Endogenous nitric oxide inhibits spinal NMDA receptor activity and pain hypersensitivity induced by nerve injury

Abstract

The role of nitric oxide (NO) in nociceptive transmission at the spinal cord level remains uncertain. Increased activity of spinal N-methyl-d-aspartate (NMDA) receptors contributes to development of chronic pain induced by peripheral nerve injury. In this study, we determined how endogenous NO affects NMDA receptor activity of spinal cord dorsal horn neurons in control and spinal nerve-ligated rats. Bath application of the NO precursor l-arginine or the NO donor S-nitroso-N-acetylpenicillamine (SNAP) significantly inhibited NMDA receptor currents of spinal dorsal horn neurons in both sham control and nerve-injured rats. Inhibition of neuronal nitric oxide synthase (nNOS) or blocking the S-nitrosylation reaction with N-ethylmaleimide abolished the inhibitory effects of l-arginine on NMDA receptor currents recorded from spinal dorsal horn neurons in sham control and nerve-injured rats. However, bath application of the cGMP analog 8-bromo-cGMP had no significant effects on spinal NMDA receptor currents. Inhibition of soluble guanylyl cyclase also did not alter the inhibitory effect of l-arginine on spinal NMDA receptor activity. Furthermore, knockdown of nNOS with siRNA abolished the inhibitory effects of l-arginine, but not SNAP, on spinal NMDA receptor activity in both groups of rats. Additionally, intrathecal injection of l-arginine significantly attenuated mechanical or thermal hyperalgesia induced by nerve injury, and the l-arginine effect was diminished in rats treated with a nNOS inhibitor or nNOS-specific siRNA. These findings suggest that endogenous NO inhibits spinal NMDA receptor activity through S-nitrosylation. NO derived from nNOS attenuates spinal nociceptive transmission and neuropathic pain induced by nerve injury.

Keywords: Dorsal horn neurons; Ion channel; Neuropathic pain; Signal transduction; Synaptic plasticity; Synaptic transmission.

Figure 1. Endogenous NO or NO donor inhibits NMDAR activity of spinal dorsal horn neurons in sham control and SNL rats

Representative recording traces (A) and mean effects (B) of 300 :M L-arginine, 100 :M TRIM, TRIM plus L-arginine, and 100 :M SNAP on currents elicited by puff application of 100 :M NMDA onto spinal dorsal horn neurons in rats that had undergone sham surgery (n = 13 neurons) or SNL (n = 12 neurons) 3 weeks ago. Data are means ∀ s.e.m. *P < 0.05 (versus respective baseline controls). #P < 0.05 (versus baseline control in the sham group). One-way ANOVA analysis followed by Tukey’s post hoc test.

Figure 2. NO inhibits NMDAR activity in spinal dorsal horn neurons independent of sGC-cGMP signaling

Summary data show the mean effects of 60 :M 8-bromo-cGMP (8-Br-cGMP), 10 :M ODQ, and ODQ plus 300 :M L-arginine on currents elicited by puff application of 100 :M NMDA onto spinal dorsal horn neurons in rats that had undergone sham surgery or SNL 3 weeks ago (n = 12 neurons per group). Data are means ∀ s.e.m. *P < 0.05 (versus respective baseline controls). #P < 0.05 (versus baseline control in the sham group). One-way ANOVA analysis followed by Tukey’s post hoc test.

Figure 3. Endogenous NO inhibits NMDAR activity in spinal dorsal horn neurons through S-nitrosylation

Original current traces (A) and mean effects (B) of 300 :M L-arginine, 100 :M NEM, and NEM plus L-arginine on currents elicited by puff application of 100 :M NMDA onto spinal dorsal horn neurons in rats that had undergone sham surgery or SNL 3 weeks ago (n = 12 neurons per group). Data are means ∀ s.e.m. *P < 0.05 (versus respective baseline controls). #P < 0.05 (versus baseline control in the sham group). One-way ANOVA analysis followed by Tukey’s post hoc test.

Figure 4. Tonic nociceptive inhibition by nNOS at the spinal cord level in sham and SNL rats

(A,B) Mean effects of intrathecal treatment with nNOS-specific siRNA (4 :g/day for 4 days) on the mRNA level of nNOS and eNOS in the dorsal spinal cord of sham control rats and SNL rats (n = 8 rats in each group). Data are presented as means ∀ s.e.m. *P < 0.05 (versus control siRNA-treated rats). Two-tailed Student’s t-test. (C) Western blot analysis and quantification of the nNOS protein level (~160 kDa) in the dorsal spinal cord of sham control rats and SNL rats (n = 6 rats in each group). GAPDH was used as a loading control. Data are means ∀ s.e.m. *P < 0.05 (versus control siRNA-treated rats). Two-tailed Student’s t-test. (D) Time course of the effects of intrathecal treatment with control siRNA or nNOS-specific siRNA (4 :g/day for 4 days) on the mechanical nociceptive withdrawal thresholds of sham and SNL rats (n = 8 rats in each group). Data are means ∀ s.e.m. *P < 0.05 (versus baseline at time 0). One-way ANOVA analysis followed by Dunnett’s post hoc test.

Figure 5. Endogenous NO derived from nNOS inhibits NMDAR activity of spinal dorsal horn neurons in sham and SNL rats

(A,B) Representative traces (A) and mean effects (B) of 300 :M L-arginine and 100 :M SNAP on currents elicited by puff application of 100 :M NMDA onto spinal dorsal horn neurons in sham (n = 15 neurons) and SNL (n = 13 neurons) rats treated with control siRNA. (C,D) Original recording traces (C) and mean effects (D) of 300 :M L-arginine and 100 :M SNAP on puff NMDA currents of spinal dorsal horn neurons in sham (n = 13 neurons) and SNL (n = 12 neurons) rats treated with nNOS-specific siRNA. Data are means ∀ s.e.m. *P < 0.05 (versus respective baseline controls). One-way ANOVA analysis followed by Dunnett’s post hoc test.

Figure 6. Endogenous NO derived from nNOS at the spinal level reduces mechanical and thermal nociception in sham and SNL rats

(A,B) Time course of the effects of intrathecal injection of 300 :g L-arginine with vehicle or 50 :g TRIM and TRIM alone on the paw withdrawal threshold in response to a noxious pressure or heat stimulus in sham control (n = 9 rats per group) and SNL (n = 10 rats per group) rats. (C,D) Time course of the effects of intrathecal injection of 300 :g L-arginine on the mechanical and thermal nociceptive withdrawal thresholds in sham and SNL rats treated with control siRNA (n = 11 rats per group) or nNOS-specific siRNA (n = 12 rats per group). Data are means ∀ s.e.m. *P < 0.05 compared with the baseline control (time 0). #P < 0.05 (versus baseline control in the control siRNA group). One-way ANOVA analysis followed by Dunnett’s post hoc test.