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 (,). Elevation of [Ca2+]i is an important regulator of multiple downstream signaling pathways and it is a major determinant of vascular smooth muscle contraction (, , ). 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 (, , ). 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) (,). The water-soluble messenger IP3, binds to specific IP3 receptors to release Ca2+from nonmitochondrial intracellular stores.