Effects of NH4CL application and removal on astrocytes and endothelial cells

Abstract

Background: Hepatic 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.

Methods: Following 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.

Results: Exposure 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.

Conclusions: This study reveals new data which may give a more complete insight into the mechanism of development and treatment of HE.

Keywords: Ammonia; Astrocytes; Calcium; Endothelial cells; Hepatic encephalopathy; Hyperammonemia; Volume; pH.

Figures

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. The morphology of the cells remained unchanged. c – An example of average B490/B440 as a function of time in astrocyte cell culture (n = 10). Application of 1 mM NH4Cl caused a rapid rise of B490/B440 followed by a slow decline. Removal of the NH4Cl by substituting it with SBS caused a rapid fall of B490/B440. T1 – time point before the substitution of the SBS with the NH4Cl bathing solution; T2 – time point at which the maximum change of B490/B440 was reached after the substitution of the SBS with the NH4Cl bathing solution; T3 – time point (at 900 s) before substituting the NH4Cl bathing solution with the SBS; T4 – time point of the maximum change of B490/B440 after substituting the NH4Cl bathing solution with the SBS

Fig. 2

NH4Cl triggers intracellular pH changes in astrocytes. a, b and c – Changes after addition of 1 mM, 5 mM and 20 mM NH4Cl plotted as trends. d, e and f – Changes after removal of 1 mM, 5 mM and 20 mM NH4Cl plotted as trends; boxplots on each side present median, upper and lower quartile, minimum and maximum and outliers. Experiments are numbered using consecutive numbers as performed. T1 – time point before the substitution of the SBS with the NH4Cl bathing solution; T2 – time point at which the maximum change of B490/B440 was reached after the substitution of the SBS with the NH4Cl bathing solution; T3 – time point (at 900 s) before substituting the NH4Cl bathing solution with the SBS; T4 – time point of the maximum change of B490/B440 after substituting the NH4Cl bathing solution with the SBS. Experiments are numbered using consecutive numbers as performed

Fig. 3

NH4Cl addition and removal stimulates [Ca2+]i changes in astrocytes. a and b – Fluorescence images, acquired using an excitation wavelength of 380 nm, of a group of astrocytes loaded with Fura-2/AM. A – Astrocytes at the beginning of the experiment. b – The same cells after being exposed to NH4Cl. The morphology of the cells remained unchanged. c – An example of average F340/F380 as a function of time in astrocyte cell culture (n = 7). T1 – time point before the substitution of the SBS with the NH4Cl bathing solution; T2 – time point at which the maximum change of F340/F380 was reached after the substitution of the SBS with the NH4Cl bathing solution; T3 – time point (at 900 s) before substituting the NH4Cl bathing solution with the SBS; T4 – time point of the maximum change of F340/F380 after substituting the NH4Cl bathing solution with the SBS

Fig. 4

NH4Cl addition and removal stimulate [Ca2+]i changes in astrocytes. a, b and c – Changes after addition of 1 mM, 5 mM and 20 mM NH4Cl plotted as trends. d, e and f – Changes after removal of 1 mM, 5 mM and 20 mM NH4Cl plotted as trends; boxplots on each side present median, upper and lower quartile, minimum and maximum and outliers. T1 – time point before the substitution of the SBS with the NH4Cl bathing solution; T2 – time point at which the maximum change of F340/F380 was reached after the substitution of the SBS with the NH4Cl bathing solution; T3 – time point (at 900 s) before substituting the NH4Cl bathing solution with the SBS; T4 – time point of the maximum change of F340/F380 after substituting the NH4Cl bathing solution with the SBS. Experiments are numbered using consecutive numbers as performed

Fig. 5

NH4Cl triggers intracellular pH changes in endothelial cells. a and b – Fluorescence images, acquired using an excitation wavelength of 490 nm, of a group of ECs loaded with BCECF/AM. A – ECs at the beginning of the experiment. b – The same cells after being exposed to NH4Cl. The morphology of the cells remained unchanged. c – An example of average B490/B440 as a function of time in EC cell culture (n = 18). T1 – time point before the substitution of the SBS with the NH4Cl bathing solution; T2 – time point at which the maximum change of B490/B440 was reached after the substitution of the SBS with the NH4Cl bathing solution; T3 – time point (at 900 s) before substituting the NH4Cl bathing solution with the SBS; T4 – time point of the maximum change of B490/B440 after substituting the NH4Cl bathing solution with the SBS; d – Changes after NH4Cl addition plotted as trends. e – Changes after removal of NH4Cl plotted as trends. Boxplots on each side present median, upper and lower quartile, minimum and maximum and outliers. Experiments are numbered using consecutive numbers as performed

Fig. 6

NH4Cl addition and removal stimulates [Ca2+]i changes in endothelial cells. a and b – Fluorescence images, acquired using an excitation wavelength of 380 nm, of a group of ECs loaded with Fura-2/AM. A – ECs at the beginning of the experiment. b – The same cells after being exposed to NH4Cl. The morphology of the cells remained unchanged. c – An example of average F340/F380 as a function of time in astrocyte cell culture (n = 18). T1 – time point before the substitution of the SBS with the NH4Cl bathing solution; T2 – time point at which the maximum change of F340/F380 was reached after the substitution of the SBS with the NH4Cl bathing solution; T3 – time point (at 900 s) before substituting the NH4Cl bathing solution with the SBS; T4 – time point of the maximum change of F340/F380 after substituting the NH4Cl bathing solution with the SBS; d – Changes after NH4Cl addition plotted as trends. e – Changes after NH4Cl removal plotted as trends. Boxplots on each side present median, upper and lower quartile, minimum and maximum and outliers. Experiments are numbered using consecutive numbers as performed

Fig. 7

Changes in volume of ECs following the addition and subsequent removal of NH4Cl. a – A group of ECs observed under confocal microscopy and presented in Imaris Section View in three perspectives. b – The same group of ECs presented as 3D objects. The volume for each cell was calculated based on voxel count. c – Relative area changes after NH4Cl addition plotted as trends. d – Relative area changes after removal of NH4Cl plotted as trends. e – Relative volume changes after NH4Cl addition plotted as trends. f – Relative volume changes after NH4Cl removal plotted as trends. Experiments are numbered using consecutive numbers as performed