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Measurement of Intracellular Free Calcium Ion Concentration in Vascular Smooth Muscle Cells: Fluorescence Imaging of Cytosolic Calcium

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Changes in intracellular free calcium ion concentration ([Ca2+ ]i ) play a major role in vascular smooth muscle cell function (1 ,2 ). Elevation of [Ca2+ ]i is an important regulator of multiple downstream signaling pathways and it is a major determinant of vascular smooth muscle contraction (1 3 ). Agonists, such as angiotensin II (Ang II), endothelin-1, and vasopressin, that mediate effects through G protein-coupled receptors, increase [Ca2+ ]i by inducing Ca2+ mobilization from intracellular sarcoplasmic/endoplasmic reticular stores, and by stimulating transplasmalemmal Ca2+ influx (4 6 ). One of the earliest measurable events resulting from Ang II stimulation of vascular smooth muscle cells, is a rapid, phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate, to produce two second messengers, 1,2 diacylglycerol and inositol 1,4,5-trisphosphate (IP3 ) (7 ,8 ). The water-soluble messenger IP3 , binds to specific IP3 receptors to release Ca2+ from nonmitochondrial intracellular stores.
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