Decrease In Intracellular ATP Levels Research Articles

Tetrandrine-induced apoptosis in rat primary hepatocytes is initiated from mitochondria: Caspases and Endonuclease G (Endo G) pathway

Tetrandrine, a bisbenylisoquinoline alkaloid isolated from the dried root of Stephenia tetrandra (S Moore), possesses a remarkable pharmacological profile. However, the mechanisms of tetrandrine hepatotoxicity remain to be elucidated. In this study, we first proved apoptosis and mitochondrial dysfunction induced by tetrandrine in Sprague–Dawley rat liver in vivo. By further assuming apoptosis as an important mechanism in tetrandrine-induced hepatotoxicity, we focused on mitochondria-initiated apoptosis in primary hepatocytes isolated from Sprague–Dawley male rats. Tetrandrine treatment led to significant release of cytochrome c and downregulation of Bcl-X L accompanied by caspase 3 activation, and ultimately, DNA fragmentation. Caspase 3 activation was markedly inhibited by cyclosporin A (CsA) and Ac-DEVD-CHO. Furthermore, Endo G, a caspase-independent apoptotic protein, was detected for its expression and DNase activity. CsA blocked the release both of Endo G and cytochrome c significantly. Additionally, the generation of reactive oxygen species (ROS) increased in a time-dependent manner corresponding with a fall in intracellular GSH content after 10 μM tetrandrine treatment in 4 h. Tetrandrine also induced mitochondrial dysfunction indicated by transition of mitochondrial transmembrane potential and decrease of intracellular ATP level. The findings indicated that the caspase-dependent mitochondrial apoptosis pathway was primarily involved in tetrandrine-induced apoptosis in rat primary hepatocytes. In addition, a caspase-independent pathway indicated by Endo G also contributed to apoptosis caused by tetrandrine. Meanwhile, ROS was proved an important inducer in this apoptosis process.

Severe Oxidative Stress Causes Inactivation of DnaK and Activation of the Redox-Regulated Chaperone Hsp33

DnaK/DnaJ/GrpE constitutes the primary chaperone machinery in E. coli that functions to protect proteins against heat-induced protein aggregation. Surprisingly, upon exposure of cells to reactive oxygen species at elevated temperature, proteins are no longer protected by the DnaK system. Instead, they bind now to the redox-regulated chaperone Hsp33, which is activated by the same conditions that inactivate DnaK. The inactivation of DnaK seems to be induced by the dramatic decrease in intracellular ATP levels that occurs upon exposure of cells to reactive oxygen species. This appears to render DnaK's N-terminal ATPase domain nucleotide depleted and thermolabile. DnaK's N terminus reversibly unfolds in vivo, and DnaK loses its ability to protect proteins against stress-induced aggregation. Now, the ATP-independent chaperone holdase Hsp33 binds to a large number of cellular proteins and prevents their irreversible aggregation. Upon return to nonstress conditions, Hsp33 becomes inactivated while DnaK reactivates and resumes its task to support protein folding.

Cadmium-induced apoptosis in oyster hemocytes involves disturbance of cellular energy balance but no mitochondrial permeability transition

Exposure to environmentally prevalent heavy metals such as cadmium can have detrimental effects on a variety of commercially and ecologically important species such as oysters. Since Cd(2+) is known to induce apoptosis in immune cells of vertebrates, we have investigated the effects of this metal on isolated oyster hemocytes, the main cellular immune defense in mollusks. Enhanced apoptosis of these cells could conceivably create immunosuppressed conditions in these organisms and result in reduced disease resistance and increased opportunistic infection, resulting in decline of their populations. Cd(2+) exposure induced apoptosis in oyster hemocytes in a dose-dependent manner in the range of 10-100 micromol l(-1), as indicated by the translocation of phosphatidylserine to the outer leaflet of the plasma membrane. At higher concentrations (200-1000 micromol l(-1)), there was no further increase in apoptosis but a significant increase in the level of necrosis. In stark contrast to vertebrate immune cells, there was no decrease in the mitochondrial membrane potential or activation of caspases in response to Cd(2+) in the apoptotic range. Surprisingly, Cd(2+) exposure in this range did cause a significant decrease in intracellular ATP levels, indicating a severe disturbance of energy metabolism. Similarly, Cd(2+) exposure of isolated mitochondria resulted in partial uncoupling of mitochondria but no difference in mitochondrial membrane potential. The results demonstrate that the important environmental pollutant Cd(2+) induces apoptosis in oyster immune cells and does so through a mitochondria/caspase-independent pathway, suggesting that a novel, perhaps ancient, apoptotic pathway is active in these cells. Furthermore, it appears that the observed decrease in ATP production during apoptosis is not due to the loss of the mitochondrial proton-motive force but is more likely to be due to inhibition of the F(0)/F(1)-ATPase and/or mitochondrial ADP/ATP or substrate transport.

Complex II Defect via Down-regulation of Iron-Sulfur Subunit Induces Mitochondrial Dysfunction and Cell Cycle Delay in Iron Chelation-induced Senescence-associated Growth Arrest

Mitochondria play a pivotal role as an ATP generator in aerobically growing cells, and their defects have long been implicated in the cellular aging process, although its detailed underlying mechanisms remain unclear. Recently, we found that, in the cellular senescent process of Chang cells induced by desferroxamine mesylate, an iron chelator, a significant decrease of intracellular ATP level was accompanied by decline in complex II activity, which preceded acquisition of the senescent phenotype. In the present study, we investigated the mechanism of how the mitochondrial ATP productivity was damaged by iron chelation and how complex II defect was involved in the senescent arrest. The ATP loss was irreversible and accompanied by sustained collapse of mitochondrial membrane potential (Delta psi m), but the ATP loss itself did not seem to be essential in progression to the senescent arrest. The Delta psi m disruption was due to decreased mitochondrial respiration, which was primarily associated with the defective complex II activity. Furthermore, we found that the declined activity of complex II was mainly due to down-regulation of protein expression of the iron-sulfur subunit, which was associated with the irreversibility of the arrest. Finally, we demonstrated that specific inhibition of complex II with 2-thenoyltrifluoroacetone induced overall delay of the cell cycle, suggesting that the delayed arrest by desferroxamine mesylate might be in part due to inhibition of complex II activity. Taken together, our results suggest that complex II might be considered as one of the primary factors to regulate mitochondrial respiratory function by responding to the cellular iron level, thereby influencing cellular growth.

Protective mechanism of reduced water against alloxan-induced pancreatic beta-cell damage: Scavenging effect against reactive oxygen species.

Reactive oxygen species (ROS) cause irreversible damage to biological macromolecules, resulting in many diseases. Reduced water (RW) such as hydrogen-rich electrolyzed reduced water and natural reduced waters like Hita Tenryosui water in Japan and Nordenau water in Germany that are known to improve various diseases, could protect a hamster pancreatic beta cell line, HIT-T15 from alloxan-induced cell damage. Alloxan, a diabetogenic compound, is used to induce type 1 diabetes mellitus in animals. Its diabetogenic effect is exerted via the production of ROS. Alloxan-treated HIT-T15 cells exhibited lowered viability, increased intracellular ROS levels, elevated cytosolic free Ca(2+) concentration, DNA fragmentation, decreased intracellular ATP levels and lowering of glucose-stimulated release of insulin. RW completely prevented the generation of alloxan-induced ROS, increase of cytosolic Ca(2+) concentration, decrease of intracellular ATP level, and lowering of glucose-stimulated insulin release, and strongly blocked DNA fragmentation, partially suppressing the lowering of viability of alloxan-treated cells. Intracellular ATP levels and glucose-stimulated insulin secretion were increased by RW to 2-3.5 times and 2-4 times, respectively, suggesting that RW enhances the glucose-sensitivity and glucose response of beta-cells. The protective activity of RW was stable at 4 degrees C for over a month, but was lost by autoclaving. These results suggest that RW protects pancreatic beta-cells from alloxan-induced cell damage by preventing alloxan-derived ROS generation. RW may be useful in preventing alloxan-induced type 1-diabetes mellitus.

Immunostimulation of rat primary astrocytes decreases intracellular ATP level

In this study we investigated the effect of immunostimulation on intracellular ATP level in rat glial cells. Rat primary astrocytes or C6 glioma cells were treated for 48 h with IFN-γ, LPS or IFN-γ plus LPS. These treatments increased NO production from the cells and a synergistic increase in NO production was observed with IFN-γ plus LPS. Intracellular ATP level was decreased to about half the control level at the highest concentration of IFN-γ (100 U/ml) plus LPS (1 μg/ml) without affecting cell viability. The level of intracellular ATP was inversely correlated with the extent of NO production from the glial cells. The increase in NO production is at least 6 h ahead of the initiation of ATP depletion, and NOS inhibitor N G-nitro- l-arginine (NNA) or Nω-nitro- l-arginine methyl ester (L-NAME) inhibited NO production and ATP depletion. Exogenous addition of peroxynitrite generator 3-morpholinosydnonimine (SIN-1) and to a lesser extent NO generator S-nitroso- N-acetylpenicillamine (SNAP) depleted intracellular ATP level in a dose-dependent manner. The results from the present study imply that immunostimulation of rat glial cells decreases the intracellular ATP level without affecting cell viability. Considering the role of astrocytes as an essential regulator of the extracellular environment in the brain, the immunostimulation-induced decrease in intracellular ATP level may participate in the pathogenesis of various neurological diseases.

The role of intracellular oxidation in death induction (apoptosis and necrosis) in human promonocytic cells treated with stress inducers (cadmium, heat, X-rays)

Treatment of U-937 human promonocytic cells with the stress inducers cadmium chloride (2 h at 200 microM), heat (2 h at 42.5 C) or X-rays (20 Gy), followed by recovery, caused death by apoptosis and stimulated caspase-3 activity. In addition, all stress agents caused intracellular oxidation, as measured by peroxide and/or anion superoxide accumulation. However, while pre-incubation with antioxidants (N-acetyl-L-cysteine or butylated hydroxyanisole) inhibited the induction of apoptosis by cadmium and X-rays, it did not affect the induction by heat-shock. Pre-incubation for 24 h with the GSH-depleting agent L-buthionine-[S,R]-sulfoximine (BSO) switched the mode of death from apoptosis to necrosis in cadmium-treated cells. By contrast, BSO only caused minor modifacions in the rate of apoptosis without affecting the mode of death in heat- and X-rays-treated cells. BSO potentiated peroxide accumulation in cells treated with both cadmium and X-rays. However, while the accumulation of peroxides was stable in the case of cadmium, it was transient in the case of X-rays. Moreover, the administration of antioxidants during the recovery period sufficed to prevent necrosis and restore apoptosis in BSO plus cadmium-treated cells. Cadmium and X-rays caused a decrease in intracellular ATP levels, but the decrease was similar in both apoptotic and necrotic cells. Taken together, these results demonstrate that (i) stress inducers cause intracellular oxidation, but oxidation is not a general requirement for apoptosis; and (ii) the duration of the oxidant state seems to be critical in determining the mode of death.

Induction of cyclosporin A-sensitive pore in mitochondria of intact neurons during uncoupling of oxidative phosphorylation.

Using primary cultures of cerebellar granule cells we showed that Ca(2+)transported into neurons under the effect of glutamate is accumulated and stored in mitochondria for a long time. Protonophore FCCP, an uncoupler of oxidative phosphorylation, stimulated the release of Ca(2+)from mitochondria in a calcium-free medium in 81% glutamate-treated cells. Cyclosporin A and ATP-synthase blocker oligomycin decreased the number of cells with FCCP-induced Ca(2+)release to 53 and 12%, respectively. Oligomycin partly prevented glutamate- and FCCP-induced decrease of intracellular ATP level.

Astroglial trophic support and neuronal cell death: influence of cellular energy level on type of cell death induced by mitochondrial toxin in cultured rat cortical neurons.

Previous in vivo and in vitro analyses have shown that both necrosis and apoptosis are involved in neuronal cell death induced by energy impairment caused by mitochondrial dysfunction. However, little is known about the key factors that determine whether the cells undergo necrosis or apoptosis. In the present study, we analyzed neuronal cell death induced by 3-nitropropionic acid (3-NP), an irreversible inhibitor of mitochondrial complex II, in a primary culture system of rat cortical neurons. The neurons were maintained for a week in coculture with astroglial cells, and then they were treated with 3-NP in the presence or absence of astroglial cells. As judged from morphological (Hoechst 33258 staining) and biochemical (DNA fragmentation and caspase activation) analyses, the cortical neurons appeared to die through an apoptotic process after 3-NP treatment in the presence of astroglial cells. However, caspase inhibitors did not suppress the 3-NP-induced cell death, suggesting the involvement of a caspase-independent pathway of 3-NP-induced neuronal cell death in the presence of astroglial cells. On the other hand, 3-NP induced necrotic cell death within 1 day in the absence of astroglial cells, following a rapid decrease in intracellular ATP level. These changes were attenuated by the presence of astroglial cells or the addition of astroglial conditioned medium. These results suggest that astroglial trophic support influences the alteration of the intracellular energy state in 3-NP-treated neurons and consequently determines the type of neuronal cell death, apoptosis or necrosis.

Oxidative glutamate toxicity involves mitochondrial dysfunction and perturbation of intracellular Ca2+ homeostasis.

Glutamate toxicity on PC12 cells is mediated by oxidative stress as a consequence of the inhibition of a cystine uptake system with depletion of GSH. In this study we report that glutamate decreases PC12 cell viability, inhibiting the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). This decrease was prevented by the antioxidants vitamin E, idebenone and L-deprenyl, which were also shown to be effective in reducing the accumulation of reactive oxygen species (ROS) in cells exposed to glutamate, decreasing the fluorescence of 2',7'-dichlorofluorescein (DCF). Incubation of PC12 cells with high glutamate concentrations induced mitochondrial dysfunction, leading to the loss of mitochondrial transmembrane potential, evaluated as a decrease in rhodamine 123 (Rh123) retention by mitochondria, and to the decrease of intracellular ATP levels. The mitochondrial dysfunction, induced by glutamate, can be involved in the observed increase of [Ca2+]i. The elevation of [Ca2+]i occurred after GSH depletion, suggesting that oxidative stress is involved in the disturbances of intracellular calcium homeostasis. In conclusion, our data indicate that glutamate, at concentrations which block cystine uptake in PC12 cells leading to GSH depletion and inducing oxidative stress, increases ROS accumulation and decreases cell survival by a mechanism involving mitochondrial dysfunction and impairment of Ca2+ homeostasis.

Acrolein toxicity: comparative in vitro study with lung slices and pneumocytes type II cell line from rats

Toxicological effects of acrolein have been studied in precision-cut rat lung slices and in L2 cells, a rat pneumocyte II cell line. These two models were cultured for 24 h with or without acrolein (0–100 μM in L2 cells; 0–200 μM in lung slices). Treatment with this pneumotoxicant produced a concentration dependent decrease in intracellular ATP levels. Acrolein concentrations higher than 50 μM induced ATP decrease in slices, while this decrease occurred from 10 μM acrolein in L2 cells. Detoxification marker evaluations showed that mostly the glutathione pathway was altered after acrolein treatment in both models. Intracellular glutathione (GSH) levels were drastically increased with an acrolein concentration of 10 μM. This increase was concomitant with glutathione- S-transferase (GST) and glutathione reductase (GRED) activities in L2 cells. After this strong increase, these enzymatic activities as well as GSH levels were quickly decreased. In precision-cut rat lung slices, the induction of the glutathione pathway was less clear-cut. A bell-shaped dose response curve was observed with a maximum for 5 μM acrolein for GST and GRED activities. These differences between acrolein toxic ranges could be explained by the presence of an active detoxification pathway in slices compared to its relative lack in L2 cells.

Risk of development of in vitro resistance to amoxicillin, clarithromycin, and metronidazole in Helicobacter pylori.

We have studied initial killing, morphological alterations, the frequency of occurrence, and the selective growth of resistant subpopulations of Helicobacter pylori during exposure to amoxicillin, clarithromycin, or metronidazole by bioluminescence assay of intracellular ATP levels, microscopy, and a viable count assay. We found an induction of spheroplasts and a decrease in intracellular ATP levels after 21 h of exposure to high concentrations of amoxicillin. During clarithromycin exposure the onset of a decrease in intracellular ATP levels started after prolonged incubation, and with the highest concentration of clarithromycin an induction of coccoid forms was seen after 68 h. Metronidazole exposure resulted in the strongest initial decrease in intracellular ATP levels, and coccoid forms were seen after 21 h of exposure to high concentrations of metronidazole. Amoxicillin caused a low-level increase in resistant subpopulations, which indicates a need for surveillance of the amoxicillin susceptibility of H. pylori in order to detect decreasing susceptibility. No increase in the numbers of resistant subpopulations was demonstrated during clarithromycin exposure. Metronidazole selected resistant subpopulations, which caused high-level resistance in H. pylori.

INHIBITION OF ATP-ACTIVATED POTASSIUM CHANNELS EXERTS PRESSOR EFFECTS AND IMPROVES SURVIVAL IN A RAT MODEL OF SEVERE HEMORRHAGIC SHOCK

Potassium channels closed by increases in intracellular ATP levels (KATP channels) have been described in vascular smooth muscle cells and other cell types. These channels are responsive to the metabolic state of the cells, and can be opened by a decrease in intracellular ATP levels and intra- or extracellular acidosis. Hemorrhagic shock is associated with early vasomotor paralysis as well as with early derangements in the intracellular metabolic status. Here we have tested whether activation of KATP channels contributes to the vasodilatation and early mortality in a rat model of severe hemorrhagic shock. In anesthetized rats hemorrhaged to a mean arterial blood pressure (MAP) of 35 mmHg, inhibition of KATP channels with glibenclamide or tolazamide (10 mg/kg i.v. bolus injection followed by an infusion of 10 mg/kg/h for 60 min), rapidly increased MAP and improved survival rate. The same dose of the KATP channel inhibitors did not cause a significant increase of MAP in animals not subjected to hemorrhage. The approach of inhibition of KATP channel activation in hemorrhagic shock is worthy of further investigations to determine whether it may represent a novel approach for early resuscitation during hemorrhage.

Effect of gossypol on cultured TM3 Leydig and TM4 Sertoli cells: 31P and 23Na NMR study.

The effects of gossypol on glucose metabolism, ATP levels and intracellular sodium levels in murine TM3 Leydig and TM4 Sertoli cell lines were investigated, and their response compared. Relative ATP levels and sodium ion levels in the two cell lines were determined by 31P and 23Na NMR spectroscopy. Short-term effect of gossypol on phosphate metabolism in immobilized and perfused cells was apparent in 31P NMR spectra only with relatively high concentrations of gossypol, corresponding to about 40 times IC50. However, incubation with low gossypol concentrations markedly affected the energetic status of the cells, especially of the TM3 cells. Although inhibition of proliferation by gossypol was stronger with the TM4 cells, the decrease of intracellular ATP level and increase of sodium ion concentration were more pronounced in the TM3 cells. The growth-inhibitory effect of (-)-gossypol was stronger than that of (+)-gossypol, with the eudesmic ratio of 2-2.5. The enantioselectivity of the effect of gossypol on the energy metabolism of TM3 and TM4 cells was low, in contrast to the in vivo antispermatogenic effect, which was reported to be solely associated with (-)-gossypol. Inhibition of energy production in somatic testicular tissue is thus unlikely to be major cause of the antispermatogenic effect of gossypol.

Deoxyglucose and reduced glutathione mimic effects of hypoxia on K+ and Ca2+ conductances in pulmonary artery cells

Hypoxia-induced pulmonary vasoconstriction (HPV) is triggered by a rise in cytosolic Ca2+ concentration ([Ca2+]i) that is partially controlled by voltage-gated Ca2+ channels. Hypoxia inhibits voltage-gated K+ (KV) channels in pulmonary artery (PA) myocytes. This depolarizes the cells, opens voltage-gated Ca2+ channels, thereby increases [Ca2+]i, and initiates HPV. In intact animals and isolated perfused lungs, metabolic inhibitors and reducing agents augment HPV. We compared the effects of hypoxia with the glycolysis inhibitor, 2-deoxy-D-glucose (2-DOG), and the reducing agent, reduced glutathione (GSH), on voltage-gated steady-state K+ currents (IK,ss) and membrane potential (Em) in cultured rat pulmonary and mesenteric arterial (MA) smooth muscle cells. Bath application of 10 mM 2-DOG (glucose-free) or 5-10 mM GSH reversibly reduced IK,ss by 25-35% in PA myocytes, with 5 mM ATP present in the pipette solution. Neither hypoxia nor 2-DOG significantly affected IK,ss in MA myocytes, but GSH did reduce IK,ss in these cells. Furthermore, hypoxia, 2-DOG, and GSH depolarized PA cells in the absence as well as in the presence of external Ca2+. Hypoxia, 2-DOG, and GSH also evoked action potentials on the top of the steady depolarization in 36-50% of PA myocytes but not in any MA myocytes; removal of external Ca2+ abolished the action potentials without affecting the steady depolarization. These effects were comparable to those produced by 4-aminopyridine (5-10 mM), a blocker of KV channels. This implies that the action potentials are attributable to Ca2+ influx through voltage-gated Ca2+ channels opened by the steady depolarization due to KV channel inhibition. In the presence of 2-DOG or GSH, hypoxia had no further effect on IK,ss or Em in PA cells; this implies that hypoxia, 2-DOG, and GSH all block the same K+ channels. The data suggest that 1) the hypoxia-induced decrease of IK,ss and the resultant depolarization in PA myocytes may be related to a local decrease of intracellular ATP level and/or a change in redox status of the membrane or cytosol and 2) extracellular Ca(2+)-dependent action potentials may be responsible for at least part of the increase in [Ca2+]i during HPV. Similarities between the effects of hypoxia, 2-DOG, and GSH on IK,ss and Em in PA myocytes, along with the dissimilar responses of PA and MA myocytes, suggest that a common mechanism may underlie the responses of PA cells to these treatments.

Regulation of GABA receptors by intracellular factors

gamma-Aminobutyric acid (GABA) directly inhibits the postsynaptic membrane through GABAA receptor Cl- channel complexes. This inhibition is promoted or blocked by various intra- and extracellular substances at the site of either receptor or channel. Recent studies focused on the intracellular inhibitory mechanisms of the GABAergic response in neurons: one is the inhibition of GABA receptors by the increase in intracellular Ca2+ and the other is inhibition through ATP receptors triggered by the decrease in intracellular ATP level. In addition, the spontaneous inhibitory postsynaptic currents (IPSCs) induced by GABA released from the nerve terminals were suppressed by activation of the GABAB receptor, which acts as a negative autoreceptor in the nerve ending. The intracellular mechanism of the suppression will be discussed.

On the role of mitochondria in cell injury caused by vanadate-induced Ca 2+ overload

Incubation of isolated rat hepatocytes with vanadate (0.25, 0.5 and 1 mM) resulted in progressive accumulation of Ca 2+ in the intracellular compartments. Vanadate- induced Ca 2+ accumulation was related to inhibition of the plasma membrane Ca 2+-extruding system, but did not involve either enhanced plasma membrane permeability to Ca 2+ or the enhanced operation of a putative Na +/Ca 2+ exchanger. After an initial rise in the cytosolic free Ca 2+ concentration, as revealed by phosphorylase activation, Ca 2+ was sequestered predominantly by the mitochondria with little contribution from the endoplasmic reticulum. As the amount of Ca 2+ in the mitochondria increased, a progressive decrease in mitochondrial membrane potential occurred, together with an impairment of the ability of these organelles to further sequester Ca 2+. Associated with this, there was a decrease in intracellular ATP level, formation of surface blebs and cytotoxicity. Addition of an uncoupler to vanadate-treated hepatocytes dramatically accelerated the appearance of plasma membrane blebs and toxicity. Our results demonstrate that under conditions in which the plasma membrane Ca 2+ pump is inhibited, mitochondria play an important role in protecting hepatocytes against damage induced by Ca 2+ overload.

Increase of phosphoribosylpyrophosphate levels in cultured L1210 leukemia cells exposed to methotrexate.

MTX‡ is a folic acid analog that binds to the enzyme dihydro-folate reductase and inhibits its activity depleting the cells of reduced folates. It blocks the synthesis of dTMP from dUMP and de novo purine biosynthesis by decreasing the availability of reduced folates1. MTX effects on cell metabolism can be reversed not only by folinic acid (5-formyltetrahydrofolic acid) but also by either TdR alone or TdR plus a purine base or nucleoside2–6 which provide for nucleotide synthesis through salvage pathways. Purine bases are converted to nucleotides by phosphoribosyltransferases in the presence of PRPP as the phosphoribosyl donor and TdR phosphorylation to dTMP by TdR kinase requires ATP. MTX has been demonstrated to cause a rapid decrease of intracellular ATP levels, which could restrict the cellular utilization of TdR7 . The purpose of our study was to evaluate the effect of MTX on the intracellular levels of PRPP in order to assess the availability of PRPP for purine salvage during MTX treatment.KeywordsFolinic AcidPurine BaseEhrlich Ascites Carcinoma CellPurine BiosynthesisL12l0 CellThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Multiple symmetric lipomatosis: a defect in adrenergic-stimulated lipolysis.

The cellularity of normal and lipomatous adipose tissue and its response to different lipolytic agents have been studied in a group of 10 patients with multiple symmetric lipomatosis (MSL). In MSL patients, fat cells from lipomatous tissue are smaller than normal, uninvolved adipocytes. Fat cells from lipomata show minimal variations in size following conspicuous increase of lipomatous masses. These findings suggest that the growth of lipomata can be attributed to the neoformation of adipocytes rather than to an enlargement in the single fat cells. The incidence of reduced glucose tolerance and of hyperlipoproteinemia is similar in MSL patients and in controls. A significant reduction in plasma free fatty acids was observed in MSL patients after a 24-h fast as well as after noradrenaline infusion. A specific insensitivity of lipomatous tissue to the lipolytic effect of noradrenaline and isoprenaline was observed in vitro, as indicated by glycerol release in the medium, whereas response to theophylline and to dibutyryl cyclic AMP was retained. The lipolytic response to catecholamines was retained. The lipolytic response to catecholamines was normal in the nonlipomatous adipose tissue of MSL patients. In basal conditions ATP concentrations were similar in normal and in lipomatous adipose tissue. However, incubation with noradrenaline induced a significant fall in intracellular ATP levels in normal tissue, whereas no variations were observed in lipomatous tissue. Theophylline, instead, induced a prompt and significant decrease in intracellular ATP levels in lipomatous tissue. These observations indicate that the block in catecholamine-stimulated lipolysis in lipomatous tissue of MSL patients can be localized at a level preceding the formation of cyclic AMP.