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

Cytosolic Ca2+ plays a critical role in the secretion of endothelium-d erived 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 va lves revealed no underlying smooth muscle cells that might influence e ndothelial cell responses or contribute to [Ca2+](i) signaling. The va lve 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 en dothelium-dependent vasodilators, including acetylcholine, with an inc rease in [Ca2+](i). Synchronized [Ca2+](i) transients were observed in the endothelial monolayer in response to agonist stimulation in K+-fr ee 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 c ells of native endothelium. In addition, this preparation affords an o pportunity for comparative studies of endothelium-related pathophysiol ogies, which can be induced experimentally in animal models.