Han DY, Nie HG, Gu X, Nayak RC, Su XF, Fu J, Chang Y, Rao V, Ji HL.

HL095435 NHLBI NIH HHS , HL87017 NHLBI NIH HHS , R01 GM085237-01A2 NIGMS NIH HHS , R01 GM085237 NIGMS NIH HHS , R03 HL095435 NHLBI NIH HHS , R01 HL087017 NHLBI NIH HHS

	Figure 1. Recovery of verapamil-reduced alveolar fluid clearance (AFC) by K+ channel openers in vivo. (A) Verapamil intratracheal application reduces alveolar fluid clearance. Verapamil (100 μM) was intratracheally delivered to mouse lung. Average AFC values in the absence of drugs (Control), in the presence of amiloride (Amiloride), verapamil (Verapamil), and both (Amiloride+Verapamil). Unpaired two-sample two-tailed Student t-test. *P < 0.05 and **P < 0.01 when compared with Control. n = 4-15. (B) Amiloride-sensitive (AS) AFC. The mean and SE values were computed as described in Methods. Unpaired two-sample two-tailed Student t-test. **P < 0.01. n = 12-15. (C) Effects of K+ channel openers on basal AFC. Unpaired two-sample two-tailed Student t-test. n = 5-15. (D) K+ channel openers restore verapamil-reduced AFC. AFC values were measured for Verapamil (100 μM) alone, + Pyrithione-Na (1 mM), + 1-EBIO (1 mM), and +Minoxidil (0.6 mM). The dashed line indicates the Control level. Unpaired two-sample two-tailed Student t-test. *P < 0.05 vs Verapamil alone. n = 4-12.
	Figure 2. Verapamil reduces short-circuit (Isc) level in H441 monolayers in a dose-dependent manner. (A) Representative Isc trace showing applications of a series of concentrations. Amiloride-sensitive Isc level (AS Ibasalsc) is the sum of verapamil-inhibitable and residual amiloride-sensitive fractions. (B) Normalized AS Isc levels (AS Iverasc/AS Ibasalsc) at each concentration were plotted as a dose-response curve. n = 6. The raw data were fitted with the Hill equation. IC50 value, 294.2 μM. (C) & (D) Verapamil on amiloride-insensitive Isc levels in amiloride pretreated cells. Representative trace (C) and corresponding average Isc levels before (Control) and after addition of amiloride and verapamil (D). n = 3. P = 0.89 for the Isc levels before and after verapamil. Paired t-test.
	Figure 3. Effects of CCB compounds on transepithelial short-circuit currents (Isc) in intact H441 monolayers. (A-C) Typical traces. 200 μM nifedipine (A), 10 μM bepridil (B) or 50 μM diltiazem (C) was added to the basolateral compartment followed by verapamil. Amiloride (100 μM, apical side) was finally applied to inhibit residual amiloride-sensitive currents. Arrows show the time point of addition. Total AS Isc is the difference between the Isc level before CCB and the amiloride-insensitive fraction, as indicated by a pair of vertical arrows. (D) CCB-sensitive fraction: CCB-inhibitable Isc/total AS Isc. One-way ANOVA. *P < 0.05 vs Verapamil. n = 3-10.
	Figure 4. Comparison of verapamil-inhibitable Isc levels in H441 monolayers when applied to the apical or basolateral compartments. (A-C) Representative Isc traces showing the effects of water (H2O), verapamil applied to basolateral (B) and apical (C) compartments. The total AS Isc levels associated with ENaC are designated by pairs of vertical arrows. (D) AS Isc levels before and after verapamil delivery to the basolateral side or apical compartment. Paired t-test for comparison of current levels before and after verapamil. *P < 0.05 and **P < 0.01. n = 4-17.
	Figure 5. Inhibition of transapical and transbasolateral Isc levels by verapamil in permeabilized H441 monolayers. (A) Representative Isc traces obtained in basolateral permeabilized H441 monolayers with amphotericin B (am B). 1 mM ouabain was added to the basolateral side to exclude any potential residual Isc across basolateral membrane. Amiloride (100 μM) was applied at the end of recording to calculate basal amiloride-sensitive (AS) Isc level as indicated between dashed lines. (B) AS Isc levels before (Basal) and after water (H2O) and verapamil (Verapamil). Paired t-test. ***P < 0.001. n = 4-8. (C) Representative Isc trace recorded in apically permeabilized H441 monolayers with amphotericin B. 100 μM amiloride was added to the apical compartment to inhibit possible residual Isc level carried by ENaC. Ouabain (1 mM) was added at the end of the recording to calculate total ouabain-sensitive (OS) Isc level. (D) Mean OS Isc levels in the absence (Basal) and presence of water (H2O) and verapamil (Verapamil). Paired t-test. *P < 0.05. n = 4.
	Figure 6. K+ channel blockers alter the inhibitory effects of verapamil in H441 cells. (A) Typical Isc traces showing the application of 100 μM verapamil alone (control), 100 μM clofilium (KV inhibitor), 20 μM tram34 (KCa3.1 inhibitor), and 100 μM glibenclamide (KATP inhibitor), respectively. These K+ channel blockers were applied to basolateral side followed by verapamil and amiloride (apical side) to compute total AS Isc. (B) Summary of average AS Isc levels. Paired t-test. *P < 0.05, **P < 0.01, *** P < 0.001 for comparison of pre- and post exposure of CCB. n = 4-17. (C) Reduced percentages of AS Isc levels by verapamil in H441 cells with and without pretreatment of K+ channel blockers. Two-sample, two-tailed t-test. *P < 0.05 vs Control. n = 4-17.
	Figure 7. Stimulatory effects of K+ channel openers subsequent to verapamil in H441 monolayers. (A) Representative traces showing response to a set of K+ channel openers from 1 μM to 1 mM in the presence of verapamil. (B) Corresponding concentration-response curves. The raw data were fitted with the Hill equation and the EC50 were 2.4, 391.8, and 1.2 μM, respectively, for pyrithione-Na, 1-EBIO, and minoxidil. (C) Summary of AS Isc levels before (Basal) and after verapamil (Verapamil) and K+ channel openers (10 μM Pyrithione-Na, 600 μM 1-EBIO, 10 μM Minoxidil). Paired t-test. *P < 0.05, **P < 0.01 for comparison of before and after K+ channel openers. n = 3-6.
	Figure 8. Compartment-dependent recovery of verapamil-inhibited Isc levels by K+ channel openers in permeabilized H441 monolayers. (A) Representative Isc traces of apical Isc level in the presence of 1 mM ouabain in the basolateral membrane permeabilized monolayers. (B) Representative Isc traces of basolateral Isc level in the presence of 100 mM amiloride in the apical permeabilized H441 monolayers. (C & D) Recovered apical (C) and basolateral (D) Isc levels by K+ channel openers. One-way ANOVA for comparing normalized Isc level post K+ channel openers with total AS or OS Isc levels. *P < 0.05 and **P < 0.01. n = 3-5.
	Figure 9. Verapamil regulates heterologously expressed human αβγ ENaC in oocytes in a Ca2+-dependent pattern. (A & B) Time-courses of whole-cell current traces at -100 mV recorded in oocytes perfused with verapamil under normal (A) and Ca2+-chelated conditions (B). 10 μM amiloride were added at the end of recording to compute basal and verapamil-inhibitable ASI levels. (C & D) Effects of verapamil on ENaC activity in control (C) and Ca2+-chelated (D) oocytes. The shift of base line in Ca2+-chelated cells (B) was corrected for comparing the current levels before (Basal) and after verapamil (Verapamil). Paired t-test. *P < 0.05.
	Figure 10. Verapamil inhibits ionophore-induced Ca2+ mobilization. Transient Ca2+ signal evoked by ionomycin was measured as relative fluorescent intensity with Fluo 4AM dye in real time with paired manner, in both control and verapamil incubated H441 cells. (A) Original traces showing on-time Ca2+ signal digitized in control (red) and verapamil exposed cells (green). (B) Maximal relative change in fluorescent intensity (Fpeak/F0). Nonparametric Mann-Whitney U-test. *P < 0.05 vs Control. n = 3. (C) The time to reach peak signal. Nonparametric Mann-Whitney U-test. *P < 0.05
	Figure 11. Schematic model for the multiple mechanisms of CCB-inhibited transepithelial Na+ transport and recovery by K+ channels. CCB compounds alter Ca2+ signal most likely via modifying Ca2+ release from cytosolic compartments. In addition, CCB compounds directly regulate K+ channels, ENaC, and Na+/K+-ATPase. Disrupted basolateral K+ recycling and apical ion transport will abrogate transalveolar salt and fluid transport. K+ channel openers significantly restore the CCB-inhibited transepithelial Na+ transport by activating K+ channel, then facilitating Na+/K+-ATPase.

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