Bupivacaine-induced cellular entry of QX-314 and its contribution to differential nerve block

Abstract

Background and purpose: Selective nociceptor fibre block is achieved by introducing the cell membrane impermeant sodium channel blocker lidocaine N-ethyl bromide (QX-314) through transient receptor potential V1 (TRPV1) channels into nociceptors. We screened local anaesthetics for their capacity to activate TRP channels, and characterized the nerve block obtained by combination with QX-314.

Experimental approach: We investigated TRP channel activation in dorsal root ganglion (DRG) neurons by calcium imaging and patch-clamp recordings, and cellular QX-314 uptake by MS. To characterize nerve block, compound action potential (CAP) recordings from isolated nerves and behavioural responses were analysed.

Key results: Of the 12 compounds tested, bupivacaine was the most potent activator of ruthenium red-sensitive calcium entry in DRG neurons and activated heterologously expressed TRPA1 channels. QX-314 permeated through TRPA1 channels and accumulated intracellularly after activation of these channels. Upon sciatic injections, QX-314 markedly prolonged bupivacaine's nociceptive block and also extended (to a lesser degree) its motor block. Bupivacaine's blockade of C-, but not A-fibre, CAPs in sciatic nerves was extended by co-application of QX-314. Surprisingly, however, this action was the same in wild-type, TRPA1-knockout and TRPV1/TRPA1-double knockout mice, suggesting a TRP-channel independent entry pathway. Consistent with this, high doses of bupivacaine promoted a non-selective, cellular uptake of QX-314.

Conclusions and implications: Bupivacaine, combined with QX-314, produced a long-lasting sensory nerve block. This did not require QX-314 permeation through TRPA1, although bupivacaine activated these channels. Regardless of entry pathway, the greatly extended duration of block produced by QX-314 and bupivacaine may be clinically useful.

Keywords: CAP; DRG; QX-314; TRPA1; TRPV1; nociception; sciatic nerve.

Figure 1

Identification of local anaesthetics that activate ruthenium red (RR)-sensitive calcium channels in dorsal root ganglia (DRG) neurons. (A) Representative recordings of neuronal calcium concentrations after stimulation with local anaesthetics. Cultured DRG neurons were stimulated for 10 s with lidocaine (3 mM), the test compound (3 mM), capsaicin (300 nM) and the test compound (3 mM) after wash of 10 μM RR. Intracellular calcium levels were determined by fura-2-based calcium imaging. (B) Statistical analysis of traces from neurons stimulated as in (A). The mean of [F₃₄₀/F₃₈₀] increases at peak amplitudes after stimulation is shown. n = 5–30 cells from three or more independent experiments ± SEM. (C) Capsaicin sensitivity for local anaesthetic responsive sensory neurons. Percentage of capsaicin sensitive cells within the local anaesthetic responsive group was calculated. (D) Concentration response analysis of procaine, bupivacaine and lidocaine-induced calcium flux in DRG neurons by a high-throughput protocol using an automated FLIPR system. n = 4 replicates from two independent experiments.

Figure 2

Bupivacaine activates heterologously expressed TRPA1 but not other relevant large-pore cation channels. (A) Representative recordings of calcium concentrations in N1E-115 neuroblastoma cells expressing mTRPA1 after stimulation with bupivacaine (3 mM, 10 s) and AITC (100 μM, 10 s). (B) Statistical analysis of Δ[F₃₄₀/F₃₈₀] at peak amplitudes in N1E-115 cells expressing mP2X3, mTRPV1, rTRPV2, mTRPV3, hTRPV4, mTRPM8 or mTRPA1 after stimulation with bupivacaine (3 mM) and the standard agonists α,β-methyl-ATP (300 μM), capsaicin (1 μM), 2-APB (100 μM), 2-APB (100 μM), 4αPDD (5 μM), menthol (200 μM) and AITC (100 μM) respectively. Shown is the mean of 14–27 cells ± SEM for each channel. (C) Representative current trace of recordings from two TRPA1-expressing HEK 293 cells during stimulation with increasing concentrations of bupivacaine. (D) Representative current trace of recording from a TRPV1-expressing HEK 293 cell during stimulation with increasing concentrations of bupivacaine (followed by capsaicin to verify expression of TRPV1). (E) Collected results (mean ± SEM) for bupivacaine-evoked currents from TRPA1- or TRPV1-expressing HEK 293 cells successively exposed to 0.1, 0.3, 1 and 3 mM bupivacaine, followed by either 100 μM AITC (for TRPA1, n = 10) or 1 μM capsaicin (for TRPV1, n = 3). Currents were normalized to the current evoked by 100 μM AITC or 1 μM capsaicin (except for TRPA1 cells in which bupivacaine produced a larger response than subsequent AITC as a result of TRPA1 inactivation, in which case normalization was to the largest current evoked by bupivacaine).

Figure 3

QX-314 can permeate into cells via TRPA1 channels. (A) QX-314 carries ionic current through TRPA1 channels heterologously expressed in N1E-115 cells. Current–voltage relation for current evoked by 100 μM AITC was determined using a 30 ms voltage ramp from +116 to −144 mV before and after application of 100 μM AITC with an external solution containing QX-314 as the sole cation (10 mM QX-314 hydroxide, 277 mM sucrose and 5 mM HEPES, pH adjusted to 7.4 with HCl). Internal solution was 135 mM N-methyl-D-glucamine, 5 mM EGTA and 10 mM HEPES, pH adjusted to 7.2 with HCl. Currents were averaged from 10 sweeps in control and then ∼4 min after application of 100 μM AITC to activate TRPA1 current. (B) Cellular uptake of QX-314 into hTRPA1 inducible HEK-293t cells. Twenty-four hours after induction of TRPA1 expression with doxycycline, HEK-293t cells were incubated with QX-314 (0.5 mM) and AITC (100 μM) for 10 min. After washout, cellular QX-314 was determined from cytosolic fractions by LC-MS/MS analysis. Note that cells that are only induced for TRPA1 expression but received vehicle (0.1% DMSO) or cells which lack TRPA1 but are AITC treated do not show increased cellular QX-314 levels. Shown is the mean of 3 (for control) or 5 incubations (for AITC and/or TRPA1) ± SEM. One-way anova and Bonferroni post-test compared with control: **P < 0.01.

Figure 4

Nociceptive and motor function block after perineural injections of QX-314 and procaine or bupivacaine in naïve rats. (A–C) Local anaesthesia induced by procaine in combination with QX-314. Procaine (2%) was injected alone or in combination with QX-314 (0.5%) into the sciatic notch of adult rats and paw withdrawal latencies after radiant heat stimulation (A), mechanical thresholds after stimulation with a pincher (B) or toe spread reflexes (n = 7) (C), were determined at the indicated time points. (D–F) Anaesthesia induced by bupivacaine in combination with QX-314. Bupivacaine (0.5%) was injected alone or in combination with QX-314 (0.5%) into the sciatic notch and behaviour determined as in (A)–(C). Data represent the mean ± SEM (n = 6 rats for bupivacaine and n = 8 for bupivacaine and QX-314) per group. Significance was calculated by the two-way repeated measures anova and Bonferroni post-test. *P < 0.05 compared with baseline.

Figure 5

Differential nerve block after perineural injections of QX-314 and procaine, or bupivacaine. To illustrate the relative duration of nociceptive and motor block, the percentage of the initial anaesthesia at the different time points was calculated from data shown in Figure 4.

Figure 6

Nociceptive and motor function after plantar incision and subsequent differential nerve block by perineural injections of QX-314 with bupivacaine 24 h after lateral plantar incision, motor and sensory function was assayed by testing grip strength (A), pinch tolerance threshold (B) and thermal response latency (‘Hargreaves' test’, C). Thereafter, rats were injected nearby the left sciatic nerve with 200 μL of vehicle (0.9% saline), bupivacaine (0.5%) or a combination of bupivacaine (0.5%) with QX-314 (0.5%). Motor and sensory measures were again assayed at 1, 2, 4, 6, 9, 12 and 24 h after perineural injections. All injections administered under isoflurane-induced general anaesthesia at time 0. Significance for the QX-314 + bupivacaine group was calculated by the two-way repeated measures anova and Bonferroni post-test. *P < 0.05 compared with post-injury baseline; n = 8 rats for all groups.

Figure 7

Prolonged and selective effects of the combination of bupivacaine and QX-314 on C-fibre CAPs in C57BL\6, TRPA1^−/− and TRPV1/A1^=/= mice. Isolated mouse sciatic nerves were superfused with bupivacaine (1 mM) and its combination with QX-314 (300 μM), and CAPs were recorded upon electrical stimulation. (A) Representative traces of CAP amplitudes of C- (upper) and A-fibres (lower) after application of bupivacaine and the combination with QX-314; insets show CAPs registered at critical time points. (B) Time constants (τ) of recovery from nerve block during wash-out of drugs in C57BL\6, with amplitudes and latencies of CAPs separately evaluated (n = 6). (C) Same as in (B) but nerves from TRPA1^−/− (left panels, n = 6/5) and TRPV1/A1^=/= mice (right panels, n = 7). Data represent means ± SEM; *P < 0.05 calculated by Wilcoxon matched pairs test for intra-individual comparisons.

Figure 8

Higher concentrations of lidocaine and bupivacaine induce QX-314 uptake into cell lines and DRG neurons independently of calcium-permeable channels. Cells were incubated with 0.5 mM QX-314 and different local anaesthetics for 10 min. After washout, QX-314 concentrations were determined in cell lysates by LC-MS/MS analysis. (A) QX-314 uptake in N1E-115 cells induced by different concentrations of lidocaine and bupivacaine. (B) 3 mM bupivacaine increases QX-314 concentrations in different cell lines. RAW macrophages, N1E-115 neuroblastoma cells and CHO cells were incubated as in (A). (C) Bupivacaine-induced cellular uptake of QX-314 does not increase significantly after TRPA1 expression. Twenty-four hours after induction of hTRPA1 expression with doxycycline, HEK-293t cells were incubated as in (A). (D) Bupivacaine increases QX-314 concentrations in DRG neurons. Primary DRG cultures from adult rats were incubated with QX-314 (0.5 mM) and bupivacaine (3 mM) ± ruthenium red (20 μM) as in (A). Data from all panels represent the mean conc. ± SEM in lysates of 3–5 culture dishes. One-way anova and Bonferroni post-test compared with control. *P < 0.05; **P < 0.01; ***P < 0.001.