Two distinct Ca2+ storage and release sites in human neutrophils

It is well established that changes in cytosolic free Ca2+ play a role in a number of neutrophil responses such as cell shape change and oxidase activation. The release of intracellular stored Ca2+ occurs with stimuli that either act by occupancy of seven membrane-spanning domain receptors or those which act by receptor crosslinking. Here, two distinct Ca2+ storage and release sites have been identified in neutrophils. Using chlortetracycline fluorescence as an indicator of Ca2+ storage, two separate Ca2+ storage sites have been identified. One site was located peripherally under the plasma membrane and the other was in the juxtanuclear space. Confocal imaging of Fluo3-loaded neutrophils demonstrated that the central Ca2+ storage site released Ca2+ in response to N-formyl-methionyl-leucyl-phenylalanine, whereas engagement and clustering of CD11b/CD18 integrins causes Ca2+ release from the peripheral stores, The release sites also correlated with organelles that stained with DiOC(6)(3). Localized phototoxicity generated by DiOC(6)(3) excitation resulted in inhibition of the release of stored Ca2+, which was selective for the stimulus used. The presence of two distinct cellular locations for these Ca2+ stores and their independent release raises the possibility that separate intracellular messengers for their release are generated.