ABSTRACT
In the past two decades, the understanding of intracellular Ca2þ homeostasis has advanced at an unprecedented pace, owing to the revolutionary discovery of Ca2þ-sensitive fluorescence indicator dyes and the tremendous progress in cell imaging techniques. Researchers are now able to study subcelullar Ca2þ regulation by resolving minute Ca2þ signals such as Ca2þ
sparklets at the submicrometer level (1) as well as imaging [Ca2þ] within intracellular organelles such as mitochondria and sarcoplasmic=endoplasmic reticulum (2-4). These technological breakthroughs allow us to begin to appreciate how the local [Ca2þ] conveys target-specific messages in an amplitude-and frequency-dependent manner (5) and how these local processes blend into the mosaic of cytosolic Ca2þ regulation and gene expression (69). The local Ca2þ release event or ‘‘Ca2þ spark’’ that originates from a cluster of ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) is one of the most thoroughly studied local Ca2þ events. It is the elementary event underlying global Ca2þ transients in striated muscles and an important modulator of membrane potential in smooth muscles. Ca2þ sparks have been recently identified and characterized in pulmonary arterial smooth muscle cells (10-14). However, most of their physiological functions in the regulation of pulmonary vascular reactivity are yet to be determined. In this
chapter, we provide an introduction to Ca2þ spark measurement by discussing (1) the general aspects of Ca2þ fluorescent indicator dyes and their calibration, (2) the detection of Ca2þ sparks using laser scanning confocal microscopy, and (3) the basic analysis of Ca2þ spark properties. We hope this will lead to wider application of the technique in the field of pulmonary circulation.
