Monitoring of cytosolic free Ca2+ in C5a-stimulated neutrophils: loss of receptor-modulated Ca2+ stores and Ca2+ uptake in granule-free cytoplasts.
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 nM, which in 2-4 min increases to 300-400 nM upon interaction with the complement fragment C5a in a concentration range of 35 pM to 1.2 microM. In the same concentration range, C5a also sequentially activates neutrophil directional migration (ED50 less than 0.5 nM), O-2 production (ED50 = 9 nM), and secretion of the contents of specific granules (ED50 = 39 nM). The selective Ca2+ ionophore ionomycin also increases cytosolic Ca2+ concentration above 1 microM under conditions where it stimulates neutrophil functions. Conversely, phorbol 12-myristate 13-acetate markedly activates secretion and O-2 production without modifying the average cytosolic Ca2+ concentration. In the presence of EGTA (Ca2+out approximately equal to 20 nM), with both C5a and ionomycin, cytosolic Ca2+ increases to less than 200 nM, and functional responses are greatly decreased. Nucleus- and granule-free neutrophil cytoplasts accumulate Ca2+ and produce O-2 when exposed to ionomycin but not to C5a. These results and other considerations suggest that (i) activation of neutrophil functions may occur after cytosolic Ca2+ has exceeded the apparent threshold level of 200 nM; (ii) 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; and (iii) the phorbol diester stimulates Ca2+-dependent pathways presumably by directly activating other mechanisms such as protein phosphorylation.
D000818:Animals; D002118:Calcium / Q000097:blood*; D002417:Cattle; D002634:Chemotaxis, Leukocyte / Q000187:drug effects; D003182:Complement C5 / Q000302:isolation & purification / Q000502:physiology*; D015936:Complement C5a; D003600:Cytosol / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D004987:Ethers / Q000494:pharmacology; D015759:Ionomycin; D007700:Kinetics; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology*; D011951:Receptors, Complement / Q000502:physiology*; D013481:Superoxides / Q000097:blood; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D003182:Complement C5; D004987:Ethers; D011951:Receptors, Complement; D013481:Superoxides; D004533:Egtazic Acid; D015759:Ionomycin; D015936:Complement C5a; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium