Chronic morphine use does not induce peripheral tolerance in a rat model of inflammatory pain

Abstract

Although opioids are highly effective analgesics, they are also known to induce cellular adaptations resulting in tolerance. Experimental studies are often performed in the absence of painful tissue injury, which precludes extrapolation to the clinical situation. Here we show that rats with chronic morphine treatment do not develop signs of tolerance at peripheral mu-opioid receptors (micro-receptors) in the presence of painful CFA-induced paw inflammation. In sensory neurons of these animals, internalization of mu-receptors was significantly increased and G protein coupling of mu-receptors as well as inhibition of cAMP accumulation were preserved. Opioid receptor trafficking and signaling were reduced, and tolerance was restored when endogenous opioid peptides in inflamed tissue were removed by antibodies or by depleting opioid-producing granulocytes, monocytes, and lymphocytes with cyclophosphamide (CTX). Our data indicate that the continuous availability of endogenous opioids in inflamed tissue increases recycling and preserves signaling of mu-receptors in sensory neurons, thereby counteracting the development of peripheral opioid tolerance. These findings infer that the use of peripherally acting opioids for the prolonged treatment of inflammatory pain associated with diseases such as chronic arthritis, inflammatory neuropathy, or cancer, is not necessarily accompanied by opioid tolerance.

Figures

Figure 1. Dose-response curves of acute i.pl. fentanyl antinociception in animals without and with hindpaw CFA inflammation.

(A) Without paw inflammation the ED50 for elevation of PPT was significantly lower in s.c. saline–pretreated than in s.c. morphine–pretreated animals (ANOVA, P < 0.001). (B) In the presence of paw inflammation, no significant difference in ED50 was detectable between chronic s.c. morphine and s.c. saline pretreatment (ANOVA, P > 0.05). SEM was occasionally smaller than symbol size. (C) Repeated pretreatment with i.pl. fentanyl (2 μg twice daily) for 4 days shifted the dose-response curve after acute i.pl. fentanyl application significantly to the right, confirming the development of peripheral opioid tolerance (ANOVA, P < 0.05). (D) In CFA animals pretreated with chronic i.pl. fentanyl, the ED50 of acute i.pl. fentanyl antinociception did not significantly change, confirming a lack of peripheral opioid tolerance (ANOVA, P > 0.05). The applied doses of i.pl. fentanyl did not change PPT in contralateral paws (data not shown), indicating that they were not systemically effective and did not activate central opioid receptors. MPE, maximum possible effect.

Figure 2. μ-Receptors on DRG neurons.

[3H]DAMGO saturation binding in DRG membranes from animals without (A) and with (F) hindpaw CFA inflammation. (A) In animals without CFA inflammation, the number of μ-receptors did not differ significantly between untreated (set at 100%) and s.c. morphine–pretreated animals (t test, P > 0.05). (F) In animals with CFA inflammation, the number of μ-receptors was significantly lower in s.c. morphine–pretreated compared with untreated animals (set at 100%) (t test; *P < 0.001). Representative immunohistochemical staining of μ-receptors in cultured DRG neurons (B, D, G, and I) and native DRG sections (C, E, H, and J) processed for confocal microscopy. In DRG from animals without CFA inflammation (B–E), μ-receptors (indicated by arrows) were primarily localized to the plasma membrane both without (B and C) and with (D and E) chronic s.c. morphine pretreatment. In animals with CFA inflammation (G–J), μ-receptors are redistributed to the cytoplasm. This endocytosis appears less pronounced without (G and H) than with (I and J) chronic s.c. morphine pretreatment. Each optical section was also individually displayed to enable analysis from the top surface to the bottom of the cell. Scale bar: 5 μM (B, D, G, and I); 10 μM (C, E, H, and J).

Figure 3. Analysis of DAMGO-stimulated (10 μM) [35S]GTPγS saturation binding in DRG membranes from animals without and with CFA-induced inflammation pretreated with daily injections of s.c. saline or morphine for 4 days.

(A) Without CFA-induced inflammation. (B) With CFA-induced inflammation. DAMGO-stimulated specific [35S]GTPγS binding was significantly lower in morphine-treated than in saline-treated animals in both cases (t test, P < 0.05). Insets: Data were plotted according to the traditional Scatchard analysis (see Supplemental Methods), in which the x axis shows specific binding and the y axis shows specific binding divided by free radioligand concentration. From the Scatchard line, Bmax G protein is the x intercept.

Figure 4. Content of cAMP in DRG cells from animals without and with hindpaw CFA inflammation pretreated with s.c. morphine.

(A–D) Basal cAMP was significantly lower in the absence of FSK in comparison with FSK treatment. Acute opioid application significantly decreased FSK-stimulated cAMP production in comparison with FSK treatment alone in DRG cells of animals with CFA (B) and CFA/morphine pretreatment (D) (ANOVA, *P < 0.01), but not in morphine–pretreated animals without CFA inflammation (C) (ANOVA, #P > 0.05). Values are expressed as percentages of FSK-stimulated (100%) cAMP levels.

Figure 5. Electron micrographs showing END immunoreactivity in inflamed paw tissue.

(A) Cells were identified as macrophages (pseudopodia of various shapes, vacuolated cytoplasm containing granules, nucleus with distinctive ring of heterochromatin around its periphery; ref. 49), (B) PMNs (relative density of intramembranous particles, membranous extensions of the plasma membrane, lysosomes, and phagocytic vacuoles; ref. 50), and (C) lymphocytes (large nucleus with heterochromatin forming a peripheral rim adjacent to the nuclear envelope with deep infolding of the nuclear membrane, thick bundles of filaments in the cytoplasm; ref. 49). Labeling for END (arrows) was confined to secretory granules grouped within membranous vesicular structures in these cells. (D and E) Higher magnifications of A and C, showing END-labeled secretory granules within membranous vesicular structures (arrows). Original magnification, ×5,000 (A–C) and ×10,000 (D and E).

Figure 6. Removal of immune cell–derived opioid peptides by CTX.

(A–D) Quantification of immune cells in inflamed paw tissue by flow cytometry. CTX treatment depleted macrophages (A), PMNs (B), T lymphocytes (C), and opioid peptide–containing cells (D). *P < 0.01. (E) [3H]DAMGO saturation binding in DRG cells from animals with paw inflammation and CTX treatment. The number of μ-receptors did not differ significantly between untreated (set at 100%) and s.c. morphine–pretreated animals (t test, P > 0.05). (F) Content of cAMP in DRG cells from CFA animals with CTX treatment. Acute opioid application (+/+) did not decrease FSK-stimulated cAMP production (+/–) after chronic s.c. morphine pretreatment (ANOVA, P > 0.05). Values are expressed as percentages of FSK-stimulated (100%) cAMP levels. (G, H, I, and J) Confocal laser scanning microscopy of cultured DRG cells (G and I) and native DRG sections (H and J) obtained from CFA animals immunosuppressed with CTX. Immunohistochemically labeled μ-receptors are primarily localized to the plasma membrane in animals treated with vehicle (G and H) and with chronic s.c. morphine (I and J) Scale bar: 5 μM (G and I); 10 μM (H and J). (K and L) Dose-response curves of acute i.pl. fentanyl antinociception in CFA animals immunosuppressed with CTX (K) or injected with i.pl. opioid peptide antibodies (L) with and without chronic s.c. morphine pretreatment. In both conditions the ED50 for elevation of PPT was significantly lower without than with s.c. morphine pretreatment (see Table 1) (CTX: t test, P < 0.05; opioid peptide antibodies: t test, P < 0.01).