Imaging Ca2+ Signals in Endothelial Cells

Ca²⁺ signals in ECs were imaged with a Revolution Andor confocal system (Andor Technology) composed of an upright Nikon microscope with a 60× water-dipping objective [numerical aperture (NA) 1.0] and an electron-multiplying CCD camera, as described previously (4 (link), 6 (link), 33 (link)). Briefly, images were acquired at 15 frames/s for arteries from GCaMP2 mice and 30 frames/s for Fluo-4–loaded arteries using Andor Revolution TL acquisition software (Andor Technology). Changes in emission associated with Ca²⁺ binding were detected by exciting at 488 nm with a solid-state laser and collecting emitted fluorescence using a 527.5/49-nm band-pass filter, with a center wavelength of 527.5 nm and a guaranteed minimum bandwidth of 49 nm. Ca²⁺ imaging experiments were performed at 36°C. TRPV4 Ca²⁺ sparklets were analyzed within a ROI defined by a 1.7-µm² box (5 × 5 pixels) positioned at a point corresponding to peak TRPV4 Ca²⁺ sparklet amplitude. In preparations from wild-type C57BL6 mice and AKAP150^−/−mice, ECs were loaded with Fluo-4 (10 µM) by incubating for 45 min at 30°C in the presence of pluronic acid (2.5 mg/ml) before imaging. The field of view was ~113 × 136 µm, corresponding to 14.5 ± 0.5 cells per field (n = 6 fields). CPA (30 µM, 15 min) was included to eliminate IP₃R-mediated Ca²⁺ signaling. The images were recorded before and 5 min after treatment with CCh, PMA, or OAG. Gö-6976 (1 µM, 10 min) was added before treatment with CCh, OAG, or PMA, and HC-067047 (1 µM, 10 min). Displayed F/F₀ traces were filtered using a Gaussian filter with a cutoff corner frequency of 3.98 Hz. Corresponding F/F₀ traces shown in figures represent the same ROIs before and after treatment. MEPs were identified as black holes in the inner elastic lamina, corresponding to an absence of autofluorescence (4 (link), 19 (link), 22 (link)). A diameter of 2 mm, the dimension of the smallest holes in autofluorescence images, was used as a low-end cutoff for defining a hole. Sparklet sites at MEPs were identified as those with peak fluorescence within 5 mm of the center of a hole in the inner elastic lamina. Changes in activity were expressed as fold change relative to controls.