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Control of inositol 1,4,5-trisphosphate-induced Ca2+ release by cytosolic Ca2+.

ABSTRACT: The synergistic action of cytosolic Ca2+ and inositol 1,4,5-trisphosphate (InsP3) in releasing intracellular Ca2+ stores has been suggested to be responsible for the complex intracellular Ca2 signals observed during hormonal stimulation of many cell types. However, the ability of cytosolic Ca2+ to potentiate Ca2+ release has recently been questioned because of the observed inhibitory effects of Ca2+ chelators used in previous studies. In the present study, EGTA and BAPTA [1,2-bis-(2-amino-phenoxy)ethane- NNN'N'-tetra-acetic acid] poorly inhibited InsP3-induced Ca2+ release from permeabilized A7r5 smooth-muscle cells. Additionally, stimulatory effects of cytosolic and luminal Ca2+ were observed either in the complete absence of Ca2+ chelator or at constant Ca(2+)-free chelator concentration. These data suggest that potentiation of InsP3-induced Ca2+ release by Ca2+ in A7r5 cells reflects an interaction between Ca2+ and InsP3 receptors, rather than a decrease in chelator-dependent inhibition. The EC50 for activation of InsP3-induced Ca2+ release by cytosolic Ca2+ was unaffected by ATP, or by changing InsP3 concentration, although InsP3-induced Ca2+ release became less sensitive to the inhibitory effects of cytosolic Ca2+ as the InsP3 concentration was elevated. Increasing H+ or Mg2+ concentration shifted the Ca(2+)-activation curve towards higher Ca2+ concentrations. These data suggest that, in addition to the InsP3-binding site, the affinity of the Ca(2+)-binding site(s) on InsP3 receptors can be modulated by intracellular cations.

SUBMITTER: Bootman MD

PROVIDER: S-EPMC1136539 | biostudies-other | 1995 Mar

REPOSITORIES: biostudies-other

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Project description:ObjectiveCaffeine reduces toxic Ca2+ signals in pancreatic acinar cells via inhibition of inositol 1,4,5-trisphosphate receptor (IP3R)-mediated signalling, but effects of other xanthines have not been evaluated, nor effects of xanthines on experimental acute pancreatitis (AP). We have determined effects of caffeine and its xanthine metabolites on pancreatic acinar IP3R-mediated Ca2+ signalling and experimental AP.DesignIsolated pancreatic acinar cells were exposed to secretagogues, uncaged IP3 or toxins that induce AP and effects of xanthines, non-xanthine phosphodiesterase (PDE) inhibitors and cyclic adenosine monophosphate and cyclic guanosine monophosphate (cAMP/cGMP) determined. The intracellular cytosolic calcium concentration ([Ca2+]C), mitochondrial depolarisation and necrosis were assessed by confocal microscopy. Effects of xanthines were evaluated in caerulein-induced AP (CER-AP), taurolithocholic acid 3-sulfate-induced AP (TLCS-AP) or palmitoleic acid plus ethanol-induced AP (fatty acid ethyl ester AP (FAEE-AP)). Serum xanthines were measured by liquid chromatography-mass spectrometry.ResultsCaffeine, dimethylxanthines and non-xanthine PDE inhibitors blocked IP3-mediated Ca2+ oscillations, while monomethylxanthines had little effect. Caffeine and dimethylxanthines inhibited uncaged IP3-induced Ca2+ rises, toxin-induced Ca2+ release, mitochondrial depolarisation and necrotic cell death pathway activation; cAMP/cGMP did not inhibit toxin-induced Ca2+ rises. Caffeine significantly ameliorated CER-AP with most effect at 25 mg/kg (seven injections hourly); paraxanthine or theophylline did not. Caffeine at 25 mg/kg significantly ameliorated TLCS-AP and FAEE-AP. Mean total serum levels of dimethylxanthines and trimethylxanthines peaked at >2 mM with 25 mg/kg caffeine but at <100 µM with 25 mg/kg paraxanthine or theophylline.ConclusionsCaffeine and its dimethylxanthine metabolites reduced pathological IP3R-mediated pancreatic acinar Ca2+ signals but only caffeine ameliorated experimental AP. Caffeine is a suitable starting point for medicinal chemistry.

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.

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Control of inositol 1,4,5-trisphosphate-induced Ca2+ release by cytosolic Ca2+.

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