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

Abstract

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 in [Ca2+]c: the intial rate of Ca2+influx was 12.4±0.5 nM/s as [Ca2+]crose monoexponentially (time constant, τ = 36 ± 2 s) to a peak (322 ± 16 nM) before declining to 109 ± 14 nM after 2000 s. Rates of Ca2+removal from the cytosol were measured throughout the overshoot by recording the monoexponential decrease in [Ca2+]cafter rapid removal of extracellular Ca2+. The time constant for recovery (τrecdecreased from 54 ± 4 s when Ca2+was removed after 10 s to its limiting value of 8.8 ± 1.0 s when it was removed after 2000 s. The time dependence of the changes in τrecindicate that an increase in [Ca2+]cis followed by a delayed (τ = 408 s) stimulation of Ca2+removal, which fully reverses (τ ~ 185 s) after Ca2+entry ceases. Numerical simulation indicated that the changes in Ca2+removal were largely responsible for the overshooting pattern of [Ca2+]c. Because prolonged (30 min) Ca2+entry did not increase the total45Ca2+content of the cells, an increased rate of Ca2+extrusion across the plasma membrane most likely mediates the Ca2+removal, and since it persists in the absence of extracellular Na+, it probably results from stimulation of a plasma membrane Ca2+pump. We conclude that delayed stimulation of a plasma membrane Ca2+pump by capacitative Ca2+entry may protect cells from excessive increases in [Ca2+]cand contribute to oscillatory changes in [Ca2+]c.