Patch-clamp experiments

Patch-clamp experiments were performed on ASMCs obtained via enzymatic digestion of freshly dissected pulmonary or cerebral arteries as described above. The inside-out (I-O) patch-clamp configuration was used to record BK channel currents in I-O membrane patches at room temperature. These I-O membrane patches were obtained by sealing cells with a 10–20-MΩ pipette and then rapidly interfacing the pipette through the bath surface to remove the cell, followed by reintroducing the pipette into the bath. Cells were bathed in a solution that consisted of 145 mmol/L KCl, 10 mmol/L PIPES, 1 mmol/L EGTA, 1 mmol/L MgCl₂, and variable CaCl₂ (range: 10⁻⁸–10⁻⁴ mol/L) and was titrated to pH 7.4 using KOH. The amounts of Ca²⁺ required to achieve desired free Ca²⁺ concentrations were calculated using Maxchelator Ca-Mg-ATP-EGTA Calculator v1.0 with constants from National Institute of Standards and Technology database 46 v8 (http://www.stanford.edu/~cpatton/CaMgATPEGTA-NIST.htm). The pipette solution contained 145 mmol/L KCl, 5 mmol/L HEPES, 1.8 mmol/L CaCl₂, and 1 mmol/L MgCl₂ and was titrated to pH 7.4 using KOH. Different patch potentials are indicated in the figure legends. The cell-attached patch-clamp configuration also was used in one series of experiments. For this study, the composition of both the bath and pipette solutions was the same as the pipette solution used for I-O patches.

To achieve a hypoxic environment for patch-clamp studies, the bath solution was bubbled with nitrogen gas in a reservoir to remove dissolved O₂. Then the hypoxic solution was drawn from the reservoir into a glass syringe before immediate, direct infusion into the patch-clamp chamber to superfuse the cells under study. An MI-730 O₂ probe (Microelectrodes, Bedford, NH) was placed near the outflow port of the patch-clamp chamber to monitor the O₂ concentration in the bath. The bath was reperfused with hypoxic solution as often as necessary to maintain a hypoxic environment (Pao₂ = 26 ± 11 mm Hg; O₂ = 3.4% ± 1.5%), which was shown earlier to inhibit K_V channels.^17,18

Patch-clamp studies were performed on an Olympus IMT-2 inverted microscope (Tokyo), using an L/M-EPC7 amplifier (List Medical, Darmstadt) and a TL-1 DMA interface (Axon Instruments/Molecular Devices, Sunnyvale, CA). Data were filtered at 1 kHz using a Frequency Devices 902 low-pass filter (Ottawa, IL) before digitization. Traces were recorded using Fetchex (ver. 6). Analysis of single-channel data was performed using Clampfit 10.3.1.5 (Molecular Devices).