cGMP produced by NO-sensitive guanylyl cyclase essentially contributes to inflammatory and neuropathic pain by using targets different from cGMP-dependent protein kinase I

Abstract

A large body of evidence indicates that the release of nitric oxide (NO) is crucial for the central sensitization of pain pathways during both inflammatory and neuropathic pain. Here, we investigated the distribution of NO-sensitive guanylyl cyclase (NO-GC) in the spinal cord and in dorsal root ganglia, and we characterized the nociceptive behavior of mice deficient in NO-GC (GC-KO mice). We show that NO-GC is distinctly expressed in neurons of the mouse dorsal horn, whereas its distribution in dorsal root ganglia is restricted to non-neuronal cells. GC-KO mice exhibited a considerably reduced nociceptive behavior in models of inflammatory or neuropathic pain, but their responses to acute pain were not impaired. Moreover, GC-KO mice failed to develop pain sensitization induced by intrathecal administration of drugs releasing NO or carbon monoxide. Surprisingly, during spinal nociceptive processing, cGMP produced by NO-GC may activate signaling pathways different from cGMP-dependent protein kinase I (cGKI), whereas cGKI can be activated by natriuretic peptide receptor-B dependent cGMP production. Together, our results provide evidence that NO-GC is crucially involved in the central sensitization of pain pathways during inflammatory and neuropathic pain.

Figure 1.

Expression of NO-GC in the spinal cord. A, B, Immunohistochemistry of the GCβ1 subunit of NO-GC in the lumbar spinal cord in wild-type mice (A) and in GC-KO mice (B) reveals specific NO-GC expression throughout the spinal cord with strong immunoreactivity in the dorsal horn and around the central canal. Scale bar, 100 μm. C, Western blot of GCβ1 (70 kDa) with spinal cord homogenates of wild-type mice (left) and GC-KO mice (right) confirms the specificity of the antibody. GAPDH (36 kDa) was used as loading control.

Figure 2.

Double labeling of NO-GC with markers in the dorsal horn of the spinal cord. A, E, I, M, GCβ1 immunoreactivity. B, F, J, N, Immunoreactivity for NK1-R, GAD67, CGRP, and IB4. C, G, K, O, Merged images with colocalization indicated in yellow. D, H, L, P, Merged images at higher magnification. The experiments revealed that NO-GC colocalizes with NK1-R and GAD67, but not with CGRP or IB4. Scale bars: A, D, H, L, P, 25 μm; E, I, M, 50 μm.

Figure 3.

Expression of NO-GC in DRGs. Triple labeling of GCβ1 (blue), caveolin-1 (red), and α-smooth muscle actin (green) demonstrates that NO-GC is expressed in satellite cells, in pericytes around capillary endothelial cells, and in smooth muscle cells, but not in DRG neurons. A, B, Wild-type mouse. C, D, GC-KO mouse. Arrows, Pericytes around capillary endothelial cells; arrowheads, satellite cells; doubled arrowheads, blood vessels. E, GCβ1-immunoreactive pericyte. Inset, Same area as in E, but GCβ1-immunoreactivity only is shown. F, GCβ1-immunoreactive small blood vessels. Inset, Same area as in F, but GCβ1-immunoreactivity only is shown. Scale bars: (in A) A, B, 50 μm; (in C) C, D, 20 μm; E, 5 μm; F, 10 μm.

Figure 4.

Inflammatory and neuropathic pain in GC-KO mice. A, B, Formalin test. A, Time course of licking behavior of the formalin-injected hindpaw. B, Sum of paw-licking time in phase 1 (0–10 min) and phase 2 (11–45 min). Note that the licking behavior in phase 2 is considerably reduced in GC-KO mice. C, Zymosan-induced mechanical hyperalgesia. Time course of paw withdrawal latency time after mechanical stimulation demonstrates reduced mechanical hyperalgesia in GC-KO mice at time points ≥5 h. D, E, SNI-induced neuropathic pain in GC-KO mice. D, Mechanical allodynia. Time course of paw withdrawal latency time after mechanical stimulation. E, Cold allodynia. Time course of latency time to shake, flinch, or lick the SNI-operated hindpaw after placement on a 15°C cold plate. Before SNI surgery, all animals stayed on the cold plate for 30 s (cutoff) without paw shaking, flinching, or licking. Data indicate that the maintenance of neuropathic pain (at time points ≥10 d), but not the induction of neuropathic pain, is impaired in GC-KO mice. All data are presented as mean ± SEM. *p < 0.05, comparing GC-KO and WT mice; n = 6–8 (GC-KO) or 8 (WT) per group.

Figure 5.

NO-, CO-, and cGMP-induced nociceptive behavior in GC-KO mice. A–C, Time course of mechanical allodynia induced by intrathecal administration of NOC-5 (NO donor, 50 nmol; A), CORM-A1 (CO-releasing molecule, 40 nmol; B), or 8-pCPT-cGMP (cGMP analog, 15 nmol; C). No allodynia was observed in GC-KO mice after administration of NOC-5 or CORM-A1, whereas 8-pCPT-cGMP-induced allodynia was intact. The hindpaw withdrawal latency times after mechanical stimulation were normalized to baseline latency times and are presented as mean percentage ± SEM. *p < 0.05, comparing GC-KO and WT mice; n = 6 per group.

Figure 6.

Double labeling of cGKI with NO-GC and NPR-B in DRGs and in the dorsal horn of the spinal cord. A, D, G, cGKI immunoreactivity, B, E, H, Immunoreactivity for GCβ1 or NPR-B. C, F, I, Merged images with colocalization being indicated in yellow. The experiments revealed that cGKI and NO-GC are not colocalized in DRGs and only partially colocalized in the spinal cord, whereas cGKI colocalizes with NPR-B in DRGs. Scale bars, 25 μm.

Figure 7.

Effect of cGKI inhibition on the nociceptive behavior induced by activators of NO-GC and natriuretic peptide-activated GCs. A, B, Time course of mechanical allodynia induced by intrathecal administration of NOC-5 (NO donor, 50 nmol), Rp-8-pCPT-cGMPS (cGKI inhibitor, 10 nmol), or the combination of NOC-5 (50 nmol) and Rp-8-pCPT-cGMPS (10 nmol) (A), and CNP (NPR-B activator, 200 ng), ANP (NPR-A activator, 200 ng), or the combination of CNP (200 ng) and Rp-8-pCPT-cGMPS (10 nmol) (B). Data indicate that the cGKI inhibitor antagonizes CNP-induced allodynia but not NOC-5-induced allodynia. The hindpaw withdrawal latency times after mechanical stimulation were measured at the indicated time points. Data were normalized to baseline latency times and are presented as mean percentage ± SEM; n = 6 or 8 per group.