Article Figures & Data
Figures
- Fig. 1.
Lack of N/OFQ-stimulated [35S]-G-TPγS binding in brain membranes of NOR−/− mice and characterization of J-113397 as an N/OFQ antagonist in [35S]GTPγS binding assay.A, Effects of various concentrations of N/OFQ on [35S]GTPγS binding in amygdala membranes of NOR+/+ and NOR−/− mice. The dose of N/OFQ (mean ± SEM) showing 50% increase in [35S]GTPγS binding was 34.9 ± 22.9 nm. B, Effects of N/OFQ or J-113397 on [35S]GTPγS binding in ddY mouse amygdala. The dose of N/OFQ showing 50% increase in [35S]GTPγS binding was 19.1 ± 7.7 nm. C, Antagonist effects of J-113397 on N/OFQ-stimulated [35S]GTPγS binding in ddY mouse amygdala. The IC50 of J-113397 for inhibition of N/OFQ (1 μm)-stimulated [35S]GTPγS binding was 423 ± 169 nm. D, E, Effects of N/OFQ or J-113397 on [35S]GTPγS binding in Sf21 cell membranes expressing ORL1 plus Gαi1β1/γ2 (D) or ORL1 plus GαoAβ1/γ2(E). F, G, Antagonist effects of J-113397 on N/OFQ-stimulated [35S]GTPγS binding in Sf21 cell membranes expressing ORL1 plus Gαi1β1/γ2 (F) or ORL1 plus GαoAβ1/γ2(G). The IC50 of J-113397 for [35S]GTPγS binding stimulated by 1 μm N/OFQ in preparations expressing Gαi1β1γ2 or GαoAβ1γ2 was 526 ± 63.4 nm (n = 3) or 426 ± 126 nm (n = 3), respectively. Data are the mean ± SEM from five to eight separate experiments.
- Fig. 2.
Lack of N/OFQ-stimulated [35S]GTPγS binding in brain sections in NOR−/− mice (A) or ddY mice treated with J-113397 (B). In both experiments, coronal sections (20 μm) were taken from NOR+/+, NOR−/− mice, or ddY mice. N/OFQ and J-113397 were used at a concentration of 1 and 10 μm, respectively.
- Fig. 3.
Characterization of J-113397 as an N/OFQ antagonist in peripheral and central nociception tests.A, B, Peripheral nociception induced by various doses of N/OFQ in NOR+/+ mice, NOR−/− mice (A), and ddY mice treated with and without J-113397 (B). N/OFQ was intraplantarly injected, and nociceptive flexor responses were observed. Results were evaluated as percent of maximal flexor response. J-113397 (10 fmol) was given intraplantarly through another cannula at 20 min before the nociception test. C, Effects of systemic injection of J-113397 on N/OFQ-induced SBL responses. J-113397 was injected subcutaneously at 30 min before N/OFQ intrathecal injection. Each point in all figures is the mean ± SEM from five to eight mice in each group. *p < 0.05 versus NOR+/+ mice or N/OFQ alone.
- Fig. 4.
Attenuation of morphine tolerance in NOR−/− mice in the tail-pinch and tail-flick tests. A, Time course of morphine analgesia in NOR+/+ or NOR−/− mice without or with morphine chronic treatment in tail-pinch test.(1) and (6) represent the results with morphine (10 mg/kg, s.c.) in naive mice and in mice that had been treated previously with daily morphine injections (10 mg/kg, s.c.) for 5 d. *p < 0.05 versus NOR+/+ mice. B, Morphine dose–analgesia curve in NOR+/+ or NOR−/− mice with or without chronic morphine pretreatment. Tail-pinch analgesia was represented by AUC units. C, D, Time course (C) and quantitative comparison (D) of morphine analgesia in tail-flick test in NOR+/+ or NOR−/− mice with or without chronic morphine pretreatment. *p < 0.05 versus NOR+/+ mice. Each point is the mean ± SEM from five to eight mice in each group.
- Fig. 5.
Attenuation of morphine tolerance in ddY mice treated with J-113397 in tail-pinch test. A, Time course of morphine analgesia in naive mice without (Veh) or with J-113397 at a dose of 30 mg/kg subcutaneously. J-113397 was given 60 min before morphine injection. B, Time course of morphine analgesia in morphine chronic mice without (Veh) or with J-113397 at a dose of 30 mg/kg subcutaneously (B). C, Effects of various doses of J-113397 (subcutaneously) on morphine tolerance.D, E, J-113397 was given intrathecally (D) or intracerebroventricularly (E) 5 min before morphine injection. *p < 0.05 versus vehicle control on the sixth day. Each point is the mean ± SEM from five to eight mice in each group.
- Fig. 6.
Attenuation of morphine tolerance in ddY mice treated with J-113397 in the tail-flick test. Details are given in the legend of Figure 5, except for the tail-flick test.
- Fig. 7.
Attenuation of naloxone-precipitated morphine withdrawal signs in NOR−/− mice or in ddY mice treated with J-113397. A–E, Comparison of the degree of withdrawal signs between NOR+/+ and NOR−/− mice. V and Mrepresent mice chronically treated with vehicle or morphine, respectively. *p < 0.05 versus morphine in NOR+/+ mice. F–J, Comparison of the degree of withdrawal signs between mice treated with vehicle (Veh) or J-113397 (10 or 30 mg/kg, s.c.), which was given 60 min before naloxone challenge. #p < 0.05 versus vehicle. Each point is the mean ± SEM from five to eight mice in each group.
- Fig. 8.
Enhancement of NOR gene expression in the spinal cord during the development of morphine tolerance and dependence. All experiments were performed in ddY mice. A, Expression of NOR or GAPDH in mice chronically treated without (V) and with morphine (M). Expression was determined by RT-PCR as described in Materials and Methods. B, Quantitative RT-PCR for NOR gene expression. NOR gene expression in the spinal cord was analyzed using GAPDH expression as a reference. The results represent the relative NOR expression to GAPDH expression in the spinal cord. Data are the mean ± SEM from four separate experiments. *p < 0.05 versus vehicle. C, Time course of NOR expression in the spinal cord during morphine (10 mg/kg, s.c.) treatments. Data are the mean ± SEM from six separate experiments. D, Quantitative RT-PCR for NOR gene expression in the spinal cord from mice treated with increasing morphine doses. The results represent the relative NOR expression to GAPDH expression in the spinal cord. Veh, Vehicle;Mor, morphine treatment. Data are the mean ± SEM from four separate experiments. *p < 0.05 versus vehicle.
