Reciprocal effects of energy utilization on palmitate oxidation and esterification in hepatocytes of fed rats

The effects of the energy-dependent process of urea synthesis from NH4Cl on the partition of [1-14C]palmitate between oxidation and esterification were examined in hepatocytes of fed rats. A high rate of urea formation from NH4Cl resulted in stimulation of total palmitate oxidation by 25 and 15% at 0.2 and 1 mM fatty acid, respectively. The stimulation of palmitate oxidation was reciprocally correlated with diminished palmitate incorporation into lipids, mainly triacylglycerols. This relationship was almost stoichiometric. NH4Cl increased the palmitate oxidation/esterification ratio from 0.72 to 1.13 and from 0.94 to 1.36 in the presence of 0.2 mM and 1 mM palmitate, respectively. The transaminase inhibitor, aminooxyacetate, strongly inhibited urea synthesis from NH4Cl, had little effect on the low beta-hydroxybutyrate/acetoacetate ratio in the presence of NH4Cl, completely reversed the changes in palmitate metabolism caused by NH4Cl and did not affect palmitate metabolism in the absence of NH4Cl. Therefore, the increased utilization of energy for urea synthesis was the causative factor by which NH4Cl stimulated total palmitate oxidation and led in consequence to its decreased esterification into lipids. Accordingly, these observations indicate that in liver cells the rate of ATP utilization is one of the determinants of triacylglycerol synthesis.     

An evaluation of the mechanisms developmentally involved on cellular and extracellular glycosaminoglycans accumulation in chick embryo skin fibroblasts.

1. Ammonium chloride, a lysosomotropic amine known to inhibit lysosomal function, was administered to 7-day cultured and 14-day chick embryo skin fibroblasts to evaluate the relationship between synthesis, degradation and uptake of glycosaminoglycans (GAG). 2. Following amine treatment, the amount of 3H-glucosamine and 35SO4 labelled cellular GAG increased, was more at 14 days than at 7 days. Hyaluronic acid (HA) incorporation was mainly interested at 7 days and that of sulphated GAG at 14 days. 3. The extracellular accumulation declined proportionally to the cellular increase of undegraded GAG. HA was mainly affected at 7 days and sulphated GAG at 14 days. 4. The amine did not change 3H-HA uptake and it was unable to inhibit its degradation. 5. The products of degradation of uptaken 3H-HA were retained inside the cell. Those released by degradation of newly synthesized GAG flowed out of the cell.     

The effects of ammonium chloride and bicarbonate on the activity of glutaminase in isolated liver mitochondria.

1. Glutamine hydrolysis in liver mitochondria was studied by measuring the production of glutamate under conditions where this compound could not be further metabolized. 2. Glutaminase activity in intact mitochondria was very low in the absence of activators. 3. Glutamine hydrolysis was markedly stimulated by NH4Cl and also by HCO3- ions. 4. The stimulation by each of these compounds was much decreased if the mitochondria were uncoupled. 5. Maximum rates of glutamine hydrolysis required the addition of phosphate. A correlation was observed between the activity of glutaminase in the presence of NH4Cl plus HCO3- and the intramitochondrial content of ATP. 6. In disrupted mitochondria, NH4Cl stimulated glutaminase to a much smaller extent than in intact mitochondria. The NH4Cl stimulation in disrupted mitochondria was much increased by the addition of ATP. KHCO3 also stimulated glutaminase activity in disrupted mitochondria, and ATP increased the magnitude of this stimulation. 7. It was concluded that maximum rates of glutaminase activity in liver mitochondria require the presence of phosphate, ATP and either HCO3- or NH4+. A comparison of the results obtained on intact and broken mitochondria indicates that these effectors have a direct effect on the glutaminase enzyme system rather than an indirect effect mediated by changes in transmembrane ion gradients or in the concentrations of intramitochondrial metabolites.     

Degradation of proteins in rat liver mitochondrial outer membrane transplanted into different cell types. Evidence for alternative processing.

The degradation of proteins in reductively [3H]methylated mitochondrial outer membrane (MOM) transplanted into cells by a poly(ethylene glycol)-mediated process has been studied. The average rate of degradation (t1/2 24-28 h) of MOM proteins transplanted into HTC cells was not the same as for endogenous MOM proteins (t1/2 56 h), mitoplast proteins (t1/2 120 h), plasma membrane proteins (t1/2 approx. 90 h) or cytosol proteins (t1/2 75 h). The degradation of transplanted MOM proteins was inhibited to the same extent (30-45%) as that of endogenous mitochondrial and plasma membrane proteins by leupeptin and NH4Cl. No inhibition of HTC cell cytosol protein degradation by NH4Cl was observed. NH4Cl differentially inhibited the degradation of endogenous MOM and mitoplast protein subunits as shown after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Proteins in MOM transplanted into tissue culture cells were degraded either with t1/2 24-28 h (MRC-5, B82 and A549 cells) or with t1/2 55-70 h (CHO-K1 and 3T3-L1 cells) similar to that of proteins in MOM transplanted into rat hepatocytes [Evans & Mayer (1983) Biochem. J. 216, 151-161]. The data suggest that membrane protein destruction is but the end part of a fundamental intracellular membrane recognition process.     

Effect of Ammonia on Dark CO2 Fixation by Anabaena Cells Treated with Methionine Sulfoximine

Dark CO₂ fixation by Anabaena cylindrica was stimulated aboutthree-fold by the addition of NH₄Cl to the cells. The ¹⁴CO₂incorporation experiments showed that ¹⁴C is most rapidly incorporatedinto aspartate and then glutamine by adding NH4CI. Glutamineaccumulated predominantly after the addition of NH₄Cl showingthat NH₄ is incorporated into glutamine by glutamine synthetase.The stimulating effect of NH4Cl on CO₂ fixation and amino acidsynthesis was suppressed by methionine sulfoximine, an inhibitorof glutamine synthetase. It was suggested that dark CO₂ fixationwas stimulated by the action of glutamine synthesis which isenhanced by ammonia. (Received February 10, 1981; Accepted April 2, 1981)     

Protective effects of 5,6,7,8-tetrahydroneopterin against X-ray radiation injury in mice

The protective effects of 5,6,7,8-tetrahydroneopterin (NH4) against radiation injury in mice were studied. (C57BL/6xA/J)F1 (B6A) mice received a single whole-body irradiation dose of 200, 400, 700 or 800 cGy of X-rays. NH4 (30 mg/kg body weight) or phosphate-buffered saline (PBS) was injected intraperitoneally into irradiated mice 10 min before and after the irradiation and again after 6 h. All mice which received the 800 cGy radiation+PBS died between 8 and 11 days after the treatment. In contrast, those which also received NH4 demonstrated a significantly prolonged survival time and 40% lived more than 5 months. Total numbers of thymocytes and spleen cells on day 5 post-irradiation were dramatically reduced in line with the radiation dose. The survival was significantly enhanced by NH4 in treated mice. The proliferation of spleen cells in mice stimulated by concanavalin A (Con A) or lipopolysaccharide (LPS) was also greater in NH4 treated mice. The immune response of survivors 5 months after 800 cGy+NH4 treatments, against Con A, LPS, allogenic mouse, and sheep red blood cells had essentially recovered to the levels of normal mice. These results indicate that NH4 had an important role in modifying radiation injury.     

Glycosylation of proteins from sugar nucleotides by whole cells. Effect of ammonium chloride treatment on mouse thymocytes.

When thymocytes are treated with iso-osmotic NH4Cl, the sugar incorporation into endogenous acceptors from labelled sugar nucleotides is largely increased compared with that in control thymocytes. This effect was obtained with labelled GDP-mannose, UDP-galactose and CMP-N-acetylneuraminic acid. The stimulation observed with NH4Cl-treated thymocytes does not involve the glycosylation of exogenous acceptors, and it was proved that the NH4Cl treatment (1) does not stimulate glycosyltransferase activities themselves, (2) does not lead to the release of soluble glycosyltransferases as the result of an extensive lysis of the thymocytes and (3) does not cause the emergence of glycosyltransferases at the cell surface. In fact, electron-microscopy observations showed that, although marked changes had occurred in the cytoplasm, the plasma membrane is sufficiently maintained to allow the cell to keep roughly its original shape and to retain the intracellular vesicles. We thus demonstrate that this stimulation is due to an enhancement of the entry of sugar nucleotides into the cell. As demonstrated by the inclusion of Trypan Blue within the cells, and the non-stimulation of glycosylation of exogenous large-molecular-mass acceptors, the effect of NH4Cl seems to be limited to the penetration of small-molecular-sized compounds through the plasma membrane. Thus NH4Cl treatment allows the labelled sugar nucleotides to penetrate the cell and to behave as the cellular pool to be utilized for glycosylation by intracellular vesicles.     

Ammonia-Induced Release of Neurotransmitters from Rat Brain Synaptosomes: Differences Between the Effects on Amines and Amino Acids

The effect of NH4Cl on release of amine and amino acid transmitters from rat brain synaptosomes was investigated. Ammonia (0.1-10 mM) stimulated the secretion of dopamine and 5-hydroxytryptamine in a dose-dependent manner, in a process which was additive with the effect of 40 mM K+, almost unaffected by withdrawal of Ca2+, and markedly decreased by increasing [H+] in the medium. The NH4Cl-induced dopamine efflux, in contrast to that caused by high [K+]e, was inhibited by benztropine. The release of gamma-aminobutyric acid, aspartate, and glutamate was unaltered by [NH4Cl] less than 5 mM, but somewhat stimulated at higher levels. Transmembrane pH gradient, acid inside, was dissipated by NH4Cl in a concentration-dependent manner and the internal alkalinization correlated with the stimulation of the rate of dopamine efflux. Transmembrane electrical potential was unaffected by [ammonia] less than 5 mM, but a small depolarization was observed at higher levels. It is postulated that ammonia-induced alkalinization of the intrasynaptic storage granules causes extrusion of amines into the cytoplasm and their subsequent leakage into the medium through a reversal of the plasma membrane transporters. A lack of correlation between the release of amino acid neurotransmitters and the dissipation of the delta pH suggests that in rat brain intrasynaptic vesicles, acidic inside, are unlikely to store substantial amounts of gamma-aminobutyric acid, aspartate, or glutamate.     

NH(4)Cl inhibition of acid secretion: possible involvement of an apical K(+) channel in bullfrog oxyntic cells

This study was undertaken to determine the mechanism by which ammonium chloride (NH(4)Cl) inhibits stimulated acid secretion in the bullfrog gastric mucosa. To this end, four possible pathways of inhibition were studied: 1) blockade of basolateral K(+) channel, 2) blockade of ion transport activity, 3) neutralization of secreted H(+) in the luminal solution, or 4) ATP depletion. Addition of nutrient 10 mM NH(4)Cl (calculated NH(3) concentration = 92.5 microM and NH(4)(+) concentration = 9.91 mM) inhibited acid secretion within 30 min. Inhibition of acid secretion did not occur by blockade of basolateral K(+) channel activity or ion transport activity or by neutralization of the luminal solution. Although ATP depletion occurred in the presence of NH(4)Cl, the magnitude of ATP depletion in 30 min was not sufficient to inhibit stimulated acid secretion. By comparing the effect of NH(4)Cl on the resistance of inhibited or stimulated tissues, we demonstrate that NH(4)Cl acts specifically on stimulated tissues. We propose that NH(4)Cl blocks activity of an apical K(+) channel present in stimulated oxyntic cells. Our data suggest that the activity of this channel is important for the regulation of acid secretion in bullfrog oxyntic cells.     

Pendrin in the mouse kidney is primarily regulated by Cl- excretion but also by systemic metabolic acidosis

The Cl(-)/anion exchanger pendrin (SLC26A4) is expressed on the apical side of renal non-type A intercalated cells. The abundance of pendrin is reduced during metabolic acidosis induced by oral NH(4)Cl loading. More recently, it has been shown that pendrin expression is increased during conditions associated with decreased urinary Cl(-) excretion and decreased upon Cl(-) loading. Hence, it is unclear if pendrin regulation during NH(4)Cl-induced acidosis is primarily due the Cl(-) load or acidosis. Therefore, we treated mice to increase urinary acidification, induce metabolic acidosis, or provide an oral Cl(-) load and examined the systemic acid-base status, urinary acidification, urinary Cl(-) excretion, and pendrin abundance in the kidney. NaCl or NH(4)Cl increased urinary Cl(-) excretion, whereas (NH(4))(2)SO(4), Na(2)SO(4), and acetazolamide treatments decreased urinary Cl(-) excretion. NH(4)Cl, (NH(4))(2)SO(4), and acetazolamide caused metabolic acidosis and stimulated urinary net acid excretion. Pendrin expression was reduced under NaCl, NH(4)Cl, and (NH(4))(2)SO(4) loading and increased with the other treatments. (NH(4))(2)SO(4) and acetazolamide treatments reduced the relative number of pendrin-expressing cells in the collecting duct. In a second series, animals were kept for 1 and 2 wk on a low-protein (20%) diet or a high-protein (50%) diet. The high-protein diet slightly increased urinary Cl(-) excretion and strongly stimulated net acid excretion but did not alter pendrin expression. Thus, pendrin expression is primarily correlated with urinary Cl(-) excretion but not blood Cl(-). However, metabolic acidosis caused by acetazolamide or (NH(4))(2)SO(4) loading prevented the increase or even reduced pendrin expression despite low urinary Cl(-) excretion, suggesting an independent regulation by acid-base status.     

Inhibition of lysosomal protein degradation inhibits the basal degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase

The effect of inhibiting lysosomal protein degradation on the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was determined using a mouse mammary cell line (TS-85) which expresses a temperature-sensitive mutation in the ubiquitin degradative pathway. Incubating cells for 18 hr in medium containing 20 mM NH4Cl did not alter total protein synthesis or cell growth, but it did inhibit the rate of total protein degradation by 19%, which is consistent with the known inhibitory effect of NH4Cl on lysosomal protein degradation. NH4Cl treatment also resulted in an increase (81% +/- 20) in HMG-CoA reductase activity. The increase in reductase activity was not correlated with changes in the phosphorylation state of the enzyme or with alteration in the relative rate of reductase synthesis. However, the basal degradation rate of the reductase was significantly inhibited, and after NH4Cl treatment, the half-life of the enzyme increased from 4.0 +/- 0.4 hr to 8.3 +/- 0.8 hr. The change in the rate of reductase degradation can account completely for the increase in reductase activity observed in NH4Cl-treated cells. The accelerated degradation of HMG-CoA reductase induced by 25-hydroxycholesterol treatment was not affected by either NH4Cl or by inactivation of the ubiquitin degradative pathway. Therefore, two different mechanisms may be responsible for the accelerated degradation and basal degradation of HMG-CoA reductase. The latter can be inhibited by NH4Cl and may imply that under basal conditions the enzyme may be degraded in lysosomes.     

Effects of chronic NH4Cl dosage and swimming exercise on bone metabolic turnover in rats

To determine the effects of ammonium chloride (NH4Cl) dosage and swimming exercise training during 4 weeks on bone metabolic turnover in rats, seven-week-old female 24 Wister-Kyoto (WKY) rats were investigated by bone status including bone mineral density (BMD) and biomechanical markers from blood and urine. Twenty-four rats (initial weight: 191.2+/-7.6 g) were randomly divided into four groups: baseline (8 weeks old) control group (n=6, BC), 4-week control group (n=6, Con), 4-week swimming exercise loading group (n=6, Swim) and 4-week chronic NH4Cl dosage group (n=6, Acid). All rats were fed an AIN93M diet (Ca: 0.5%, P: 0.3%), and both Con and Swim groups were pair-fed by feeding volume of the NH4Cl dosage group. The acid group only received 0.25 M NH4Cl distilled water ad libitum. At the end of the experimental period, rats were sacrificed with blood drawn and femur and tibia were removed for analysis of bone mineral density (BMD) by dual energy X-ray absorptiometry (DEXA). In the Swim group, 24-hour urinary deoxypiridinoline (Dpd) excretion, reflecting bone resorption, was significantly increased (p<0.05) with a tendency towards decrease of BMD (N.S.), and body weight and abdominal fat weight were decreased in approximately 7% (p<0.05) and 58% (p<0.001), as compared with age matched Con rats. In the Acid group, 24-hour urinary calcium (Ca) and phosphorus (P) excretion were increased approximately 2.1-fold (p<0.05) and 2.0-fold (p<0.01), respectively, with increase of kidney weight as much as in the Con groups. Serum Ca and P concentration, as well as urinary Dpd excretion were, however, not significantly changed. These results suggest that blood Ca and P concentrations in the chronic acidosis condition during the 4-weeks might be maintained by hypercalciuria and hyperphosphaturia with kidney disorder, and swimming exercise training leads to decrease in BMD with stimulation of bone resorption and reduction of body fat.     

Effects of amino acids, ammonia and leupeptin on protein synthesis and degradation in isolated rat hepatocytes.

Protein synthesis in isolated rat hepatocytes, as measured by the incorporation of [14C]-valine at constant specific radioactivity, proceeded at a rate of 0.3-0.5%/h in an unsupplemented medium, i.e. only about one-tenth the rate of protein degradation (4%/h). Leupeptin, which inhibits lysosomal protein degradation (previously found to be 75% of the total degradation in hepatocytes), had no effect on protein synthesis, showing that endogenous protein degradation supplied amino acids in excess of the substrate requirements for protein synthesis. The inhibition of protein synthesis by NH4Cl (another inhibitor of lysosomal protein degradation) as well as the stimulation by a physiological amino acid mixture must therefore represent indirect effects, either on general energy metabolism, or on unknown regulatory processes.     

Nutrition and carbon metabolism of Methanococcus voltae.

Methanococcus voltae is a heterotrophic, H2-oxidizing methanogenic bacterium. In complex medium, this bacterium has a doubling time of 1.2 h at its temperature optimum of 38 degrees C. In defined medium, optimal growth is obtained with 0.75 mM isoleucine, 0.75 mM leucine, 2.5 mM acetate, 5 mM NH4Cl, 84 mM MgSO4, 0.4 M NaCl, 1 mM CaCl2, 10 microM Fe2O3, and 0.2 microM NiCl2. In addition, pantothenate, sodium selenate, and cobalt stimulate growth. Optimal growth is obtained between pH 6.0 and 7.0 with either H2 or formate as the electron donor. The volatile fatty acids 2-methylbutyrate and isovalerate can substitute for isoleucine and leucine, respectively. Cellular carbon is derived from acetate (31%), isoleucine (22%), leucine (25%), and carbon dioxide (23%). The amino acids and fatty acids are incorporated almost exclusively into protein. A comparison of the incorporation of U-14C-amino acids and 1-14C-fatty acids indicated that the fatty acids are degraded during incorporation into cell protein. The distribution of carbon from the amino acids suggests that acetyl coenzyme A is not a major intermediate in the degradation of these compounds. Thus, M. voltae may convert isoleucine and leucine to other amino acids by a unique mechanism. The lipid carbon is derived largely from acetate. Thus, the isoprenoid lipids are synthesized de novo from acetate rather than by degradation of leucine. The carbon in the nucleic acids is derived from carbon dioxide (45%), the C-1 of acetate (25%), the C-2 of acetate (22%), and isoleucine and leucine (7%). This labeling pattern is consistent with known biochemical pathways.     

Influence of dexamethasone phosphate upon the DNA- and the NAD-metabolism of concanavaline a stimulated T-lymphocytes

1. Glucocorticoids have a decisive function in the immune system. In this paper, special attention is paid to the DNA and the NAD metabolism in T-lymphocytes of mice stimulated by Con A under the influence of dexamethasone phosphate. 2. Nicotinamide increases the incorporation of [3H]thymidine into the DNA of T-cells in dependence on the concentration. There is a similar but less pronounced effect with 1-methylnicotinamide. 3. Dexamethasone phosphate even at 10(-9) M inhibits the incorporation of [3H]thymidine into DNA. 4. The incorporation of [3H]thymidine into the DNA is reduced after preincubation of the T-cells with 6-aminonicotinamide or with 3-acetylpyridine. 5. Dexamethasone phosphate decreases the content of NAD in the T-cells. 6. The activity of the ADPR transferase increases after addition of Con A. Presence of nicotinamide stimulates the effect of Con A on this enzyme. This is not the case with 1-methylnicotinamide. The enzyme is inhibited drastically by dexamethasone phosphate. 7. It may be concluded that the NAD-adenoribosylation metabolism is markedly influenced by the mitogen Con A and by dexamethasone phosphate.     

Biogenesis of lysosomes in marshall cells and in cells of the male reproductive system

The mechanism of plasma membrane trafficking and degradation is still poorly understood. This investigation deals with the biogenesis of lysosomes during endocytic flow in Marshall cells and in various cell types of the male reproductive system. Marshall cells were exposed to ammonium chloride (NH4Cl) and leupeptin after labeling with cationic ferritin. In some experiments, the treated cells were immunogold labeled with anti-prosaposin antibody. NH4Cl and leupeptin are lysosomotropic agents that affect the endosomal-lysosomal progression. Testes, efferent ducts and epididymis from mouse mutants with defects affecting plasma membrane degradation were also used to analyze this process. NH4Cl produced a retention of cationic ferritin in endosomes and hindered the endosomal/lysosomal progression. Leupeptin did not affect this process. NH4Cl decreased the labeling of prosaposin in endosomes and lysosomes, while leupeptin increased the labeling of prosaposin in lysosomes. The number of lysosomes per cytoplasmic area was higher in treated cells than in controls. These findings suggest that leupeptin affected lysosomes whereas NH4Cl affected both endosomes and lysosomes. The endosomal and lysosomal accumulation of prosaposin induced by the treatment with NH4Cl and leupeptin indicated that the site of entry of prosaposinwas both the lysosome and endosome. Electron microscopy (EM) of tissues from mouse mutants with defects affecting plasma membrane degradation substantiated these observations. The EM analysis revealed a selective accumulation of multivesicular bodies (MVBs) and the disappearance of lysosomes, in testicular fibroblasts, nonciliated cells of the efferent ducts and principal cells of the epididymis, suggesting that MVBs are precursors of lysosomes. In conclusion: (1) endosomes and MVBs are a required steps for degradation of membranes; (2) endosomes and MVBs are precursors of lysosomes; and (3) endosomes, MVBs, and lysosomes appear to be transient organelles.     

Ammonium chloride inhibits basal degradation of newly synthesized collagen in human fetal lung fibroblasts

The objective of this work was to characterize basal degradation of newly synthesized collagen in human fetal lung fibroblasts. Analysis of 22 separate determinations showed that in cells incubated under normal conditions, the level of intracellular degradation was normally distributed with a mean of 15.2% and a standard deviation of 2.6%. Within each experiment, however, the uncertainty (standard deviation) in determining degradation was very small, usually less than 1.5%. Consideration of the large variation between experiments and the ability of our analytic technique to detect small, but "statistically significant," differences between groups within the same experiment led us to formulate two criteria for determining whether degradation measured in cultures exposed to some agent differs in a "biologically significant" way from degradation measured in control cultures. These criteria were used to evaluate the effects of the following proteinase inhibitors on basal degradation: NH4Cl, which increases the pH of subcellular compartments that are normally acidic; and leupeptin and Na-p-tosyl-L-lysine chloromethyl ketone (TLCK), which are inhibitors of lysosomal cathepsins (B and L) that degrade collagen. NH4Cl (16 mM) lowered degradation to an extent that was both statistically and biologically significant, but neither leupeptin nor TLCK affected degradation. The effect of NH4Cl on degradation was independent of its inhibitory effects on production of collagen, protein, and ATP. These results suggest that basal degradation occurs in, or beyond, an acidic (i.e., NH4Cl-sensitive) but nonlysosomal compartment of the cell, and that NH4Cl inhibits processing within, or transport to, that compartment. This is the first report of an agent that inhibits basal degradation of newly synthesized collagen in soft tissue fibroblasts.     

NBD-cholesterol incorporation by rat macrophages and lymphocytes: a process dependent on the activation state of the cells

The time-course of incorporation of NBD-cholesterol by macrophages (Ma) and lymphocytes (LY) obtained from untreated and thioglycollate-injected (thio) rats was investigated. NBD-cholesterol incorporation was also examined in Ma obtained from untreated rats and stimulated in vitro by lipopolysaccharide (LPS) and phorbol-myristate acetate (PMA). The same measurement was performed in LY from untreated rats stimulated by addition of LPS and concanavalin A (Con A) into the culture medium. Thio-treated Ma showed high fluorescence intensity after 1 h of incubation with NBD-cholesterol. Ma submitted concomitant to LPS and NBD-cholesterol showed low fluorescence intensity, as well as Ma stimulated with PMA. Ma from untreated and LPS pre-treated rats showed a similar time-course of incorporation. LY from thio-treated rats showed lower incorporation of NBD-cholesterol in comparison to LY from untreated rats. Incorporation was reduced when LPS was added concomitantly with NBD-cholesterol. On the other hand, LY pre-treated with LPS for 48 h showed a very high incorporation of NBD-cholesterol. Con A treatment did not cause a significant effect on NBD-cholesterol incorporation. The findings presented herein led us to conclude that the uptake of NBD-cholesterol by Ma and LY is markedly affected by the activation state of the cells.     

Urea synthesis and CO2/HCO3- compartmentation in isolated perfused rat liver

With physiological portal HCO3- and CO2 concentrations of 25mM and 1.2mM in the perfusate, respectively, acetazolamide inhibited urea synthesis from NH4Cl in isolated perfused rat liver by 50-60%, whereas urea synthesis from glutamine was inhibited by only 10-15%. A decreased sensitivity of urea synthesis from glutamine to acetazolamide inhibition was also observed when the extracellular HCO3- and CO2 concentrations were varied from 0-50mM and 0-2.4mM, respectively. Stimulation of intramitochondrial CO2 formation at pyruvate dehydrogenase with high pyruvate concentrations (7mM) was without effect on the acetazolamide sensitivity of urea synthesis from NH4Cl. Urea synthesis was studied under conditions of a limiting HCO3- supply for carbamoyl-phosphate synthesis. In the absence of externally added HCO3- or CO2, when 14CO2 was provided intracellularly by [U-14C]glutamine or [1-14C]-glutamine oxidation, acetazolamide had almost no effect on label incorporation into urea, whereas label incorporation from an added tracer H14CO3- dose was inhibited by about 70%. 14CO2 production from [U-14C]glutamine was about twice as high as from [1-14C]glutamine, indicating that about 50% of the CO2 produced from glutamine is formed at 2-oxoglutarate dehydrogenase. The fractional incorporation of 14CO2 into urea was about 13% with [1-14C]-as well as with [U-14C]glutamine. Addition of small concentrations of HCO3- (1.2mM) to the perfusate increased urea synthesis from glutamine by about 70%. This stimulation of urea synthesis was fully abolished by acetazolamide. The carbonate-dehydratase inhibitor prevented the incorporation of added HCO3- into urea, whereas incorporation of CO2 derived from glutamine degradation was unaffected. Without HCO3- and CO2 in the perfusion medium, when 14CO2 was provided by [1-14C]-pyruvate oxidation, acetazolamide inhibited urea synthesis from NH4Cl as well as 14C incorporation into urea by about 50%. Therefore carbonate-dehydratase activity is required for the utilization of extracellular CO2 or pyruvate-dehydrogenase-derived CO2 for urea synthesis, but not for CO2 derived from glutamine oxidation. This is further evidence for a special role of glutamine as substrate for urea synthesis.     

Role of monochloramine in the oxidation of erythrocyte hemoglobin by stimulated neutrophils

Stimulation of the oxygen (O2) metabolism of isolated human neutrophilic leukocytes resulted in oxidation of hemoglobin of autologous erythrocytes without erythrocyte lysis. Hb oxidation could be accounted for by reduction of O2 to superoxide (O-2) by the neutrophils, dismutation of O-2 to yield hydrogen peroxide (H2O2), myeloperoxidase-catalyzed oxidation of chloride (Cl-) by H2O2 to yield hypochlorous acid (HOCl), the reaction of HOCl with endogenous ammonia (NH+4) to yield monochloramine ( NH2Cl ), and the oxidative attack of NH2Cl on erythrocytes. NH2Cl was detected when HOCl reacted with the NH+4 and other substances released into the medium by neutrophils. The amount of NH+4 released was sufficient to form the amount of NH2Cl required for the observed Hb oxidation. Oxidation was increased by adding myeloperoxidase or NH+4 to increase NH2Cl formation. Due to the volatility of NH2Cl , Hb was oxidized when neutrophils and erythrocytes were incubated separately in a closed container. Oxidation was decreased by adding catalase to eliminate H2O2, dithiothreitol to reduce HOCl and NH2Cl , or taurine to react with HOCl or NH2Cl to yield taurine monochloramine . NH2Cl was up to 50 times more effective than H2O2, HOCl, or taurine monochloramine as an oxidant for erythrocyte Hb, whereas HOCl was up to 10 times more effective than NH2Cl as a lytic agent. NH2Cl contributes to oxidation of erythrocyte components by stimulated neutrophils and may contribute to other forms of neutrophil oxidative cytotoxicity.