We describe a high temporal resolution confocal spot microfluorimetry setup which makes possible the detection of fluorescence transients elicited by Ca2+ indicators in response to large (50-200 microM), short duration (< 100 ns), free [Ca2+] transients generated by laser flash photolysis of DM-nitrophen (DM-n; caged Ca2+). The equilibrium and kinetic properties of the commercially available indicators Fluo-3, Rhod-2, CalciumOrange-5N (COr-5N) and CalciumGreen-2 (CGr-2) were determined experimentally. The data reveal that COr-5N displays simple, fast response kinetics while, in contrast, Fluo-3, Rhod-2 and CGr-2 are characterized by significantly slower kinetic properties. These latter indicators may be unsuitable for tracking Ca2+ signaling events lasting only a few milliseconds. A model which accurately predicts the time course of fluorescence transients in response to rapid free [Ca2+] changes was developed. Experimental data and model predictions concur only when the association rate constant of DM-n is approximately 20 times faster than previously reported. This work establishes a quantitative theoretical framework for the study of fast Ca2+ signaling events and the use of flash photolysis in cells and model systems.
The development of mechanical force in skeletal muscle fibres is brought about by rapid increases in the intracellular calcium concentration (Ca2+ transients) which can be detected by optical methods. Local stimulation experiments and ultrastructural evidence suggest that, at a microscopic level, these Ca2+ transients are generated by the release of Ca2+ ions from the terminal cisternae of the sarcoplasmic reticulum in response to the depolarization of the transverse tubules (t-tubules). Nevertheless, to date, there is no functional information on the exact location at which Ca2+ release takes place. The present experiments were designed to obtain direct evidence about dynamic changes in localization and microscopic distribution of Ca2+ in a single sarcomere using two independent novel methodologies: confocal spot detection of Ca2+ transients and Ca2+ imaging with pulsed laser excitation.
To the best of our knowledge, this is the first study in which SR-Ca2+ transients are recorded in the intact heart, revealing a previously unknown participation of SR on cytosolic Ca2+ overload upon reperfusion in the intact beating heart. Additionally, the associated shortening of phase 2 of the AP may provide a clue to explain early reperfusion arrhythmias.
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