Rat liver folate metabolism can provide an independent functioning of associated metabolic pathways.

Folate metabolism in mammalian cells is essential for multiple vital processes, including purine and pyrimidine synthesis, histidine catabolism, methionine recycling, and utilization of formic acid. It remains unknown, however, whether these processes affect each other via folate metabolism or can function independently based on cellular needs. We addressed this question using a quantitative mathematical model of folate metabolism in rat liver cytoplasm. Variation in the rates of metabolic processes associated with folate metabolism (i.e., purine and pyrimidine synthesis, histidine catabolism, and influxes of formate and methionine) in the model revealed that folate metabolism is organized in a striking manner that enables activation or inhibition of each individual process independently of the metabolic fluxes in others. In mechanistic terms, this independence is based on the high activities of a group of enzymes involved in folate metabolism, which efficiently maintain close-to-equilibrium ratios between substrates and products of enzymatic reactions.

Na+ and K+ ion imbalances in Alzheimer's disease.

Alzheimer's disease (AD) is associated with impaired glutamate clearance and depressed Na(+)/K(+) ATPase levels in AD brain that might lead to a cellular ion imbalance. To test this hypothesis, [Na(+)] and [K(+)] were analyzed in postmortem brain samples of 12 normal and 16 AD individuals, and in cerebrospinal fluid (CSF) from AD patients and matched controls. Statistically significant increases in [Na(+)] in frontal (25%) and parietal cortex (20%) and in cerebellar [K(+)] (15%) were observed in AD samples compared to controls. CSF from AD patients and matched controls exhibited no differences, suggesting that tissue ion imbalances reflected changes in the intracellular compartment. Differences in cation concentrations between normal and AD brain samples were modeled by a 2-fold increase in intracellular [Na(+)] and an 8-15% increase in intracellular [K(+)]. Since amyloid beta peptide (Aß) is an important contributor to AD brain pathology, we assessed how Aß affects ion homeostasis in primary murine astrocytes, the most abundant cells in brain tissue. We demonstrate that treatment of astrocytes with the Aß 25-35 peptide increases intracellular levels of Na(+) (~2-3-fold) and K(+) (~1.5-fold), which were associated with reduced levels of Na(+)/K(+) ATPase and the Na(+)-dependent glutamate transporters, GLAST and GLT-1. Similar increases in astrocytic Na(+) and K(+) levels were also caused by Aß 1-40, but not by Aß 1-42 treatment. Our study suggests a previously unrecognized impairment in AD brain cell ion homeostasis that might be triggered by Aß and could significantly affect electrophysiological activity of brain cells, contributing to the pathophysiology of AD.

Distribution of methionine between cells and incubation medium in suspension of rat hepatocytes.

Methionine is an essential amino acid involved in many significant intracellular processes. Aberrations in methionine metabolism are associated with a number of complex pathologies. Liver plays a key role in regulation of blood methionine level. Investigation of methionine distribution between hepatocytes and medium is crucial for understanding the mechanisms of this regulation. For the first time, we analyzed the distribution of methionine between hepatocytes and incubation medium using direct measurements of methionine concentrations. Our results revealed a fast and reversible transport of methionine through the cell membrane that provides almost uniform distribution of methionine between hepatocytes and incubation medium. The steady-state ratio between intracellular and extracellular methionine concentrations was established within a few minutes. This ratio was found to be 1.06±0.38, 0.89±0.26, 0.67±0.16 and 0.82±0.06 at methionine concentrations in the medium of 64±19, 152±39, 413±55, and 1,204±104 µmol/L, respectively. The fast and uniform distribution of methionine between hepatocytes and extracellular compartments provides a possibility for effective regulation of blood methionine levels due to methionine metabolism in hepatocytes.

Muscarinic receptor regulation of osmosensitive taurine transport in human SH-SY5Y neuroblastoma cells.

The ability of G protein-coupled receptors to regulate osmosensitive uptake of the organic osmolyte, taurine, into human SH-SY5Y neuroblastoma cells has been examined. When monitored under isotonic conditions and in the presence of physiologically relevant taurine concentrations (1-100 microM), taurine influx was mediated exclusively by a Na(+)-dependent, high-affinity (K(m) = 2.5 microM) saturable transport mechanism (V(max) = 0.087 nmol/mg protein/min). Reductions in osmolarity of > 20% (attained under conditions of a constant NaCl concentration) resulted in an inhibition of taurine influx (> 30%) that could be attributed to a reduction in V(max), whereas the K(m) for uptake remained unchanged. Inclusion of the muscarinic cholinergic agonist, oxotremorine-M (Oxo-M), also resulted in an attenuation of taurine influx (EC(50) approximately 0.7 microM). Although Oxo-M-mediated inhibition of taurine uptake could be observed under isotonic conditions (approximately 25-30%), the magnitude of inhibition was significantly enhanced by hypotonicity (approximately 55-60%), a result that also reflected a reduction in the V(max), but not the K(m), for taurine transport. Oxo-M-mediated inhibition of taurine uptake was dependent upon the availability of extracellular Ca(2+) but was independent of protein kinase C activity. In addition to Oxo-M, inclusion of either thrombin or sphingosine 1-phosphate also attenuated volume-dependent taurine uptake. The ability of Oxo-M to inhibit the influx of taurine was attenuated by 4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid, an inhibitor of the volume-sensitive organic osmolyte and anion channel. 4-[(2-Butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid also prevented receptor-mediated changes in the efflux and influx of K(+) under hypoosmotic conditions. The results suggest that muscarinic receptor activation can regulate both the volume-dependent efflux and uptake of taurine and that these events may be functionally coupled.

An allosteric mechanism for switching between parallel tracks in mammalian sulfur metabolism.

Methionine (Met) is an essential amino acid that is needed for the synthesis of S-adenosylmethionine (AdoMet), the major biological methylating agent. Methionine used for AdoMet synthesis can be replenished via remethylation of homocysteine. Alternatively, homocysteine can be converted to cysteine via the transsulfuration pathway. Aberrations in methionine metabolism are associated with a number of complex diseases, including cancer, anemia, and neurodegenerative diseases. The concentration of methionine in blood and in organs is tightly regulated. Liver plays a key role in buffering blood methionine levels, and an interesting feature of its metabolism is that parallel tracks exist for the synthesis and utilization of AdoMet. To elucidate the molecular mechanism that controls metabolic fluxes in liver methionine metabolism, we have studied the dependencies of AdoMet concentration and methionine consumption rate on methionine concentration in native murine hepatocytes at physiologically relevant concentrations (40-400 microM). We find that both [AdoMet] and methionine consumption rates do not change gradually with an increase in [Met] but rise sharply (approximately 10-fold) in the narrow Met interval from 50 to 100 microM. Analysis of our experimental data using a mathematical model reveals that the sharp increase in [AdoMet] and the methionine consumption rate observed within the trigger zone are associated with metabolic switching from methionine conservation to disposal, regulated allosterically by switching between parallel pathways. This regulatory switch is triggered by [Met] and provides a mechanism for stabilization of methionine levels in blood over wide variations in dietary methionine intake.

Doxorubicin pharmacokinetics in lymphoma patients treated with doxorubicin-loaded eythrocytes.

Doxorubicin-loaded erythrocytes (DLE) were administrated to 15 lymphoma patients. Antibiotic peak concentration in blood decreased by 55%, doxorubicin circulated several times longer, and the area under the concentration-time curve increased 5 times if compared with standard doxorubicin administration. The DLE was well tolerated by patients.

Analysis of pathological defects in methionine metabolism using a simple mathematical model.

Derangements in methionine metabolism are a hallmark of cancers and homocystinuria, an inborn error of metabolism. In this study, the metabolic consequences of the pathological changes associated with the key pathway enzymes, methionine adenosyl transferase (MAT), glycine N-methyl transferase (GNMT) and cystathionine beta-synthase (CBS) as well as an activation of polyamine metabolism, were analyzed using a simple mathematical model describing methionine metabolism in liver. The model predicts that the mere loss of allosteric regulation of CBS by adenosylmethionine (AdoMet) leads to an increase in homocysteine concentration. This is consistent with the experimental data on the corresponding genetic defects, which specifically impair allosteric activation but not basal enzyme activity. Application of the characteristics of transformed hepatocytes to our model, i.e., substitution of the MAT I/III isozyme by MAT II, loss of GNMT activity and activation of polyamine biosynthesis, leads to the prediction of a significantly different dependence of methionine metabolism on methionine concentrations. The theoretical predictions were found to be in good agreement with experimental data obtained with the human hepatoma cell line, HepG2.

Pharmacokinetics of erythrocyte-bound daunorubicin in patients with acute leukemia.

BACKGROUND: The objective of the present study was in vitro and in vivo investigation of erythrocytes as vehicles for anthracycline antibiotics. MATERIAL/METHODS: The kinetics of daunorubicin binding with erythrocytes was studied in blood and in washed erythrocyte suspensions from healthy donors and patients with acute leukemia. The effect of daunorubicin on erythrocyte deformability was studied using cell filtration through membranes with 3 microm-diameter cylindrical pores. Erythrocyte-bound daunorubicin (EBD), prepared by equilibrating anticoagulated autologous blood with the antibiotic, was administered (45 or 60 mg/m2 body surface) to 14 leukemic patients as part of the 7+ 3 or RACOP courses. The pharmacokinetics of daunorubicin and its tolerability were studied. RESULTS: Human erythrocytes bound daunorubicin (rubomycin) in citrated whole blood or in washed saline suspension. The equilibrium erythrocyte/medium daunorubicin concentration ratios (attained in 30-60 min at 37 degrees C) averaged 2.9 +/- 0.5 (n=13) in blood and 5.7 +/- 0.6 (n=8) in suspension (p<0.001), without any significant difference between the erythrocytes of donors and patients with acute drug-resistant leukemia or leukemic relapses. Incubation of patient blood with daunorubicin (0.5 mg/ml cells) did not affect erythrocyte deformability (filterability). After intravenous administration, the peak drug concentration and its elimination rate were lower for EBD than for free daunorubicin. The patients tolerated EBD better than its standard free form. In nine patients who received three EBD infusions, side effects were less frequent than in those treated with free daunorubicin. CONCLUSIONS: Our results indicate that daunorubicin-loaded erythrocytes are promising for clinical application and deserve further clinical study.

What determines the intracellular ATP concentration.

Analysis is made of the mechanisms that control the intracellular ATP level. The balance between energy production and expenditure determines the energy charge of the cell and the ratio of [ATP] to the adenylate pool. The absolute ATP concentration is determined by the adenylate pool, which, in its turn, depends on the balance between the rates of AMP synthesis and degradation. Experimental data are discussed that demonstrate an increase in the adenylate pool in response to activation of energy-consuming processes. A hypothesis is proposed according to which variation in the adenylate pool and absolute ATP concentration affords a cell the possibility of additional control over processes fulfillinguseful work. A mechanism involved in this regulation is described using human erythrocytes as an example. The hypothesis explains why different metabolic pathways (protein and DNA syntheses, polysaccharide synthesis, and lipid synthesis) use different trinucleotides (GTP, UTP, and CTP, respectively) as an energy source. This allows the cell to independently control these metabolic processes by varying the individual nucleotide pools.