General anesthetics activate a nociceptive ion channel to enhance pain and inflammation

Abstract

General anesthetics (GAs) have transformed surgery through their actions to depress the central nervous system and blunt the perception of surgical insults. Counterintuitively, many of these agents activate peripheral nociceptive neurons. However, the underlying mechanisms and significance of these effects have not been explored. Here, we show that clinical concentrations of noxious i.v. and inhalation GAs excite sensory neurons by selectively activating TRPA1, a key ion channel in the pain pathway. Further, these GAs induce pain-related responses in mice that are abolished in TRPA1-null animals. Significantly, TRPA1-dependent neurogenic inflammation is greater in mice anesthetized with pungent compared with nonpungent anesthetics. Thus, our results show that TRPA1 is essential for sensing noxious GAs. The pronociceptive effects of GAs combined with surgical tissue damage could lead to a paradoxical increase in postoperative pain and inflammation.

Fig. 1.

Volatile GAs activate TRPA1. (A) Representative current traces during application of isoflurane (0.9 mM, 2.9 MAC) in HEK293 cells expressing TRPM8, TRPV1, or TRPA1. Positive responses were elicited by menthol (1 mM), capsaicin (1 μM), or AITC (100 μM). (B) Isoflurane (0.9 mM) evoked inward currents in AITC-sensitive sensory neurons (n = 11). (C) Isoflurane activates TRPA1 in a dose-dependent manner with an EC₅₀ of 180 ± 20 μM (n = 4–7) and a Hill coefficient of 1.6 ± 0.2. At 2.7 mM isoflurane the response is reduced reflecting an additional blocking mechanism. (Inset) Example of washout of isoflurane; scale bars: 100 pA and 5 s. (D) Isoflurane (0.25 mM) and desflurane (0.9 mM) activate single TRPA1 channels in outside-out patches from HEK293 cells (no activity was observed in mock-transfected cells). The V_m was +50 mV. All-points histogram from 2-s data segments are shown on the right. (E) The mean currents (fraction of isoflurane) evoked by 0.9 mM concentrations of halothane, sevoflurane, and desflurane. Data are mean from five to six experiments.

Fig. 2.

Noxious i.v. GAs activate TRPA1. (A and B) In HEK293 cells, propofol and etomidate (100 μM) selectively activate TRPA1 without affecting TRPM8 or TRPV1 currents (V_m = −50 mV, n = 6–8). (C) I–V relationship for responses to propofol and AITC (1 mM, n = 7). (D) Dose-dependent activation by propofol (0.3–300 μM, n = 4–6). (E) Propofol (100 μM) activates single TRPA1 channels in an outside-out patch (n = 3, V_m = +40 mV). All-points histograms reveal a decrease in unitary conductance from 108 to 94 pS. (F and G) Propofol (100 μM) evoked inward currents and depolarized AITC-sensitive DRG neurons (n = 6). Currents were blocked by camphor (0.5 mM).

Fig. 3.

GAs excite DRG neurons via TRPA1 (A and C) (Left) Representative Ca²⁺ transients evoked by desflurane (1.5 mM, 3 MAC), propofol (100 μm), and AITC (1 mM) in DRG neurons obtained from wild-type mice. (Right) The percentage of neurons responsive to desflurane (n = 123), propofol (n = 63), and AITC. (B and D) (Left) Representative Ca²⁺ transients evoked by desflurane propofol and capsaicin (100 nM) in DRG neurons obtained from TRPA1-null mice. (Right) The number of DRG neurons responsive to desflurane (n = 125), propofol (n = 120), or capsaicin.

Fig. 4.

Volatile anesthetics interact directly with TRPA1 channels. (A and B) Activation of TRPA1 by hexanol (3 mM), octanol (1 mM), and decanol (0.6 mM) (n = 4). (C) Octanol (1.8 mM) and isoflurane (0.9 mM) modulate TRPA1 in a nonadditive fashion. (D) Activation of TRPM8, TRPV1, and TRPA1 currents at −50 mV by isoflurane (0.9 mM) and octanol (1 mM), compared with maximal stimulation with menthol (1 mM), capsaicin (1 μM), and AITC (1 mM), respectively (n = 4–6). (E) Propofol (100 μM) and octanol (1.8 mM) produce an additive response at TRPA1. (F) Mean effects of octanol (1.8 mM) on isoflurane (0.9 mM) and propofol (100 μM)-evoked currents (n = 5–6), *, P < 0.01.

Fig. 5.

TRPA1 mediates propofol-evoked, pain-related behavior. (A) Topical application of propofol (50%) to the nasal epithelium evokes nocifensive behavior in wild-type (n = 5) and TRPV1-null (n = 4) mice (see Movie S1). (B) Propofol-induced nociception is abolished in TRPA1^−/− animals (n = 5); *, P < 1E-6 compared with TRPA1^+/− littermates (n = 5). (C and D) Integrated EMG activity from semitendinosus muscle of TRPA1^+/− and TRPA1^−/− mice after injection of 30 μl of propofol (500 μM) or capsaicin (50 μM, 5 min later) into the femoral artery (n = 3 for both).

Fig. 6.

AITC-induced ear swelling is greater during anesthesia with isoflurane compared with sevoflurane. (A and B) AITC (0.6%, 20 μl) was applied to one ear of mice and the contralateral ear received mineral oil alone. Animals were anesthetized with 1.2 MAC of isoflurane or sevoflurane for 60 min followed by 60 min of recovery. Data show the change in ear thickness from baseline (both groups, n = 7) *, P < 0.01, AITC+Isoflurane versus other groups ANOVA; †, P < 0.05 for isoflurane alone versus sevoflurane alone. (C) Pungent (isoflurane and desflurane) but not smooth (methoxyflurane and sevoflurane) VGAs (0.5–0.65 mM) enhance currents evoked by AITC (10 μM) in TRPA1-expressing oocytes (n = 3–4 for each point). *, P < 0.05 versus AITC alone.