Journal: Pulmonary Circulation

Article Title: Functional coupling of TRPV4, IK, and SK channels contributes to Ca 2+ -dependent endothelial injury in rodent lung

doi: 10.1086/680166

Figure Lengend Snippet: Whole-cell voltage-clamp recordings in PMVECs. A, Voltage-clamp recording protocol. Cells were held at their resting potential and stepped to −80 mV for 100 ms before a voltage ramp of −80 to +60 mV (200 ms), a voltage step to +30 mV (100 ms), and return to resting potential. B, Representative whole-cell current densities elicited by the voltage-clamp protocol shown in A. After stable baseline recordings (control), sequential additions of paxilline (1 μM), TRAM-34 (1 μM), and apamin (200 nM) selectively blocked BK, IK, and SK channels, respectively. C–E, BK (C), IK (D), and SK (E) currents were isolated from digital subtraction of the voltage-ramp portion of traces in B. F, Normalized BK, IK, and SK channel current density to whole-cell current density, calculated from the steady-state current density recordings at +30 mV. BK, IK, SK: large-, intermediate-, and small-conductance KCa (Ca2+-activated potassium) channels, respectively; PMVECs: pulmonary microvascular endothelial cells; TRAM-34: 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole.

Article Snippet: TRAM-34 was from Alomone Labs. All other chemicals were obtained from Fisher Scientific (Pittsburgh, PA).

Techniques: Isolation

Journal: Pulmonary Circulation

Article Title: Functional coupling of TRPV4, IK, and SK channels contributes to Ca 2+ -dependent endothelial injury in rodent lung

doi: 10.1086/680166

Figure Lengend Snippet: TRPV4 channel activation increases IK and SK channel current densities. A, Representative whole-cell current densities elicited by the 200-ms −80 to +60 mV voltage-ramp protocol. Following stable baseline recordings, GSK (50 nM) was used to activate TRPV4 channels. Sequential applications of paxilline (500 nM), TRAM-34 (1 μM), and apamin (200 nM) were used to selectively inhibit BK, IK, and SK channels, respectively. B, Bar graph showing BK, IK, and SK channel current densities, in the presence of GSK, normalized to baseline whole-cell current density (control in A). Normalized results were calculated from the steady-state current density recordings at +30 mV. Dashed lines indicate normalized averages of each channel current density in the absence of GSK, as shown in Figure 2F. Asterisks indicate statistical significance (P < 0.05). C–F, The effect of GSK (C), paxilline (D), TRAM-34 (E), and apamin (F) on whole-cell current density isolated from digital subtraction of the traces shown in A. Averaged membrane capacitance was 23.8 ± 3.2 pF. BK, IK, SK: large-, intermediate-, and small-conductance KCa (Ca2+-activated potassium) channels, respectively; GSK: GSK1016790A; TRAM-34: 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole; TRPV4: transient receptor potential vanilloid 4.

Article Snippet: TRAM-34 was from Alomone Labs. All other chemicals were obtained from Fisher Scientific (Pittsburgh, PA).

Techniques: Activation Assay, Isolation

Journal: Pulmonary Circulation

Article Title: Functional coupling of TRPV4, IK, and SK channels contributes to Ca 2+ -dependent endothelial injury in rodent lung

doi: 10.1086/680166

Figure Lengend Snippet: TRPV4 channel inhibition reduces IK and SK channel current densities. A, Representative whole-cell current density elicited by the 200-ms −80 to +60 mV voltage-ramp protocol. Following stable baseline recordings (control), HC (500 nM) was used to inhibit TRPV4 channels. Sequential applications of paxilline (500 nM), TRAM-34 (1 μM), and apamin (200 nM) were used to selectively inhibit BK, IK, and SK channels, respectively. B, Bar graph showing BK, IK, and SK channel current densities in the presence of HC, normalized to baseline whole-cell current density (control in A). Normalized results were calculated from the steady-state current density recordings at +30 mV. Dashed lines indicate normalized averages of each channel current density in the absence of HC, as shown in Figure 2F. Asterisks indicate statistical significance (P < 0.05). C–F, The effect of HC (C), paxilline (D), TRAM-34 (E), and apamin (F) on whole-cell current density isolated from digital subtraction of the traces shown in A. Averaged membrane capacitance was 22.3 ± 4.5 pF. BK, IK, SK: large-, intermediate-, and small-conductance KCa (Ca2+-activated potassium) channels, respectively; HC: HC067047; TRAM-34: 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole; TRPV4: transient receptor potential vanilloid 4.

Article Snippet: TRAM-34 was from Alomone Labs. All other chemicals were obtained from Fisher Scientific (Pittsburgh, PA).

Techniques: Inhibition, Isolation

Journal: Pulmonary Circulation

Article Title: Functional coupling of TRPV4, IK, and SK channels contributes to Ca 2+ -dependent endothelial injury in rodent lung

doi: 10.1086/680166

Figure Lengend Snippet: Role of KCa channels in the 14,15-EET-induced permeability response. Permeability increased significantly with 14,15-EET (3 μM, n = 5) in lungs from wild-type mice. This response was attenuated in low-Ca2+ buffer and was restored by Ca2+ add-back. Pretreatment of lungs with iberiotoxin (IbTx, 100 nM, n = 5) had no effect on the Kf response to 14,15-EET. In contrast, pretreatment of lungs with the combination of charybdotoxin (ChTx, 100 nM) and apamin (300 nM) to block all KCa channels (n = 4), with apamin alone to block SK channels (300 nM, n = 5), or with TRAM-34 (1 μM, n = 5) to block IK channels significantly attenuated this response. Note that 14,15-EET had no effect in any group in low-Ca2+ buffer. Asterisks indicate P < 0.05; only groups with 14,15-EET alone or 14,15-EET in presence of IbTx showed significant increases in permeability (response in low vs. normal Ca2+ or baseline [BL] vs. normal Ca2+), 2-way ANOVA. BK, IK, SK: large-, intermediate-, and small-conductance KCa (Ca2+-activated potassium) channels, respectively; 14,15-EET: 14,15-epoxyeicosatrienoic acid; KCa: Ca2+-activated potassium; TRAM-34: 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole.

Article Snippet: TRAM-34 was from Alomone Labs. All other chemicals were obtained from Fisher Scientific (Pittsburgh, PA).

Techniques: Permeability, Blocking Assay