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Intrathecal (i.t.) injection of morphine-3-glucuronide (M3G), a major metabolite of morphine without analgesic actions, produces severe hindlimb scratching followed by biting and licking in mice. The M3G-induced behavioral response was inhibited dose-dependently by pretreatment with an antisera against dynorphin. However, the selective κ-opioid receptor antagonist, nor-BNI did not prevent the M3G-induced behavioral response. Dynorphin is rapidly degraded by a dynorphin-converting enzyme (cystein protease), to leucine-enkephalin (Leu-ENK). The M3G-induced behavioral response was inhibited dose-dependently by pretreatment with the antisera against Leu-ENK. We also showed that M3G co-administered with Leu-ENK-converting enzyme inhibitors, phosphoramidon and bestatin produced much stronger behavioral responses than M3G alone. Furthermore, the M3G-induced behavioral responses were inhibited dose-dependently by i.t. co-administration of the non-selective δ-opioid receptor antagonist, naltrindole or the selective δ2-opioid receptor antagonist, naltriben, whereas the selective δ1-opioid receptor antagonist, BNTX had no effect. An i.t. injection of M3G also produced a definite activation of ERK in the lumbar dorsal spinal cord. Western blotting analysis revealed that antisera against dynorphin, antisera against Leu-ENK, naltrindole or naltriben resulted in a significant blockade of ERK activation induced by M3G in the spinal cord. Taken together, these results suggest that M3G-induced nociceptive responses and ERK activation may be triggered via δ2-opioid receptors activated by Leu-ENK, which is formed from dynorphin in the spinal cord., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Intrathecal (i.t.) injection of leucine-enkephalin (Leu-ENK), co-administered with peptidase inhibitors, phosphoramidon (an endopeptidase 24.11 inhibitor), and bestatin (a general aminopeptidase inhibitor), produced behaviors consisting of the biting and/or licking of the hindpaw and the tail along with hindlimb scratching directed toward the flank, which peaked at 10-15 min after an injection. This characteristic behavior was not observed in mice treated with i.t. Leu-ENK alone. We also investigated the effect of the extracellular signal-regulated kinase (ERK) in spinal processing of nociception induced by i.t. co-administration of Leu-ENK with phospharamidon and bestatin. Western blot analysis of phospho-ERK (pERK) showed a significant increase of pERK2 in the lumbar spinal cord in response to i.t. Leu-ENK co-injected with peptidase inhibitors. The MAP kinase-ERK inhibitor, U0126 dose-dependently attenuated the nociceptive behavior and spinal ERK activation to i.t. Leu-ENK co-injected with peptidase inhibitors. Furthermore, the nociceptive behavior and spinal ERK activation evoked by i.t. Leu-ENK in combination with peptidase inhibitors were inhibited by co-administration of the non-selective δ-opioid receptor antagonist, naltrindole, the selective δ2-opioid receptor antagonist, naltriben, the non-competitive N-methyl-D-aspartate (NMDA) antagonist, MK-801 or the non-selective nitric oxide synthase inhibitor, L-NAME, the selective nNOS inhibitor, N(ω)-propyl-L-arginine or the selective iNOS inhibitor, W1400, but not by the selective δ1-receptor antagonist, BNTX (7-benzylidenenaltrexone). These results suggest that spontaneous nociceptive behaviors produced by i.t. co-administration of Leu-ENK with peptidase inhibitors may be induced by an activation of the glutamate-NO-ERK pathway through the δ2-opioid receptor in the dorsal spinal cord., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Nociceptin/orphanin FQ (N/OFQ), the endogenous ligand for the N/OFQ peptide (NOP) receptors, has been shown to be metabolized into some fragments. We examined to determine whether intrathecal (i.t.) N/OFQ (1-13), (1-11) and (1-7) have antinociceptive activity in the pain-related behavior after intraplantar injection of capsaicin. The i.t. administration of N/OFQ (0.3-1.2 nmol) produced an appreciable and dose-dependent inhibition of capsaicin-induced paw-licking/biting response. The N-terminal fragments of N/OFQ, (1-13) and (1-11), were antinociceptive with a potency lower than N/OFQ. Calculated ID₅₀ values (nmol, i.t.) were 0.83 for N/OFQ, 2.5 for N/OFQ (1-13) and 4.75 for N/OFQ (1-11), respectively. The time-course effect revealed that the antinociceptive effects of these N-terminal fragments lasted longer than those of N/OFQ. Removal of amino acids down to N/OFQ (1-7) led to be less potent than N/OFQ and its fragments, (1-13) and (1-11). Antinociception induced by N/OFQ or N/OFQ (1-13) was reversed significantly by i.t. co-injection of [Nphe¹]N/OFQ (1-13)NH₂, a peptidergic antagonist for NOP receptors, whereas i.t. injection of the antagonist did not interfere with the action of N/OFQ (1-11) and (1-7). Pretreatment with the opioid receptor antagonist naloxone hydrochloride did not affect the antinociception induced by N/OFQ and its N-terminal fragments. These results suggest that N-terminal fragments of N/OFQ are active metabolites and may modulate the antinociceptive effect of N/OFQ in the spinal cord. The results also indicate that N/OFQ (1-13) still possess antinociceptive activity through NOP receptors., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Intrathecal (i.t.) injection of morphine-3-glucuronide (M3G), a major metabolite of morphine without analgesic actions, produces a severe hindlimb scratching followed by biting and licking in mice. The pain-related behavior evoked by M3G was inhibited dose-dependently by i.t. co-administration of tachykinin NK(1) receptor antagonists, sendide, [D-Phe(7), D-His(9)] substance P(6-11), CP-99994 or RP-67580 and i.t. pretreatment with antiserum against substance P. The competitive NMDA receptor antagonists, D-APV and CPP, the NMDA ion-channel blocker, MK-801 or the competitive antagonist of the polyamine recognition site of NMDA receptor ion-channel complex, ifenprodil, produced inhibitory effects on i.t. M3G-evoked nociceptive response. The NO-cGMP-PKG pathway, which involves the extracellular signal-regulated kinase (ERK), has been implicated as mediators of plasticity in several pain models. Here, we investigated whether M3G could influence the ERK activation in the NO-cGMP-PKG pathway. The i.t. injection of M3G evoked a definite activation of ERK in the lumbar dorsal spinal cord, which was prevented dose-dependently by U0126, a MAP kinase-ERK inhibitor. The selective nNOS inhibitor N(omega)-propyl-l-arginine, the selective iNOS inhibitor W1400, the soluble guanylate cyclase inhibitor ODQ and the PKG inhibitor KT-5823 inhibited dose-dependently the nociceptive response to i.t. M3G. In western blotting analysis, inhibiting M3G-induced nociceptive response using these inhibitors resulted in a significant blockade of ERK activation induced by M3G in the spinal cord. Taken together, these results suggest that activation of the spinal ERK signaling in the NO-cGMP-PKG pathway contributes to i.t. M3G-evoked nociceptive response.

Abstract: Previous research has demonstrated that substance P N-terminal fragments produced by the action of several different enzymes in the spinal cord could reduce nociception when injected intrathecally (i.t.) into mice. The present study examined the possible involvement of spinal extracellular signal-regulated protein kinase (ERK), a mitogen-activated protein kinase (MAPK), in i.t. substance P (1–7)-induced antinociception as assayed by the capsaicin test. The i.t. injection of substance P (1–7) (20–80 nmol) into mice resulted in a dose-dependent attenuation of paw-licking/biting behavior induced by intraplantar injection of capsaicin, which was reversed by co-injection of [d-Pro2, d-Phe7]substance P (1–7), a d-isomer and antagonist of substance P (1–7). In Western blot analysis, intraplantar injection of capsaicin (400 and 1600 ng/paw) produced an increase of ERK phosphorylation in the dorsal spinal cord, whereas expression of p38 and c-Jun N-terminal kinase (JNK) phosphorylation was unchanged by capsaicin treatment. In parallel to the behavioral results, i.t. substance P (1–7) inhibited capsaicin-induced EKR phosphorylation, which was reversed by [d-Pro2, d-Phe7]substance P (1–7), a substance P (1–7) antagonist. Both nociceptive behavioral response and spinal ERK activation induced by intraplantar capsaicin were reduced by U0126, an upstream inhibitor of ERK phosphorylation. Taken together, these findings suggest that the activation of ERK, but not p38 and JNK MAPKs in the spinal cord, contributes to intraplantar capsaicin-induced nociception, and that blocking ERK activation via substance P (1–7) binding sites may provide significant antinociception at the spinal cord level. [Copyright &y& Elsevier]

Abstract: Intrathecal (i.t.) administration of morphine at a high dose of 60nmol into the spinal lumbar space in mice produces a severe hindlimb scratching followed by biting and licking. Nitric oxide (NO) is thought to play an important role in signal transduction pathways that enhance nociceptive transmission in the spinal cord. The present study was designed to determine whether high-dose i.t. morphine could influence the activation of the extracellular signal-regulated kinase (ERK), a mitogen-activated protein (MAP) kinase in neuronal nitric oxide synthase (nNOS) and inducible NOS (iNOS) activation. Both 7-NI and TRIM, selective inhibitors of nNOS, resulted in a dose-dependent inhibition of high-dose i.t. morphine-induced behavior. The selective iNOS inhibitor W1400 in relatively large doses inhibited in a non dose-dependent manner. The i.t. injection of morphine evoked a definite activation of ERK in the lumbar dorsal spinal cord. Behavioral experiments showed that U0126 (0.5–2.5nmol), a MAP kinase-ERK inhibitor, dose-dependently attenuated the behavioral response to i.t. morphine. In mice treated with high-dose morphine, 7-NI was very effective in blocking ERK activation, whereas W1400 had no effect. Taken together, these results suggest that the behavioral response to high-dose i.t. morphine may be triggered by the nNOS-ERK pathway in the dorsal spinal cord. [Copyright &y& Elsevier]