Digitizer through pclamp 10

Whole-cell patch-clamp recordings were performed with MultiClamp 700B amplifier and Digidata 1440A digitizer (Axon Instrument) at room temperature. The micropipettes were fabricated from borosilicate glass tubes (O.D.: 1.5 mm, I.D.: 0.86 mm, BF150−86−7.5, Shutter Instrument) with a flaming brown micropipette puller (P−80/PC, Shutter Instrument). The intracellular solution was comprised of 142 mM K-gluconate, 2 mM KCl, 0.2 mM ethylene glycol tetraacetic acid (EGTA), 4 mM MgATP, 10 mM HEPES, 7 mM Na₂-phosphocreatine. The pH was adjusted to 7.4. The extracellular solution was comprised of 125 mM NaCl, 2 mM KCl, 2 mM MgCl₂, 2 mM CaCl₂, 25 mM HEPES, and 51 mM D-glucose. The pH was adjusted to 7.4. Cells were perfused with a gravity-fed perfusion system at a rate of 1−2 mL/min before stimulation. Pulse sequences were formed by a Digitizer through pClamp 10.2 (Molecular Devices). Signals were sampled at 10 kHz and were then low-pass filtered at 1 kHz. The total membrane capacitance was estimated from the surface area of the cell, according to the previous report⁴⁷ . Due to its short lifetime, NO donor was rapidly injected into the extracellular solution with gel-loading pipette tips (Fisher Scientific).