Freshly isolated third-fourth-order mesenteric arteries from WT and Rsk2KO mice were incubated with 2 μM BCECF-AM (Thermo Fisher Scientific, B1170) at 37°C for 20 min and extensively washed with Hepes-Krebs solution, mounted in a pressure arteriography chamber (The Instrumentation and Model Facility, University of Vermont, Burlington, VT, USA). Andor Revolution WD (with Borealis) spinning disc confocal imaging system was used to record intracellular pH. pH measurements were made by determining the ratio of emission intensity recorded using a 525/36-nm bandpass filter when the dye is excited at ~488-nm solid-state laser (pH dependent) compared to the emission intensity when the dye is excited at its isosbestic point of ~435 nm (non–pH dependent) with a light-emitting diode light source (pE-4000, CoolLED Ltd., Andover, UK), as described previously (64). After loading, vessels were equilibrated at 37°C for 15 min at 20 mmHg and superfused (flow rate, ~3 ml/min) with Hepes-Krebs (pH 7.3) with the lumen filled with Krebs-Hepes solution. pH images were recorded over 300 ms period taken every minute. Three samples were recorded at 20 mmHg, and five samples were recorded at 60 mmHg. Upon Na+ removal by replacing Hepes-Krebs buffer with Hepes-Krebs Na+-free buffer (NaCl was substituted with equimolar concentration of N-methyl-d-glucamine; Sigma-Aldrich, catalog number: M2004; pH 7.3). Seven samples were recorded, and upon reintroduction of Na+ with Krebs-Hepes solution, 10 samples were recorded. At the end of each experiment, an in situ calibration curve was established. Arteries were treated with nigericin (Sigma-Aldrich, catalog number: N7143) and exposed to high K+ depolarizing Hepes-Krebs solution of varying pH (6.3, 6.6, 6.9, 7.2, and 7.5) to equilibrate internal and external pH (65). Background signal was collected in a mesenteric artery not loaded with BCECF-AM at 525 nm after excitation at 435 and 488 nM. pH was calculated as the ratio of 435/488 nm signal with background signal subtracted for each wavelength and pH interpreted from the calibration curve.

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