Article Figures & Data
Figures
- Figure 1.
Morphology, expression of Nox isoforms, and superoxide production in DRG of Nox1+/Y and Nox1−/Y. a, DRG sections stained with o-toluidine blue; b, immunohistochemistry with NeuN (green) and anti-TRPV1 antibody (red); c, expression of Nox isoforms in DRG. The level of each Nox mRNA was determined by real-time PCR. d, Extracellular superoxide production in DRG neurons measured with lucigenin in the absence or presence of DPI (10 μm). WT (wild-type) and KO (knock-out) represent control littermate (Nox1+/Y) and Nox1-deficient (Nox1−/Y) mice, respectively. Values were obtained from four samples. RLU, Relative light units.
- Figure 2.
Thermal and chemical nociception in Nox1+/Y and Nox1−/Y. a, Latency for hindpaw licking and/or jumping in hotplate tests; b, the licking and/or biting time 0–10 and 20–50 min after Formalin was injected to the hindpaw. WT (wild-type) and KO (knock-out) represent control littermate (Nox1+/Y) and Nox1-deficient (Nox1−/Y) mice, respectively. Values were obtained from 8 to 10 animals per group. *p < 0.05, compared with WT.
- Figure 3.
Carrageenan-induced inflammatory pain is attenuated in Nox1−/Y. a, Development of edema after intraplantar injection of carrageenan; b, the hindpaw withdrawal latency for thermal stimuli; c, the pain score determined with the von Frey test. Thermal and mechanical hyperalgesia were significantly attenuated in Nox1−/Y. Filled and open squares indicate ipsilateral and contralateral hindpaws of Nox1+/Y, and filled and open circles indicate ipsilateral and contralateral hindpaws of Nox1−/Y (KO, for knock-out), respectively. Values were obtained from eight to nine animals per group. **p < 0.01, compared with the ipsilateral hindpaw of Nox1+/Y (WT, for wild-type).
- Figure 4.
Augmentation of the capsaicin-induced calcium increase by chemical mediators is blunted in Nox1−/Y DRG neurons. a, Functional characterization of TRPV1. Capsaicin, an agonist of TRPV1, dose dependently increased intracellular calcium levels in DRG neurons of both genotypes. Values were obtained from four to six experiments. b, Effects of forskolin (10 μm) or PMA (100 nm) on capsaicin-induced increase in intracellular calcium. c, Effects of bradykinin (10 μm) or serotonin (30 μm) on capsaicin-induced increase in intracellular calcium. Forskolin (FSK), PMA, bradykinin (BK), or serotonin (5-HT) was applied before the addition of capsaicin (Cap). Values were obtained from four to eight experiments. *p < 0.05; **p < 0.01.
- Figure 5.
PMA-induced translocation of PKC is attenuated in Nox1−/Y DRG neurons. a, Representative photographs and fluorescence intensity histograms of PKC. Top row demonstrates localization of PKCε in DRG neurons. The histogram is taken at a line bisecting the neural soma. Scale bars, 10 μm. Bottom row demonstrates quantitative measurements of the fluorescence intensity of PKCε, PKCα, and PKCδ. **p < 0.01. Values were obtained from 18 to 30 neurons. b, Effects of ROS-scavenging agents on PMA-induced PKCε translocation. Three hours before the addition of 100 nm PMA, 300 mU/ml SOD–PEG (SOD), 300 mU/ml catalase–PEG (Cat.), 10 mm N-acetyl cysteine (NAC), or 1 mm DTT was applied. Values were obtained from 15 to 25 neurons. *p, < 0.05, **p < 0.01, compared with PMA alone. WT (wild-type) and KO (knock-out) represent control littermate (Nox1+/Y) and Nox1-deficient (Nox1−/Y) mice, respectively.
- Figure 6.
Involvement of the redox state of sulfhydryl residues in the C1A domain of PKCε. a, Determination of free sulfhydryl residues in PKCε and deletion mutants after H2O2 treatment. HEK293 cells transfected with a GFP-fused full-length PKCε or deletion mutant lacking the C1A (ΔC1A) or C1B (ΔC1B) domain were exposed to 100 μm H2O2 for 5 min. Top row demonstrates iodoacetyl-PEO2 biotin-bound PKCε (I-bound), which corresponds to the enzyme containing reduced cysteine residues. Bottom row indicates the total PKCε level in the lysate. Right demonstrates quantitative densitometric analysis of the ratio of I-bound to total PKCε. Results are representative of five independent experiments. *p < 0.05, **p < 0.01, compared with control. N.S., Not significant. b, Translocation of PKCε induced by H2O2 or PMA. CHO cells transfected with full-length PKCε–GFP or ΔC1A–GFP were stimulated with 100 μm H2O2 for 2 min or with 1 μm PMA for 10 min. Scale bar, 10 μm. Representative photographs of three experiments are shown (left). Right demonstrates quantitative measurements of the fluorescence intensity of PKCε–GFP or ΔC1A–GFP. **p < 0.01. Values were obtained from 9 to 18 neurons.
Tables
Locomotor activity Coordinated movement Counts Latency to fall (s) 8 rpm 12 rpm 16 rpm Wild types 1504.0 ± 35.0 286.7 ± 13.2 193.5 ± 30.4 182.1 ± 30.5 Knock-outs 1504.5 ± 43.7 291.0 ± 9.0 222.6 ± 30.5 206.6 ± 30.3 -
Values were obtained from 11–12 animals per group.
-
