Reactive oxygen species are second messengers of neurokinin signaling in peripheral sensory neurons

Abstract

Substance P (SP) is a prominent neuromodulator, which is produced and released by peripheral damage-sensing (nociceptive) neurons; these neurons also express SP receptors. However, the mechanisms of peripheral SP signaling are poorly understood. We report a signaling pathway of SP in nociceptive neurons: Acting predominantly through NK1 receptors and G(i/o) proteins, SP stimulates increased release of reactive oxygen species from the mitochondrial electron transport chain. Reactive oxygen species, functioning as second messengers, induce oxidative modification and augment M-type potassium channels, thereby suppressing excitability. This signaling cascade requires activation of phospholipase C but is largely uncoupled from the inositol 1,4,5-trisphosphate sensitive Ca(2+) stores. In rats SP causes sensitization of TRPV1 and produces thermal hyperalgesia. However, the lack of coupling between SP signaling and inositol 1,4,5-trisphosphate sensitive Ca(2+) stores, together with the augmenting effect on M channels, renders the SP pathway ineffective to excite nociceptors acutely and produce spontaneous pain. Our study describes a mechanism for neurokinin signaling in sensory neurons and provides evidence that spontaneous pain and hyperalgesia can have distinct underlying mechanisms within a single nociceptive neuron.

Fig. 1.

Functional expression of NKR in sensory neurons. (A and B) Ca²⁺ imaging in small-diameter TRPV1⁺ DRG neurons loaded with fura-2 AM. (A) Capsaicin (CAP, 50 nM) was added to the bathing solution as indicated by the black bars. Before the fourth application of CAP, an NKR agonist was added to the bathing solution as indicated by the orange shaded area. “CAP MAX“ indicates a saturating dose of capsaicin (1 µM). Each trace represents an example of the effect of four different NKR agonists; the key is shown in B. All NKR agonists were applied at a concentration of 1 µM. SP is an agonist of all three NKR; Sar-met SP is an agonist of NK1, β-ala NKA is an agonist of NK2; and senktide is an agonist of NK3. Data are presented as the fluorescence ratio (340/380 nm, R) normalized to the initial ratio at time = 0 s (R₀). (B) Mean data from A normalized to the size of the first CAP peak response (CAP1). Only cells that showed sensitization of the CAP4 response were included. ***Significant difference between CAP3 and CAP4 peak response (P < 0.001; paired t test). (C) Proportion of DRG neurons responding with a rise in cytosolic Ca²⁺ in response to NKR agonists (Left) or with a sensitization of TRPV1 (Right). Significant difference in the proportions of Ca²⁺ responders and TRPV1 sensitizers is shown by **P < 0.01 and ***P < 0.001 (χ² test). Ca²⁺ responders: SP, n = 43/336; Sar-met SP, n = 20/435; β-ala NKA; NK2, n = 28/228; senktide, n = 7/218. TRPV1 sensitization: SP, n = 32/83; Sar-met SP, n = 46/121; β-ala NKA, n = 51/233; senktide, n = 36/218. (D) As in C but using TG neurons and fluo-4 as the Ca²⁺ indicator dye. Ca²⁺ responders: SP, n = 39/200; Sar-met SP, n = 10/124; β-ala NKA, n = 15/124; senktide, n = 18/124. TRPV1 sensitization: SP, n = 23/51; other NKR agonists were not tested in TG neurons.

Fig. 2.

NKR triggering in sensory neurons induces PLC activation but does not induce strong Ca²⁺ release from intracellular stores. (A and B) Translocation of the PIP₂/IP₃ probe PLCδ-PH-GFP in the transfected DRG neuron in response to SP. (A) Low-resolution epifluorescence image of the transfected DRG neuron (Upper Left). (Scale bar, 100 μm.) Other images are confocal micrographs of the same neuron before (basal), during (SP), and after (Wash) application of 1 μM SP; the neuron shown is representative of 9/34 cells tested. (Scale bars, 10 μm). (B) Time course of the cytosolic fluorescence intensity measurements from the cell shown in A. (C) Sample trace showing the relative size of Ca²⁺ transient elicited by SP (1 µM) or BK (1 µM) in DRG neurons measured using fura-2 AM. (D) Mean data from experiments as in C for DRG neurons. Number of cells is stated inside bars. ***Significant difference between groups (P < 0.001; unpaired t test). (E) As in D, but for TG neurons measured using Fluo-4.

Fig. 3.

SP augments M current and increases the AP firing threshold in small-diameter sensory neurons. (A–C) Sample perforated patch-clamp recordings from TG neurons. M current is plotted as the magnitude of the deactivating tail current when stepping from −30 to −60 mV (I_deac); bath application of SP (1 µM) and the M channel inhibitor XE991 (3 µM) is indicated by the black bars. Insets show current traces recorded at the time points indicated (–4). (D) Proportion of DRG and TG neurons responding with an increase, decrease, or no effect in M current in response to SP. The number of cells is shown within the pie charts. (E) Whole-cell current-clamp recording from a DRG neuron. The effect of SP is shown after 15 min exposure. Inset shows the current injection protocol. (F and G) Changes in membrane voltage (V_m) (F) and AP firing threshold (G) after 15-min bath perfusion of SP (1 µM). Each point represents one experiment (n = 17). **Significant difference between groups (P < 0.01; unpaired t test).

Fig. 4.

SP increases M current through oxidative modification. (A) Sample time course of the effect of SP on M current in a DRG neuron. Note that augmentation was not washable but was reversed by DTT (1 mM). Inset shows current traces recorded at the time points indicated (1–5). (B) (Left) Mean data from experiments as in A expressed as percent change in M current (XE991-sensitive fraction of I_deac) from basal state. (Right) In a separate series of experiments DTT alone was shown to have no effect on M current, and SP had no effect in the presence of DTT. The number of experiments is shown within the bars. (C) Sample time course of the effect of H₂O₂ (100 µM) on M current in DRG neurons; other labeling is as in A. (D) Mean data from experiments as in C. ***Significant difference from basal (P < 0.001; one-way ANOVA).

Fig. 5.

SP induces intracellular release of ROS in a subset of DRG neurons. (A) Sample recording from DRG neurons loaded with the ROS-sensitive dye CM-H₂DCFDA. Each line represents an individual DRG neuron. Data are represented as fluorescence at 488 nm (F) normalized to initial fluorescence at time = 0 (F₀) and corrected for the dye auto-oxidation (see Materials and Methods). (B) (Top) Bright-field (Left), fluorescent confocal 3D reconstruction (Z-stack; Center), and a zoomed-in single-plane fluorescent confocal micrograph (Right) of cultured DRG neurons transfected with the mitochondrial O₂^•− sensor mt-cpYFP. (Middle) Sample time course of the effect of SP (1 μM) on the mt-cpYFP fluorescence within the areas of interest (AOI) depicted by colored boxes in the image on the right. (Bottom) Individual frames of the blue AOI during the flash of the O₂^•− release (shown is a 5-s sequence recorded at three frames/s). The colors of the traces in the time course correspond to the colors of the AOIs. (C) Mean time course of the normalized (F/F₀) total cellular fluorescence of five responsive DRG neurons during the application of 1 μM SP. Error bars are indicated in dark gray. *Significant difference in mean F/F₀ at t = 150 s (just before SP application) and t = 330 s (peak effect) (P < 0.05; paired t test).

Fig. 6.

Augmentation of M current is mediated by mitochondrial ROS. (A) Sample time course of the effect of antimycin A (Ant A; 25 μM) on M current measured using the perforated patch-clamp technique. As with SP and H₂O₂, augmentation was not washable but was reversed by DTT (1 mM). (B) Mean data from A expressed as percent change in M current (XE991-sensitive fraction of I_deac) from basal state. *P < 0.05 compared with basal state (one-way ANOVA). (C) Sample time course of the effect of FCCP (1 μM) on M current. (D) Mean data from C expressed as percent change in M current [XE991-sensitive (3 µM) fraction of I_deac] from basal state. The number of cells is presented inside bars. ***P < 0.001 compared with basal state (one-way ANOVA). (E) Rate of oxygen consumption by intact, isolated rat DRG cells normalized to initial O₂ flux. Columns from left to right are basal respiration (Routine); O₂ flux following the addition of 1 μL vehicle (distilled water; Control), 1 μM SP, and 2 μg/mL oligomycin. All measurements were made when O₂ flux had stabilized and are corrected for nonmitochondrial respiration. Bars show mean data; n = 5; *P < 0.05 (one-way ANOVA followed by Bonferroni’s post hoc test) (F) As in E, but in glucose-free medium containing 10 mM sodium succinate and the mitochondrial complex I inhibitor rotenone (1 μM). Columns from left to right are initial O₂ flux; flux following the addition of 1μM SP; and flux following the addition of 2 μg/mL oligomycin. All data are corrected for nonmitochondrial respiration and are normalized to initial O₂ flux. Bars show mean data; n = 10; *P < 0.05 (one-way ANOVA followed by Bonferroni’s post hoc test).

Fig. 7.

Overexpressed NKR couple to G_q/11. (A and B) SP-induced translocation of the PIP₂/IP₃ probe PLCδ-PH-GFP (A) or the Ca²⁺-insensitive DAG probe PKCγ-C1-GFP (B) in CHO cells transfected with NK1. Upper panels show confocal micrographs of the transfected cell before (basal) and during the application of 1 µM SP. Lower panels show the corresponding surface intensity plots (warm colors indicate greater pixel intensity). (C) Calcium imaging from CHO cells transfected with NK1, NK2, or NK3. Data represent mean ± SEM (n = 22–27 as indicated) and are normalized to the fluorescence ratio at time 0 (R/R₀). SP (1 µM) was added to the bathing solution as indicated by the black bar. (D) Perforated patch-clamp recording from CHO cells overexpressing Kv7.2, Kv7.3, and NK1. (Left) Time course of inhibition of M current by SP (1 µM) and XE991 (XE, 3 µM) measured as the whole-cell current at 0 mV. (Right) Individual current traces at the time points 1–4; voltage protocol is shown above the traces. (E) Ca²⁺ imaging of a DRG neuron transfected with NK1. Drugs [1 µM SP; 30 mM KCl (30 K); and 1 µM capsaicin (CAP)] were added to the bathing solution as indicated by the black bars. (F) Perforated patch-clamp recording from a DRG neuron transfected with NK1. Endogenous M current is plotted as the I_deac upon stepping the membrane voltage from −30 to −60 mV, 1 µM SP; 3 µM XE991. Inset shows current traces at the time points indicated (1–4).

Fig. 8.

Intraplantar injection of SP causes minimal spontaneous pain but robust thermal hyperalgesia. (A) Thermal hyperalgesia was measured as the time (s) until hind paw withdrawal following application of a heat source (Hargreaves’ apparatus). Measurements were taken at baseline (before injection) and 10, 20, and 30 min after 50 μL intraplantar injection of 10 µM SP, 10 µM BK, or vehicle. Mean hind paw withdrawal latencies are shown ± SEM; n = 4. A significant difference from vehicle control is seen at each time point (*P < 0.05; **P < 0.01; two-way ANOVA.) (B) Spontaneous pain was measured as the time spent demonstrating nocifensive behavior (licking, biting, and lifting the hind paw) following a 50-μL intraplantar injection of 10 µM SP (n = 8), 10 µM BK (n = 8), or vehicle (n = 10). ***Significant difference from control (P < 0.001; one-way ANOVA).

Fig. P1.

Signaling cascades generated in nociceptive neurons by substance P (SP) and bradykinin (BK). B₂R, bradykinin receptor subtype 2; CaCC, Ca²⁺-activated Cl⁻ channels; DAG, diacylglycerol; ER, endoplasmic reticulum; G_i/o and G_q/11, G protein alpha subunit i/o or q/11 types, respectively; M, M-type K⁺ channel; NK1, neurokinin receptor subtype 1; PIP₂, phosphatidylinositol 4,5-bisphosphate; IP₃, inositol 1, 4, 5-trisphosphate; PKC, protein kinase C; PLC, phospholipase C; PM, plasma membrane; TRPV1, transient receptor potential cation channel, subfamily V, member 1. “+” indicates activation and “−” depicts inhibition.