Bradykinin Induces TRPV1 Exocytotic Recruitment in Peptidergic Nociceptors

Abstract

Transient receptor potential vanilloid I (TRPV1) sensitization in peripheral nociceptors is a prominent phenomenon that occurs in inflammatory pain conditions. Pro-algesic agents can potentiate TRPV1 activity in nociceptors through both stimulation of its channel gating and mobilization of channels to the neuronal surface in a context dependent manner. A recent study reported that ATP-induced TRPV1 sensitization in peptidergic nociceptors involves the exocytotic release of channels trafficked by large dense core vesicles (LDCVs) that cargo alpha-calcitonin gene related peptide alpha (αCGRP). We hypothesized that, similar to ATP, bradykinin may also use different mechanisms to sensitize TRPV1 channels in peptidergic and non-peptidergic nociceptors. We found that bradykinin notably enhances the excitability of peptidergic nociceptors, and sensitizes TRPV1, primarily through the bradykinin receptor 2 pathway. Notably, bradykinin sensitization of TRPV1 in peptidergic nociceptors was significantly blocked by inhibiting Ca(2+)-dependent neuronal exocytosis. In addition, silencing αCGRP gene expression, but not substance P, drastically reduced bradykinin-induced TRPV1 sensitization in peptidergic nociceptors. Taken together, these findings indicate that bradykinin-induced sensitization of TRPV1 in peptidergic nociceptors is partially mediated by the exocytotic mobilization of new channels trafficked by αCGRP-loaded LDCVs to the neuronal membrane. Our findings further imply a central role of αCGRP peptidergic nociceptors in peripheral algesic sensitization, and substantiate that inhibition of LDCVs exocytosis is a valuable therapeutic strategy to treat pain, as it concurrently reduces the release of pro-inflammatory peptides and the membrane recruitment of thermoTRP channels.

Keywords: TRPV1; exocytosis; inflammation; neuropeptides; nociceptors.

FIGURE 1

Effect of BK on modulating excitability of neonatal rat peptidergic IB4^- and non-peptidergic IB4⁺ neurons. (A) Representative of the RMP change in a peptidergic (red) and a non-peptidergic (blue) neuron upon exposure to 1 μM BK. (B) Effect of BK on the RMP in from peptidergic and non-peptidergic neuron before and after 1 μM BK application. Each point represents a neuron from three independent cultures. Statistical analysis was performed using the non-parametric Wilconson Rank sum test.

FIGURE 2

Effect of BK on evoked responses of IB4^- and IB4⁺ soma to depolarizing currents. Representative traces of APs evoked by injecting a depolarizing current of 100 pA (100 ms) and 300 pA (1 s) delivered to the soma of IB4^- (A,C) and IB4⁺ (B,D) neurons before (Vehicle) and after instillation to 1 μM BK.

FIGURE 3

Sensitization of TRPV1 activity by BK, BKR1 and BKR2 receptor agonists. (A,B) Representative traces of capsaicin (cps = 500 nM, 15 s) evoked APs in neonatal rat DRG neurons upon applying buffer (Control) or 1 μM of BK between the second (P2) and third (P3) vanilloid pulse, respectively. (C) Representative recordings of 1 μM Sar-[D- Phe⁸]-des-Arg⁹-Bradykinin (BKR1), and (D) 1 μM BKR2 ([Phe⁸ψ(CH-NH)-Arg⁹]-Bradykinin; BKR2) inducing potentiation of capsaicin evoked neuronal excitability in rat nociceptors. (E) BK receptor agonists evoked fold potentiation (ratio P3/P2) of TRPV1 mediated neuronal firing activity. (F) BK evoked fold potentiation (ratio P3/P2) of TRPV1 mediated neuronal firing activity in the presence of BK receptor antagonists (1 μM R715 for BKR1, 1 μM HOE140 for BKR2). Data were analyzed as paired values through comparison of the responses of each electrode in the 30 s time interval upon stimulation. Data are expressed as mean ± SEM. The numbers above the bars represents the total number of electrodes that responded. Number of cultures ≥3. Statistical analysis was performed by one-way ANOVA followed by Bonferroni post hoc test (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001).

FIGURE 4

Peptide DD04107 inhibits BK-sensitized TRPV1-mediated nociceptor excitability. (A) Representative traces of capsaicin-evoked APs upon applying BK between the second (P2) and third (P3) vanilloid pulses in neonatal rat DRG neurons exposed to vehicle, DD04107 and DD04107^RDM peptides. (B) Mean spike frequency of capsaicin evoked AP firing under control (buffer) and BK-treated conditions in the presence of vehicle and peptides DD04107 and DD04107^RDM. (C,D) Fold potentiation (ratio P3/P2) of BK-induced potentiation of TRPV1 evoked neuronal firing in the presence of peptide DD04107 and DD04107^RDM respectively. Capsaicin (cps = 500 nM, 15 s) and BK (1 μM, 8 min) were used. DD04107 and DD04107^RDM (20 μM) were pre-incubated for 1 h at 37°C. Mean spike frequency was calculated from recordings displayed in (A). Data were analyzed as paired values through comparison of the responses of each electrode in the 30 s time interval upon stimulation. Data are expressed as mean ± SEM. The numbers above the bars represents the total number of electrodes that responded. Number of independent cultures ≥3. Statistical analysis was performed by one-way ANOVA repeated measures with Bonferroni’s post hoc test and Unpaired Student’s t-test (^∗p < 0.05, ^∗∗p < 0.01).

FIGURE 5

Effect of non-palmitoylated DD04107 on BK-induced sensitization of TRPV1 channel on rat peptidergic and non-peptidergic DRG neurons. (A,B) Representative showing the effect of 1 μM BK on the ionic currents elicited by capsaicin (cps = 1 μM, 10 s) in IB4^- and IB4⁺ nociceptors, respectively. (C,D) Effect of 100 μM non-palmitoylated DD04107 peptide (denoted as Peptide) on the BK-induced potentiation of capsaicin-evoked ionic currents in IB4^- and IB4⁺ nociceptors, respectively. (E) Fold potentiation (ratio P3/P2) of capsaicin-evoked ionic currents by BK in cultures exposed to vehicle and peptide. Cells were held at -60 mV. Peptide was given through the patch pipette and incubated for 10 min after forming the seal. Data are expressed as mean ± SEM. The numbers above the bars denote the total neurons registered. Number of independent cultures = 4. Statistical analysis was performed using two-way ANOVA with Bonferroni’s post-test (^∗p < 0.05, ^∗∗p < 0.01).

FIGURE 6

BK-induced potentiation of TRPV1 evoked neuronal firing in mice nociceptors is sensitive to DD04107. (A) Representative MEA recordings of capsaicin induced action potentials (APs) and desensitization (Top), and potentiation by BK between the second (P2) and third (P3) vanilloid pulse (Bottom) in mice nociceptors. (B) Representative recordings of BK-induced sensitization of capsaicin-evoked neuronal excitability in mice nociceptors preincubated with 20 μM of DD04107. (C) Mean spike frequency of capsaicin induced AP firing in WT nociceptors. Capsaicin (cps = 500 nM, 15 s) and BK (1 μM, 8 min) were used. DD04107 was preincubated for 1 h at 37°C. Mean spike frequency was calculated from recordings displayed in (A,B). Data were analyzed as paired values through comparison of the responses of each electrode in the 30 s time interval upon stimulation Data are expressed as mean ± SEM, n = 4 independent cultures. Statistical analysis was performed by one-way ANOVA followed by Bonferroni post hoc test. ^∗p < 0.05.

FIGURE 7

BK-induced TRPV1 sensitization in αCGRP^-/- nociceptors. (A) Representative traces of capsaicin-evoked APs in primary nociceptor cultures of DRGs from αCGRP^-/- mice upon exposure to buffer (Top) or BK (Bottom) between the second (P2) and third (P3) vanilloid pulse. (B) Mean spike frequency of capsaicin-evoked AP firing under control and BK treated conditions. Capsaicin (cps = 500 nM, 15 s) and BK (1 μM, 8 min) were used. Mean spike frequency was calculated from recordings displayed in (A). Data were analyzed as paired values through comparison of the responses of each electrode in the 30 s time interval upon stimulation. Data are expressed as mean ± SEM. The numbers above the bars represents the total number of electrodes that responded. Number of independent cultures ≥4. Statistical analysis was performed by one way ANOVA repeated measures with Bonferroni’s post hoc test.

FIGURE 8

BK-induced TRPV1 sensitization in Tac1^-/- nociceptors. (A) Representative traces of capsaicin-evoked APs in primary nociceptor cultures of DRGs from Tac1^-/- mice upon exposure to buffer (Top) or BK (Bottom) between the second (P2) and third (P3) vanilloid pulse. (B) Mean spike frequency of capsaicin-evoked AP firing under control and BK treated conditions. Capsaicin (cps = 500 nM, 15 s) and BK (1 μM, 8 min) were used. Mean spike frequency was calculated from recordings displayed in (A). Data were analyzed as paired values through comparison of the responses of each electrode in the 30 s time interval upon stimulation. Data are expressed as mean ± SEM. The numbers above the bars represents the total number of electrodes that responded. Number of independent cultures ≥3. Statistical analysis was performed by one-way ANOVA repeated measures with Bonferroni’s post hoc test (^∗∗∗p < 0.001).