Nitric Oxide in the Spinal Cord Is Involved in the Hyperalgesia Induced by Tetrahydrobiopterin in Chronic Restraint Stress Rats

Abstract

It has been well recognized that exposure to chronic stress could increase pain responding and exacerbate pain symptoms, resulting in stress-induced hyperalgesia. However, the mechanisms underlying stress-induced hyperalgesia are not yet fully elucidated. To this end, we observed that restraint as a stressful event exacerbated mechanical and thermal hyperalgesia, accompanied with up-regulation of nitric oxide (NO) (P < 0.001), GTP cyclohydrolase 1 (GCH1) (GCH1 mRNA: P = 0.001; GCH1 protein: P = 0.001), and tetrahydrobiopterin (BH4) concentration (plasma BH4: P < 0.001; spinal BH4: P < 0.001) on Day 7 in restraint stress (RS) rats. Intrathecal injection of N ^ω-nitro-L-arginine methyl ester (L-NAME), a non-specific NO synthase inhibitor, or N-([3-(aminomethyl)phenyl]methyl) ethanimidamide, a special inhibitor of inducible NO synthase (iNOS), for seven consecutive days attenuated stress-induced hyperalgesia and decreased the production of NO (P < 0.001). Interestingly, 7-nitro indazole, a special inhibitor of neuronal NO synthase, alleviated stress-induced hyperalgesia but did not affect spinal NO synthesis. Furthermore, intrathecal injection of BH4 not only aggravated stress-induced hyperalgesia but also up-regulated the expression of spinal iNOS (iNOS mRNA: P = 0.015; iNOS protein: P < 0.001) and NO production (P < 0.001). These findings suggest that hyperalgesia induced by RS is associated with the modulation of the GCH1-BH4 system and constitutively expressed spinal iNOS. Thus, the GCH1-BH4-iNOS signaling pathway may be a new novel therapeutic target for pain relief in the spinal cord.

Keywords: GTP cyclohydrolase 1; inducible nitric oxide synthase; spinal cord; stress-induced hyperalgesia; tetrahydrobiopterin.

FIGURE 1

Mechanical or thermal nociceptive thresholds in rats with or without chronic RS. (A–C) Nociceptive behavior tests including PWMT (RS effect: F_1,22 = 192.322, P < 0.001; observation intervals: F_3,66 = 22.641, P < 0.001; interaction: F_3,66 = 16.798, P < 0.001), PWTL (RS effect: F_1,22 = 141.084, P < 0.001; observation intervals: F_2,44 = 4.606, P = 0.015; interaction: F_2,44 = 12.632, P < 0.001), and TFL (RS effect: F_1,22 = 5.457, P = 0.029; observation intervals: F_2,44 = 0.002, P = 0.998; interaction: F_2,44 = 2.552, P = 0.089). Data are shown as mean ± SEM (n = 12). *Significant difference with respect to control groups (two-way ANOVA with repeated measures in nociceptive behavior tests, followed by Bonferroni post hoc test or Dunnett’s T3 test if necessary). *P < 0.05. RS, restraint stress; PWMT, paw withdrawal mechanical threshold; PWTL, paw withdrawal thermal latency; TFL, tail-flick latency; NO, nitric oxide; iNOS, inducible nitric oxide synthase.

FIGURE 2

The expression of spinal GCH1 and the production of plasma and spinal BH4 in the rats with or without chronic RS. (A,B) The expression of spinal GCH1 mRNA (P = 0.001) and spinal GCH1 protein (P = 0.001) in the RS rats and control rats. (C) The concentration of plasma BH4 (P < 0.001) and spinal BH4 (P < 0.001) in the RS and control rats. *Significant difference with respect to control groups (Student’s t test). **P < 0.01 and ***P < 0.001. GCH1, GTP cyclohydrolase 1; BH4, tetrahydrobiopterin; RS, restraint stress.

FIGURE 3

Effects of either DAHP or BH4 intrathecal administration on nociceptive behavior in rats subjected to chronic RS. Chronic RS rats were consecutively administered with either DAHP (6 mg/kg) or BH4 (1 μg/ml, 10 μl) as the study protocol in Figure 1. (A–C) Nociceptive behavior tests including PWMT (treatment: F_2,23 = 73.995, P < 0.001; observation intervals: F_3,69 = 45.069, P < 0.001; interaction: F_6,69 = 11.544, P < 0.0001); (B) treatment: F_2,23 = 51.543, P < 0.001; observation intervals: F_2,46 = 3.942, P = 0.026; interaction: F_4,46 = 0.761, P = 0.556; (C) treatment: F_2,23 = 50.134, P < 0.001; observation intervals: F_2,46 = 6.665, P = 0.003; interaction: F_4,46 = 0.257, P = 0.904. Data are shown as mean ± SEM (n = 8–10). *Significant difference with respect to vehicle groups (two-way ANOVA with repeated measures in nociceptive behavior tests, followed by Bonferroni post hoc test or Dunnett’s T3 test if necessary, *P < 0.05). BH4, tetrahydrobiopterin; RS, restraint stress; PWMT, paw withdrawal mechanical threshold.

FIGURE 4

The expression of spinal NO in the rats subjected or not subjected to chronic RS and effect of L-NAME on the nociceptive behavior and spinal NO in the rat models of RS. (A) The expression of spinal NO in the rats subjected or not subjected to chronic RS, P < 0.001. (B) Effects of either DAHP or BH4 intrathecal administration on spinal NO in rats subjected to chronic RS. Chronic RS rats received chronic intrathecal treatment with either DAHP (6 mg/kg) or BH4 (1 μg/ml, 10 μl) from the first day of establishing chronic RS until the end of the experiment, F_2,15 = 67.94, P < 0.001. (C–E) Effect of L-NAME intrathecal administration on nociceptive behavior in rats subjected to chronic RS; chronic RS rats that received chronic intrathecal treatment with L-NAME (30 μg/μl, 10 μl) 30 min prior to BH4 (1 μg/ml, 10 μl), L-NAME (30 μg/μl, 10 μl), and BH4 (1 μg/ml, 10 μl) treatment. Nociceptive behavior tests including PWMT (treatment: F_3,33 = 47.94, P < 0.001; observation intervals: F_3,99 = 49.132, P < 0.001; interaction: F_9,99 = 16.453, P < 0.0001), PWTL (treatment: F_3,33 = 34.594, P < 0.001, observation intervals: F_2,66 = 1.131, P = 0.329; interaction: F_6,66 = 3.15, P = 0.009), and TFL (treatment: F_3,33 = 22.943, P < 0.001; observation intervals: F_2,66 = 1.598, P = 0.21; interaction: F_6,66 = 2.904, P = 0.014). (F) Effect of intrathecal administration of L-NAME on the expression of spinal NO, F_3,20 = 51.906, P < 0.001. *Significant difference with respect to control or vehicle groups (two-way ANOVA with repeated measures). *P < 0.05 and ***P < 0.001. NO, nitric oxide; RS, restraint stress; L-NAME, N^ω-nitro-L-arginine methyl ester; NO, nitric oxide; BH4, tetrahydrobiopterin; PWMT, paw withdrawal mechanical threshold; PWTL, paw withdrawal thermal latency; TFL, tail-flick latency.

FIGURE 5

The expression of three isoforms of NOS in the spinal cord in the chronic RS or control rats. (A,B) The expression of spinal iNOS mRNA (P = 0.045) and protein (P = 0.046) in the rats subjected or not subjected to chronic RS. (C,D) The expression of spinal eNOS mRNA (P = 0.937) and protein (P = 0.449) in the rats subjected or not subjected to chronic RS. (E,F) The expression of spinal nNOS mRNA (P = 0.3) and protein (P = 0.565) in the RS rats or control rats. Data are presented as mean ± SEM (n = 4), *P < 0.05. NOS, nitric oxide synthase; RS, restraint stress; iNOS, inducible nitric oxide synthase; eNOS, endothelial nitric oxide synthase; nNOS, neuronal nitric oxide synthase.

FIGURE 6

Effect of either DAHP or BH4 intrathecal administration on the expression of three spinal NOS isoforms. (A,B) The expression of spinal iNOS mRNA (F_2,9 = 6.869, P = 0.015) and protein (F_2,9 = 123.036, P < 0.001) in rats treated with DAHP or BH4. (C,D) The expression of spinal eNOS mRNA (F_2,9 = 0.879, P = 0.448) and protein (F_2,9 = 0.081, P = 0.923) in rats treated with DAHP or BH4. (E,F) The expression of spinal nNOS mRNA (F_2,9 = 0.223, P = 0.804) and protein (F_2,9 = 0.131, P = 0.879) in rats treated with DAHP or BH4. Data are shown as mean ± SEM (n = 4). *Significant difference with respect to vehicle groups (one-way ANOVA followed by Bonferroni post hoc test or Dunnett’s T3 test if necessary). *P < 0.05 and **P < 0.01. BH4, tetrahydrobiopterin; NOS, nitric oxide synthase; iNOS, inducible nitric oxide synthase; eNOS, endothelial nitric oxide synthase; nNOS, neuronal nitric oxide synthase.

FIGURE 7

Effect of specific 1400 W or 7-NI on nociceptive behavior and spinal NO in rats subjected to chronic RS, and the cell type specificity of iNOS. Chronic RS rats were consecutively treated with either 1400 W (1 μg/ml, 10 μl) or 7-NI (40 μg/ml, 10 μl). (A–C) Nociceptive behavior tests including PWMT (treatment: F_2,25 = 20.961, P < 0.001; observation intervals: F_3,75 = 4.045, P = 0.016; interaction: F_6,75 = 9.247, P < 0.0001), PWTL (treatment: F_2,25 = 15.561, P < 0.001; observation intervals: F_2,50 = 2.038, P = 0.154; interaction: F_4,50 = 1.831, P = 0.158), and TFL (treatment: F_2,25 = 4.898, P = 0.016; observation intervals: F_2,50 = 3.174, P = 0.064; interaction: F_4,50 = 1.1, P = 0.362). (D) Effect of 1400 W or 7-NI on the expression of spinal NO, F_2,15 = 27.36, P < 0.001. Data are shown as mean ± SEM (n = 8–10). *Significant difference with respect to vehicle groups (two-way ANOVA with repeated measures in nociceptive behavior tests and one-way ANOVA in spinal NO measurement followed by Bonferroni post hoc test or Dunnett’s T3 test if necessary), *P < 0.05 and ***P < 0.001. (E) In the superficial layer of the spinal dorsal horn (lamina I–III), iNOS-positive cells were increased in the RS rats compared with control rats. (F) Confocal images of iNOS immunostaining (green) and its colocalization with astrocytes (GFAP, red), but not with microglia (Iba1, red) or neurons (Neun, red) in the superficial spinal dorsal horns (lamina I–III, n = 3 in each group). Scale bar = 100 μm. 7-NI, 7-nitro indazole; NO, nitric oxide; RS, restraint stress; iNOS, inducible nitric oxide synthase; PWMT, paw withdrawal mechanical threshold; PWTL, paw withdrawal thermal latency; iNOS, inducible nitric oxide synthase.

FIGURE 8

Experimental design. RS, restraint stress; PWMT, paw withdrawal mechanical threshold; PWTL, paw withdrawal thermal latency; TFL, tail-flick latency; BH4, tetrahydrobiopterin; L-NAME, N^ω-nitro-L-arginine methyl ester; DAHP, 2,4-diamino-6-hydroxypyrimidine; 7-NI, 7-nitro indazole; 1400W, N-([3-(aminomethyl)phenyl]methyl)ethanimidamide.