naloxone, CTOP, ICI 174,864, nor-BNI, and NaCl were injected concomitantly with fi-EP (1 pg) into inflamed paws. PPT were measured 5 min postinjection. Fourteen separate groups (n = 5-6) of rats were used. Data analysis was analogous to experiment 4.

Discussion
Experiment 1 demonstrates that CWS can produce antinociceptive effects against noxious pressure in both normal and FCA-treated rats. These effects are clearly enhanced in inflamed paws compared to contralateral noninflamed paws or to control animals. The similarity of this phenomenon to that observed after systemic administration of exogenous opioid agonists in this model (Stein et al., 1988b) prompted us to put forth the hypothesis that the recruitment of peripheral opioid receptorspecific mechanisms accounts for the augmented antinociceptive effect of CWS in inflamed tissue. We tested this hypothesis by examining whether this effect was reversible by naloxone injected locally into the inflamed paw.
Experiment 2 demonstrates that the antinociceptive effect produced by CWS in inflamed paws can be reversed by locally applied naloxone but not by equivalent doses given systemically. Moreover, this antagonism is stereospecific and dose-dependent. Taken together, these findings are consistent with the notion that CWS can activate local opioid receptor-specific mechanisms in inflamed tissue.
The question arises as to whether antiinflammatory and/or vasoconstrictive effects contribute to this unilateral antinociception. Both of these are unlikely, the former since paw volume did not change during CWS and the latter since temperature decreased to the same extent in both hindpaws and, in contrast to the antinociceptive effect, this drop was not reversible by naloxone. Therefore, we are inclined to invoke a neural mechanism. As discussed extensively in a previous report (Stein et al., 1989a), the most likely location of these peripheral receptors seems to be the primary afferent neuron. supported both by biochemical studies demonstrating opioid ities of the endogenous opioid peptides (HGllt, 1986), one would binding (LaMotte et al., 1976;Fields et al., 1980;Ninkovic et consider cleavage products from either the POMC or the proenal., 1982) or opioid modulation of substance P release (Yaksh, kephalin A system, such as @EP or the enkephalins, as the most 1988) and by electrophysiological data demonstrating opioid-likely candidates. To further delimit the possibilities, we applied specific effects (Werz and Macdonald, 1982;Frank, 1985;Rus-antisera against the major representatives of the 3 opioid famsell et al., 1987) on sensory neurons. It should be borne in mind, ilies in experiment 5. The results show that both the specific however, that by varying the specific parameters of CWS (e.g., antibody to P-EP and 3-E7, which exhibits complete immutemperature, duration) it may well be possible to activate central noreactivity with P-EP , but not antisera opioid and/or nonopioid antinociceptive systems in addition to against ME or DYN, can abolish CWS-induced antinociception these peripheral mechanisms.
in inflamed paws. Previously, we have shown that it is possible to differentiate between the types of peripheral opioid receptors mediating antinociception in this model (Stein et al., 1989a). Therefore, experiment 4 sought to identify the receptor type(s) involved. The results demonstrate that the antinociceptive effect of CWS on inflamed paws can be reversed dose-dependently by antagonists selective for p-and 6-, but not for K-receptors. Exogenous K-agonists, however, can produce peripheral antinociceptive effects in inflammation pointing to the presence of K-reCeptOrS (Joris et al., 1987;Stein et al., 1988bStein et al., , 1989a. Thus, it is conceivable that endogenous K-ligands may be activated by other environmental stimuli. Nevertheless, the above findings indicate that opioid agonists with activity at p-and/or 6-but not at K-receptors mediate the CWS effect. Inferring from the well-documented binding affin-These findings support the contention that M-and &receptors in the inflamed paw may be activated by P-EP released during CWS. This hypothesis, however, is based on the assumption that both /3-EP and opioid receptors display similar binding characteristics in inflamed tissue as in conventional assays (HBllt, 1986). That this may not be correct was already discussed in a previous report (Stein et al., 1989a): First, the anatomical circumstances in peripheral tissue must be considered. Before reaching its presumed locus of action, the peripheral sensory nerve axon or its terminal, an opioid molecule has to traverse several connective tissue and/or lipid membranous barriers. Each peripheral nerve axon is encased in a Schwann cell and, in the case of A6 fibers, in several layers of lipoid myelin. The nerve fiber lies embedded in the surrounding subcutaneous connective tissue. As in the case of local anesthetics, the milieu (pH) of this Antagonists were injected into the inflamed paw and antisera intravenously at 0 min. PPT in gm (means + SE; n = 5-6 per group). L = left, noninflamed paw; R = right, inflamed paw. environment may crucially influence activity and penetration of the agonist through these layers by altering the relative fraction of agent present in its charged or uncharged forms (Savarese and Covino, 1986). This is of special importance in the acidic conditions of inflamed tissue. Second, changes in tissue pH may result in alteration of receptor conformation which may entail a transformation from desensitized to active state, or vice versa. Thus, several critical factors have to be considered and one has to be careful in drawing parallels to more customary assays. For these reasons, we carried out the last experiment investigating the actions of exogenously applied @-EP. The data clearly demonstrate that intraplantar administration of &EP can produce an antinociceptive effect in inflamed paws. This effect is dose-dependent and reversible by naloxone, CTOP, and ICI 174,864 but not by nor-BNI. Moreover, the dose ranges for the antagonists to block the effect of fi-EP are very similar to those required to abolish the effect of CWS on inflamed paws. Apparently, P-EP displays activity at both p-and a-receptors, which is in line with studies on its central application in viva (Shook et al., 1988;Bals-Kubik et al., 1990) as well as on in vitro assays (Wiister et al., 1979;Shook et al., 1988).
Taken together, the present evidence strongly indicates that the endogenous ligand that is released during CWS and activates p-and d-receptors in inflamed paws is most likely &EP.
One important question remains unanswered: What is the anatomical source for the endogenous opioids released during CWS? The pituitary is the richest source of P-EP and various stimuli can cause the release of pituitary opioids (Guillemin et al., 1977). Beyond that, however, opioid peptides have been detected in primary sensory neurons (Botticelli et al., 1981;Przewlocki et al., 1983;Weihe et al., 1988). Studies addressing this issue have commenced in our laboratory (Stein et al., 1989b;Parsons et al., 1990).
In summary, we have demonstrated that peripherally located opioid receptors in inflamed tissue can be activated by endogenous opioids released during exposure to environmental stimuli and mediate a decrease of nociception. One of the most likely endogenous ligands appears to be fi-EP.