Figure 1.
Chemical structure of S3969 and NGCC.
Figure 2.
Calcium responses in hTRPV1-expressing cells stimulated with capsaicin (Cap) and NGCC.
(A) Cells expressing hTRPV1 were loaded with Fura-2 AM and Ca2+ images were obtained at 0 and 60 s after stimulation with Cap or NGCC. The non-specific blocker of TRP channels, RR (30 µM), and the specific TRPV1 antagonist, CPZ (5 µM), were added to test the selectivity of Cap and NGCC. Representative ratiometric images are shown after treatment with Cap and NGCC. (B) As a control, the Ca2+ response was monitored in non-hTRPV1-expressing HEK 293T cells treated with Cap or NGCC. (C) The effects of Cap or NGCC treatment were quantified using Calcium-4 in a cell-based assay in the presence or absence of 30 µM RR or 5 µM CPZ. Experiments were repeated in triplicate and data points represent the means ± SEM (n = 3).
Figure 3.
Activation of TRPV1 by capsaicin and NGCC.
(A) TRPV1 current was activated by capsaicin (100 nM) and reversibly blocked by TRPV1 antagonist, CPZ (1 µM) in HEK293T cells transiently transfected with hTRPV1. NGCC (1 mM) induced an inward current in a capsaicin-sensitive cell. Neither capsaicin nor NGCC evoked the currents in non-transfected cells (n = 3). Dashed lines indicate zero current. (B) NGCC-induced currents were blocked by CPZ in hTRPV1-expressing HEK293T cells. (C) Summary of normalized current density in cells expressing hTRPV1 (n = 4). (D) Current density of the first pulse was normalized to 1.0. NGCC triggered inward currents in a dose-dependent manner in hTRPV1-expressing HEK293T cells (n = 3). Results are presented as the mean ± SEM.
Figure 4.
Effects of S3969 or NGCC on αβγ hENaC-expressing cells.
Cells expressing αβγ-hENaC were loaded with FMP blue-dye and the effects of S3969 or NGCC were quantitatively evaluated using membrane potential assay. S3969 depolarized membrane potential on αβγ hENaC-expressing cells and benzamil (Bz) effectively inhibited S3969 activity. NGCC showed no effect on αβγhENaC-expressing cells. Experiments were repeated in triplicate and data points represent the means ± SEM (n = 3–4).
Figure 5.
Effects of NGCC in amiloride-sensitive ASIC1a.
(A) Rapid change of extracellular pH from 7.4 to 6.0 for 10 seconds induced an inward current in HEK293T cells transiently transfected with mASIC1a. Current density of pH 6.0-evoked inward currents is 5.52±1.33 pA/pF (n = 3) and 118.1±33.0 pA/pF (n = 3) in non-transfected cells and mASIC1a-expressing cells, respectively. (B) mASIC1a currents were triggered by extracellular pH drop from 7.4 to 6.0. However, ASIC1a channels are not sensitive to NGCC (1 mM). The time interval between each stimulation is 120 seconds. Normalized current density was measured in cells expressing mASIC1a (n = 5). Results are presented as the mean ± SEM.
Figure 6.
Calcium responses in hTRPA1-expressing cells stimulated with AITC and NGCC.
(A) hTRPA1-expressing cells were loaded with Fura-2 AM and Ca2+ images were obtained at 0 and 60 s after stimulation with AITC or NGCC. The selectivity of AITC and NGCC was tested by adding the non-specific blocker of TRP channels, RR (30 µM), or a specific TRPA1 antagonist, HC-030031 (100 µM). Representative ratiometric images are shown after treatment with AITC and NGCC. (B) As a control, the Ca2+ response to AITC or NGCC treatment was monitored in mock-transfected Flp-In 293 cells. (C) AITC and NGCC treatment showed dose-dependent effects in hTRPA1-expressing cells. The effects of AITC or NGCC treatment in the presence or absence of 30 µM RR or 100 µM HC-030031 were quantified using Calcium-4 in a cell-based assay. Experiments were repeated in triplicate and data points represent the means ± SEM (n = 3).
Figure 7.
Activation of hTRPA1 by AITC and NGCC.
(A) AITC (100 µM) induced an inward current in hTRPA1-expressing cells. AITC-induced currents were partially inhibited by TRPA1 antagonist, HC-030031 (100 µM) and recovered after wash out. NGCC triggered an inward current in a AITC-sensitive cell. Neither AITC nor NGCC evoked the currents in non-transfected cells (n = 3). (B) NGCC-induced currents were blocked by HC-030031 in hTRPA1-expressing cells. (C) Summary of normalized current density in cells expressing hTRPA1 (n = 4). (D) NGCC triggered inward currents in a dose-dependent manner in hTRPA1-expressing cells (n = 4). Results are presented as the mean ± SEM.