Overshooting cytosolic Ca2+ signals evoked by capacitative Ca2+ entry result from delayed stimulation of a plasma membrane Ca2+ pump

The effect of capacitative Ca2+ entry on cytosolic free Ca2+ concentration ([Ca2+](c)) was examined in calf pulmonary artery endothelial cells treated with thapsigargin. Restoration of extracellular Ca2+ evoked an overshoot i n [Ca2+](c): the intial rate of Ca2+ influx was 12.4+/-0.5 nM/s as [Ca2+](c ) rose monoexponentially (time constant, tau = 36 +/- 2 s) to a peak (322 /- 16 nM) before declining to 109 +/- 14 nM after 2000 s. Rates of Ca2+ rem oval from the cytosol were measured throughout the overshoot by recording t he monoexponential decrease in [Ca2+](c) after rapid removal of extracellul ar Ca2+. The time constant for recovery (tau(rec) decreased from 54 +/- 14 s when Ca2+ was removed after 10 s to its limiting value of 8.8 +/- 1.0 s w hen it was removed after 2000 s. The time dependence of the changes in tau( rec) indicate that an increase in [Ca2+](c) is followed by a delayed (tau = 408 s) stimulation of Ca2+ removal, which fully reverses (tau similar to 1 85 s) after Ca2+ entry ceases. Numerical simulation indicated that the chan ges in Ca2+ removal were largely responsible for the overshooting pattern o f [Ca2+](c),. Because prolonged (30 min) Ca2+ entry did not increase the to tal Ca-45(2+) content of the cells, an increased rate of Ca2+ extrusion acr oss the plasma membrane most likely mediates the Ca2+ removal, and since it persists in the absence of extracellular Na+, it probably results from sti mulation of a plasma membrane Ca2+ pump. We conclude that delayed stimulati on of a plasma membrane Ca2' pump by capacitative Ca2+ entry may protect ce lls from excessive increases in [Ca2+](c), and contribute to oscillatory ch anges in [Ca2+](c).