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

Journal: Journal of cellular physiology

Article Title: Stimulation of vascular smooth muscle cell proliferation by stiff matrix via the IK Ca channel-dependent Ca 2+ signaling.

doi: 10.1002/jcp.30349

Figure Lengend Snippet: FIGURE 3 Stiff substrate‐induced IKCa channel upregulation is required for increased A7r5 VSMC proliferation. (a) Summary of the effects of treatment with 100 nM TRAM34 on stiff substrate‐induced cell proliferation from three independent experiments. (b) Representative western blots showing siRNA‐mediated knockdown of the IKCa protein expression (top) and summary of the data from five independent experiments (bottom). (c) Summary of stiff substrate‐induced cell proliferation in siRNA‐transfected cells from three independent experiments. *p < .05 and **p < .01 compared between 0.21 and 1.72 MPa without any treatment or compared between 1.72 MPa with or without treatment with TRAM34, respectively. IKCa, intermediate‐conductance Ca2+‐activated K+; siRNA, small interfering RNA; VSMC, vascular smooth muscle cell

Article Snippet: In some cases, cells were cultured in medium containing 100 nM TRAM34 (Santa Cruz) to block the IKCa channel, 1 mM ethylene glycol‐bis(β‐aminoethyl ether)‐N,N,N′,N′‐tetraacetic acid (EGTA) to reduce extracellular Ca2+ concentration, 10 μM BAPTA‐AM (AbMole Bioscience) to chelate intracellular Ca2+ concentration, or 20 μM PD98059 (Cell Signaling Technology) to inhibit ERK, respectively.

Techniques: Western Blot, Knockdown, Expressing, Transfection, Small Interfering RNA

Journal: Journal of cellular physiology

Article Title: Stimulation of vascular smooth muscle cell proliferation by stiff matrix via the IK Ca channel-dependent Ca 2+ signaling.

doi: 10.1002/jcp.30349

Figure Lengend Snippet: FIGURE 4 Stiff substrate induces IKCa‐ dependent increase in [Ca2+]i in A7r5 VSMCs. (a) Representative single‐cell images showing intracellular Ca2+ concentration in cells cultured on the soft and stiff substrate under indicated conditions. Treatment with 100 nM TRAM34 was made during culture media and also in extracellular recording solution. (b) Summary of the relative Fluo4 fluorescence intensity, indicative of the [Ca2+]i, in individual cells under the condition shown in (a), with 375 cells examined for each case. **p < .01. IKCa, intermediate‐conductance Ca2+‐activated K+; VSMC, vascular smooth muscle cell

Article Snippet: In some cases, cells were cultured in medium containing 100 nM TRAM34 (Santa Cruz) to block the IKCa channel, 1 mM ethylene glycol‐bis(β‐aminoethyl ether)‐N,N,N′,N′‐tetraacetic acid (EGTA) to reduce extracellular Ca2+ concentration, 10 μM BAPTA‐AM (AbMole Bioscience) to chelate intracellular Ca2+ concentration, or 20 μM PD98059 (Cell Signaling Technology) to inhibit ERK, respectively.

Techniques: Concentration Assay, Cell Culture, Fluorescence

Journal: Journal of cellular physiology

Article Title: Stimulation of vascular smooth muscle cell proliferation by stiff matrix via the IK Ca channel-dependent Ca 2+ signaling.

doi: 10.1002/jcp.30349

Figure Lengend Snippet: FIGURE 6 Stiff substrate‐induced ERK activation is required for increased A7r5 VSMC proliferation. Representative western blots showing stiff substrate‐induced ERK activation in cells without or with treatment with 100 nM TRAM34 (a) or in culture media containing no or 10 μM BAPTA‐AM (b). (c) Summary of the mean data from three independent experiments. (d) Summary of the effects of treatment with 20 μM PD98059 on stiff substrate‐induced cell proliferation from three independent experiments. *p < .05 and **p < .01. ERK, extracellular signal‐regulated kinase; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; VSMC, vascular smooth muscle cell

Article Snippet: In some cases, cells were cultured in medium containing 100 nM TRAM34 (Santa Cruz) to block the IKCa channel, 1 mM ethylene glycol‐bis(β‐aminoethyl ether)‐N,N,N′,N′‐tetraacetic acid (EGTA) to reduce extracellular Ca2+ concentration, 10 μM BAPTA‐AM (AbMole Bioscience) to chelate intracellular Ca2+ concentration, or 20 μM PD98059 (Cell Signaling Technology) to inhibit ERK, respectively.

Techniques: Activation Assay, Western Blot

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: R-(+)-WIN55212-2 protects pericytes from ischemic damage and restores retinal microcirculatory patency after ischemia/reperfusion injury.

doi: 10.1016/j.biopha.2023.115197

Figure Lengend Snippet: Fig. 1. R-(+)-WIN55212–2 regulated the tone of pericyte on retinal capillaries via activation of BKCa. (A-C) Representative images and summarized data showed effects of iberiotoxin on R-(+)-WIN55212–2 induced regulation on pericytes and capillaries. (C-K) Effects of the apamin, triarylmethane-34 (TRAM 34), Gliben clamide and 4-Aminopyridine (4-AP) on R-(+)-WIN55212–2 induced pericyte and capillaries variation. *P < 0.05, * *P < 0.01 and * * *P < 0.001. Scale bar: 10 µm. The blue lines indicate the lumen diameter. The red circles indicate the outlines of pericytes. NA, noradrenalin; IBTX, iberiotoxin; WIN, R-(+)-WIN55212–2; TRAM 34, triarylmethane-34; Gb, Glibenclamide; 4-AP, 4-Aminopyridine.

Article Snippet: The large conductance Ca2+activated potassium (BKCa) channel inhibitor iberiotoxin (IBTX), the small-conductance Ca2+-sensitive K+ channel (SKCa) inhibitor apamin, the intermediate-conductance Ca2+-activated K+ channel (IKCa) inhibitor triarylmethane-34 (TRAM-34), the ATP-sensitive potassium channel (K(ATP)) inhibitor glibenclamide, voltage-gated potassium channel (VGKC) inhibitor 4-aminopyridine (4-AP), the inducible nitric oxide synthase (iNOS) inhibitor S-methyl-isothiourea (SMT) were purchased from Selleck Chemicals (Houston, TX, USA), and the neuronal NOS inhibitor 7-Nitroindazole (7-NI) were purchased from MedChemExpress ( New Jersey, USA).

Techniques: Activation Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: R-(+)-WIN55212-2 protects pericytes from ischemic damage and restores retinal microcirculatory patency after ischemia/reperfusion injury.

doi: 10.1016/j.biopha.2023.115197

Figure Lengend Snippet: Fig. 7. Signaling pathway of R-(+)-WIN55212–2 protective effect on retinal I/R injury. A, B. Representative western blot bands and quantitative analysis of cannabinoid receptors 1 receptor expression. C, D. Quantitative analysis of overall nitric oxide (NO) content and endothelium derived NO in control, vehicle pre treated ischemic-reperfusion (I/R) groups and R-(+)-WIN55212–2 pretreated ischemic-reperfusion (I/R) groups. E. Quantitative analysis of cyclic guanosine monophosphate in control, vehicle pretreated ischemic-reperfusion (I/R) groups and R-(+)-WIN55212–2 pretreated ischemic-reperfusion (I/R) groups. F, G. Representative western blot bands and quantitative analysis of large conductance Ca2+-activated potassium channel -αsubunit expression. *P < 0.05, * *P < 0.01 and * * *P < 0.001. CB1R, cannabinoid receptors 1 receptor; NC, normal control; I/R, ischemic-reperfusion; WIN, R-(+)-WIN55212–2; NO, nitric oxide; eNO, endo thelium derived nitric oxide; iNOS, inducible nitric oxide synthase; cGMP, cyclic guanosine monophosphate; BKCa, large conductance Ca2+-activated potas sium channel.

Article Snippet: The large conductance Ca2+activated potassium (BKCa) channel inhibitor iberiotoxin (IBTX), the small-conductance Ca2+-sensitive K+ channel (SKCa) inhibitor apamin, the intermediate-conductance Ca2+-activated K+ channel (IKCa) inhibitor triarylmethane-34 (TRAM-34), the ATP-sensitive potassium channel (K(ATP)) inhibitor glibenclamide, voltage-gated potassium channel (VGKC) inhibitor 4-aminopyridine (4-AP), the inducible nitric oxide synthase (iNOS) inhibitor S-methyl-isothiourea (SMT) were purchased from Selleck Chemicals (Houston, TX, USA), and the neuronal NOS inhibitor 7-Nitroindazole (7-NI) were purchased from MedChemExpress ( New Jersey, USA).

Techniques: Western Blot, Expressing, Derivative Assay, Control