A quantitative investigation into the dependence of Ca²⁺ mobilisation on changes in inositol 1,4,5‐trisphosphate levels in the stimulated neutrophil

Abstract

The coupling of N‐formyl‐methionyl‐leucyl‐phenylalanine (fMet‐Leu‐Phe) receptor stimulation to Ca2+ mobilisation has been investigated in the human neutrophil by measuring the concentration‐effect curves for inositol 1,4,5‐trisphosphate (IP3) formation and Ca2+ mobilisation. fMet‐Leu‐Phe‐dependent mobilisation of intracellular Ca2+ has been monitored in fluo‐3‐loaded human neutrophils by measuring increases in the cytoplasmic free Ca2+ concentration ([Ca2+]i) in the presence of extracellular EGTA. Fluo‐3 was used in preference to fura‐2 because it was found to be more sensitive to the high Ca2+ levels seen in stimulated neutrophils. fMet‐Leu‐Phe induced a rapid mobilisation of intracellular Ca2+ (EC50 = 2.9 ± 0.1 nm) and increased [Ca2+]i to a maximum of 1286 ± 184 nm. The amount of IP3 in fMet‐Leu‐Phe‐stimulated neutrophils was determined by competition with [3H]‐IP3 for a specific IP3 binding protein isolated from bovine adrenocortical microsomes. Basal IP3 levels of 13.3 ± 2.0 pmol per 107 cells were increased nearly 4 fold by maximally effective concentrations of fMet‐Leu‐Phe. The EC50 for the IP3 response (95 ± 18 nm) was much higher than that for mobilisation of intracellular Ca2+, such that only a doubling in the concentration of IP3 was required to fully mobilise intracellular Ca2+. As a result of this relationship IP3 production was more sensitive than Ca2+ mobilisation to inhibition by demethoxyviridin, an inhibitor of phospholipase activation.

Endogenous adenosine regulates neutrophil pro-inflammatory activities by cyclic AMP-dependent accelerated clearance of cytosolic calcium

“…Following preincubation, the cells were activated with 1 mM FMLP (stimulated cells) or an equal volume of HBSS (unstimulated cells) in a final volume of 2 ml, after which the reactions were terminated and the IP 3 extracted by the addition of 0.4 ml of 20% perchloric acid at time 0, 5 and 10 s after the addition of the stimulant. These incubation times coincide with the peak IP 3 responses of FMLP-activated neutrophils which were determined in a series of preliminary experiments and are in agreement with previous reports on the time course of the IP 3 responses elicited by FMLP-activation of neutrophils [27,28]. Following a 20 min incubation on ice, the tubes were centrifuged at 2000 ¥ g for 15 min and the supernatants removed and titered to pH 7.5 with 5N KOH, followed by centrifugation at 2000 ¥ g for 15 min to remove precipitated KClO 4 .…”

“…Secondly, we have observed (data not included) that the threshold concentration of U 73122, a selective inhibitor of PLC, for inhibition of the peak cytosolic Ca 2+ response of FMLP-activated neutrophils is 2.5 mM, with no detectable acceleration of clearance of the cation observed with this, as well as with both higher and lower concentrations of the inhibitor. Thirdly, the effects of ADA on prolongation of Ca 2+ transients in FMLP-activated neutrophils are most striking at around 1 -2 min after addition of the chemoattractant, a time at which the IP 3 response has returned to basal values [27,28]. Finally, the ADA-mediated prolongation of elevated cytosolic Ca 2+ was almost completely abolished by inclusion of the extracellular Ca 2+ -chelating agent, EGTA, emphasising that the primary involvement of cAMP in FMLP-activated neutrophils appears to be in promoting Ca 2+ sequestration/resequestration.…”

Complex relationship between Ins(1,4,5)P3 accumulation and Ca2+‐signalling in a human neuroblastoma revealed by cellular differentiation

“…Agonist potency for muscarinic receptor‐mediated [Ca 2+ ] i elevation was greater than that for Ins(1,4,5)P 3 elevation as previously reported in these cells ( Willars & Nahorski, 1995a ) and for other agonists in other cell types ( Thompson et al ., 1991 ; McArdle et al ., 1996 ; Yu & Hinkle, 1997 ). This potency difference was not, however, maintained following differentiation although in these cells the potency difference was present for bradykinin receptor‐mediated responses.…”

Effects of SCA40 on human isolated bronchus and human polymorphonuclear leukocytes: comparison with rolipram, SKF94120 and levcromakalim

“…In the human neutrophil the concentration-response curve for FMLP-stimulated Ca2" mobilization lies to the left of those for other FMLP-dependent effects (Rossi et al, 1985;Thompson et al, 1991). Thus, FMLP (30 nM) produces maximal mobilization of intracellular Ca2" (Thompson et al, 1991) but represents only the EC50 for a number of functional responses (this study; Hatzelman et al, 1990). This difference in sensitivity may explain the finding that SCA40 was more effective against responses at the functional level than against full mobilization of intracellular Ca2"…”