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. 2006 Mar 21;103(12):4699-704.
doi: 10.1073/pnas.0508005103. Epub 2006 Mar 14.

Modulation of sensory neuron mechanotransduction by PKC- and nerve growth factor-dependent pathways

Amalia Di Castro  1 , Liam J Drew, John N Wood, Paolo Cesare
Affiliations

Modulation of sensory neuron mechanotransduction by PKC- and nerve growth factor-dependent pathways

Amalia Di Castro et al. Proc Natl Acad Sci U S A. .

Abstract

Many sensations of pain are evoked by mechanical stimuli, and in inflammatory conditions, sensitivity to such stimuli is commonly increased. Here we used cultured sensory neurons as a model of the peripheral terminal to investigate the effects of inflammatory signaling pathways on mechanosensitive ion channels. Activation of two of these pathways enhanced transduction in a major population of nociceptors. The proinflammatory neurotrophin nerve growth factor caused an up-regulation of mechanically activated currents via a transcriptional mechanism. Activators of PKC, given in vitro and in vivo, also caused an increase in mechanically activated membrane current and behavioral sensitization to mechanical stimulation, respectively. The effect of activating PKC was inhibited by tetanus toxin, suggesting that insertion of new channels into the cell membrane is involved in sensitization. These results reveal previously undescribed mechanisms by which PKC and nerve growth factor synergistically enhance the response of nociceptors to mechanical stimuli, suggesting possible targets for pain treatment.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Activation of PKC increases MA current amplitude in neonatal sensory neurons. (a) Examples of voltage-clamp recordings of neurons responding to incrementing mechanical stimuli (2–14 μm) in control conditions (Left) and incubated overnight with PMA (10 nM; Right). (Calibration: vertical, 0.8 nA; horizontal, 100 ms.) (b) Average peak response to 14 μm mechanical stimulus in control conditions, overnight Fsk, and overnight PMA. (c) Overnight PMA increased MA current amplitude in a concentration dependent manner (one-way ANOVA, P < 0.001): control (1.07 ± 0.18 nA; n = 13), 0.1 nM (1.91 ± 0.55 nA; n = 5), 1 nM (2.72 ± 0.82 nA; n = 5; P = 0.04 vs. control), and 10 nM (3.64 ± 0.33 pA; n = 11; P < 0.01 vs. control). (d) MA currents evoked by increasing membrane displacements (from 4 to 14 μm) in control neurons (○, 14-μm step: 0.62 ± 0.30 nA; n = 6), neurons treated overnight with 10 nM PMA (▵, 2.28 ± 0.68 nA; n = 6; P = 0.05 vs. control), and neurons treated for 1 h with 10 nM PMA (▿, 1.67 ± 0.22 nA; n = 6; P = 0.02 vs. control). (e) Average peak response to 14-μm mechanical stimulation in control neurons and in those treated with either PMA alone or PMA plus bisindolylmaleimide I (1 μM, applied for 15 min before PMA). (f) Neurons treated with PMA had significantly larger MA currents than neurons treated with α-PMA, the inactive enantiomer. (g) The efficacy of ruthenium red in blocking MA currents was unchanged before and after PMA treatment, whereas amiloride was ineffective in both conditions. (h) Example traces showing current inhibition by ruthenium red. (Calibration: top vertical, 0.2 nA; bottom vertical, 1 nA; horizontal, 100 ms.)
Fig. 2.
Fig. 2.
MA currents are differentially regulated by PKC and trophic factors in distinct neuronal subpopulations from adult rats. (a) Up-regulation of MA currents by PMA depends on pretreatment with NGF. Graph shows average, maximal MA currents in control conditions (and after 1-h incubation in 30 nM PMA). (b) Cell diameter frequency distribution of all NGF-deprived (filled columns; n = 43) and NGF-treated (open columns; n = 61) neurons used in a. (c) Average peak MA current of PMA-treated cells seen in a, grouped according to cell size (<35 μm, 35–40 μm, and >40 μm). Open columns, with NGF; filled columns, no NGF. (d) Comparison of MA current amplitude, in control conditions and after PMA treatment, in IB4+ neurons and IB4− neurons. (e) Cell diameter distribution of IB4+ (filled columns; n = 40) and IB4− (empty columns; n = 99) neurons used in d. (f) Average peak MA currents in neurons treated with PMA (from d) grouped according to cell diameter (<35 μm, 35–40 μm, and >40 μm) and IB4 labeling (filled columns, IB4+, empty columns, IB4−).
Fig. 3.
Fig. 3.
Regulation of adult nociceptor mechanosensitivity by neurotrophins and PMA. (a) Average peak currents in IB4-labeled, PMA-treated neurons (30 nM for 1 h). Either NGF or GDNF was applied overnight to culture medium. (b) Kinetics of MA currents are unchanged after PMA treatment. In a population of IB4− neurons, peak current amplitudes and total charge transfer increased at a similar rate after PMA, adaptation was unchanged. (c) Example traces showing current adaptation and the parameters that were used for kinetic analysis: total charge transfer was measured between points a and b; arrows show residual current at the end of the stimulus used to calculate adaptation. (Calibration: vertical, 0.3 nA; horizontal, 100 ms.) (d) Examples of current-clamp recordings made from mechanically stimulated neurons (8-μm membrane deflection): neuron cultured in control conditions (Left) and a neuron incubated for 1 h with 30 nM PMA (Right). (Calibration: vertical, 20 mV; horizontal, 100 ms.) (e) Probability of neurons firing either a single action potential (open columns) of repetitively (filled columns) in response to mechanical stimulation in control conditions (10% and 0%, respectively; n = 10) or incubated for 1 h with 1 μM Fsk (43% and 14%; n = 7), 30 nm PMA (44% and 11%; n = 9), or both (69% and 46%; n = 13).
Fig. 4.
Fig. 4.
Mode of action of NGF and time course of cellular and behavioral effects of PMA. (a) MA currents in IB4−, PMA-treated neurons (30 nM for 1 h). Neurons were grown without NGF, or NGF was applied before PMA for either 1 h or 8 h. Mean MA current amplitude recorded after 8 h NGF without PMA was 0.24 ± 0.06 nA (n = 20, P = 0.01 vs. NGF 8 h plus PMA). (b) Cycloheximide (Chx) and actinomycine D (Act) inhibit the effect of NGF on MA currents in IB4− neurons. Cells were treated with NGF for 8 h either alone or with Chx (10 μg/ml) or Act (5 μg/ml, each was added 1 h before NGF), and then 30 nM PMA was added for 1 h. (c) Effect of NGF on short-term (9–12 h) neonatal cultures. Shown are current amplitudes evoked by incrementing mechanical stimuli in IB4− neurons (no PMA added). Currents recorded in neurons without NGF (○) were significantly smaller than those in cells cultured in NGF (▵). (d) Average peak MA currents in IB4− neurons incubated with PMA for 10 min or 1 h. (e) Augmentation of MA currents by PKC activation is maintained after PMA is removed. Shown are current amplitudes evoked by incrementing mechanical stimuli in IB4− neurons. Currents recorded in control conditions (○) were significantly smaller than those in cells treated with PMA for 1 h and then washed for 4 h before recording (▵). (f) PMA caused a dose-dependent decrease in PWT after intraplantar injection. All doses of PMA induced a decrease in PWT 1 h after injection. Three hours after injection, hyperalgesia induced by 160 and 1,600 pmol per paw was maintained, whereas PWTs in rats given the lowest dose returned to near control values (81.0 ± 17.7% of control, P = 0.54) (n = 4 in each group).
Fig. 5.
Fig. 5.
PKC activation up-regulates MA currents by a mechanism involving insertion of new channels in the membrane. (a) TeNT inhibits sensitization of MA currents by PMA. Shown are average responses to increasing mechanical stimulation (up to 14 μm) in neonatal neurons. PMA alone up-regulates MA currents (▿; peak: 1.38 ± 0.16 nA, n = 24; P < 0.005 vs. control), but after an 8-h incubation in TeNT (20 nM), PMA had no effect on current amplitude (◇; mean at 14 μm: 0.79 ± 0.18 nA, n = 24; P < 0.05 vs. PMA-treated cells). Peak control current was 0.64 ± 0.16 nA (○, n = 16). (b) Schematic representation of the proposed mechanisms underlying peripheral sensitization to mechanical stimulation. NGF, acting via trkA receptors, increases transcription of MA channels, which are packaged into vesicles. Activation of PKC by proinflammatory signals induces fusion of the vesicles and insertion of new mechanosensitive channels into the cell membrane. (c) Epifluorescence images taken 5 min (Left) and 20 min (Right) after FM1-43, a marker of vesicle recycling, was applied to neurons bathed in control extracellular solution (horizontal bar: 40 μm.) (d) Quantitative analysis of the mean fluorescence (arbitrary units) in 11 neurons whose images were taken as described in c (•, 5 min; ○, 20 min), measured along a 40-μm segment (indicated by yellow line in c). Segments were drawn such that each cell’s profile could be superimposed for comparison. (e) As in c, but FM1-43 was coapplied with 30 nM PMA. (f) Analysis as in d, but neurons (n = 12) were treated with PMA as in e.
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