Etomidate and propylene glycol activate nociceptive TRP ion channels

Abstract

Background: Etomidate is a preferred drug for the induction of general anesthesia in cardiovascular risk patients. As with propofol and other perioperatively used anesthetics, the application of aqueous etomidate formulations causes an intensive burning pain upon injection. Such algogenic properties of etomidate have been attributed to the solubilizer propylene glycol which represents 35% of the solution administered clinically. The aim of this study was to investigate the underlying molecular mechanisms which lead to injection pain of aqueous etomidate formulations.

Results: Activation of the nociceptive transient receptor potential (TRP) ion channels TRPA1 and TRPV1 was studied in a transfected HEK293t cell line by whole-cell voltage clamp recordings of induced inward ion currents. Calcium influx in sensory neurons of wild-type and trp knockout mice was ratiometrically measured by Fura2-AM staining. Stimulated calcitonin gene-related peptide release from mouse sciatic nerves was detected by enzyme immunoassay. Painfulness of different etomidate formulations was tested in a translational human pain model. Etomidate as well as propylene glycol proved to be effective agonists of TRPA1 and TRPV1 ion channels at clinically relevant concentrations. Etomidate consistently activated TRPA1, but there was also evidence for a contribution of TRPV1 in dependence of drug concentration ranges and species specificities. Distinct N-terminal cysteine and lysine residues seemed to mediate gating of TRPA1, although the electrophile scavenger N-acetyl-L-cysteine did not prevent its activation by etomidate. Propylene glycol-induced activation of TRPA1 and TRPV1 appeared independent of the concomitant high osmolarity. Intradermal injections of etomidate as well as propylene glycol evoked severe burning pain in the human pain model that was absent with emulsification of etomidate.

Conclusions: Data in our study provided evidence that pain upon injection of clinical aqueous etomidate formulations is not an unspecific effect of hyperosmolarity but rather due to a specific action mediated by activated nociceptive TRPA1 and TRPV1 ion channels in sensory neurons.

Keywords: Etomidate; TRPA1; TRPV1; anesthetic; human pain model; injection pain; nociception; propylene glycol; sensory neuron; transient receptor potential ion channel.

Figures

Figure 1.

Etomidate activates and desensitizes mTRPA1. (a) Representative current traces of etomidate-evoked inward currents in HEK293t cells transiently expressing mTRPA1. To prevent desensitization only one concentration was tested on each cell. Cells were held at −60 mV, etomidate was applied until the current had reached a steady state. (b) Representative current traces evoked by three consecutive applications of 1000 µM etomidate, each applied at intervals of 2 min. (c) Concentration–response curve for etomidate-evoked inward currents in mTRPA1-expressing HEK293t cells; 6 to 10 cells were tested at each concentration. Data were fitted to the Hill equation. (d) Representative etomidate-evoked inward current blocked by the TRPA1 antagonist AP-18. 300 µM etomidate was applied until the current had reached a steady state, followed by a combined application of etomidate and 50 µM AP-18. (e) Normalized current amplitudes ± SEM measured in (d). Currents were normalized to the peak inward current during the steady state. TRP: transient receptor potential.

Figure 2.

Etomidate activates rTRPV1. (a) Representative current traces evoked by 0.01–2500 µM etomidate in rTRPV1-expressing HEK293t cells. To prevent desensitization, only one concentration was tested on each cell. Cells were held at −60 mV, etomidate was applied for at least 20 s. Note the changed scales at 500 and 2500 µM etomidate. (b) Concentration–response curve for etomidate-evoked inward currents in rTRPV1-expressing HEK293t cells; between 6 and 29 cells were tested at each concentration. Note the bell-shaped curve progression suggesting channel block at etomidate concentrations > 1 µM. (c) Representative capsaicin-evoked inward current of a rTRPV1-expressing HEK293t cell blocked by 100 µM etomidate. Capsaicin (100 nM) was applied until the current had reached a steady state followed by a combined application of capsaicin and 100 µM etomidate. (d) Etomidate did not activate rTRPV2-, rTRPV3-, rTRPV4- or rTRPM8-expressing HEK293t cells. Cells were held at −60 mV, etomidate was applied for at least 20 s. Note the leak current-stabilizing effect of etomidate on rTRPM8. Channel expression was verified by a subsequent application of the TRPV2 and TRPV3 agonist 2-APB, the TRPV4 agonist 4αPDD and the TRPM8 agonist menthol (not shown); at least four cells were tested for each TRP channel. TRP: transient receptor potential.

Figure 3.

Activation of hTRPA1 by etomidate involves N-terminal cysteine and lysine residues. (a) Representative current traces of etomidate-evoked inward currents in HEK293t cells transiently expressing hTRPA1. To prevent desensitization, only one concentration was tested on each cell. Cells were held at −60 mV, etomidate was applied until the current had reached a steady state. (b) Concentration–response curve for etomidate-evoked inward currents in hTRPA1- and hTRPA1-C621S/C641S/C665S (3C)-expressing HEK293t cells; 6 to 10 cells were tested at each concentration. Data were fitted to the Hill equation. Note the plateau followed by a second slope at concentrations over 100 µM in the curve of hTRPA1. (c) Representative current traces of the hTRPA1 mutants C621S/C641S/C665S (3C) and C621S/C641S/C665S/K710R (3CK) evoked by 1000 µM etomidate. Cells were held at −60 mV, etomidate was applied for at least 25 s. In 3CK-expressing cells, channel expression was verified by a subsequent and effective application of the TRPA1 agonist 2-APB (not shown), while the functionality was tested using a single application of 2-APB 1000 µM per cell. TRP: transient receptor potential; WT: wild type; 2-APB: 2-amino-phenyl borane.

Figure 4.

Etomidate induces an increase in [Ca2+]i in sensory neurons. (a) Etomidate 300 µM lead to an increase in [Ca2+]i in capsaicin- and AITC-sensitive DRG neurons from C57BL/6 mice during blockade of GABAA-receptors by 100 µM PTX. Etomidate and GABA (30 µM) were applied for 30 s, AITC (100 µM) for 20 s, and capsaicin (0.3 µM) for 10 s. (b) Etomidate 300 µM induced an increase in [Ca2+]i in DRG neurons from C57BL/6 mice. PTX 100 µM reduced etomidate-induced Ca2+ responses. Co-application of PTX and the TRPA1-antagonist AP-18 (15 µM) completely inhibited etomidate-induced Ca2+ increase. Etomidate was applied for 30 s, application of PTX either alone or in combination with AP-18 was started 30 s before and ended 120 s after etomidate. (c) Etomidate 300 µM did not lead to an increase in [Ca2+]i in DRG neurons from Trpa1−/−-mice during blockade of GABAA receptors by 100 µM PTX. Although neurons did not respond to 100 µM AITC applied as a control, 0.3 µM capsaicin evoked an immediate increase in [Ca2+]i in a subpopulation of neurons. Etomidate was applied for 30 s, AITC for 20 s, and capsaicin for 10 s. (d) Etomidate (100 µM)-evoked Ca2+ increases in DRG neurons from C57BL/6 mice correlated better to responses evoked by 30 µM GABA than to responses evoked by 100 µM AITC. Note the inverse correlation at an etomidate concentration of 600 µM. Panels provide the product–momentum correlation coefficient r. (e) Concentration–response curves for etomidate-evoked [Ca2+]i increases in DRG neurons derived from C57BL/6 mice (with and without 100 µM PTX) and Trpa1−/− mice. Etomidate concentrations from 10 to 2000 µM were tested, the curves were fitted to the Hill equation. TRP: transient receptor potential; PTX: picrotoxin.

Figure 5.

Propylene glycol (PG) activates hTRPA1 and rTRPV1. (a) Representative current traces of PG-evoked inward currents in non-transfected (MOCK) HEK293t cells or cells transiently expressing hTRPA1. To prevent desensitization, only one concentration was tested on each cell. Cells were held at −60 mV, PG was applied until the current had reached a steady state. (b) Concentration–response curve for PG-induced inward currents in hTRPA1-expressing HEK293t cells; 5 to 13 cells were tested at each concentration. Data were fitted to the Hill equation. (c) Representative PG-evoked inward current in an hTRPA1-expressing HEK293t cell blocked by the TRPA1 antagonist HC-030031. PG 10% was applied until the current had reached a steady state followed by a combined application of PG and 50 µM HC-030031. (d) Representative inward current of 10% PG in HEK293t cells expressing rTRPV1. Cells were held at −60 mV, PG was applied until the current had reached a steady state. (e) Representative PG-evoked inward current in an rTRPV1-expressing HEK293t cell blocked by the selective TRPV1 antagonist BCTC. PG 10% was applied until the current had reached a steady state followed by a combined application of PG and 10 µM BCTC. (f) Representative current in response to 1.4 M glucose (osmolarity corresponds approximately to the osmolarity of 10% PG). Note the loss of seal integrity during application of glucose. TRP: transient receptor potential.

Figure 6.

Etomidate and PG induce release of CGRP. (a) 10-fold diluted clinical formulations of Etomidat-® Lipuro and Hypnomidate® (both 0.8 mM etomidate) did not induce relevant CGRP release from isolated sciatic nerves of C57BL/6 mice. CGRP release was also not caused by 0.8 mM etomidate in SIF with dimethyl sulfoxide as solubilizer. A 10-fold higher etomidate concentration (8 mM) evoked significant release of CGRP as did a 35% PG solution in SIF. (b) Etomidat-induced CGRP release was significantly reduced in sciatic nerves from Trpa1−/−-, Trpv1−/−-, and Trpv1/Trpa1=/=-knockout mice. TRP: transient receptor potential; PG: propylene glycol; CGRP: calcitonin gene-related peptide.

Figure 7.

Etomidate formulations induce pain and axon reflex vasodilatation in human volunteers. (a) 100 µl Etomidat-® Lipuro (8.2 mM etomidate) or Lipofundin® injected intradermally in volar forearms of volunteers did not induce pain. 100 µl Hypnomidate® (8.2 mM etomidate) or 35% PG in Ringer’s solution caused high values on the numerical rating scale (NRS) in all volunteers indicating severe pain. An 8.2 mM etomidate formulation with methanol as solubilizer caused highest values on the NRS; methanol alone in Ringer`s solution produces significantly lower values on the NRS. (b) Measurements of regional skin perfusion by LDI 2 min after injection. Intradermal injections of 100 µl of Etomidat-® Lipuro, Lipofundin®, Hypnomidate® or 35% PG in Ringer’s solution caused local vasodilatation. Hypnomidate® and 35% PG in Ringer’s solution produced larger flare responses than lipid emulsion-based formulations after 2 min. Left-hand panels display baseline skin perfusion before injection. (c) Quantitative analyses of the flare sizes as area in cm2 developing over time based on the data from (b), showing wider and extended flare reactions upon Hypnomidate® and PG than after Etomidat-® Lipuro. NRS: numerical rating scale.