Lymph vessels were subjected to enzymatic isolation as described for flow cytometry, except after incubation in 500 µl of solution III, a drop of solution was visualized on an inverted microscope to ensure initial release of LMCs from LVs. The tube containing the LVs was then placed on ice, and a glass bulb pipette was used to remove solution III and replace it with solution I, which was free of enzymes. Gentle trituration resulted in the further separation of LMCs from residual LV tissue. The resulting suspension of LMCs was transported to a patch-clamp station, and aliquots of LMCs were transferred to a chamber mounted on the stage of an Olympus IMT-2 inverted microscope (Olympus Corporation, Shinjuki City, Tokyo, Japan) and allowed to settle before superfusion with high-K+ bath solution of the following composition (in millimole per liter): 145 KCl, 5 HEPES, 1.8 CaCl2, and 1 MgCl2, titrated to pH 7.4 with KOH. Patch pipettes were pulled from borosilicate glass (number 7052; Garner Glass Co., Claremont, CA), fire-polished to a tip resistance of 5–8 MΩ, and filled with solution consisting of (in millimole per liter): 145 KCl, 5 HEPES, 1.8 CaCl2, and 1 MgCl2, titrated to pH 7.4 with KOH. Slight suction was used to form a high-resistance seal (>5 GΩ) between LMC membranes and the pipette tip to establish the cell-attached recording mode for recording of unitary K+ currents. The patch potential was maintained at −50 mV for 2 minutes to record single-channel K+ currents in drug-free bath solution. Subsequently, the chamber was filled with new bath solution containing 1 μmol/l cromakalim to activate KATP channels. Cromakalim-induced single-channel currents were recorded for 2–4 minutes at −50 mV and then in 10-mV steps using 2-minute increments at patch potentials between −70 and +50 mV. Current signals were amplified by an EPC-7 patch-clamp amplifier (List Medical Electronic, Darmstadt, Germany) connected to a TL-1 DMA Interface driven by pClamp 6.0 software (Axon Instruments, Foster City, CA) running an IBM-compatible computer for data acquisition and digitization. Analog to digital conversion was accomplished at a sampling rate of 5 kHz after signals were passed through an eight-pole Bessel filter with a cutoff frequency of 1 kHz to enable real-time visualization of small current amplitudes. After an average current-voltage relationship was generated by plotting unitary current amplitudes as a function of patch potential using Clampfit 10.3 software (Molecular Devices, San Jose, CA) and GraphPad Prism 7 software (GraphPad Software, San Diego, CA), the average unitary conductance of cromakalim-sensitive channels was calculated at negative patch potentials associated with linearity and then again at positive patch potentials associated with inward rectification. In additional studies, the patch potential was maintained at −50 mV in drug-free bath solution for 2 minutes prior to filling the chamber with solution containing either 0.1 μmol/l minoxidil sulfate or 3 μmol/l diazoxide. Recordings at a patch potential of −50 mV were resumed to determine whether these concentrations of KCOs, which caused half inhibition of calculated flow in cannulated LVs, also activated a similar single-channel current as cromakalim in rat mesenteric LMCs.

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