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. 2011 Jun;79(6):1023-30.
doi: 10.1124/mol.110.069922. Epub 2011 Mar 15.

Rapid and contrasting effects of rosiglitazone on transient receptor potential TRPM3 and TRPC5 channels

Yasser Majeed  1 , Yahya Bahnasi, Victoria A L Seymour, Lesley A Wilson, Carol J Milligan, Anil K Agarwal, Piruthivi Sukumar, Jacqueline Naylor, David J Beech
Affiliations

Rapid and contrasting effects of rosiglitazone on transient receptor potential TRPM3 and TRPC5 channels

Yasser Majeed et al. Mol Pharmacol. 2011 Jun.

Abstract

The aim of this study was to generate new insight into chemical regulation of transient receptor potential (TRP) channels with relevance to glucose homeostasis and the metabolic syndrome. Human TRP melastatin 2 (TRPM2), TRPM3, and TRP canonical 5 (TRPC5) were conditionally overexpressed in human embryonic kidney 293 cells and studied by using calcium-measurement and patch-clamp techniques. Rosiglitazone and other peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists were investigated. TRPM2 was unaffected by rosiglitazone at concentrations up to 10 μM but was inhibited completely at higher concentrations (IC(50), ∼22.5 μM). TRPM3 was more potently inhibited, with effects occurring in a biphasic concentration-dependent manner such that there was approximately 20% inhibition at low concentrations (0.1-1 μM) and full inhibition at higher concentrations (IC(50), 5-10 μM). PPAR-γ antagonism by 2-chloro-5-nitrobenzanilide (GW9662) did not prevent inhibition of TRPM3 by rosiglitazone. TRPC5 was strongly stimulated by rosiglitazone at concentrations of ≥10 μM (EC(50), ∼30 μM). Effects on TRPM3 and TRPC5 occurred rapidly and reversibly. Troglitazone and pioglitazone inhibited TRPM3 (IC(50), 12 μM) but lacked effect on TRPC5, suggesting no relevance of PPAR-γ or the thiazolidinedione moiety to rosiglitazone stimulation of TRPC5. A rosiglitazone-related but nonthiazolidinedione PPAR-γ agonist, N-(2-benzoylphenyl)-O-[2-(methyl-2-pyridinylamino)ethyl]-l-tyrosine (GW1929), was a weak stimulator of TRPM3 and TRPC5. The natural PPAR-γ agonist 15-deoxy prostaglandin J(2), had no effect on TRPM3 or TRPC5. The data suggest that rosiglitazone contains chemical moieties that rapidly, strongly, and differentially modulate TRP channels independently of PPAR-γ, potentially contributing to biological consequences of the agent and providing the basis for novel TRP channel pharmacology.

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Figures

Fig. 1.
Fig. 1.
Fast and reversible inhibition of TRPM3 activity by rosiglitazone. a, two-dimensional structure of rosiglitazone (rosi) with the thiazolidinedione (TZD) moiety highlighted. b–e, data were generated by Ca2+ measurement (b) or whole-cell patch-clamp (c–e) in cells overexpressing TRPM3 (Tet+) unless specified by Tet− (in b). b, example parallel comparisons of 5 μM PregS responses in the presence of 10 μM rosi or vehicle applied for 15-min before PregS application and maintained throughout the recordings (N = 4). c, time-series plot showing the effect of bath-applied 5 μM PregS and then the addition of 10 μM rosi. Shown are outward and inward currents sampled during the voltage ramp. d, typical I-V relationships for the PregS-induced currents immediately before (PregS) and after the response to 10 μM rosi (+10 rosi). e, for experiments of the type shown in c, the mean residual PregS-evoked current after application of rosi normalized to the current amplitude immediately before application of rosi (n = 9).
Fig. 2.
Fig. 2.
Concentration-dependence of TRPM3 inhibition by rosiglitazone. Data were generated by Ca2+ measurement. a, example direct comparison of the effects of different concentrations of rosiglitazone (rosi) on TRPM3 activity evoked by 20 μM nifedipine. Rosi was applied 15 min before nifedipine and was continuously present thereafter. There was no response to nifedipine in the absence of TRPM3 expression (data not shown). b, mean normalized data for experiments of the type exemplified in a. Signals were measured 90 s (black circles) and 240 s (white circles) after nifedipine application (n/N = 3/12). The Hill equation fitted to the 90-s data points had an IC50 of 9.52 μM and slope of 1.34. c, mean concentration-response data for rosi inhibition of TRPM3 activated by 5 μM PregS (black circles; IC50, 4.6 μM; slope, 1.1) or TRPM2 activated by 1 mM H2O2 (white squares; IC50, 22.5 μM; slope, 3.4) (n/N = 3/12 for each data point). b and c, statistical comparisons (*, P < 0.05) were made against paired solvent controls (i.e., 100%, no effect) and measured 90 s after application of the channel stimulator.
Fig. 3.
Fig. 3.
Stimulation of TRPC5 activity by rosiglitazone. Data were from cells overexpressing TRPC5 (Tet+) unless specified by Tet−. a, typical Ca2+ measurement experiment showing the effect of 100 μM rosiglitazone (rosi) in TRPC5-expressing cells (N = 4 each). b, example whole-cell patch-clamp experiment for currents at +80 and −80 mV, showing the effect of bath-applied 100 μM rosi. c, I-V relationship for the rosi-induced current of b. d, concentration-dependent stimulation of TRPC5 determined after 500-s (white circles) and 15-min (black circles) applications of rosi in Ca2+ measurement experiments (n/N = 3/18 each). The curve is a fitted Hill equation to the 15-min data (EC50, 31.1 μM; slope, 1.65). Also included are whole-cell patch-clamp data for responses to rosi at −80 mV (white triangles) and +80 mV (white squares) (n = 5).
Fig. 4.
Fig. 4.
Differential requirement for the thiazolidinedione moiety. Data were generated by Ca2+ measurement in cells over-expressing TRPM3 (b–e) or TRPC5 (f). a, two-dimensional structures of troglitazone (tro) and pioglitazone (pio). b and c, mean data for effects of tro (b) and pio (c) on TRPM3 stimulated by 5 μM PregS. Statistical comparisons were made with paired solvent controls. The fitted Hill equations gave IC50 values of 11.97 μM (slope, 2.91) and 12.10 μM (slope, 2.11) for tro and pio, respectively (n/N = 3/12 for each point). d, example experiment showing the effect of 15-min treatment with 30 μM tro on TRPM3 stimulated by 20 μM nifedipine. e, mean normalized data for experiments with tro (as exemplified in d) and 30 μM pio (n/N = 3/24 for each point). f, mean Ca2+ responses evoked by 100 μM rosi, 100 μM tro, 100 μM pio, or the vehicle control (dimethylsulfoxide) in cells overexpressing TRPC5 (n/N = 3/12 each).
Fig. 5.
Fig. 5.
Insensitivity of the rosiglitazone effect on TRPM3 to PPAR-γ antagonism. Data were generated by Ca2+ measurement in cells overexpressing TRPM3 (Tet+). a, cells were pretreated with 50 μM GW9662 (or vehicle) for 24 h before experiments and the GW9662 was maintained during the recordings. Pretreatment with 10 μM rosiglitazone (rosi) was as described for Fig. 1b, and PregS was applied at 5 μM. b, mean data for experiments exemplified in a, showing the effect of GW9662 alone (+ GW9662), rosi alone (+ rosi), or GW9662 with rosi (+ GW9662 + rosi) on the PregS-induced response (n/N = 3/12 for each condition).
Fig. 6.
Fig. 6.
Insensitivity of TRPM3 and TRPC5 to 15-deoxy-Δ12, 14-prostaglandin J2 (15d-PGJ2). a, two-dimensional structure of 15d-PGJ2. Data were generated in cells overexpressing TRPM3 (b–d) or TRPC5 (e). b and c, example time-series for a whole-cell voltage-clamp experiment showing the effect of bath-applied 10 μM 15d-PGJ2 on currents elicited by 5 μM PregS. c, I-V relationship for PregS-evoked currents before (PregS) and after application of 15d-PGJ2 (+10 15d-PGJ2). d, mean data for the experiments exemplified in b and analyzed 5 and 10 min after 15d-PGJ2 application (n = 7). e, Ca2+ measurement data comparing the amplitude of the signals elicited by 10 μM 15d-PGJ2 or 100 μM Gd3+ in cells overexpressing TRPC5 (Tet+) and control (Tet−) cells (n/N = 3/24 for each point).
Fig. 7.
Fig. 7.
Effect of a nonthiazolidinedione PPAR-γ agonist (GW1929) on TRPM3. Data were generated by Ca2+ measurement in cells overexpressing TRPM3 (Tet+) or control (Tet−) cells. a, two-dimensional structure of GW1929. b, example parallel comparisons of 5 μM PregS responses in the presence of 50 μM GW1929 or vehicle (Ctrl) applied for 15 min before PregS application and maintained throughout the recordings (N = 8). Note the small increase in basal Ca2+ (indicated by arrows) in the presence of GW1929. c and d, mean data for experiments exemplified in (b), showing the effect of pretreatment with GW1929 on the PregS response (c) and GW1929-evoked increase in basal Ca2+ in Tet+ (TRPM3) but not Tet− cells (d) (n/N = 3/24 each).
Fig. 8.
Fig. 8.
Effect of GW1929 on TRPC5. a–e, data were generated by Ca2+ measurement (a and b) or whole-cell patch-clamp recording (c–e). a, example experiment showing the effect of application of 100 μM GW1929 or vehicle (Ctrl) on cells overexpressing TRPC5. b, mean data for the peak and sustained components of the GW1929-evoked Ca2+ response in cells overexpressing TRPC5 (Tet+) or control (Tet−) cells (n/N = 3/12 each). c, example time-series plot from a whole-cell voltage-clamp experiment showing the effect of bath-applied 50 μM GW1929, 30 μM Gd3+ and then 75 μM 2-aminoethoxydiphenylborate (2-APB). d, example I-V relationship from the experiment in c. e, mean currents induced by 50 μM GW1929 or 30 μM Gd3+ in cells overexpressing TRPC5 (Tet+) (n = 5). Statistical analysis was of the response to GW1929 relative to its vehicle (pre-GW1929) control.
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