Active metabolites of dipyrone induce a redox-dependent activation of the ion channels TRPA1 and TRPV1

Abstract Introduction: The nonopioid analgesic and antipyretic dipyrone (metamizol) is frequently used worldwide. Dipyrone is a prodrug, and the metabolites 4-N-methylaminoantipyrine (MAA) and 4-aminoantipyrine (AA) seem to induce analgesia and antipyresia in part by inhibiting cyclooxygenase. In mice, however, the analgesic effect of dipyrone also seems to depend on the ion channel TRPA1. In this study, we explored the effects of dipyrone and its active metabolites on recombinant and native TRPA1 and TRPV1 channels. Methods: Constructs human (h) TRPA1 and TRPV1 were expressed in HEK293 cells, and dorsal root ganglion neurons were isolated from adult mice. Effects of dipyrone, MAA, and AA were explored by means of whole-cell patch clamp recordings and ratiometric calcium imaging. Results: Dipyrone failed to activate both hTRPA1 and hTRPV1. However, both MAA and AA evoked small outwardly rectifying membrane currents and an increase of intracellular calcium in cells expressing hTRPA1 or hTRPV1. MAA also sensitized both channels and thus potentiated inward currents induced by carvacrol (hTRPA1) and protons (hTRPV1). MAA-induced activation was inhibited by the antioxidant 10-mM glutathione included in the pipette, and the mutant constructs hTRPA1-C621/C641/C665S and hTRPV1-C158A/C391S/C767S were insensitive to both MAA and AA. Mouse dorsal root ganglion neurons exhibited a marginal calcium influx when challenged with MAA. Conclusion: The metabolites MAA and AA, but not dipyrone itself, activate and sensitize the nociceptive ion channels TRPA1 and TRPV1 in a redox-dependent manner. These effects may be relevant for dipyrone-induced analgesia and antipyresia.


Introduction
Dipyrone (metamizol) is an effective analgesic and antipyretic that is rapidly hydrolysed and converted into the metabolites MAA and AA in vivo, both known to inhibit cyclooxygenase (Fig. 1). 5,8,11,13,15 Dipyrone itself inhibits activation of the irritant receptor TRPA1 by reactive compounds, and dipyrone-induced analgesia is abrogated in mice lacking TRPA1. 12 TRPA1 is also critical for analgesia and hypothermia induced by acetaminophen, presumably due to an activation of TRPA1 by reactive metabolites. 1,6 Both MAA and AA are reactive and may thus modify redox-sensitive proteins. 13 Both TRPA1 and the capsaicin receptor TRPV1 are redoxsensitive, a property which in both ion channels mainly depend on N-terminal cysteines. 2,4,14 Considering that dipyrone is rapidly hydrolysed to MAA after intake, we hypothesized that the MAA and AA might as well modulate TRPA1 and TRPV1. Therefore, we examined the effects of dipyrone, MAA, and AA on recombinant and native TRPA1 and TRPV1 channels.

Cell culture
Stable cell lines with hTRPA1 or hTRPA1 were cultured and used as described previously. 3 HEK-293 t cells were cultured in DMEM (D-MEM; Gibco, BRL Life Technologies, Karlsruhe Germany) with 10% FBS (Biochrom, Berlin, Germany) and  Representative current traces in hTRPV1-HEK293 cells during 2 consecutive applications of pH 6.0. One micromolar MAA was applied for about 5 minutes between the 2 applications. (L) Average inward currents elicited by the second application of pH 6.0, expressed as average "-fold increase" calculated by normalizing the peak currents amplitudes of the second carvacrol-induced current with the first one. n.s., not significant; Student paired t test. *P , 0.05.

Electrophysiology
Whole-cell patch clamp was performed using the Patchmaster Software (HEKA Electronics, Lambrecht, Germany) and a HEKA USB 10 amplifier. Pipette solution was prepared using (in mM) NaCl 140, KCl 5, MgCl 2 , CaCl 2 1.2, HEPES 10, and glucose 10, pH 7.4. Calcium-free extracellular solution was prepared using (in mM) NaCl 140, KCl 5, MgCl 2 2, EGTA 5, HEPES 10, and glucose 10, pH 7.4. Data were sampled at 10 kHz and filtered at 2 kHz. The Fitmaster Software (HEKA Electronics) as well as Origin 7.0273 and Origin 8.5.1 (Origin Lab, Northampton, MA) were used for data analysis. Paired Student t test was performed for statistical analyses on dependent variables. P , 0.05 was regarded statistically significant.

MAA evokes a calcium influx in mouse dorsal root ganglion neurons
We finally asked if TRPA1 and TRPV1 account for a MAAevoked calcium-influx in mouse DRG neurons. MAA (10 mM) did not evoke a substantial calcium influx in DRG neurons, neither in neurons which responded briskly to carvacrol and/or capsaicin (n 5 568), nor in neurons with no or only very small responses to either carvacrol or capsaicin (n 5 225). The minimal increase in intracellular calcium occurring throughout the application of MAA in neurons expressing TRPA1 and TRPV1 did not seem to be substantially reduced by the simultaneous inhibition of TRPA1 (A967079, 10 mM) and TRPV1 (BCTC 100 nM) (Fig. 4B, n 5 271).

Discussion
Although associated with severe side effects such as agranulocytosis and with a yet unclear pharmacological mechanism of action, dipyrone is commonly used as a first-choice nonopioid analgesic. 7 The analgesic effect of dipyrone is strongly reduced in mice lacking TRPA1, 12 and it was suggested that this effect is due to a dipyrone-induced inhibition of TRPA1. Our data reveal that both MAA and AA gate instead of inhibiting both TRPA1 and TRPV1. This effect seems to be redox dependent and thus involves N-terminal cysteines, which are known to account for gating of both channels by oxidants. 2,4,9 Although the relevance of these cellular data is yet to be explored, both MAA and AA were already suggested to be required for dipyrone-induced analgesia and antipyresia in rodents. 5,11 Dipyrone is rapidly hydrolyzed into MAA and then converted into AA after intake, and the proteinunbound plasma levels of MAA and AA at therapeutic dosages of dipyrone are well above the concentrations found to gate TRPA1 and TRPV1. 5,8,15 Furthermore, the inhibitory effects of MAA and AA on neurons of the rostral ventromedial medulla are reduced by inhibition of TRPV1. 10 Similar to how acetaminophen was suggested to induce analgesia by inducing a presynaptic inhibition by activating TRPA1 in central nerve terminals, 1 it seems possible that an activation and sensitization-in addition to an inhibition-of TRPA1 and TRPV1 are relevant for dipyroneinduced analgesia and antipyresia. Further studies are needed to substantiate this somewhat controversial hypothesis.

Disclosures
The authors have no conflict of interest to declare.