Patch clamp electrophysiology

Mesenteric arteries were digested in dissociation solution (80 mmol/L Na glutamate, 55 mmol/L NaCl, 10 mmol/L HEPES pH 7.3, 6 mmol/L KCl, 2 mmol/L MgCl₂, 0.5 mg/mL human albumin, 50 μmol/L CaCl₂ and 10 mmol/L glucose) containing Papain (Worthington, MA) 1 mg/mL and Dihdroerythritol 0.5 mg/mL for 25 min at 37°C. Following this, 0.5 mg/mL elastase, 2 mg/mL collagenase (Type 4) and 1 mg/mL trypsin inhibitor were added to the dissociation solution and the digestion was continued for a further 10 min. The artery was then dissected into segments and gently triturated in Ca²⁺‐free dissociation solution. The isolated smooth muscle cells, still in Ca²⁺‐free dissociation solution, were then transferred to the electrophysiology chamber and left for 30 min to adhere to the bottom (a glass coverslip). Outward K⁺ currents were measured using the whole‐cell, perforated‐patch configuration of the patch‐clamp technique using an Axopatch 200A amplifier (Axon Instruments, Union City, CA). Amphotericin (200 μg/mL) was used to enable perforation. Composition of the extracellular solution: 134 mmol/L NaCl, 6 mmol/L KCl, 1 mmol/L MgCl₂, 2 mmol/L CaCl₂, 7 mmol/L glucose, and 10 mmol/L HEPES. (pH 7.4). Composition of the pipette solution: 110 mmol/L potassium aspartate, 30 mmol/L KCl, 10 mmol/L NaCl, 1 mmol/L MgCl₂ and 10 mmol/L HEPES (pH 7.2). Patch clamp protocols were also performed using the traditional whole cell configuration, in order to delineate whether adiponectin could mediate effects once there had been dialysis of the cytoplasm through the pipette. For these protocols, the pipette solution contained 110 mmol/L K Aspartate, 30 mmol/L KCl, 10 mmol/L NaCl, 1 mmol/L MgCl2, 10 mmol/L HEPES, 5 mmol/L EGTA, 2.5418 mmol/L CaCl2 (200 nmol/L free Ca²⁺ at pH 7.2 at 25°C). For all experiments, currents were filtered at 1KHz and digitized at 4 kHz. Protocols to measure Kv and BK channel currents were performed in the presence of ryanodine (20 mmol/L). The BK channel current was estimated as the decrease in current following application of paxilline (1 μmol/L). Voltage step protocols started from a holding potential of −50 mV prior to 250 msec steps from −40 mV to +50 mV with 10 mV increments (10 steps, for protocol see Figure 3D). Voltage ramps measured current secondary to a linear increase in voltage from −100 mV to +100 mV over 250 msec with a holding potential of −50 mV (Figure 3E). Spontaneous transient outward currents (STOCs) were measured in extracellular solution (composition above) without ryanodine. Currents were analyzed using Clampfit.