Abstract
We have undertaken a detailed study of the mechanisms of maintenance of intracellular Ca2+ homeostasis in human polymorphonuclear neutrophils (PMN) and its implications for phagocytosis and IgG Fc receptor (FcR) signaling. When PMN were incubated in Ca(2+)-free medium, cytoplasmic calcium concentration ([Ca2+]i) was markedly depressed and intracellular stores were depleted of calcium. [Ca2+]i in these depleted cells increased within 1 min when PMN were placed in medium containing Ca2+ and then decreased to a level close to the normal basal [Ca2+]i, replenishing the intracellular Ca2+ pools. LaCl3 prevented entry of Ca2+ into Ca(2+)-depleted PMN, but the calcium channel blockers nifedipine, diltiazem, and verapamil did not. Nifedipine and diltiazem but not verapamil inhibited the movement of Ca2+ from cytosol to intracellular stores. Nifedipine and diltiazem inhibited the normal increase in [Ca2+]i from aggregated IgG binding to FcR and also prevented formyl-methionyl-leucyl-phenyl-alanine (fMLP)-induced [Ca2+]i rise. Verapamil had no effect on either an fMLP- or IgG-mediated increase in [Ca2+]i. Consistent with this, nifedipine and diltiazem inhibited fMLP-stimulated phagocytosis (which is dependent on an increase in [Ca2+]i) when PMN had repleted intracellular stores. In contrast, LaCl3 inhibited fMLP-stimulated ingestion only in PMN which had intracellular store depleted. None of these compounds had any effect on phorbol dibutyrate-stimulated ingestion (which is independent of a [Ca2+]i rise). In summary, these data show that Ca2+ is in rapid equilibrium between intracellular and extracellular compartments in PMN. Exchange of cytoplasmic Ca2+ with the extracellular space is inhibited by LaCl3, while exchange of Ca2+ between the cytosol and intracellular stores is inhibited by the dihydropyridine nifedipine and the benzothiazepine diltiazem. These data suggest that these drugs, which are known to regulate some plasma membrane Ca2+ channels in excitable cells, can also regulate Ca2+ release from intracellular stores in PMN and that this regulation may have significant effects on PMN function.
Highlights
We have undertaken a detailed study of the mecha- conditions which simulate an inflammatory site [1,2,3]
We have shown that in neutrophils, fMLP-stimulated phagocytosis is dependent on a rise of [Ca"], while phorbol esters or plateletactivatingfactor-stimulated ingestion proceeds normally without [Ca"]; rise [8].Interestingly, the increase in [Ca2+Ii the movement of Ca2+ from cytosol to intracellular necessary for fMLP-stimulated ingestion arises from Fc stores
We found that release of crease in [Ca2+Jfirom aggregated IgG binding to Fc receptor (FcR) Ca2+from intracellular storesto thecytoplasm was inhibited and prevented formyl-methionyl-leucyl-phenyl- in whole polymorphonuclear neutrophils (PMN) by the dihydropyridine nifedipine and by alanine-induced[Ca2+Iriise
Summary
These drugs,which are known to regulatesome plasma membrane Ca2+ channels in excitable cells, can . The abbreviations used are: PMN, human polymorphonuclear neutrophils; PDB, phorbol dibutyrate; EIgG,IgG-opsonized sheep erythrocyte; fMLP, formyl-methionyl-leucyl-phenylalanine;HBSS, Hanks' balanced salt solution; HBSS", HBSS with 1.5 mM Ca2+and 1.5 mM M F ; [Ca2+Ii,cytoplasmic calcium concentration; agg-IgG, aggregated IgG; Tg, thapsigargin; IP3, inositol 1,4,5-trisphosphate; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid HEPES, 4(2-hydroxyethyl)-l-piperazineethanesulfoniaccid. For some experiments with calcium channel blockers, PMN kept in HBSS were treated with 1 mM LaCl3before adding stimulant, CaZ+M, $+, and EIgG. Nonspecific binding was determined in the presence of 10 mM EGTA
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