Effects of NH4CL application and removal on astrocytes and endothelial cells
© The Author(s) 2016
Received: 22 July 2015
Accepted: 29 December 2015
Published: 23 August 2016
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.
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.
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.
This study reveals new data which may give a more complete insight into the mechanism of development and treatment of HE.
KeywordsHepatic encephalopathy Hyperammonemia Ammonia Astrocytes Endothelial cells Volume Calcium pH
Hepatic encephalopathy (HE) comprises a broad spectrum of neurological and psychiatric dysfunctions seen in patients with liver disease. It can reflect diverse conditions, such as a reversible metabolic encephalopathy, brain atrophy, brain edema or a combination of them. Prevalent hypotheses suggest that a number of metabolic factors, present at the same time, are responsible for the development of HE. Neurotoxins, alteration of the blood-brain barrier (BBB), impairment of neurotransmission, altered brain energy metabolism and systemic response to infections and neuroinflammation are all factors playing a role in the pathogenesis of HE [1–3].
Ammonia is the most studied and well-described neurotoxin that precipitates HE [3–5], since an increase in brain ammonia level plays a critical role in its manifestation . Although the correlation between venous ammonia levels and the severity of HE is still uncertain, lowering the blood ammonia concentration is the basis of present treatment of HE [6, 7].
Strong evidence suggests that astrocytes, which form the predominant cellular compartment in the brain , are the principal neural cells affected by ammonia toxicity . Hyperammonemia (HA) is known to induce astrocyte swelling, which may be a key event in the development of HE . HA, especially the acute elevation of ammonia level, leads to brain edema [11, 12]. Mechanisms underlying astrocyte swelling are not fully understood, although the metabolism of ammonia in astrocytes and the production of glutamine, which increases intracellular osmolarity, appear to be involved [13, 14]. Many studies have also shown that one of the earliest events in astrocytes exposed to ammonia is a rise in [Ca2+]i [9, 13, 15, 16] that correlates with ammonia concentration . Changes in astrocyte [Ca2+]i may also play an important role modulating brain microcirculation . However, little is known about how the removal of NH4Cl, as a treatment of HE, influences the affected cells .
Apart from affecting central nervous system cells directly, ammonia may also affect the blood–brain barrier (BBB) . The associated endothelial cells (ECs) are the first cells in contact with blood containing ammonia during HA [20, 21]. Although the BBB is, structurally, made of endothelial cells connected by tight junctions and resting on the basal lamina, its function is far more complex, since it is influenced by interactions between endothelial cells, pericytes, smooth muscle cells and astrocytes and, occasionally, neuronal terminals . The BBB is therefore difficult to study, and the effects of ammonia on endothelial cells, whether isolated or in contact with adjacent cells, have not been studied extensively. Data from the literature are controversial due to the use of different models and different NH4Cl concentrations. Hyperammonemia did not produce brain edema in experimental animals in vivo , but it caused profound swelling of brain slices exposed to ammonia . Another phenomenon that has been suggested as the causative agent in the development of brain edema is change in the cerebral blood flow, resulting in altered intracranial blood volume [21, 25–27]. Both mechanisms are strongly influenced by the state of the capillary endothelial cells .
Changes of pH and [Ca2+]i in astrocytes following the application of ammonia have been studied [13, 18], but there is no information on the acute effect of ammonia removal. The aim of the present study therefore was to further analyze pH and [Ca2+]i changes after the acute removal of ammonia in order to give a more complete insight into the mechanisms of HE treatment. Additionally, the role of changes in EC morphology in the development of brain edema has been evaluated by analyzing changes in pH, [Ca2+]i and volume in ECs following the addition and subsequent removal of ammonia.
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. Ca2+-free solutions were prepared by substituting the CaCl2 with 2 mM EGTA as a calcium chelator. For the ammonia solutions, NH4Cl substituted the equivalent amount of NaCl in SBS in order to maintain the same osmolarity. Although the exact concentration of NH4 + in the cerebrospinal fluid of patients with HA is not known, NH4Cl levels in the arterial blood of patients with liver dysfunction reach roughly 1 mM and in patients with primary urea cycle disorders NH4Cl concentration can reach more extreme values , but values higher than 20 mM are improbable. In animal models of acute hyperammonemia, the NH4Cl concentration range was between 1.5 and 5 mM [23, 24]. To be able to compare our results to data from other authors [13, 25] we used 1 mM, 5 mM and 20 mM NH4Cl bathing solutions.
Fluorophores Fura-2/AM, BCECF/AM and FM 1-43FX were obtained from Molecular Probes, Inc., USA. Minimum Essential Medium Eagle (MEM Eagle), Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12), fetal bovine serum (FBS) and N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) were from GIBCO, Invitrogen Corp, USA. ATP, thapsigargin, penicillin and streptomycin were from Sigma-Aldrich Co.
Animals were kept on a 12 h light-dark cycle in a temperature-controlled colony room at 22–24 °C with free access to rodent pellets and tap water. They were handled according to the NIH Guide for the Care and Use of Laboratory Animals, and all experiments were carried out in accordance with the European Council Directive of November 24th, 1986 (86/609/EEC) and Slovenian legislation. The number of animals was kept at a minimum by preparing primary cultures in a central laboratory for researchers working on astrocytes and neurons. For our experiments astrocyte cultures were prepared from the cerebral cortex of 36 animals. For each series of experiments the cells from three Wistar Hannover rats were pooled. A slightly different method from that described by Čarman-Kržan  was used. 1–3 day old rats were anesthetized, decapitated and the brains removed aseptically. The meninges were removed and the tissue dissociated by passage through sterile Nitex nylon screens (75 μm mesh size) into 10 ml of culture medium composed of DMEM/F12 (1:1), 10 % (vol/vol) FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. The cell suspension was diluted and plated into 75 cm2 tissue-culture flasks. Cells were grown at 37 °C in a water-saturated air environment containing 5 % CO2 until confluence. The culture medium was changed every three days. The cell cultures were shaken at 200 rpm overnight and the culture medium changed the following day. The procedure was repeated 3 times to remove small process-bearing cells on the protoplasmic cell layer (mainly oligodendrocyte progenitors and microglia). The cell cultures were trypsinized and plated onto 100-mm culture dishes and further cultured for 7–10 days. Under these conditions, astroglial-enriched cultures were obtained (95–98 % of cells showed immunoreactivity for glial fibrillary acidic protein). Analyses were carried out on astrocytes plated on poly-L-lysine-coated coverslips at a concentration of 50 000 cells per ml that had been grown for 5 days until reaching a sub-confluent state .
Endothelial cell cultures
We used the endothelial cell line T24 (ECs) derived from human bladder. Cells were grown in cell flasks in cell medium at 37 °C in an environment of water-saturated air/5 % CO2. Every 3 – 4 days (depending on cell density and state of cell confluence) the cell medium was removed and the cell cultures were trypsinized and again plated in cell flasks with cell medium under the same conditions. For experimental purposes ECs were plated on a coverslip.
Measurement of intracellular H+ and intracellular Ca2+ concentrations
Fluorescence imaging was performed using an Axiovert S100 microscope (Zeiss) with Fluar 10x/0.5 and Fluar 40x/1.3 oil objectives combined with a Polychromator II (T.I.L.L. Photonics GmbH, Germany) light source and Coolsnap HQ2 camera (Photometrics). The Visiview 2.0.1 (Visitron Systems GmbH, Germany) computer software was used to acquire images and to measure intensities at different wavelengths. Before the beginning of any experiment the viability of cells on coverslips was checked under a light microscope. The cells were incubated in 0.5 μM BCECF/AM in SBS at room temperature (21 °C) in the dark for 15 – 20 min, then rinsed with SBS and again incubated in SBS for 10 – 15 min for complete de-esterification of intracellular AM esters. Upon completion of labeling, the coverslips with the cells were placed on a coverslip holder on the microscope. A group of cells without visible deformities was located under a light microscope. These cells were studied with microspectrofluorometry, using excitation wavelengths of 490 nm and 440 nm; emitted radiation was passed through a filter that passes wavelengths higher than 530 nm. The images acquired at the two excitation wavelengths were stored at intervals of 5 s to capture the rapid changes during the first minute after application or removal of NH4Cl and then at intervals of 60 s for another 9 min. Each cell was outlined manually and its fluorescence intensity under excitation at 490 and 440 nm measured. The background fluorescence intensity was subtracted, and an intensity ratio for each cell calculated. The ratio of fluorescence intensities at 490 nm to that at 440 nm (B490/B440) was used to determine [H+]i. During experiments SBS was exchanged as appropriate for a solution with a different concentration of ammonia which, after 10 min, was again changed for SBS .
To determine [Ca2+]i, astrocytes were labeled with Fura-2/AM at 4 μM. The protocol was similar to that for [H+]i measurement, except that excitation wavelengths of 340 nm (F340) and 380 nm (F380) and a filter passing wavelengths higher than 510 nm were used . The ratio (F340/F380) was used to determine [Ca2+]i.
Measurement of cell volume
The imaging system for volume analysis comprised a laser confocal microscope Leica TCS SP (Leica, Heidelberg, Germany) with HC PL Fluotar 20x0.5 and PL APO Fluotar 100x1.4 oil objectives and Leica Confocal Software 2.0 (Leica Microsystems Heidelberg GmbH, Germany). Bitplane Imaris 7.4.2 software (Bitplane AG, Switzerland) was used for image analysis. Before the beginning of each experiment, the viability of cells on coverslips was checked under a light microscope. The cells were incubated in 5 μM membrane stain FM 1-43FX at room temperature (21 °C) in the dark for 15 min on the microscope holder and then rinsed with SBS. A group of cells without visible deformities was first identified under a light microscope. 3D images of cells were acquired using confocal microscopy with an excitation wavelength of 488 nm (He/Ne laser). Acquired spectra between 555 nm and 585 nm were used. Series of 32 slices at 100x magnification, with a resolution of 512 x 512, were recorded. 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. Relative volume changes were expressed as % change of the original volume.
Measurement of cell areas
Images of those cells acquired during the experiments while measuring [H+]i were further analyzed with the ImageJ (National Institutes of Health) computer software for measuring cell areas. The contrast of the images was enhanced to better determine cell borders. Each visible cell was then outlined at the beginning of the experiment, before and after each solution exchange and at the end of the experiment. Changes of areas were then calculated.
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. The directions of changes are presented as trend plots. 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 also before and after the removal of NH4Cl. Group analysis was then performed by combining the rank sum tests of all experiments . Weighted means (and pooled SDs) were also calculated for all experiments to assess the average relative change of fluorescence ratio values after each type of treatment. Cell volume and area changes after the application and after the removal of NH4Cl were analyzed using the same statistical approach.
N is the number of experiments in one group of experiments (one coverslip = one experiment) and n is the total number of cells studied. All statistical analyses were performed using R computer software. Changes were considered significant at p < 0.01. All numerical results in the text are expressed as weighted means ± pooled standard deviation.
Results and discussion
NH4Cl triggers intracellular pH changes in astrocytes
The results clearly show that the exposure of cells to NH4Cl leads to a rapid increase of pHi followed by a slow decline and that the removal of extracellular NH4Cl leads to a rapid pHi fall and a slow recovery. All pHi changes correlate with the extracellular NH4Cl concentration. The results are consistent with the previously described mechanism of intracellular pHi shifts with NH4Cl [31, 32].
Addition and removal of NH4Cl stimulates changes of [Ca2+]i in astrocytes
Since the rise in [Ca2+]i following a rise of pHi cannot be explained by an increase of binding sites on cytoplasmic proteins, it must be due either to the influx of Ca2+ through the cell membrane or to its release from intracellular storage . To check the possibility of Ca2+ entry into the cells, changes in [Ca2+]i were compared following the exposure of astrocytes to NH4Cl in SBS and to astrocytes in the Ca2+-free bathing solution. No statistically significant differences were observed (p = 0.21), suggesting that intracellular Ca2+ stores must be responsible for the rapid increase in [Ca2+]i following exposure to NH4Cl. In a Ca2+-free bathing solution, exposure to 20 mM NH4Cl resulted in an increase of F340/F380 of 54.6 % ± 19.6 % (p < 0.01; N = 4; n = 46).
The suggestion that Ca2+ is released from intracellular stores was further tested by depleting the intracellular stores. Thapsigargin (500 nM), which blocks Ca2+ transport into the endoplasmic reticulum (ER) and prevents the refilling of calcium stores, and ATP (100 μM), which stimulates Ca2+ release from ER stores, were added  to the Ca2+-free bathing solution before the start of the experiment. Addition of 20 mM NH4Cl to the astrocytes pretreated with thapsigargin and ATP resulted in a decrease in F340/F380 of 26.5 % ± 8.5 % (p < 0.01, N = 12; n = 88). After intracellular Ca2+ stores were depleted, the fall in [H+]i following the addition of NH4Cl resulted in release of H+ from cytoplasmic proteins. The newly freed protein-binding sites were filled by intracellular Ca2+, resulting in a reduction in [Ca2+]i.
The release of Ca2+ from intracellular stores demonstrated after alkalinization of astrocytes by NH4Cl is consistent with reported results . Further experiments were therefore performed to determine how the removal of extracellular NH4Cl and the acidification of astrocytes influence the intracellular Ca2+.
After the astrocytes were incubated for 10 min in a solution containing NH4Cl, the solution was rapidly substituted by SBS, at the same time measuring F340/F380. A temporary rise in F340/F380 was observed that was lower than the rise of F340/F380 seen during the addition of NH4Cl (Figs. 3c and 4).
The exchange of the 1 mM NH4Cl solution with SBS resulted in a rise in F340/F380 of 12.3 % ± 19.5 % (p < 0.01; N = 7; n = 93). The exchange of bathing solutions of 5 mM and 20 mM NH4Cl resulted in rises of 20.4 % ± 16.5 % (p < 0.01; N = 13; n = 171) and 30.4 % ± 19.1 % (p < 0.01; N = 18; n = 266) respectively (Fig. 4d, e and f). The fall of pH always preceded the increase of intracellular Ca2+. These results show that acidification of astrocytes leads to a transient rise in [Ca2+]i, the magnitude being dependent on the concentration of NH4Cl in the bathing solution before its washout. This is similar to the results obtained on cultured astrocytes caused by alkalinization and acidification using the weak base trimethylamine . The same authors have shown that, in a different experimental setup using astrocytes within brain slices, alkalinization and acidification by the application and washout of 10 mM trimethylamine does not cause any changes in intracellular Ca2+ . The difference in the results may, at least in part, be due to the use of brain slices and not isolated astrocytes. Evidently the study on isolated cells is only a minor step in understanding the complex interactions between cells within the nervous system.
To determine the source of the increase of Ca2+ described above, changes of F340/F380 were also recorded following the removal of NH4Cl in the experiments in the bathing solution without Ca2+, and in the experiments on cells pretreated with the combination of thapsigargin and ATP in Ca2+-free solution.
A transient rise in F340/F380 (47.9 % ± 10.0 % (p < 0.01; N = 4; n = 46) was observed, even in the Ca2+-free bathing solution, suggesting that extracellular Ca2+ does not play a significant role in the detected rise of [Ca2+]i. Even after depleting intracellular stores with thapsigargin and ATP, the removal of NH4Cl led to an increase in F340/F380 of 79.3 % ± 27.6 % (p < 0.01; N = 12; n = 88). The [Ca2+]i rise is probably a result of the release of Ca2+ from cytoplasmic proteins.
Changes in astrocyte volume on addition and removal of NH4Cl
After determining the changes in H+ and Ca2+ concentrations in astrocytes that occurred on substituting SBS with 20 mM NH4Cl, then resubstituting the bathing solution back with SBS, changes in cell volume following these acute events were investigated. Volume changes immediately and approximately 10 min after acute exchange of the bathing solutions were determined by confocal microscopy. However, no significant changes in astrocyte volume during the experiment were detected, which is consistent with other studies, in which a progressive increase of the cell volume of astrocytes exposed to NH4Cl was observed only after 1–3 days . Our data are also in agreement with the observation that hyperammonemia does not produce brain edema in vivo .
NH4Cl triggers intracellular pH changes in endothelial cells
Endothelial cells are an essential part of the blood brain barrier, and their function is regulated by astrocytes as well as by metabolites, cytokines and other biologically active molecules [22, 35]. These endothelial cells are involved in the regulation of transport of molecules between the blood and the cerebrospinal fluid, and their function appears to serve specific roles within the nervous system. Endothelial cells in the brain are difficult to study due to their small size and their complex interaction with other cells such as pericytes, smooth muscle cells and astrocytes. Primary endothelial cells from other sources and endothelial cell lines differ from those in the brain; therefore the results obtained on endothelial cell lines must be interpreted cautiously.
Addition and removal of NH4Cl stimulates changes in [Ca2+]i in endothelial cells
Intracellular Ca2+ stores were further depleted by the addition of thapsigargin to a final concentration of 500 nM and ATP to a final concentration of 100 μM  before measuring changes of [Ca2+]i. After the addition of NH4Cl, a small increase in F340/F380 (3.9 % ± 2.9 %; p < 0.01; N = 4; n = 73) was observed but, after its removal, a rise in F340/F380 (43.0 % ± 17.0 %; p < 0.01; N = 4; n = 73) similar to that observed in ECs in Ca2+-free solution without intracellular Ca2+ stores being depleted (p = 0.092) was measured.
Our results suggest that the addition of NH4Cl to ECs triggers a transient increase in [Ca2+]i that is attenuated in ECs in Ca2+-free bathing solution and almost abolished in ECs after the depletion of intracellular Ca2+ stores in the Ca2+-free bathing solution. However, acute removal of NH4Cl from the extracellular solution provoked a transient rise in [Ca2+]i which could not be abolished, either by substituting the SBS with Ca2+-free bathing solution or by further depleting intracellular Ca2+ stores, suggesting that the increase in [Ca2+]i is due to a release of Ca2+ from cytoplasmic proteins.
The volume of ECs changes following the addition and subsequent removal of NH4Cl
Endothelial cells may play a major role in vasogenic mechanisms of HE. It has been shown that brain slices swell after exposure to 10 mM or higher concentrations of NH4Cl . In our experiments astrocyte volume did not change significantly even after exposure to 20 mM NH4Cl. Therefore experiments on ECs were performed to test the hypothesis that ammonia triggers volume changes in ECs. EC volume changes could result in brain blood flow changes and consequently in changes of intracerebral blood volume or cerebral edema [21, 25–27].
Volume changes of ECs were measured after the administration and subsequent removal of NH4Cl, using confocal microscopy and computer software to calculate maximum volume changes. The time points at which the solutions were exchanged were the same as in the previous experiments when pH and [Ca2+]i were measured. Exposure of EC to different solutions was relatively short, and it is not possible to exclude further volume changes of ECs that might have appeared after a longer exposure to NH4Cl.
Acute addition of NH4Cl elicits a transient rise of intracellular Ca2+ concentration in astrocytes and ECs. This is consistent with the results of previous studies . The acute removal of NH4Cl leads to a transient rise of intracellular Ca2+ in both cell types, and the observed increase of [Ca2+]i is shown to be due to release of Ca2+ from cytoplasmic proteins. The present study also shows that the addition and removal of NH4Cl results in an increase and decrease respectively of EC volume. Such effects were, however, absent in astrocytes. Moreover, the removal of NH4Cl, which is the basis of modern treatment of HE, resulted in a decrease in EC volume despite an accompanying transient increase in [Ca2+]i. Further studies are needed to determine the connection between these events.
ATP, adenosine-5’-triphosphate; BBB, blood–brain barrier; [Ca2+]i, intracellular calcium concentration; ECs, endothelial cells; [H+]i, intracellular H+ concentration; HA, hyperammonemia; HE, hepatic encephalopathy; pHi, intracellular pH; SBS – standard bathing solution.
This work was supported by grant P3-0019 from the Slovenian Research Agency. We thank Dr. Damijana Mojca Jurič for kindly preparing and maintaining astrocyte cultures, which made our experiments possible, and Prof. Roger Pain for reading the manuscript.
MB participated in the design of the study, analysis of the results and he performed pH, Ca2+ and volume measurements. AV participated in the design of the study and analysis of the results, including statistical analysis and graphical presentations of the results. DŠ was responsible for the design of the study, analysis of the results, discussion, preparation and finalization of the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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