Peripheral effects of morphine and expression of μ-opioid receptors in the dorsal root ganglia during neuropathic pain: nitric oxide signaling

Abstract

Background: The local administration of μ-opioid receptor (MOR) agonists attenuates neuropathic pain but the precise mechanism implicated in this effect is not completely elucidated. We investigated if nitric oxide synthesized by neuronal (NOS1) or inducible (NOS2) nitric oxide synthases could modulate the local antiallodynic effects of morphine through the peripheral nitric oxide-cGMP-protein kinase G (PKG)-ATP-sensitive K+ (KATP) channels signaling pathway activation and affect the dorsal root ganglia MOR expression during neuropathic pain.

Results: In wild type (WT) mice, the subplantar administration of morphine dose-dependently decreased the mechanical and thermal allodynia induced by the chronic constriction of the sciatic nerve (CCI), which effects were significantly diminished after their co-administration with different subanalgesic doses of a selective NOS1 (N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]-N'-nitroguanidine tris(trifluoroacetate) salt; NANT), NOS2 (L-N(6)-(1-iminoethyl)-lysine; L-NIL), L-guanylate cyclase (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; ODQ), PKG ((Rp)-8-(para-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate; Rp-8-pCPT-cGMPs) inhibitor or a KATP channel blocker (glibenclamide). The evaluation of the expression of MOR in the dorsal root ganglia from sham-operated and sciatic nerve-injured WT, NOS1 knockout (KO) and NOS2-KO mice at 21 days after surgery demonstrated that, although the basal mRNA and protein levels of MOR were similar between WT and both NOS-KO animals, nerve injury only decreased their expression in WT mice.

Conclusions: These results suggest that the peripheral nitric oxide-cGMP-PKG-KATP signaling pathway activation participates in the local antiallodynic effects of morphine after sciatic nerve injury and that nitric oxide, synthesized by NOS1 and NOS2, is implicated in the dorsal root ganglia down-regulation of MOR during neuropathic pain.

Figure 1

Antiallodynic effects of morphine. Effects of the subplantar administration of different doses (logarithmic axis) of morphine or vehicle on the mechanical (A) and thermal allodynia (B) induced by CCI in the ipsilateral paw of WT mice at 21 days after surgery. Morphine was administered 20 min before starting behavioral testing. Data are expressed as mean values of maximal possible effect (%) for mechanical allodynia and inhibition (%) for thermal allodynia ± SEM (5-6 animals for dose). In both tests, for each dose, * P <0.05 and *** P <0.001 denote significant differences between morphine and vehicle treated animals (Student's t test).

Figure 2

Reversion of the antiallodynic effects of morphine. Reversal of the effects of morphine (400 nmol) on the mechanical (A) and thermal (B) allodynia induced by CCI in the ipsilateral paw of WT mice, at 21 days after CCI, by the subplantar co-administration of a selective MOR antagonist (CTAP; 108.7 nmol) or a peripheral non-selective opioid receptor antagonist (NX-ME; 42.6 nmol). The effects of the subplantar administration of vehicle, CTAP (108.7 nmol) or NX-ME (42.6 nmol) administered alone are also shown. Data are expressed as mean values of maximal possible effect (%) for mechanical allodynia and inhibition (%) for thermal allodynia ± SEM (5-6 animals for each group). For each test, * represents significant differences compared to the other groups (P <0.05; one way ANOVA, followed by the Student Newman Keuls test).

Figure 3

Role of the peripheral nitric oxide synthesized by NOS1 and NOS2 in the antiallodynic effects of morphine. Mechanical (A, C) and thermal (B, D) antiallodynic effects of the subplantar co-administration of morphine (400 nmol) plus vehicle or different doses of NANT (17.0 - 50.9 nmol; A, B) or L-NIL (44.7 - 134.1 nmol; C, D) in the ipsilateral paw of sciatic nerve-injured WT mice at 21 days after surgery. The effects of the subplantar administration of vehicle and the maximal doses of NANT (50.9 nmol) or L-NIL (134.1 nmol) injected alone are also shown. All drugs were administered 20 min before starting behavioral testing. Data are expressed as mean values of the maximal possible effect (%) for mechanical allodynia and as inhibition (%) for thermal allodynia ± SEM (5-6 animals per group). For each behavioral test and selective inhibitor assayed, * P <0.05 denotes significant differences vs. group treated with morphine plus vehicle (one way ANOVA followed by Student Newman Keuls test) and + P <0.05 denotes significant differences vs. group treated with vehicle (one way ANOVA followed by the Student Newman Keuls test).

Figure 4

Role of the peripheral nitric oxide-cGMP-PKG-KATP signaling pathway in the antiallodynic effects of morphine. Mechanical (A, C, E) and thermal (B, D, F) antiallodynic effects of the subplantar co-administration of morphine (400 nmol) plus vehicle or different doses of ODQ (4.0 - 13.3 nmol; A, B), Rp-8 (1.2 - 4.1 nmol; C, D) or glibenclamide (Glib; 20.2 - 60.7 nmol; E,F) in the ipsilateral paw of sciatic nerve-injured WT mice at 21 days after surgery. The effects of the subplantar administration of vehicle and the maximal doses of ODQ (13.3 nmol), Rp-8 (4.1 nmol) or glibenclamide (60.7 nmol) injected alone are also shown. All drugs were administered 20 min before starting behavioral testing. Data are expressed as mean values of the maximal possible effect (%) for mechanical allodynia and as inhibition (%) for thermal allodynia ± SEM (5-6 animals per group). For each behavioral test and selective inhibitor assayed, * P <0.05 denotes significant differences vs. group treated with morphine plus vehicle (one way ANOVA followed by the Student Newman Keuls test) and + P <0.05 denotes significant differences vs. group treated with vehicle (one way ANOVA followed by Student Newman Keuls test).

Figure 5

Dorsal root ganglia expression of MOR in WT, NOS1-KO and NOS2-KO mice. Relative mRNA (A) and protein (B) expression of MOR in the ipsilateral dorsal root ganglia from sham-operated or sciatic nerve-injured WT, NOS1-KO and NOS2-KO mice. Data are expressed as mean values ± SEM (4-5 samples per group). For each genotype, * P <0.05 denotes significant differences between sciatic nerve-injured and sham-operated mice (Student's t test). For each experimental group, different letters (a, b) denote significant differences between genotypes (P <0.05; one way ANOVA followed by the Student Newman Keuls test).