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Abstract
Emptying the intracellular calcium stores of fura-2-loaded human neutrophils by treatment with the endomembrane ATPase inhibitor thapsigargin leads to a maintained increase of [Ca2+]i by Ca2+ entry through a store-operated Ca2+ entry pathway. Under these conditions, [Ca2+]i was reduced transiently by N-formyl-methionyl-leucyl-phenylalanine (fMLP) and permanently by phorbol 12,13-dibutyrate (PDB). Platelet-activating factor (PAF) had no effect. The fMLP- and PDB-induced [Ca2+]i decreases were not due to stimulated Ca2+ efflux but to inhibition of store-operated Ca2+ entry pathway. PDB and fMLP, but not PAF, inhibited the entry of Ca2+, Mn2+, and Ba2+ in thapsigargin-treated cells. This inhibition was dependent on [Ca2+]i, barely detectable at [Ca2+]i of 50 nM and increasingly strong and fast to appear at 170 and 630 nM. Inhibition of entry by fMLP was complete within 5-10s, disappeared within 2-3 min, and was partially prevented by staurosporin (100 nM). Inhibition by PDB was equally fast, but no recovery was detected within 5 min, and it was fully prevented by staurosporin. The inhibitory effect of fMLP had similar characteristics when PAF was used instead of thapsigargin to induce the entry of Ca2+ or Mn2+. We conclude that fMLP, but not PAF, is able to produce a transient inhibition of store-operated Ca2+ entry pathway, probably mediated by protein kinase C. This action could be part of a general homeostatic mechanism designed to moderate [Ca2+]i increases induced by some agonists.
Highlights
Emptying the intracellular calciumstores of fura-2- the termstore-operated Ca2+entry pathway (SOCP),’ to refer loaded human neutrophils by treatment with the en- to this Ca2+entry mechanism? domembrane ATPase inhibitor thapsigargin leatdosa In addition to the agonist-induced rise in [Ca2+];,there is maintained increase of [Ca2+], by Cean2tr+y through a evidence that some agonists induce homeostatic procstore-operated Caz+entry pathway
In thapsigargin-treated human neutrophils, fMLP anpdhorbol ester, The agonist-induced increase of cytosolic Ca2+concentra- but not platelet activating factor (PAF), decrease [Ca2+Iiby tion ([Ca2+]J in human neutrophils iosften composed of two phases: (i) anearly and transient [Ca2+]pi eak due to inositol 1,4,5-trisphosphate-mediatedCa2+release from the intracellular Ca2+stores, and (ii) a sustained [Ca2+];increase due to increased Ca“ entry through the plasma membrane [1,2,3,4,5]
To test for the possible involvement of protein kinase C in the inhibition of SOCP, we have investigated the effects of the protein kinase C inhibitor staurosporinon the fMLP- and PDB-induced inhibition of the increase in [Ca2+Jin experiments similar to those of Fig. 7
Summary
Human neutrophilswere obtained from blood of healthy volunteers anticoagulated by mixing 6:l (v/v) with acid citrate-dextrose. Dextran (T500,Pharmacia LKBBiotechnology Inc.) was added to obtain a final concentration of 1.3%.After 45 min a t room temperature, the upper phase containing no red cells wasremoved and centrifuged (300 X g,10 rnin). 1. Comparison of the effects of fMLP, PDB, and PAF standard medium containing (in mM): NaCl, 145; KCl, 5; MgCI2,1; on [Ca*+], in control (leftpanels) and thapsigargin-treated. Neutrophils were loaded with fura-2 by incubation with 2-4 p~ cubated in 1mM Ca2+-containingmedium, and the [Ca2+]iwas monfura-Z/AM for 30 min at room temperature in standard incubation itored from the ratio of fura-2 fluorescences excited at 340 and 380. 0.5 FM thapsigargin was added at t = 0 in the figure and, 10 min later, either 1 p M fMLP (upper panel), 100 nM PDB (center panel)o, r 36 nM PAF (lowerpanel) were added.
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