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 redox-sensitive, 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.
Dipyrone, reduced glutathione (Sigma-Aldrich, Taufkirchen, Germany), 4-N-methylaminoantipyrine, and 4-aminoantipyrine (Sanofi-Aventis, Frankfurt, Germany). L-dithiothreitol, capsaicin, and carvacrol (Sigma-Aldrich). Allyl isothiocyanate (AITC; Merck, Darmstadt, Germany). BCTC (4-(3-Chloro-2-pyridinyl)-N-[4-(1,1-dimethylethyl)phenyl]-1-piperazinecarboxamide) and HC030031 (Tocris, Bristol, United Kingdom).
2.2. 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 100 µg/mL penicillin–streptomycin (Gibco). For transient transfection, cDNAs were transfected with the nanofectin transfection kit (PAA, Pasching, Austria). Cysteine modifications in hTRPA1 and hTRPV1 were introduced by using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Waldbronn, Germany). All cysteine exchanges were verified by DNA sequencing (GATC, Konstanz, Germany). Experiments were performed in accordance with the requirements of the local authorities (Hannover, Niedersachsen, Germany).
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, MgCl2, CaCl2 1.2, HEPES 10, and glucose 10, pH 7.4. Calcium-free extracellular solution was prepared using (in mM) NaCl 140, KCl 5, MgCl2 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.
2.4. Ratiometric [Ca2+]i measurements
Coverslips were incubated with 4-µM Fura-2-AM and 0.02% pluronic for 45 minutes and then mounted on an inverse microscope (Axio observer D1; Zeiss, Jena, Germany). Cells were superfused with (in mM) NaCl 145, KCl 5, CaCl2 1.25, MgCl2 1, glucose 10, and HEPES 10, pH 7.4. Images were exposed for 20 and 40 ms, respectively, and acquired at 1 Hz with a CCD camera (Cool SNAP EZ; Photometrics, Puchheim, Germany). Data were recorded using VisiView 2.1.1 software (Visitron Systems GmbH, Puchheim, Germany).
2.5. Dorsal root ganglion culture
Dorsal root ganglion (DRG) neurons were isolated from adult wild-type C57/BL6 mice. After surgical preparation, ganglia were incubated for 1 hour at 37°C in DMEM (Invitrogen, Darmstadt, Germany) containing 0.6 mg/mL collagenase (type XI) and 3 mg/mL protease (both, Sigma Aldrich, Seelze, Germany) before dissociated neurons were plated onto coverslips coated with poly-D-lysine (0.1 mg/mL for 30 minutes). Cells were cultured (37°C and 5% CO2) in serum-free TNB-100 basal medium (Biochrom AG), supplemented with penicillin/streptomycin 100 U/mL.
3.1. MAA and AA, but not dipyrone, activate hTRPA1 and hTRPV1
Dipyrone up to 1 mM did not evoke changes in intracellular calcium (n = 262, Fig. 1B) or increased membrane currents (Fig. 1C, D, n = 7) in HEK293 cells expressing hTRPA1. By contrast, 1 µM of MAA induced a calcium influx (Fig. 1E, n = 92) as well as outwardly rectifying membrane currents monitored during voltage ramps ranging from −100 to +100 mV within 500 ms (Fig. 1F, n = 5). This effect did not obey a clear concentration dependency (Fig. 1G, n = 5–7 for each concentration). AA also induced an increase of intracellular calcium (Fig. 1H, n = 109) and membrane currents (Fig. 1I, n = 7) in cells with hTRPA1. Again, this effect lacked a clear concentration dependency (Fig. 1J, n = 5–8). MAA-evoked membrane currents were inhibited by the selective TPRA1-inhibitor HC-030031 (Fig. 1K, n = 6), confirming that the observed effects are generated by hTRPA1. As is demonstrated in Figures 1L and M, both MAA (1 µM) and AA (100 nM) also induced a significant potentiation of carvacrol-induced currents in hTRPA1 cells (MAA: 7 ± 2-fold, n = 7; AA: 5 ± 4-fold, n = 5; P < 0.05, paired t tests).
Similar to hTRPA1, hTRPV1 was not activated by dipyrone (Fig. 2A, n = 264, Fig. 2B, C, n = 8). However, MAA induced both an increase in intracellular calcium (Fig. 2D, n = 345), and robust membrane currents (Fig. 2E, F, n = 10) in cells expressing hTRPV1. We also observed an activation of hTRPV1 by AA (Fig. 2G, n = 229, Fig. 2H, I, n = 6). The TRPV1-antagonist BCTC (100 nM) inhibited MAA-induced activation (n = 6). MAA also induced a significant potentiation of proton (pH 6.0)-induced currents in hTRPV1 cells (Fig. 2K, L, 9 ± 3-fold, n = 6; P < 0.05, paired t test).
3.2. MAA activates hTRPA1 and hTRPV1 in a redox-dependent manner
We next asked if MAA might gate TRPA1 and TRPV1 through redox modification. N-terminal cysteines confer both TRPA1 and TRPV1 their redox sensitivity.2,4,9 Indeed, MAA failed to evoke a calcium influx (Fig. 3A, n = 77) as well as membrane currents (Fig. 3B, C, n = 7) in cells expressing the redox-insensitive mutant hTRPA1-C621S/C641S/C665S. In case of hTRPV1, the redox-insensitive mutant hTRPV1-C158S/C391S/C767S also failed to generate both MAA-induced calcium influx (Fig. 3D, n = 135) and membrane currents (Fig. 3E, F, n = 6). Furthermore, carvacrol-induced membrane currents were not potentiated by MAA on the mutant hTRPA1-C621S/C641S/C665S (0.8 ± 0.1-fold increase; n = 5, Fig. 3G, I). Accordingly, proton-induced membrane currents were not potentiated by MAA on hTRPV1-C158S/C391S/C767S (75 ± 0.1-fold increase; n = 4, Fig. 3H, I). To substantiate that MAA gates TRPA1 and TRPV1 through oxidation, the antioxidant glutathione (GSH, 10 mM) was added to the pipette solution. Indeed, MAA was completely ineffective on both hTRPV1 and hTRPV1 in presence of intracellular GSH (Fig. 3J, K, n = 5 each).
3.3. MAA evokes a calcium influx in mouse dorsal root ganglion neurons
We finally asked if TRPA1 and TRPV1 account for a MAA-evoked calcium-influx in mouse DRG neurons. MAA (10 µM) did not evoke a substantial calcium influx in DRG neurons, neither in neurons which responded briskly to carvacrol and/or capsaicin (n = 568), nor in neurons with no or only very small responses to either carvacrol or capsaicin (n = 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 µM) and TRPV1 (BCTC 100 nM) (Fig. 4B, n = 271).
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 protein-unbound 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 dipyrone-induced analgesia and antipyresia. Further studies are needed to substantiate this somewhat controversial hypothesis.
The authors have no conflict of interest to declare.
The Department of Anaesthesiology and Intensive Care Medicine, Hannover Medical School, supported the study.
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