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

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.