NH4Cl Treatment Research Articles

Potassium restriction, high protein intake, and metabolic acidosis increase expression of the glutamine transporter SNAT3 (Slc38a3) in mouse kidney

Kidneys produce ammonium to buffer and excrete acids through metabolism of glutamine. Expression of the glutamine transporter Slc38a3 (SNAT3) increases in kidney during metabolic acidosis (MA), suggesting a role during ammoniagenesis. Potassium depletion and high dietary protein intake are known to elevate renal ammonium excretion. In this study, we examined SNAT3, phosphate-dependent glutaminase (PDG), and phosphoenolpyruvate carboxykinase (PEPCK) regulation during a control (0.36%) or low-K(+) (0.02%) diet for 7 or 14 days or a control (20%) or high-protein (50%) diet for 7 days. MA was induced in control and low-K(+) groups by addition of NH(4)Cl. Urinary ammonium excretion increased during MA, after 14-day K(+) restriction alone, and during high protein intake. SNAT3, PDG, and PEPCK mRNA abundance were elevated during MA and after 14-day K(+) restriction but not during high protein intake. SNAT3 protein abundance was enhanced during MA (both control and low K(+)), after 14-day low-K(+) treatment alone, and during high protein intake. Seven-day dietary K(+) depletion alone had no effect. Immunohistochemistry showed SNAT3 staining in earlier parts of the proximal tubule during 14-day K(+) restriction with and without NH(4)Cl treatment and during high protein intake. In summary, SNAT3, PDG, and PEPCK mRNA expression were congruent with urinary ammonium excretion during MA. Chronic dietary K(+) restriction, high protein intake, and MA enhance ammoniagenesis, an effect that may involve enhanced SNAT3 mRNA and protein expression. Our data suggest that SNAT3 plays an important role as the glutamine uptake mechanism in ammoniagenesis under these conditions.

Increased proline loading to phloem and its effects on nitrogen uptake and assimilation in water‐stressed white clover (Trifolium repens)

The aim of this study was to investigate the physiological significance of increased proline loading to phloem caused by water-deficit stress in relation to nitrogen (N) uptake and assimilation. N uptake and N assimilation were quantified by 15N tracing in well-watered (control) and water deficit-stressed white clover (Trifolium repens). De novo proline synthesis and proline loading to the phloem were also compared between treatments. The relationships among proline concentrations in phloem exudates, N uptake, and assimilation of newly absorbed N were assessed. The newly synthesized proline in the phloem exudates increased rapidly after 3 d of water deficit. The water-deficit treatment significantly reduced the maximum nitrate reductase activity (NRA), and also attenuated de novo synthesis of amino acids and proteins in the roots. The increase in proline concentrations in phloem exudates was closely related to reductions in NRA in the roots, N uptake, and the assimilation of newly absorbed N. The accumulation of proline induced in roots by exogenous proline and NH4Cl treatments was closely associated with the decrease in NRA. These results indicate that increased proline transport to roots via phloem caused by water deficit has a significant influence on the down-regulation of N uptake and the assimilation of newly absorbed N.

Evaluation of four methods for processing human cord blood and subsequent study of the expansion of progenitor stem cells isolated using the best method

The application of cord blood (CB)-derived hematopoietic cells in allogeneic transplantation has been restricted because of the unavailability of adequate cell numbers from one unit of CB and their delayed engraftment in recipients. The main purpose of this study was to develop an economic process for isolating nucleated cells from CB and expanding enriched CD133(+) cells. Four protocols for processing CB, using different combinations of density-gradient centrifugation, hydroxyethyl starch (HES) and NH4Cl treatment, were compared regarding the yields of CD45(+), CD34(+)/CD133(+) and colony-forming cells. CD133-enriched cells were expanded for 10 days in the presence of hematopoietic growth factor-supplemented medium. The performance of the culture was evaluated by analyzing colony-forming potential, fold-expansion of primitive hematopoietic stem cells (HSC) and lineage commitment by flow cytometry. A two-step treatment of CB with HES followed by NH4Cl resulted in the highest recovery of CD45(+) (84.3%) and CD34(+) (69.1%) cells compared with that present in umbilical CB. A suspension culture of CD133-enriched cells resulted in an average 150-fold increase in nucleated cells, of which CD133(+), CD34(+) and CD34(+) CD38 cell expansions were 17.2+/-1.3, 40+/-4.6, and 6.9+/-1.1 fold, respectively. The total myeloid colony-forming cell count was almost 3800-fold higher than that present in the processed CB. The highest yields of nucleated and progenitor stem cells were obtained with a two-step processing of CB. The CD133(+) cells obtained by this method are expected to yield enough hematopoietic progenitors for potential allogeneic transplantation.

Induction of four glutamine synthetase genes in brain of rainbow trout in response to elevated environmental ammonia

The key strategy for coping with elevated brain ammonia levels in vertebrates is the synthesis of glutamine from ammonia and glutamate, catalyzed by glutamine synthetase (GSase). We hypothesized that all four GSase isoforms (Onmy-GS01-GS04) are expressed in the brain of the ammonia-intolerant rainbow trout Oncorhynchus mykiss and that cerebral GSase is induced during ammonia stress. We measured GSase activity and the mRNA expression of Onmy-GS01-GS04 in fore-, mid- and hindbrain and liver, as well as ammonia concentrations in plasma, liver and brain of fish exposed to 9 or 48 h of 0 (control) or 670 micromol l(-1) NH(4)Cl (75% of the 96 h-LC(50) value). The mRNA of all four GSase isoforms were detected in brain (not liver). After 9 h of NH(4)Cl exposure, brain, liver and plasma ammonia content were elevated by two- to fourfold over control values. Midbrain, hindbrain and liver GSase activities were 1.3- to 1.5-fold higher in ammonia-exposed fish relative to control fish. Onmy-GS01-GS04 mRNA levels in brain (not liver) of ammonia-exposed fish (9 h) were significantly elevated by two- to fourfold over control values. After 48 h of the NH(4)Cl treatment, ammonia content and GSase activity, but not mRNA levels, in all tissues examined remained elevated compared to control fish. Taken together, these findings indicate that all four GSase isoforms are constitutively expressed in trout brain and are inducible under high external ammonia conditions. Moreover, elevation of GSase activities in fore-, mid- and hindbrain in response to environmental ammonia underlines the importance of brain GSase in the ammonia-stress response.

NH 4+ modulates renal tubule amantadine transport independently of intracellular pH changes

A bicarbonate-dependent organic cation transporter, unique from rOCT1 and rOCT2, primarily mediates amantadine uptake into renal proximal tubules. We examined whether intracellular pH regulates bicarbonate-dependent amantadine transporter function in these tubules. NH 4Cl treatment resulted in immediate intracellular alkalinization of tubules for up to 30 s followed by gradual acidification that was maximal at 5 min. Proximal tubule amantadine uptake was similarly inhibited (60%) by NH 4Cl during both the early intracellular alkalinization and later acidification phases. Sodium propionate treatment resulted in immediate intracellular acidification of proximal tubules without inhibiting amantadine uptake. NH 4Cl inhibition of bicarbonate-dependent amantadine uptake was dose-dependent, competitive and sex-dependent. NH 4Cl, NH 4NO 3, (NH 4) 2SO 4 and (NH 4) 2HPO 4 inhibited amantadine uptake into proximal tubules similarly. NH 4Cl also stimulated efflux of amantadine and tetraethylammonium from preloaded proximal tubules, suggesting mediation of a facilitated process. These data suggest the potential for direct modulation of organic cation transporters by NH 4 + in rat kidney proximal tubules.

Effects of NH4Cl intake on renal growth in rats: role of MAPK signalling pathway

There is a surprising lack of experimental data on the mechanisms of NH4Cl-induced chronic metabolic acidosis which causes kidney hypertrophy. The NH4Cl treatment results in an absolute increase in kidney mass. Despite findings to indicate a close interaction between NH4Cl-induced chronic metabolic acidosis and renal enlargement, the role of the stimulated serine kinase cascade, mediated by the stepwise activation of extracellular signal-regulated kinase (ERK) signalling, on kidney hypertrophy has not yet been investigated. To test this hypothesis, the present study was undertaken to further explore the possible involvement of mitogen-activated protein kinase (MAPK) signalling pathway in renal growth in chronic NH4Cl-treated rats by western blot analysis. Our major findings are as follows: (1) Urinary sodium excretion significantly increased during the early phases of NH4Cl-induced acidosis, (2) This occurrence is associated with sustained renal hypertrophy, and (3) sustained basal phosphorylation of IRS-1, Shc, and MAPK/ERKs in acidotic kidneys. The present study confirms that NH4Cl-induced acidosis causes disturbances in renal sodium handling. In addition, these findings demonstrate a sustained pre-stimuli activation of kidney MAPK/ERKs signalling pathways in the NH4Cl-treated rats that may correlate with an increased rate of kidney hypertrophy and a transient renal tubule inability to handle sodium. Thus, the altered renal electrolyte handling may result from a reciprocal relationship between the level of renal tubule metabolic activity and ion transport. In addition, the study shows that the appropriate regulation of tyrosine kinase protein phosphorylation, and its downstream signal transduction pathway, plays an important role on renal growth in the NH4Cl-treated rats.

BACE Is Degraded via the Lysosomal Pathway

Amyloid plaques are formed by aggregates of amyloid-beta-peptide, a 37-43-amino acid fragment (primarily Abeta(40) and Abeta(42)) generated by proteolytic processing of the amyloid precursor protein (APP) by beta- and gamma-secretases. A type I transmembrane aspartyl protease, BACE (beta-site APP cleaving enzyme), has been identified to be the beta-secretase. BACE is targeted through the secretory pathway to the plasma membrane where it can be internalized to endosomes. The carboxyl terminus of BACE contains a di-leucine-based signal for sorting of transmembrane proteins to endosomes and lysosomes. In this study, we set out to determine whether BACE is degraded by the lysosomal pathway and whether the di-leucine motif is necessary for targeting BACE to the lysosomes. Here we show that lysosomal inhibitors, chloroquine and NH(4)Cl, lead to accumulation of endogenous and ectopically expressed BACE in a variety of cell types, including primary neurons. Furthermore, the inhibition of lysosomal hydrolases results in the redistribution and accumulation of BACE in the late endosomal/lysosomal compartments (lysosome-associated membrane protein 2 (LAMP2)-positive). In contrast, the BACE-LL/AA mutant, in which Leu(499) and Leu(500) in the COOH-terminal sequence (DDISLLK) were replaced by alanines, only partially co-localized with LAMP2-positive compartments following inhibition of lysosomal hydrolases. Collectively, our data indicate that BACE is transported to the late endosomal/lysosomal compartments where it is degraded via the lysosomal pathway and that the di-leucine motif plays a role in sorting BACE to lysosomes.

Caveola-Dependent Endocytic Entry of Amphotropic Murine Leukemia Virus

Early results suggested that the amphotropic murine leukemia virus (A-MLV) does not enter cells via endocytosis through clathrin-coated pits and this gammaretrovirus has therefore been anticipated to fuse directly with the plasma membrane. However, here we present data implicating a caveola-mediated endocytic entry route for A-MLV via its receptor Pit2. Caveolae belong to the cholesterol-rich microdomains characterized by resistance to nonionic detergents such as Triton X-100. Extraction of murine fibroblastic NIH 3T3 cells in cold Triton X-100 showed the presence of the A-MLV receptor Pit2 in detergent-insoluble microdomains. Using coimmunoprecipitation of cell extracts, we were able to demonstrate direct association of Pit2 with caveolin-1, the structural protein of caveolae. Other investigations revealed that A-MLV infection in contrast to vesicular stomatitis virus infection is a slow process (t(1/2) approximately 5 h), which is dependent on plasma membrane cholesterol but independent of NH4Cl treatment of cells; NH4Cl impairs entry via clathrin-coated pits. Furthermore, expression of dominant-negative caveolin-1 decreased the susceptibility to infection via Pit2 by approximately 70%. These results show that A-MLV can enter cells via a caveola-dependent entry route. Moreover, increase in A-MLV infection by treatment with okadaic acid as well as entry of fusion-defective fluorescent A-MLV virions in NIH 3T3 cells further confirmed our findings and show that A-MLV can enter mouse fibroblasts via an endocytic entry route involving caveolae. Finally, we also found colocalization of fusion-defective fluorescent A-MLV virions with caveolin-1 in NIH 3T3 cells. This is the first time substantial evidence has been presented implicating the existence of a caveola-dependent endocytic entry pathway for a retrovirus.

Role of oxidative stress in the ammonia-induced mitochondrial permeability transition in cultured astrocytes

Ammonia is a neurotoxin that has been strongly implicated in the pathogenesis of hepatic encephalopathy (HE) and other neurological disorders, and astrocytes are thought to be the principal target of ammonia toxicity. While the precise mechanisms of ammonia neurotoxicity remain to be more clearly defined, altered bioenergetics and oxidative stress appear to be critical factors in its pathogenesis. It has recently been demonstrated that pathophysiological concentrations of ammonia induce the mitochondrial permeability transition (MPT) in cultured astrocytes, a process associated with mitochondrial dysfunction, and frequently caused by oxidative stress. This study investigated the potential role of oxidative stress in the induction of the MPT by ammonia. Accordingly, the effect of various antioxidants on the induction of the MPT by ammonia in cultured astrocytes was examined. Astrocytes were subjected to NH 4Cl (5 mM) treatment for 2 days with or without various antioxidants. The MPT was assessed by quantitative fluorescence imaging for the mitochondrial membrane potential (Δ Ψ m), employing the potentiometric dye TMRE; by changes in mitochondrial calcein fluorescence and by 2-deoxyglucose-6-phosphate (2-DG-6-P) changes in mitochondrial permeability. Astrocytes treated with ammonia significantly dissipated the Δ Ψ m, which was blocked by the MPT inhibitor, cyclosporin A, caused a decrease in mitochondrial calcein fluorescence and increased 2-DG-6-P permeability into mitochondria. All of these findings are consistent with induction of the MPT. Pretreatment with SOD, catalase, desferroxamine, Vitamin E, PBN and the nitric oxide synthase inhibitor, N G-nitro- l-arginine methyl ester ( l-NAME), completely blocked the ammonia-induced MPT. These data provide strong evidence that oxidative stress is involved in the induction of the MPT by ammonia, and suggest that oxidative stress and the subsequent induction of the MPT contribute to the pathogenesis of HE and other hyperammonemic disorders.

Appetite-suppressing effects of ammonia exposure in rainbow trout associated with regional and temporal activation of brain monoaminergic and CRF systems

To assess whether the brain's monoaminergic and/or corticotropin-releasing factor (CRF) systems may be involved in mediating the appetite-suppressing effects of high environmental ammonia levels, we exposed rainbow trout to one of four NH4Cl treatments (0, 500, 750, 1000 micromol l(-1)) for 24 or 96 h and monitored changes in food intake, brain serotonin (5-HT) and dopamine (DA) activity, CRF and urotensin I (UI) mRNA levels, and plasma cortisol levels. Food intake decreased in a dose-dependent manner after 24 h of ammonia exposure and partially recovered in all groups after 96 h. Ammonia also elicited dose-dependent increases in serotonergic activity in the hypothalamus (HYP), telencephalon (TEL) and posterior brain (PB). Whereas the increase in serotonergic activity was timed with the 24 h food intake inhibition, TEL and PB serotonergic activity increased after 96 h. In the PB, exogenous ammonia also elicited dose-dependent increases in dopaminergic activity after both 24 and 96 h of exposure. Transient increases in TEL CRF and UI mRNA levels, HYP UI mRNA levels, and plasma cortisol concentrations were evidence that the hypothalamic-pituitary-interrenal (HPI) stress axis was primarily stimulated in the first 24 h of ammonia exposure when food intake was depressed. Overall, the transient nature of the appetite suppression during chronic ammonia exposure, and the time-dependent changes in brain monoaminergic and CRF systems, implicate 5-HT, DA, CRF and UI as potential mediators of the appetite-suppressing effects of ammonia. Among these anorexigenic signals, our results specifically identify hypothalamic 5-HT as a potentially key neurobiological substrate for the regulation of food intake during exposure to high external ammonia concentrations.

Lactate induces tumour necrosis factor‐α, interleukin‐6 and interleukin‐1β release in microglial‐ and astroglial‐enriched primary cultures

Hyperammonaemia has deleterious effects on the CNS in patients with liver dysfunction. Cellular mechanisms underlying the effects of hyperammonaemia are largely unknown, although astrocytes have been the main target of interest. This study investigated how treatment with NH4Cl and lactate, which increase in the brain as a consequence of hyperammonaemia, affects cells in primary rat cultures enriched in either astrocytes or microglia. Morphological changes were studied over time using light microscopy. Release of the proinflammatory cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-6 and IL-1beta was measured using ELISA. NH4Cl was found to induce vacuole formation in both culture systems. Lactate treatment altered astrocytic appearance, resulting in increased space between individual cells. Microglia adopted a round morphology with either NH4Cl or lactate treatment. Lactate, but not NH4Cl, induced release of TNF-alpha and IL-6 in both astroglial- and microglial-enriched cultures, while IL-1beta was released only in microglial cultures. Cytokine release was higher in the microglial- than in the astroglial-enriched cultures. Additionally, the astroglial-enriched cultures containing approximately 10% microglial cells released more cytokines than cultures containing about 5% microglial cells. Taken together, our data suggest that most TNF-alpha, IL-6 and IL-1beta release comes from microglia. Thus, microglia could play an important role in the pathological process of hyperammonaemia.

Adsorption Characteristics of Lead-, Barium- and Hydrogen-Rich Clinoptilolite Mineral

The carbon dioxide and water vapour adsorption properties of local clinoptilolite-rich material, both as the original and as lead-, barium- and hydrogen-rich forms, were examined. The lead- and barium-rich forms were prepared by treatment of the original clinoptilolite with Pb(NO3)2 and BaCl2 respectively, while the hydrogen-rich form was prepared by NH4Cl and heat treatment. Water and CO2 adsorption experiments were conducted in a volumetric system under static conditions, with low-pressure adsorption data being used for the characterization of the natural, Pb-rich, Ba-rich and H-rich clinoptilolite samples. Although the existence of barium-exchange was not noted, an appreciable decrease in CO2 adsorption was observed with the Pb-rich and H-rich forms due to a decrease in the electrostatic interaction between the surface and the adsorbate. Application of the Dubinin–Astakhov equation to the water adsorption data established the existence of micropores of different sizes that exhibited different adsorption mechanisms.

Role of oxidative stress in the ammonia‐induced mitochondrial permeability transition in cultured astrocytes

Ammonia is the principal neurotoxin implicated in the pathogenesis of hepatic encephalopathy (HE). The mechanisms of ammonia toxicity are unclear, but appear to involve oxidative stress and mitochondrial dysfunction. Astrocytes appear to be cell mainly affected. We have previously shown that ammonia increases the production of reactive oxygen species (ROS), and that it also induces the mitochondrial permeability transition (MPT) in cultured astrocytes. Since ROS is a factor commonly implicated in the induction of the MPT, we investigated the role of oxidative stress in the induction of the MPT by ammonia. We therefore examined the effects of various antioxidants on the induction by ammonia of the MPT in cultured astrocytes. Astrocytes were subjected to NH4Cl (5 mm) treatment for two days alone or with various antioxidants. The MPT was assessed by 2‐deoxyglucose (2‐DG) permeability into mitochondria, and by changes in the mitochondrial membrane potential (ΔΨm) by confocal microscopy using the potentiometric dye JC‐1. Astrocytes treated with ammonia significantly increased 2‐DG permeability (90%, p < 0.01) and concomitantly dissipated the ΔΨm (60%, p < 0.01), consistent with the induction of the MPT. The antioxidants superoxide dismutase (by 50%, p < 0.05), vitamin E (by 80%, p < 0.01), and the spin trapping agent PBN (by 80%, p < 0.01), all significantly blocked the increase in 2‐DG permeability and the dissipation of the ΔΨm produced by ammonia. These data demonstrate the induction of the MPT by ammonia and suggest the involvement of ROS in this process. Increased oxidative stress and subsequent induction of the MPT by ammonia in astrocytes may contribute to the pathogenesis of HE.

Transformation of Nitrogen Fertilizers in Greenhouse Experiments

The primary (1 st year) and the after-effects (2 nd , 3 rd year) of N fertilizers (KNO 3 , NH 4 Cl) on the soil-plant-atmosphere system were studied in a three-year greenhouse pot experiment with and without maize plants. The two- and three-year balances of the fertilizer N uptake and gaseous N losses were also analyzed. The cumulative values of the gaseous losses showed a similar trend in all years, significant differences were not obtained. On the basis of the three-year balance, the gaseous loss in the planted and unplanted pots was 18-22% and about 37-39%, respectively. Consequently, there was a 50% decrease in denitrificated gaseous losses of fertilizer N due to plant N uptake. The cumulative gaseous loss, calculated by the difference method, was significantly higher in cases of KNO 3 applications than in NH 4 Cl treatments, as an assumed consequence of the intensive denitrification. It was found that the gaseous loss was not influenced by soil moisture. In contrast to the gaseous losses, the values of plant N uptake and soil mineral N content showed significant differences in the years studied, as a result of the quick transformation of mineral N to organic N, the non-complete homogenization of the total soil amount, the seasonal climatic differences in the greenhouse during the years studied, and consequently the different microbiological activity. The plant N uptake was found to depend significantly on the fertilizer N form. Results obtained by the difference method and the 15 N-tracer technique were very similar. In the case of KNO 3 treatment and higher soil moisture (WHC = 80%) plant N uptake was more intensive, ranging between 48-57% (calculated by the difference method), and 35-51% (calculated by the 15 N- tracer method) in the first year (1993). It can be concluded that 60-100% of the fertilizer N was used from the soil by plant uptake and gaseous losses, which depends mainly on the treatments and the soil moisture during the first year. These values changed between 7-17% in the 1 st year after-effect and between 1-5% in the 2 nd year after-effect.

Ammonium ion, ethylene, and NaCl-induced senescence of detached rice leaves

The possibility that NH4 + accumulation is linkedto the senescence of detached rice (Oryza sativa) leavesinduced by NaCl was investigated. NaCl was effective in promoting senescenceandin increasing NH4 + content of detached rice leaves.NaCl-promoted senescence is mainly due to the effect of both Na+ andCl- ions. NaCl had no or slight effect on relative water content,suggesting that an osmotic effect is unlikely to be a major factor contributingto senescence of these leaves. NaCl-induced NH4 +accumulation was due to enhanced nitrate reduction and decreased glutaminesynthetase activity. Exogenous NH4Cl, which caused an accumulationofNH4 + in detached rice leaves, also promoted senescence.Itwas found that an increase in NH4 + content preceded theoccurrence of senescence caused by NaCl. Results also show that NaCl-promotedsenescence is unlikely to be due to the lack of glutamate, glutamine,aspartate,and asparagine. The current results suggest that NH4 +accumulation is linked to NaCl-induced rice leaf senescence. Since ethylene isknown to be a potent promoter of leaf senescence, we also investigated the roleof ethylene in the regulation of NH4 +-promoted senescenceof detached rice leaves. NaCl or NH4Cl treatment resulted in adecrease of ethylene production. Evidence was presented to show thatNH4 + accumulation in detached rice leaves does not changetissue sensitivity to ethylene. Clearly, the possible involvement of ethyleneinNH4 +-promoted senescence is excluded.

Cell wall peroxidase activity, hydrogen peroxide level and NaCl-inhibited root growth of rice seedlings

The changes in cell-wall peroxidase (POD) activity and H2O2 level in roots of NaCl-stressed rice seedlings and their correlation with root growth were investigated. Increasing concentrations of NaCl from 50 to 150 mM progressively reduced root growth and increased ionically bound cell-wall POD activity. NaCl had no effect on covalently bound cell-wall POD activities. The reduction of root growth by NaCl is closely correlated with the increase in H2O2 level. Exogenous H2O2 was found to inhibit root growth of rice seedlings. Since ammonium and proline accumulation are associated with root growth inhibition caused by NaCl, we determined the effects of NH4Cl or proline on root growth, cell-wall POD activity and H2O2level in roots. External application of NH4Cl or proline markedly inhibited root growth, increased cell-wall POD activity and increased H2O2 level in roots of rice seedlings in the absence of NaCl. An increase in cell-wall POD activity and H2O2 level preceded inhibition of root growth caused by NaCl, NH4Cl or proline. NaCl or proline treatment also increased NADH-POD and diamine oxidase (DAO) activities in roots of rice seedlings, suggesting that NADH-POD and DAO contribute to the H2O2 generation in the cell wall of NaCl- or proline-treated roots. NH4Cl treatment increased NADH-POD activity but had no effect on DAO activity, suggesting that NADH-POD but not DAO is responsible for H2O2 generation in cell wall of NH4Cl-treated roots.

Responses in primary astrocytes and C6-glioma cells to ammonium chloride and dibutyryl cyclic-AMP.

Elevated brain ammonia levels are a major factor in the genesis of hepatic encephalopathy (HE). The mechanism of ammonium chloride (NH4Cl) neurotoxicity involves interruption of oxidative metabolism. This leads to decreased levels of ATP concentration and subsequent glial fibrillary acidic protein (GFAP) degradation of astrocytes and fibrous C6-glioma cells. Our study investigates NH4Cl toxicity by evaluating changes in ATP concentration and mitochondrial function as well as by evaluating alterations in GFAP expression. NH4Cl induced decreases in ATP were detected after 15 minutes in C6-glioma cells and 24 hours in both cell types. Mitochondrial function, assessed by MTT (2-4,5-dimethylthiazol A-yl)-2, 5-diphenyltetrazolium bromide) assay, was impaired in both cell types at 24 hours following NH4Cl treatment. GFAP was also significantly decreased in both cell types. Morphologic and metabolic toxicities were greater in C6-glioma cells. The data clearly indicate that NH4Cl interrupts oxidative metabolism. The greater toxicity seen in C6-glioma cells may be due to their greater dependence on oxidative metabolism. Lastly, the decrease in GFAP is probably a consequence of diminished ATP.

Ceroid/lipofuscin-loaded human fibroblasts show decreased survival time and diminished autophagocytosis during amino acid starvation.

To test whether heavy accumulation of ceroid/lipofuscin can disturb important functions of the lysosomal system, AG-1518 human fibroblasts, ceroid/lipofuscin-loaded (following prolonged culture at normobaric hyperoxia) or not, were exposed to amino acid starvation. Ceroid/lipofuscin-loading resulted in decreased cellular survival. Also, there was an inverse relationship between amounts of ceroid/lipofuscin and the survival time of individual cells within the same cultures. Ceroid/lipofuscin-loaded fibroblasts displayed diminished autophagocytotic capacity, as demonstrated by electron microscopy and by treatment of cell cultures with NH4Cl (which inhibits autophagocytotic degradation by increasing intralysosomal pH) for 1 week before ensuing starvation. The latter treatment increased survival of control cells (due to deposition of nondegraded autophagocytosed material before start of starvation), but not that of ceroid/lipofuscin-loaded cells. Moreover, when NH4Cl treatment was combined with starvation, both groups of cells showed approximately the same shortened survival times, testifying to the causal relationship between diminished autophagocytosis and decreased survival of starving ceroid/lipofuscin-loaded cells. We hypothesize that large amounts of undegradable ceroid/lipofuscin within the acidic vacuolar compartment may interfere with lysosomal function, resulting in poor renewal of long-lived proteins and worn-out/damaged organelles, decreased adaptability, and cell death.

Regulation of cation transport pathways and glycolytic enzyme activity by alterations in red cell volume.

In the presence of NH4Cl and hypotonic solutions, Rana balcanica red cells respond by increasing their volume. The stimulation of cellular volume by hypotonicity is more rapid than that of NH4Cl, while the maximum value is less than that observed in the presence of NH4Cl. Depending on the cause of swelling, (nct uptake of NH4Cl or decrease in external osmolality) cells show specific responses. The NH4Cl treatment causes a significant increase in intracellular Na+, from 5.14 +/- 0.78 to 29.84 +/- 0.47 mmoles l-1 cell, while hypotonicity leads to a significant decrease of this cation, to 3.85 +/- 0.25 mmoles l-1 cell in relation to the control, after 30 min of incubation of Rana balcanica erythrocytes. In addition, amiloiride significantly reverses the NH4Cl effect with respect to intracellular Na+. Both treatments cause a significant K+ loss in comparison with controls. Two glycolytic enzymes glyceraldehyde phosphate dehydrogenase (GAPDH) and pyruvate kinase (PK) of Rana balcanica haemolysate were found to respond to the NH4Cl effect by significantly decreasing their activity.

Polarized targeting of epithelial cell proteins in thyrocytes and MDCK cells.

Polarized trafficking signals may be interpreted differently in different cell types. In this study, we have compared the polarized trafficking of different proteins expressed endogenously in primary porcine thyroid epithelial cells to similar proteins expressed in MDCK cells. As in MDCK cells, NH4Cl treatment of filter-grown thyrocytes caused mis-sorted soluble proteins to exhibit enhanced secretion to the apical medium. In independent studies, thrombospondin 1 (a thyroid basolaterally secreted protein) was secreted basolaterally from MDCK cells. Likewise, the 5'-deiodinase (a thyroid basolateral membrane protein) encoded by the DIO1 gene was also distributed basolaterally in transfected MDCK cells. Consistent with previous reports, when the secretion of human growth hormone (an unglycosylated regulated secretory protein) was examined from transfected MDCK cells, the release was nonpolarized. However, transfected thyrocytes secreted growth hormone apically in a manner dependent upon zinc addition. Moreover, two additional regulated secretory proteins expressed in thyrocytes, thyroglobulin (the major endogenous glycoprotein) and parathyroid hormone (an unglycosylated protein expressed transiently), were secreted apically even in the absence of zinc. We hypothesize that while cellular mechanisms for interpreting polarity signals are generally similar between thyrocytes and MDCK cells, thyrocytes allow for specialized packaging of regulated secretory proteins for apical delivery, which does not require glycosylation but may involve availability of certain ions as well as appropriate intracellular compartmentation.