CYTOSOLIC [CA2+] MEASUREMENTS IN ENDOTHELIUM OF RABBIT CARDIAC VALVES USING IMAGING FLUORESCENCE MICROSCOPY

Cytosolic Ca2+ plays a critical role in the secretion of endothelium-derived factors. A new preparation that allows fluorescence imaging of intracellular free Ca2+ concentration ([Ca2+](i)) in endothelial cells of rabbit cardiac valves is described. Electron micrographs of the valves revealed no underlying smooth muscle cells that might influence endothelial cell responses or contribute to [Ca2+](i) signaling. The valve leaflets, which were <100 mu m in diameter, were visualized using a specially designed chamber and a long working distance fluorescence objective. The semilunar valves (pulmonary and aortic) responded to endothelium-dependent vasodilators, including acetylcholine, with an increase in [Ca2+](i). Synchronized [Ca2+](i) transients were observed in the endothelial monolayer in response to agonist stimulation in K+-free solutions. The ability to monitor changes in [Ca2+](i) in a native endothelial monolayer provides a more realistic assessment of stimulus-response coupling within individual cells and communication between cells of native endothelium. In addition, this preparation affords an opportunity for comparative studies of endothelium-related pathophysiologies, which can be induced experimentally in animal models.