Identification and characterisation of Ca2+ sparks in intact retinal arterioles

Abstract

Abstract: : Purpose: Ca²⁺ sparks are localised Ca²⁺ transients that are thought to inhibit constriction in small resistance arteries through activation of BK(Ca) channels. We have recorded Ca²⁺ sparks in microvascular smooth muscle cells (MVSM) of intact retinal arterioles for the first time, and have begun to characterise their spatiotemporal properties and functional relevance. Methods: Sprague Dawley rats (200–300g) were anaesthetised using CO₂ and killed by cervical dislocation in accordance with ARVO guidelines. Arterioles were mechanically dispersed from fresh retinae using a fire–polished Pasteur pipette and incubated with 10µM Fluo–4AM for 2 hours. Changes in [Ca²⁺]_i were imaged in segments of microvessels (9–17 cells) using confocal scanning laser microscopy in X–Y (2 images/s) or line scan (500 scans/s) mode. Raw fluorescence data were analysed using Image J (NIH). Results: Under resting conditions X–Y scans revealed both discretely localised, spontaneous near–membrane Ca²⁺ events resembling Ca²⁺ sparks, and more global and prolonged Ca²⁺ transients that from time to time led to cell contraction. Line scan data showed that Ca²⁺ sparks were similar to those previously described in other types of smooth muscle with an amplitude (rise in F/F₀) of 0.81±0.036 (mean±SE; n=102), Full Duration Half Maximum (FDHM) of 23.62±1.2ms (n=102), Full Width Half Maximum (FWHM) of 1.25±0.05µm (n=96) and frequency of 0.56±0.06Hz (60 cells). Visual inspection of line scan traces demonstrated that individual sparks were often grouped and that their fusion occasionally triggered more global and prolonged Ca²⁺ elevations. Ca²⁺ sparks persisted during these periods of elevated [Ca²⁺]_i, but exhibited a smaller amplitude (0.69±0.035; n=78; p<0.05, Student’s t–test), larger FWHM (1.67±1.12µm; n=65; p<0.001) and a higher frequency (2.86±025Hz; 50 cells; p<0.001). Conclusions: We conclude that retinal arterioles generate Ca²⁺ sparks whose characteristics divide them into two populations. Sparks summate to produce sustained Ca²⁺–transients, and contrary to current paradigms these may play an important excitatory role in initiating vasomotion.

Keywords: calcium • imaging/image analysis: non–clinical • blood supply