Abstract
BackgroundHepatic encephalopathy (HE) is a complex disorder associated with increased ammonia levels in the brain. Although astrocytes are believed to be the principal cells affected in hyperammonemia (HA), endothelial cells (ECs) may also play an important role by contributing to the vasogenic effect of HA.MethodsFollowing acute application and removal of NH4Cl on astrocytes and endothelial cells, we analyzed pH changes, using fluorescence imaging with BCECF/AM, and changes in intracellular Ca2+ concentration ([Ca2+]i), employing fluorescence imaging with Fura-2/AM. Using confocal microscopy, changes in cell volume were observed accompanied by changes of [Ca2+]i in astrocytes and ECs.ResultsExposure of astrocytes and ECs to 1 – 20 mM NH4Cl resulted in rapid concentration-dependent alkalinization of cytoplasm followed by slow recovery. Removal of the NH4Cl led to rapid concentration-dependent acidification, again followed by slow recovery. Following the application of NH4Cl, a transient, concentration-dependent rise in [Ca2+]i in astrocytes was observed. This was due to the release of Ca2+ from intracellular stores, since the response was abolished by emptying intracellular stores with thapsigargin and ATP, and was still present in the Ca2+-free bathing solution. The removal of NH4Cl also led to a transient concentration-dependent rise in [Ca2+]i that resulted from Ca2+ release from cytoplasmic proteins, since removing Ca2+ from the bathing solution and emptying intracellular Ca2+ stores did not eliminate the rise. Similar results were obtained from experiments on ECs. Following acute application and removal of NH4Cl no significant changes in astrocyte volume were detected; however, an increase of EC volume was observed after the administration of NH4Cl, and EC shrinkage was demonstrated after the acute removal of NH4Cl.ConclusionsThis study reveals new data which may give a more complete insight into the mechanism of development and treatment of HE.
Highlights
Hepatic encephalopathy (HE) is a complex disorder associated with increased ammonia levels in the brain
NH4Cl triggers intracellular pH changes in astrocytes Extracellular application of NH4Cl triggered a rapid rise in B490/B440 (Fig. 1c)
This can be explained by a rapid influx of NH3, consuming intracellular H+ for NH4+ formation, Fig. 1 NH4Cl triggers intracellular pH changes in astrocytes. a and b – Fluorescence images, acquired using an excitation wavelength of 490 nm, of a group of astrocytes loaded with BCECF/AM. a – Astrocytes at the beginning of the experiment. b – The same cells after being exposed to NH4Cl
Summary
Solutions All solutions were prepared immediately prior to the experiment. The standard bathing solution (SBS) consisted of 150 mM NaCl, 5.4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES and 10 mM glucose; the pH was adjusted to 7.4 with NaOH. Using the Bitplane Imaris 7.4.2 computer software, 3D objects were created from the acquired images and the volume of each cell measured, based on object-containing voxels, at specific time points during the experiment. Statistical methods Analysis of 101 experiments dealing with changes of fluorescence ratio, area and volume before and after the addition of NH4Cl and before and after its removal showed identical directions of changes. We performed a nonparametric Wilcoxon signed rank test for each experiment, to test the hypothesis that there is no change in values of fluorescence ratio recorded before and after the addition of NH4Cl, and before and after the removal of NH4Cl. Group analysis was performed by combining the rank sum tests of all experiments [30]. All numerical results in the text are expressed as weighted means ± pooled standard deviation
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