Effect of 11,12-EET on the membrane potential of wild-type and BKβ₁^−/− pulmonary artery smooth muscle cells.

<p>(A) Representative blots showing the expression of the α and β₁ subunits of the BK in cultured pulmonary artery smooth muscle cells. (B) Original tracing showing the effect of 11,12-EET (10 µmol/L) on the fluorescence emission ratio of Di-8-ANEPPS-loaded pulmonary artery smooth muscle cells. (C) Effect of 11,12-EET (10 µmol/L) on the membrane potential of wild-type (WT) and BKβ₁^−/− pulmonary artery smooth muscle cells in the presence of solvent (Sol) or iberiotoxin (IbTx, 300 nmol/L). Experiments were performed in the presence of diclofenac (10 µmol/L) and L-NA (300 µmol/L). The bar graph summarizes data obtained in 4–8 independent experiments; **P<0.01, ***P<0.001 versus WT+Sol.</p

Effect of iberiotoxin on the sensitivity of the acute hypoxic vasoconstriction to sEH inhibition.

<p>Hypoxia-induced increases in pulmonary arterial pressure (ΔPAP) were assessed in the presence of solvent (Sol), ACU (3 µmol/L) and iberiotoxin (IbTx, 300 nmol/L) in isolated lungs from (A) wild-type (WT) or (B) BKβ₁^−/− mice. All experiments were performed in the presence of diclofenac and L-NA. The graphs summarize data obtained in 7–13 independent experiments; **P<0.01 versus Sol; §P<0.05, §§P<0.001 versus WT+ACU.</p

11,12-EET-induced association of the BKα and β₁ subunits.

<p>Representative blot and densitometric analysis showing the co-precipitation of BKα with BKβ1 from HEK293 cells overexpressing either one or both BK subunits and stimulated with 11,12-EET (10 µmol/L) for 2–10 minutes. The graph summarizes data from 6 independent experiments; *P<0.05 versus the unstimulated control (CTL).</p

Effect of sEH inhibition on hypoxic pulmonary vasoconstriction in wild-type and BKβ₁^−/− mice.

<p>Hypoxia-induced increases in pulmonary arterial pressure (ΔPAP) were assessed in the presence of solvent (Sol) or ACU (3 µmol/L) in isolated lungs from wild-type (WT) or BKβ₁^−/− mice. Experiments were performed in (A) the absence and (B) the presence of diclofenac (10 µmol/L) and L-NA (300 µmol/L). The graphs summarize data obtained in 4–15 independent experiments; *P<0.05, **P<0.01 versus Sol; §§§P<0.001 versus WT+ACU.</p

Effect of 11,12-EET on the mitochondrial membrane potential.

<p>(A) Representative tracing of the 11,12-EET (3 µmol/L)-induced changes in JC-1 fluorescence in pulmonary artery endothelial cells from wild-type (WT) cells in the absence or presence of iberiotoxin (IbTx, 300 nmol/L) and from BKβ₁^−/− cells. (B) Mitochondrial membrane depolarization by 11,12-EET in WT and BKβ₁^−/− pulmonary artery smooth muscle cells in the presence of solvent (Sol) or IbTx. (C) Mitochondrial membrane depolarization by 11,12-EET in WT cells in the presence of Sol, 14,15-EEZE (10 µmol/L), or Rp-cAMPS (10 µmol/L). All experiments were performed in the presence of diclofenac (10 µmol/L) and L-NA (300 µmol/L). The bar graphs summarize data obtained in 4–10 independent experiments; *P<0.05, ***P<0.001 versus WT+EET.</p