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Luminal Ca2+ regulates passive Ca2+ efflux from the intracellular stores of hepatocytes.

ABSTRACT: Ca2+ uptake into the intracellular stores of permeabilized hepatocytes was entirely dependent on ATP and substantially inhibited by either ionomycin or thapsigargin, although both were required for complete inhibition. Unidirectional efflux of 45Ca2+ after removal of ATP from cells loaded to steady state (1.60+/-0.12 nmol/10(6) cells) was monoexponential and occurred with a half-time of 103+/-10 s. However, the 45Ca2+ content of the stores did not return to their pre-ATP level, but reached a plateau at 0.12+/-0.04 nmol/10(6) cells. A similar amount of Ca2+ was trapped within the stores when Ca2+ uptake was prevented by thapsigargin and chelation of Ca2+; at all temperatures between 2 degreesC and 37 degreesC; and after stores had first been loaded with unlabelled Ca2+. Simultaneous addition of inositol 1,4,5-trisphosphate (InsP3) and inhibition of Ca2+ uptake reduced the amount of trapped Ca2+ to a level consistent with InsP3 rapidly and more completely emptying a fraction of the stores that could be only partially emptied by the passive leak. After dilution of the specific activity of the 45Ca2+ under conditions that maintained the steady-state activities of the pumps and leaks, the stores rapidly lost their entire 45Ca2+ content. We conclude that the channel responsible for mediating the leak of Ca2+ abruptly closes when the luminal [Ca2+] of the intracellular stores falls below a critical threshold corresponding to about 7% of their steady-state loading. Whereas InsP3 is capable of completely emptying a fraction of the stores, regulation of the passive leak by luminal [Ca2+] is likely to prevent it from completely emptying them; such regulation may ensure that the many other functions of Ca2+ within the endoplasmic reticulum are not compromised.

SUBMITTER: Beecroft MD

PROVIDER: S-EPMC1219706 | biostudies-other | 1998 Sep

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.

Project description:Fura-2-loaded human platelets were used to study Ca2+ release from intracellular compartments, as well as Ca2+ influx from the extracellular space. We investigated the response towards the endoperoxide/thromboxane-receptor agonist. U46619, and the inhibitor of the endoplasmic-reticulum Ca(2+)-ATPase, thapsigargin. U46619 dose-dependently depleted intracellular Ca2+ stores, followed by active sequestration of released Ca2+. Ca2+ influx induced by U46619 largely relies on receptor occupancy. Removing the thromboxane analogue from its receptor by using the endoperoxide/thromboxane-receptor antagonist BM 13177 largely blunted U46619-mediated Ca2+ influx. The Ca(2+)-ATPase inhibitor thapsigargin evoked a gradual rise in intracellular Ca2+, which was potentiated by a preceding activation of platelets with the receptor agonist U46619. This agonist-sensitizing effect also depends on receptor occupancy. Removing U46619 from its receptor by addition of the endoperoxide/thromboxane-receptor antagonist BM13177 suppressed the sensitizing effect completely. Furthermore, interrupting downstream receptor signalling events by raising intracellular levels of cyclic nucleotides (cyclic AMP, cyclic GMP) again suppressed the U46619-sensitizing effect on thapsigargin-induced Ca2+ release. This study indicates that the process of Ca2+ release followed by resequestration in response to a platelet agonist by its own is not sufficient to produce the sensitizing effect. Rather, a continuously occupied receptor triggering sustained downstream signalling events seems to be required for sensitization. The presence of a receptor agonist may induce an increased cycling of Ca2+ between the agonist-responsive and the thapsigargin-dischargeable compartment, leading to faster and more intense accumulation of Ca2+ in the cytosolic compartment after inhibition of the Ca(2+) ATPase. Suggestively, receptor occupancy increases the Ca(2+)-releasing potency of thapsigargin by coupling the thapsigargin-sensitive Ca(2+)-storing compartments with an agonist-responsive compartment that exhibits a high leakage rate in stimulated platelets.

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Luminal Ca2+ regulates passive Ca2+ efflux from the intracellular stores of hepatocytes.

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