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 microns 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.
Laskey
R E
RE
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Florida 33101.
Adams
D J
DJ
van Breemen
C
C
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D011188:Potassium / Q000494:pharmacology; D011664:Pulmonary Valve / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D011817:Rabbits
D000109:Acetylcholine; D011188:Potassium; D002118:Calcium