Identification and Spatiotemporal Characterization of Spontaneous Ca2+Sparks and Global Ca2+Oscillations in Retinal Arteriolar Smooth Muscle Cells

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Abstract
Purpose. To identify spontaneous Ca2+ sparks and global Ca2+ oscillations in microvascular smooth muscle (MVSM) cells within intact retinal arterioles and to characterize their spatiotemporal properties and physiological functions. methods. Retinal arterioles were mechanically dispersed from freshly isolated rat retinas and loaded with Fluo-4, a Ca2+-sensitive dye. Changes in [Ca2+]i were imaged in MVSM cells in situ by confocal scanning laser microscopy in x-y mode or line-scan mode. results. The x-y scans revealed discretely localized, spontaneous Ca2+ events resembling Ca2+ sparks and more global and prolonged Ca2+ transients, which sometimes led to cell contraction. In line scans, Ca2+ sparks were similar to those previously described in other types of smooth muscle, with an amplitude (ΔF/F0) of 0.81 ± 0.04 (mean ± SE), full duration at half maximum (FDHM) of 23.62 ± 1.15 ms, full width at half maximum (FWHM) of 1.25 ± 0.05 μm, and frequency of 0.56 ± 0.06 seconds−1. Approximately 35% of sparks had a prolonged tail (>80 ms), similar to the Ca2+“embers” described in skeletal muscle. Sparks often summated to generate global and prolonged Ca2+ elevations on which Ca2+ sparks were superimposed. These sparks occurred more frequently (2.86 ± 025 seconds−1) and spread farther across the cell (FWHM = 1.67 ± 0.08 μm), but were smaller (ΔF/F0 = 0.69 ± 0.04). conclusions. Retinal arterioles generate Ca2+ sparks with characteristics that vary during different phases of the spontaneous Ca2+-signaling cycle. Sparks summate to produce sustained Ca2+ transients associated with contraction and thus may play an important excitatory role in initiating vessel constriction. This deserves further study, not least because Ca2+ sparks appear to inhibit contraction in many other smooth muscle cells.