Dataset Information

Control of Ca2+ influx in human neutrophils by inositol 1,4,5-trisphosphate (IP3) binding: differential effects of micro-injected IP3 receptor antagonists.

ABSTRACT: Neutrophils signal Ca2+ changes in response to occupancy of G-protein-linked receptors such as the formylated peptide receptor. This Ca2+ signal is composed of two parts, inositol 1,4,5-trisphosphate (IP3)-triggered release of Ca2+ from an intracellular store and Ca2+ influx. In order to probe the relationship between these events, cytosolic free Ca2+ changes in neutrophils were monitored after micro-injection of agents which inhibit IP3 binding. Micro-injection of heparin into neutrophils totally inhibited both formylmethionyl-leucylphenylalanine-induced Ca2+ release and the subsequent Ca2+ influx. This effect was not due to prior depletion of Ca2+ stores. Furthermore, micro-injection with anti-IP3-receptor antibody also inhibited Ca2+ release. However, anti-IP3-receptor antibody and another high-molecular-mass IP3-binding antagonist, heparin-albumin conjugate, failed to inhibit the accompanying Ca2+ influx. It was concluded that two IP3-binding sites exist in neutrophils: one accessible by both heparin and the high-molecular-mass inhibitors of IP3 binding and responsible for Ca2+ release, and another inaccessible to high-molecular-mass molecules and responsible for Ca2+ influx.

SUBMITTER: Davies-Cox EV

PROVIDER: S-EPMC1221721 | biostudies-literature | 2001 Apr

REPOSITORIES: biostudies-literature

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Control of Ca2+ influx in human neutrophils by inositol 1,4,5-trisphosphate (IP3) binding: differential effects of micro-injected IP3 receptor antagonists.

Davies-Cox E V EV Laffafian I I Hallett M B MB

The Biochemical journal 20010401 Pt 1

Neutrophils signal Ca2+ changes in response to occupancy of G-protein-linked receptors such as the formylated peptide receptor. This Ca2+ signal is composed of two parts, inositol 1,4,5-trisphosphate (IP3)-triggered release of Ca2+ from an intracellular store and Ca2+ influx. In order to probe the relationship between these events, cytosolic free Ca2+ changes in neutrophils were monitored after micro-injection of agents which inhibit IP3 binding. Micro-injection of heparin into neutrophils total ...[more]

PMID: 11256958

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Project description:AIMS:Phospholamban (PLB) is the key regulator of the cardiac Ca2+ pump (SERCA2a)-mediated sarcoplasmic reticulum Ca2+ stores. We recently reported that PLB is highly concentrated in the nuclear envelope (NE) from where it can modulate perinuclear Ca2+ handling of the cardiomyocytes (CMs). Since inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) mediates nuclear Ca2+ release, we examined whether the nuclear pool of PLB regulates IP3-induced nuclear Ca2+ handling. METHODS AND RESULTS:Fluo-4 based confocal Ca2+ imaging was performed to measure Ca2+ dynamics across both nucleus and cytosol in saponin-permeabilized CMs isolated from wild-type (WT) or PLB-knockout (PLB-KO) mice. At diastolic intracellular Ca2+ ([Ca2+]i?=?100?nM), the Fab fragment of the monoclonal PLB antibody (anti-PLB Fab) facilitated the formation and increased the length of spontaneous Ca2+ waves (SCWs) originating from the nuclear region in CMs from WT but not from PLB-KO mice. We next examined nuclear Ca2+ activities at basal condition and after sequential addition of IP3, anti-PLB Fab, and the IP3R inhibitor 2-aminoethoxydiphenyl borate (2-APB) at a series of [Ca2+]i. In WT mice, at 10?nM [Ca2+]i where ryanodine receptor (RyR2) based spontaneous Ca2+ sparks rarely occurred, IP3 increased fluorescence amplitude (F/F0) of overall nuclear region to 1.19?±?0.02. Subsequent addition of anti-PLB Fab significantly decreased F/F0 to 1.09?±?0.02. At 50?nM [Ca2+]i, anti-PLB Fab not only decreased the overall nuclear F/F0 previously elevated by IP3, but also increased the amplitude and duration of spark-like nuclear Ca2+ release events. These nuclear Ca2+ releases were blocked by 2-APB. At 100?nM [Ca2+]i, IP3 induced short SCWs originating from nucleus. Anti-PLB Fab transformed those short waves into long SCWs with propagation from the nucleus into the cytosol. In contrast, neither nuclear nor cytosolic Ca2+ dynamics was affected by anti-PLB Fab in CMs from PLB-KO mice in all these conditions. Furthermore, in WT CMs pretreated with RyR2 blocker tetracaine, IP3 and anti-PLB Fab still increased the magnitude of nuclear Ca2+ release but failed to regenerate SCWs. Finally, anti-PLB Fab increased low Ca2+ affinity mag-fluo 4 fluorescence intensity in the lumen of NE of nuclei isolated from WT but not in PLB-KO mice. CONCLUSION:PLB regulates nuclear Ca2+ handling. By increasing Ca2+ uptake into lumen of the NE and perhaps other perinuclear membranes, the acute reversal of PLB inhibition decreases global Ca2+ concentration at rest in the nucleoplasm, and increases Ca2+ release into the nucleus, through mechanisms involving IP3R and RyR2 in the vicinity.

Dataset Information

Control of Ca2+ influx in human neutrophils by inositol 1,4,5-trisphosphate (IP3) binding: differential effects of micro-injected IP3 receptor antagonists.

Publications

Control of Ca2+ influx in human neutrophils by inositol 1,4,5-trisphosphate (IP3) binding: differential effects of micro-injected IP3 receptor antagonists.

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