Effect of U46619 and L-<i>cis</i>-diltiazem on [Ca²⁺]_i in the primary cultured aortic smooth muscle cells.

<p>Cells were pretreated with 300 nM nifedipine to block L-type Ca²⁺ channels. <b>A</b>, <b>B</b>. Representative fluorescence images of cultured smooth muscle cells before (<b>A</b>) and after (<b>B</b>) 100 nM U46619. <b>C</b>. Summary of data showing the inhibitory effect of L-<i>cis</i>-diltiazem (100 nM) on U46619-induced [Ca²⁺]_i rises. Shown were the percentage of cells displaying U46619-induced [Ca²⁺]_i rises before and after U46619 challenge, Mean ± SE (n = 5). * p<0.05 as compared with the control.</p

Effect of LY83583 on U-46619-induced vasoconstriction in an endothelium-denuded small mesenteric artery segment.

<p><b>A</b>. A representative trace of the tension developed in the ring upon various treatments. <b>B</b>. A dose response curve showing the concentration dependent effect of LY83583 on contractions induced by U-46619 in the presence of L-type voltage-gated Ca²⁺ channels blocker. Mean ± SE (n = 4).</p

Representative traces of the tension developed in endothelium-denuded small mesenteric arteries.

<p><b>A</b>. Addition of 1 mM BAPTA in Ca²⁺-free KHS prevented the U46619-induced contraction. <b>B</b>. Nifedipine significantly inhibited the U46619-induced contraction. <b>C</b>. 1 µM nifedipine was added to block the contraction induced by high K⁺ solution. After stabilization, U46619 was added to recontract the vessel, followed by the addition of 3 mM EGTA. Inset of C. similar to C, except that the U46619-induced contraction was followed by replacing the bath solution by a Ca²⁺-free KHS. n = 5.</p

The anion selectivity of synthetic Cl⁻ channel 1 in HEK 293 cells.

<p>(<b>a</b>) The ramp stimulation protocol for whole-cell recording. (<b>b</b>) The data summary for reverse potential changes when external solutions were changed from symmetric 150 mM Cl⁻ to 150 mM F⁻, Br⁻, NO₃⁻ and I⁻, respectively. All data are means ± s.e. n = 3–4. (c–f) Zoomed representative traces showing changes in reverse potential when external solutions were changed from 150 mM Cl⁻ (black traces) to 150 mM (<b>c</b>) F⁻, (<b>d</b>) Br⁻, (<b>e</b>) NO₃⁻ and (<b>f</b>) I⁻ (red traces), respectively.</p

Effect of L-<i>cis</i>-diltiazem on the contraction induced by U46619.

<p><b>A</b>. A representative trace of the tension developed in an endothelium-denuded small mesenteric artery. 1 µM Nifedipine was added to inhibit L-type Ca²⁺ channels. U46619 (100 nM) was added to recontract the vessel, which is followed by cumulative doses of L-<i>cis</i>-diltiazem. N = 5. <b>B</b>. A dose response curve showing the concentration dependent effect of L- and D-<i>cis</i>-diltiazem on contractions induced by U46619 after the blockage of L-type voltage-gated Ca²⁺ channels. Mean ± SE (n = 5). <b>C</b>. A representative trace of the tension developed in the ring showing that nicardipine had no additional effect on U-46619-induced contractions after the blockage of L-type voltage-gated Ca²⁺ channels by nifedipine. N = 4.</p

Chemical structure and single channel currents of compound 1 in HEK 293 cells.

<p>(<b>a</b>) Chemical structure of compound <b>1</b>. (<b>b</b>) Representative traces showing single channel currents with DMSO control (top trace) and the application of 1 µM compound <b>1</b> (lower four traces) at −60 mV in HEK 293 cells. “C” represents the base line in which the channel is in the close state.</p

Synthetic Cl⁻ channel formed by compound 1 restored cell Cl⁻ permeability in CF cells.

<p>(<b>a</b>) Representative traces showing whole-cell currents in CF cells. The left panel shows the stimulation protocol for whole-cell recording, and the middle panel shows DMSO control currents. The right panel shows whole-cell currents after the application of 1 µM compound <b>1</b>. (<b>b</b>) The current–voltage relationships obtained in the absence (○) and presence (•) of 1 µM compound <b>1</b> and after washout (▴) with the control bath solution for 30 min in CuFi-8 cells. (<b>c</b>) Summary of whole-cell currents at ±80 mV induced by forskolin (2 µM) and compound <b>1</b> (1 µM) in CuFi-8 and NuLi-1 cells. All data are means ± s.e. n = 5–6.</p

Electrophysiological properties of the ion channels formed by compound 1 in HEK 293 cells.

<p>(<b>a</b>) Representative traces showing whole-cell currents in HEK 293 cells. The upper panel shows the stimulation protocol for whole-cell recording, and the lower left panel shows DMSO control currents. The right panel shows whole-cell currents after the application of 1 µM compound <b>1</b>. (<b>b</b>) Current–voltage relationships obtained in the absence (○) and presence (•) of 1 µM compound 1 and after washout (▴) with the control bath solution for 30 min in HEK 293 cells. (<b>c</b>) Representative traces showing the whole-cell ramp currents recorded in symmetric 150 mM NMDG-Cl (black trace) and asymmetric NMDG-Cl with 150 mM in internal and 50 mM in external solutions (red trace). All data are means ± s.e. n = 5–6.</p

Effect of extracellular Ca²⁺ on H₂O₂-induced [Ca²⁺]_i rises in aortic ECs and MAECs.

<p><b>A and B</b>. Representative traces of [Ca²⁺]_i rises in response to 5 mM H₂O₂ in the primary cultured aortic ECs (A) and MAECs (B) that were bathed in N-PSS (1 mM Ca²⁺), 0.5Ca²⁺-PSS (0.5 mM Ca²⁺) or 0Ca²⁺-PSS (nominal Ca²⁺-free). Fluorescence intensity before H₂O₂ application was normalized to 1 as F0. <b>C and D</b>. Summary of the maximal [Ca²⁺]_i changes to H₂O₂ as expressed in F1/F0. Mean±SEM of 4 to 8 independent experiments (10 to 15 cells per experiment). *, <i>P</i><0.05 as compared to N-PSS.</p