Diclofenac, a nonsteroidal anti-inflammatory drug, is an antagonist of human TRPM3 isoforms

Abstract

The effects of diclofenac (Dic), an acetic acid derivative-type nonsteroidal anti-inflammatory drug, were examined on the function of transient receptor potential (TRP) melastatin (TRPM) 3 (TRPM3) in human embryonic kidney 293 cell-line (HEK293) cells with recombinant human TRPM3 isoforms (TRPM31325, TRPM3-3, TRPM3-9, and TRPM3-S) and in a neuroblastoma cell line human neuroblastoma IMR-32 cells (IMR-32 cells) derived from human peripheral neurons. TRPM3 responses evoked by pregnenolone sulfate (PregS) were effectively inhibited by Dic in a concentration-dependent manner in Ca(2+) measurement and electrophysiological assays. The apparent IC 50 for PregS-induced Ca(2+) response of TRPM31325, TRPM3-3, and TRPM3-9 was calculated to be 18.8, 42.5, and 7.1 μmol/L, respectively. The TRPM3-dependent Ca(2+) responses evoked by nifedipine, another TRPM3 agonist, were also significantly inhibited by Dic. In contrast, aceclofenac, an acetoxymethyl analog of Dic, had no effects on PregS-induced TRPM3 responses. Constitutive channel activity of TRPM3-S without TRPM3 agonists was substantially inhibited by Dic, ruling out the possibility of interaction of Dic against TRPM3 agonists to the channel binding sites. Moreover, Dic reversibly inhibited TRPM3 single-channel activity recorded in excised outside-out patches without affecting the channel conductance. In differentiated neuronal IMR-32 cells with endogenous TRPM3, Dic inhibited PregS-evoked Ca(2+) responses with an apparent IC 50 of 17.1 μmol/L. Taken together, our findings demonstrate that Dic inhibits human TRPM3 without interacting with the channel pore.

Keywords: Diclofenac; TRPM3; neuroblastoma; pain.

Figure 1

PregS‐evoked TRPM3‐mediated responses. (A) Scheme of human TRPM3 isoforms used in this study. (B) The ΔCa²⁺ _i induced by the application of PregS at a concentration range between 1 and 100 μmol/L (3–300 μmol/L for TRPM3‐9) is summarized as concentration–response relationships (square symbols, six independent experiments for TRPM3₁₃₂₅; circle symbols, six independent experiments for TRPM3‐3; triangle symbols, 10 independent experiments for TRPM3‐9). (C–E) Current and voltage (I‐V) relationships of TRPM3₁₃₂₅ (C), TRPM3‐3 (D), and TRPM3‐9 (E) evoked by ramp voltage command pulses were shown in the absence and presence of 30 μmol/L PregS. Ramp voltage command pulses for 400 msec were applied every 10 sec at a holding potential of −10 mV. (F) Comparison of PregS‐evoked current amplitude in HEK‐TRPM3s cells. Bars represent the mean ± SEM from 9 to 10 cells. * versus TRPM3₁₃₂₅ and TRPM3‐3. PregS, pregnenolone sulfate; TRPM, transient receptor potential melastatin; HEK, human embryonic kidney.

Figure 2

The effect of Dic on PregS‐induced Ca²⁺ responses of HEK‐TRPM3s cells. (A–C) PregS at 10 μmol/L (for HEK‐TRPM3₁₃₂₅ [A] and HEK‐TRPM3‐3 [B]) or 30 μmol/L (for HEK‐TRPM3‐9 [C]) was applied twice and 30 μmol/L Dic was added before the second application of PregS. (D) Change in PregS‐induced Ca²⁺ response by Dic at a concentration range between 1 and 100 μmol/L is summarized as concentration–response relationships. The amplitude of change in the second PregS‐induced Ca²⁺ response with Dic was normalized to that in the first without Dic (square symbols, six independent experiments for TRPM3₁₃₂₅; circle symbols, six independent experiments for TRPM3‐3; triangle symbols, six independent experiments for TRPM3‐9). Data were fitted to a sigmoid curve to determine the apparent IC ₅₀ of Dic against PregS‐induced Ca²⁺ response of each TRPM3 isoform. Dic, diclofenac; PregS, pregnenolone sulfate; HEK, human embryonic kidney; TRPM, transient receptor potential melastatin.

Figure 3

(A–C) Change in current amplitude of TRPM3₁₃₂₅ at −80 mV and +80 mV was plotted against time (A). I‐Vs evoked by ramp voltage command pulses were also shown in the control and with or without 10 and 30 μmol/L Dic in the presence of 30 μmol/L PregS (B). Ramp voltage command pulses for 400 msec were applied every 10 sec at a holding potential of −10 mV. Comparison of relative change in PregS‐evoked current amplitude by 10 and 30 μmol/L Dic in HEK‐TRPM3₁₃₂₅ cells (C). Peak current amplitude at −80 mV and +80 mV was pooled and summarized in a bar graph. Bars represent the mean ± SEM from eight cells. (D–F) As for (A–C) except TRPM3₁₃₂₅ to TRPM3‐3. Bars represent the mean ± SEM from four to six cells. (G–I) As for (A–C) except TRPM3₁₃₂₅ to TRPM3‐9. Bars represent the mean ± SEM from five cells. ** versus without Dic. ## versus with 10 μmol/L Dic. TRPM, transient receptor potential melastatin; Dic, diclofenac; PregS, pregnenolone sulfate; HEK, human embryonic kidney.

Figure 4

Nif‐induced TRPM3‐mediated Ca²⁺ response and the effect of Dic. (A) The ΔCa²⁺ _i evoked by the application of Nif at a concentration range between 1 and 100 μmol/L is summarized as concentration–response relationships (square symbols, six independent experiments for TRPM3₁₃₂₅; circle symbols, six independent experiments for TRPM3‐3; triangle symbols, six independent experiments for TRPM3‐9). (B–D) Nif at 30 μmol/L was applied twice and 30 μmol/L Dic was added before the second application of Nif. (E) Change in Nif‐induced Ca²⁺ response by Dic at a concentration range between 1 and 100 μmol/L is summarized as concentration–response relationships. The amplitude of change in the second Nif‐induced Ca²⁺ response with Dic was normalized to that in the first without Dic (square symbols, six to eight independent experiments for TRPM3₁₃₂₅; circle symbols, four to seven independent experiments for TRPM3‐3; triangle symbols, five to seven independent experiments for TRPM3‐9). Data were fitted to a sigmoid curve to determine the apparent IC ₅₀ of Dic against Nif‐induced Ca²⁺ response of TRPM3₁₃₂₅ and TRPM3‐9 isoforms. Nif, nifedipine; TRPM, transient receptor potential melastatin; Dic, diclofenac.

Figure 5

No effects of a glycolic acid ester of Dic (Afc) on TRPM3 and Dic on TRPM2, TRPM8, and TRPV4. (A) Chemical structures of Dic and Afc. (B and C) Effects of 100 μmol/L Afc on PregS‐induced Ca²⁺ response in HEK‐TRPM3₁₃₂₅ cells: a representative trace (B) and summary of change in Ca²⁺ response in the absence and presence of Afc (C, three independent experiments). (D) Summary of effects of 100 μmol/L Dic on the Ca²⁺ response to TRPM2 (four independent experiments), TRPM8 (five independent experiments), and TRPV4 (four independent experiments). Hydrogen peroxide (H₂O₂; 1 mmol/L), menthol (Men; 100 μmol/L), and GSK1016790 (GSK; 3 nmol/L) were applied to evoke TRPM2‐, TRPM8‐, and TRPV4‐mediated response, respectively. Dic, diclofenac; Afc, aceclofenac; TRPM, transient receptor potential melastatin; TRPV, transient receptor potential vanilloid; PregS, pregnenolone sulfate; HEK, human embryonic kidney.

Figure 6

Inhibitory mechanisms of Dic on TRPM3. (A) Effects of Dic on membrane currents in a wild‐type control HEK293 cell. (B and C) Effects of Dic on constitutive spontaneous TRPM3‐S‐mediated currents, which were recorded in the absence of TRPM3 agonists. Change in current amplitude of TRPM3‐S at −80 mV and +80 mV was plotted against time (B). I‐Vs evoked by ramp voltage command pulses were shown in the absence (cont) and presence of 10 and 30 μmol/L Dic in a wild‐type control (A) and a TRPM3‐S expressing HEK293 cell (C). Ramp voltage command pulses for 400 msec were applied every 10 sec at a holding potential of −10 mV. A dash‐line in (A) exhibits leak‐like currents. (D) Comparison of change in current amplitude in the absence of TRPM3 agonists by 10 and 30 μmol/L Dic among HEK‐TRPM3‐S, HEK‐TRPM3₁₃₂₅, and wild‐type control HEK293 cells. To confirm the expression of TRPM3 channels in HEK‐TRPM3‐S and HEK‐TRPM3₁₃₂₅ cells, 30 μmol/L PregS was applied to these cells. Bars represent the mean ± SEM from four to six cells. (E–G) Effects of Dic on PregS‐evoked single TRPM3₁₃₂₅ channel currents, which were recorded in excised outside‐out patches. Change in peak channel current amplitude of TRPM3₁₃₂₅ at −80 mV and +80 mV was plotted against time (E). I‐Vs evoked by ramp voltage command pulses were also shown (F) in the absence of both PregS and Dic (cont), and presence of 30 μmol/L PregS with (+30 μmol/L Dic) or without Dic (30 μmol/L PregS). Ramp voltage command pulses for 400 msec were applied every 10 sec at a holding potential of −10 mV. (G) The amplitude of peak PregS‐evoked single‐channel currents at +80 mV was summarized before and during application of 10 and 30 μmol/L Dic. Bars represent the mean ± SEM from five cells. ** versus without Dic. # versus with 10 μmol/L Dic. Dic, diclofenac; TRPM, transient receptor potential melastatin; HEK293, human embryonic kidney 293 cell‐line; PregS, pregnenolone sulfate.

Figure 7

Channel conductance and open probability of TRPM3₁₃₂₅ with or without Dic. (A) A representative single‐channel current trace in an outside‐out patch excised from a HEK‐TRPM3₁₃₂₅ cell. The channel activity was increased by 30 μmol/L PregS and thereafter 10 and 30 μmol/L Dic were applied. During application of PregS and PregS plus Dic, the holding potential was changed from +70 to +80, +90, and +100 mV to determine the channel conductance. In lower panels, traces at +70 and +90 mV were shown in a higher time resolution (B). The “c” indicates the channel close level. I‐Vs of PregS‐induced single‐channel currents with or without Dic (C, PregS; five cells, +10 μmol/L Dic; four cells +30 μmol/L Dic; four cells). The channel conductance was calculated to fit a set of data in each cell to a linear regression line. (D) Amplitude histograms at +80 mV for 5 sec in the presence of 30 μmol/L PregS, 30 μmol/L PregS plus 10 μmol/L Dic, and 30 μmol/L PregS plus 30 μmol/L Dic from the same cell as shown in (A). The bin width is 0.163 pA. (E) The open probability of PregS‐evoked single‐channel currents was summarized before and during application of 10 and 30 μmol/L Dic. Bars represent the mean ± SEM from three to four cells. * versus without Dic. TRPM, transient receptor potential melastatin; Dic, diclofenac; HEK, human embryonic kidney; PregS, pregnenolone sulfate.

Figure 8

Induction of TRPM3 in differentiated human neuroblastoma IMR‐32 cells and effects of Dic. Non‐neuronal IMR‐32 cells were differentiated to nIMR‐32 cells with BrdU treatment (see Materials and Methods). (A) To confirm quantitative induction of TRPM3, TRPM3 mRNA transcripts were compared between IMR‐32 and nIMR‐32 cells (six independent experiments). ** versus IMR‐32. (B and C) The Ca²⁺ response (ΔCa²⁺ _i) to 30 μmol/L PregS was shown in representative three IMR‐32 and nIMR‐32 cells, and the pooled data were summarized as a bar graph (C, four independent experiments for IMR‐32 and nIMR‐32 cells, respectively). ** versus IMR‐32. (D and E) Cumulative application of PregS between 1 and 300 μmol/L to nIMR‐32 cells. Typical traces (D, one responsive and another nonresponsive cell to PregS) and summarized data of responsive cells (E, four independent experiments) were shown. (F) Application of 3–100 μmol/L Dic to nIMR‐32 cells significantly inhibited PregS‐induced Ca²⁺ response. Bars represent the mean ± SEM (five to eight independent experiments). Data were fitted to a sigmoid curve to determine the apparent IC ₅₀ of Dic against PregS‐induced Ca²⁺ responses. TRPM, transient receptor potential melastatin; IMR‐32, human neuroblastoma IMR‐32; Dic, diclofenac; nIMR‐32, neuronal IMR‐32; BrdU, 5‐bromo‐2′‐deoxyuridine; PregS, pregnenolone sulfate.