Mechanisms of calcium transport in human endothelial cells subjected to hyperthermia

摘要

We have probed the mechanisms by which severe heat alters cytosolic calcium ion concentrations (Cai) in individual cultured human endothelial cells (ATCC #1998). Cells adhering to glass coverslips were heated to as high as 50 deg C and Cai determined by means of a fluorescence laser imaging system using the calcium-sensitive dye, indo-l, in the presence of thapsigargin, and in Na-free and Ca-free media. Baseline Cai varied between 175-225 nM. When cells were heated to 50 deg C in a complete Ca-containing medium, there was first a transient fall in Cai, then a rapid rise of 50-100 nM in Cai, followed by a slower, secondary rise of 50-75 nM. Depleting the intracellular calcium stores with thapsigargin blocked both the transient fall and the secondary rise in Cai. Placement of the cells into a Ca-free medium blocked both the transient fall and the initial rapid rise, while use of a Na-free buffer prevented the initial rapid rise only. These data suggest that in human endothelial cells, extreme heat accelerates the CaATPase pumps of the intracellular Ca stores causing the transient fall in Cai which is soon followed by activation of the reverse mode of the Na/Ca exchanger to cause the initial rapid rise in Cai. The Ca-release channels of the intracellular stores become activated by heat to cause the secondary, slow rise in Cai. This preliminary work indicates that the application of heat to cultured cells can be a useful probe to examine the kinetics and unmask mechanisms of intracellular Ca fluctuations.