Capacitative Ca2+ entry is graded with degree of intracellular Ca2+ store depletion in bovine vascular endothelial cells

Abstract

1. In endothelial cells, release of Ca2+ from endoplasmic reticulum (ER) Ca2+ stores activates Ca2+ influx via the capacitative Ca2+ entry (CCE) pathway. In cultured bovine pulmonary artery endothelial cells, we investigated the relationship between intracellular Ca2+ store load and CCE activity, as well as the kinetics of CCE activation and deactivation, by simultaneously measuring changes in [Ca2+]i and unidirectional manganese (Mn2+) entry through the CCE pathway. 2. Submaximal concentrations of ATP caused quantal release of Ca2+ from the ER, resulting in a dose-dependent depletion of Ca2+ stores and acceleration of Mn2+ entry. Mn2+ entry rate, as a measure of CCE activity, was graded with the amount of released Ca2+. Maximal activation of CCE did not require complete store depletion. 3. Slow depletion of the ER by exposure to the ER Ca2+ pump inhibitor cyclopiazonic acid resulted in a delayed activation of CCE, revealing a temporal dissociation between release of Ca2+ from intracellular stores and activation of CCE. 4. During [Ca2+]i oscillations, at frequencies higher than 0.5 spikes min-1, each Ca2+ spike resulted in a progressive acceleration of CCE without leading to oscillations of Ca2+ entry. In contrast, low frequency [Ca2+]i oscillations were paralleled by transient CCE that was activated and deactivated with each Ca2+ spike, resulting in an oscillatory pattern of Ca2+ entry. 5. It is concluded that CCE is a rapidly activating process which is graded with store depletion and becomes fully activated before complete depletion. The duration of CCE activation correlates with the degree of store depletion and the time that is required to refill depleted stores. Overall, a mechanism of graded CCE prevents exhaustion of intracellular Ca2+ reserves and provides an efficient way to respond to variable degrees of intracellular store depletion.