Ca2+ signals in cultured smooth muscle cells grown from abdominal aortae from 2-month-old normal and Rsk2KO mice and in third-order branches of mesenteric arteries. These cells were used to determine the role of RSK2 in regulating NHE-1 activity stimulated by a brief acid load because they allowed continuous Ca2+ monitoring during the brief transient change in pH. Cells were imaged using an Andor Revolution WD (with Borealis) spinning disc confocal imaging system (Andor Technology, Belfast, UK), composed of an upright Nikon microscope with a 40× water-dipping objective (numerical aperture, 0.8) and an electron-multiplying charge-coupled device camera (63). For Ca2+ imaging of smooth muscle cells, cells were plated on glass bottom culture dishes (MatTek Corporation) and maintained in AmnioMAX medium plus supplement. Cells were incubated with Fluo-4 AM (Thermo Fisher Scientific) for 1 hour and washed with Hepes-Krebs solution, and Ca2+ fluorescence was recorded as follows: (i) Hepes-Krebs, (ii) Hepes-Krebs–buffered 80 mM Na acetate (27), (iii) Hepes-Krebs, (iv) Hepes-Krebs–buffered 80 mM Na acetate in the presence of cariporide, (v) Hepes-Krebs, and (vi) ionomycin. Equimolar Na acetate replaced NaCl in the control solution, and CaCl2 was increased to 1.37 mM to keep [Ca2+] constant. Ca2+ concentrations were measured in a 1.7-μm2 region encompassing multiple confluent cells. Images were recorded from 12 to 22 cells in a field, with 999 frames taken for each field, and the mean and SEM were calculated for each condition. Fluorescence at saturating Ca2+ was measured after ionomycin treatment and used to calculate [Ca2+] (66). Mean Fluo-4 fluorescent intensities were as follows: WT control, NaAC, and ionomycin treatment = 536, 559, and 665, respectively, and Rsk2KO = 544, 530, and 637, respectively. For Ca2+ imaging of arteries, third-fourth-order branches of mesenteric arteries (~100 μm internal diameter at 80 mmHg) were isolated into Hepes-Krebs, loaded with Fluo-4 AM (10 μM) for 1 hour, washed for 30 min with Hepes-Krebs, cannulated, and incubated without or with 10 μM ryanodine to inhibit ryanodine receptors, and fluorescent events were recorded. Images were recorded at 60 frames s−1 under the following conditions: basal pressure at 20 mmHg, 60 mmHg, and 60 mmHg plus cariporide (30 μM). Fluo-4–bound Ca2+ was detected by exciting at 488 nm with a solid-state laser and collecting emitted fluorescence using a 527.5- to 49-nm bandpass filter.

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