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Mechanism of long-range Ca2+ signalling in the nucleus of isolated rat hepatocytes.

ABSTRACT: Ca2+ regulates a wide range of cell proteins, in both the cytosol and nucleus. It enters the nucleus from stores along the nuclear envelope, but how it then spreads through the nuclear interior is unknown. Here we used high-speed confocal line-scanning microscopy to examine the propagation of Ca2+ waves across nuclei in isolated rat hepatocytes. Nuclear Ca2+ waves began at the nucleus/cytosol border as expected, then spread across the nucleus at less than half the speed of cytosolic Ca2+ waves. High concentrations of caffeine slowed Ca2+ waves in the cytosol but not in the nucleus. We developed a mathematical model based on diffusion to analyse these data, and the model was able to describe the nuclear but not cytosolic Ca2+ waves that were experimentally observed. These findings suggest that Ca2+ waves cross the nucleus by simple diffusion, which is distinct from the reaction-diffusion mechanism by which Ca2+ waves propagate across the cytosol. Since the range of messenger action for Ca2+ in the cytosol is much smaller than the distance across the nucleus, this also suggests that the unique environment and geometry of the nuclear interior may permit this simple mechanism of Ca2+ wave propagation to control Ca2+-mediated processes in a relatively large region despite Ca2+ release pools that are spatially limited.

SUBMITTER: Fox JL

PROVIDER: S-EPMC1218696 | biostudies-other | 1997 Sep

REPOSITORIES: biostudies-other

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Project description:BackgroundRecombinant human erythropoietin alpha (rHu-EPO) has been reported to protect the liver of rats and mice from ischemia-reperfusion injury. However, direct protective effects of rHu-EPO on hepatocytes and the responsible signalling pathways have not yet been described. The aim of the present work was to study the protective effect of rHu-EPO on warm hypoxia-reoxygenation and cold-induced injury to hepatocytes and the rHu-EPO-dependent signalling involved.MethodsLoss of viability of isolated rat hepatocytes subjected to hypoxia/reoxygenation or incubated at 4 degrees C followed by rewarming was determined from released lactate dehydrogenase activity in the absence and presence of rHu-EPO (0.2-100 U/ml). Apoptotic nuclear morphology was assessed by fluorescence microscopy using the nuclear fluorophores H33342 and propidium iodide. Erythropoietin receptor (EPOR), EPO and Bcl-2 mRNAs were quantified by real time PCR. Activation of JAK-2, STAT-3 and STAT-5 in hepatocytes and rat livers perfused in situ was assessed by Western blotting.ResultsIn contrast to previous in vivo studies on ischemia-reperfusion injury to the liver, rHu-EPO was without any protective effect on hypoxic injury, hypoxia-reoxygenation injury and cold-induced apoptosis to isolated cultured rat hepatocytes. EPOR mRNA was identified in these cells but specific detection of the EPO receptor protein was not possible due to the lack of antibody specificity. Both, in the cultured rat hepatocytes (10 U/ml for 15 minutes) and in the rat liver perfused in situ with rHu-EPO (8.9 U/ml for 15 minutes) no evidence for EPO-dependent signalling was found as indicated by missing effects of rHu-EPO on phosphorylation of JAK-2, STAT-3 and STAT-5 and on the induction of Bcl-2 mRNA.ConclusionTogether, these results indicate the absence of any protective EPO signalling in rat hepatocytes. This implies that the protection provided by rHu-EPO in vivo against ischemia-reperfusion and other causes of liver injury is most likely indirect and does not result from a direct effect on hepatocytes.

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Mechanism of long-range Ca2+ signalling in the nucleus of isolated rat hepatocytes.

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