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Stimulation of inositol trisphosphate formation in hepatocytes by vasopressin, adrenaline and angiotensin II and its relationship to changes in cytosolic free Ca2+.

ABSTRACT: At maximally effective concentrations, vasopressin (10(-7) M) increased myo-inositol trisphosphate (IP3) in isolated rat hepatocytes by 100% at 3 s and 150% at 6 s, while adrenaline (epinephrine) (10(-5) M) produced a 17% increase at 3 s and a 30% increase at 6 s. These increases were maintained for at least 10 min. Both agents increased cytosolic free Ca2+ [( Ca2+]i) maximally by 5 s. Increases in IP3 were also observed with angiotensin II and ATP, but not with glucagon or platelet-activating factor. The dose-responses of vasopressin and adrenaline on phosphorylase and [Ca2+]i showed a close correspondence, whereas IP3 accumulation was 20-30-fold less sensitive. However, significant (20%) increases in IP3 could be observed with 10(-9) M-vasopressin and 10(-7) M-adrenaline, which induce near-maximal phosphorylase activation. Vasopressin-induced accumulation of IP3 was potentiated by 10mM-Li+, after a lag of approx. 1 min. However the rise in [Ca2+]i and phosphorylase activation were not potentiated at any time examined. Similar data were obtained with adrenaline as agonist. Lowering the extracellular Ca2+ to 30 microM or 250 microM did not affect the initial rise in [Ca2+]i with vasopressin but resulted in a rapid decline in [Ca2+]i. Brief chelation of extracellular Ca2+ for times up to 4 min also did not impair the rate or magnitude of the increase in [Ca2+]i or phosphorylase a induced by vasopressin. The following conclusions are drawn from these studies. IP3 is increased in rat hepatocytes by vasopressin, adrenaline, angiotensin II and ATP. The temporal relationships of its accumulation to the increases in [Ca2+]i and phosphorylase a are consistent with it playing a second message role. Influx of extracellular Ca2+ is not required for the initial rise in [Ca2+]i induced by these agonists, but is required for the maintenance of the elevated [Ca2+]i.

SUBMITTER: Charest R

PROVIDER: S-EPMC1144811 | biostudies-other | 1985 Apr

REPOSITORIES: biostudies-other

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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.

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Stimulation of inositol trisphosphate formation in hepatocytes by vasopressin, adrenaline and angiotensin II and its relationship to changes in cytosolic free Ca2+.

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