Monitoring of cytosolic free Ca2+ in C5a-stimulated neutrophils: loss of receptor-modulated Ca2+ stores and Ca2+ uptake in granule-free cytoplasts.

Abstract

The cytosolic concentration of free Ca2+ in bovine neutrophils was monitored by using the intracellular Ca2+ indicator quin2, 2-{[2-bis(acetylamino)5-methylphenoxy]methyl-6-methoxy-8-bis(acetylamino)}quinoline. Neutrophils at rest have a cytosolic Ca2+ concentration of 85 .+-. 5 M, which in 2 to 4 min increases to 300 to 400 nM upon interaction with the complement fragment C5a [complement component 5a] in a concentration range of 35 pM to 1.2 .mu.M. In the same concentration range, C5a also sequentially activates neutrophil directional migration (ED50 < 0.5 nM), O2- production (EC50 = 9 nM) and secretion of the contents of specific granules (EC50 = 39 nM). The selective Ca2+ ionophore ionomycin also increases cytosolic Ca2+ concentration > 1 .mu.M under conditions where it stimulates neutrophil functions. Phorbol 12-myristate 13-acetate markedly activates secretion and O2- production without modifying the average cytosolic Ca2+ concentration. In the presence of EGTA [ethylene glycol-bis-(.beta.-aminoethylether)N,N,N'',N''-tetraacetic acid] .**GRAPHIC**. .apprxeq. 20 nM) with both C5a and ionomycin, cytosolic Ca2+ increases to < 200 nM and functional responses are greatly decreased. Nucleus- and granule-free neutrophil cytoplasts accumulate Ca2+ and produce O2- when exposed to ionomycin but not to C5a. Activation of neutrophil functions may occur after cytosolic Ca2+ has exceeded the apparent threshold level of 200 nM. C5a receptor-mediated activation of Ca2+ influx may require cooperation between the neutrophil surface and some cytoplasmic organelle and/or redistribution of the C5a-receptor complexes on the cell surface. The phorbol diester stimulates Ca2+-dependent pathways presumably by directly activating other mechanisms such as protein phosphorylation.