Peripheral antinociceptive effects of exogenous and immune cell-derived endomorphins in prolonged inflammatory pain

Abstract

Endomorphins (EMs) are endogenous selective mu-opioid receptor agonists. Their role in inflammatory pain has not been fully elucidated. Here we examine peripheral antinociception elicited by exogenously applied EM-1 and EM-2 and the contribution of EM-containing leukocytes to stress- and corticotropin-releasing factor (CRF)-induced antinociception. To this end, we applied behavioral (paw pressure) testing, radioligand binding, immunohistochemistry, and flow cytometry in rats with unilateral hindpaw inflammation induced with Freund's adjuvant. EMs injected directly into both hindpaws produced antinociception exclusively in inflamed paws. This was blocked by locally applied mu-receptor-selective (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2) but not kappa-receptor-selective (nor-binaltorphimine) antagonists. Delta-receptor antagonists (naltrindole and N,N-diallyl-Tyr-Aib-Aib-Phe-Leu) did not influence EM-1-induced but dose-dependently decreased EM-2-induced antinociception. Antibodies against beta-endorphin, methionine-enkephalin, or leucine-enkephalin did not significantly change EM-2-induced antinociception. Both EMs displaced binding of [3H]-[D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin to mu-receptors in dorsal root ganglia (DRG). Using [3H]-naltrindole or [(125)I]-[D-Pen2,5]-enkephalin, no detectable delta-binding was found in DRG of inflamed hindlimbs. Numerous beta-endorphin-containing and fewer EM-1- and EM-2-containing leukocytes were detected in subcutaneous tissue of inflamed paws. Leukocyte-depleting serum decreased the number of immigrating opioid-containing immune cells and attenuated swim stress- and CRF-induced antinociception in inflamed paws. Both forms of antinociception were strongly attenuated by anti-beta-endorphin and to a lesser degree by anti-EM-1 and anti-EM-2 antibodies injected into inflamed paws. Together, exogenously applied and immune cell-derived EMs alleviate prolonged inflammatory pain through selective activation of peripheral opioid receptors. Exogenous EM-2 in addition to mu-receptors also activates peripheral delta-receptors, which does not involve actions via other opioid peptides.

Figure 1.

Antinociception elicited by exogenous EM-1, EM-2, and β-endorphin (END) in inflamed paws. Left, Dose-dependent antinociceptive effects at 5 min after intraplantar injections (p < 0.001, ANOVA, linear regression). Middle, Time course of EM-1- and EM-2-induced antinociception. *p < 0.001, Bonferroni’s test. Right, Lack of EM-1- and EM-2-induced antinociception after systemic (subcutaneous) injection at the neck (p > 0.05, two-way repeated-measures ANOVA). Data are expressed as means ± SEM.

Figure 2.

Opioid receptor selectivity of exogenous EM-1- and EM-2-induced antinociception. Left, EM-1-induced antinociception was dose dependently blocked by μ-receptor (CTOP) but not δ-receptor (ICI 174,864) or κ-receptor (norBNI) selective antagonists (p < 0.001, ANOVA, linear regression; p > 0.05, ANOVA, respectively). Middle, EM-2-induced antinociception was dose dependently blocked by μ-receptor (CTOP) and attenuated by δ-receptor (ICI 174,864 and naltrindole) selective antagonists (p < 0.001, ANOVA, linear regression). EM-2-induced antinociception was not significantly changed by a κ-receptor (norBNI) selective antagonist (p > 0.05, ANOVA). Right, EM-2-induced antinociception was not significantly changed by antibodies against Met-enkephalin (anti-Met-ENK), Leu-enkephalin (anti-Leu-ENK), and β-endorphin (anti-END) (p > 0.05, ANOVA). Receptor antagonists and peptide antibodies were coadministered intraplantarly into inflamed paws with either EM-1 or EM-2. PPTs were measured 5 min after injections. Dashed lines represent baseline paw-pressure thresholds of representative groups and are 40 ± 1.2 g. Data are expressed as means ± SEM.

Figure 3.

Representative immunohistochemical images showing the effects of leukocyte-depleting serum (intravenously) on the number of cells expressing β-endorphin (END), EM-1, and EM-2 in inflamed subcutaneous paw tissue. The control group was treated with nonimmune serum (intravenously). All three peptides are localized in granulocytes (1) and mononuclear cells (2). Scale bar, 20 μm.

Figure 4.

Effects of antibodies against EM-1 (anti-EM-1), EM-2 (anti-EM-2), and β-endorphin (anti-END) injected (intraplantarly) into inflamed paws on swim stress-induced antinociception. Left, Dose dependency of anti-opioid peptides injected separately (p = 0.007 for anti-EM-1; p = 0.002 for anti-EM-2; p < 0.001 for anti-END, ANOVA, linear regression). Right, Comparison of single injections of anti-END, anti-EM-1, and anti-EM-2 with concomitant injection of anti-EM-1 and anti-EM-2. * indicates significant differences versus control, and ⁺ indicates significant differences versus anti-END (p < 0.05, Bonferroni’s test). Data are means ± SEM.

Figure 5.

Effects of antibodies against EM-1 (anti-EM-1), EM-2 (anti-EM-2), and β-endorphin (anti-END) injected (intraplantarly) into inflamed paws on CRF-induced antinociception. Left, Dose dependency of anti-opioid peptides injected separately (p = 0.02 for anti-EM-1; p = 0.016 for anti-EM-2; p < 0.001 for anti-END, ANOVA, linear regression). Right, Comparison of single injections of anti-END, anti-EM-1, and anti-EM-2 with concomitant injection of anti-EM-1 and anti-EM-2. * indicates significant differences versus control, ⁺ indicates significant differences versus anti-END, and ^# indicates a significant difference versus anti-EM-1 (p < 0.05, Bonferroni’s test). Data are means ± SEM.