Spreading of porcine kidney epithelial cells under normal conditions and under the effect of inhibitors of energy metabolism. II. Behavior of cellular organelles

In the present work the behavior of mitochondria and lysosomes during cell spreading has been investigated in normal conditions and under ATP-synthesis inhibitors: sodium aside and N,N-dicyclohexylcarbodiimide (DCCD). In the control culture, microtubules run along the stable edge and perpendicular to the leading edge in most of spreading cells. As a whole, microtubules form a dense network in these cells. However, the radial cells contain bundles of microtubules, radiating from the perinuclear area or form circular arrays around the nucleus. The microtubule network is more dense under inhibitory treatment, than in control conditions. In the control culture the spherical cells display numerous small mitochondria (staining with Rhodamine 123). In the process of cell spreading some elongated mitochondria appear, most of them being localized in the perinuclear area. The mitochondria of cells with radial microtubule organization are directed towards the cell periphery, while in cells with circular bundles of microtubules the mitochondria are localized chaotically. Under DCCD treatment the mitochondria retain the staining for 2-3 h. In the spreading cells, round mitochondria may be distributed all over the cytoplasm. In the presence of sodium aside the mitochondria are not stained. However, by means of phase contrast microscopy some disoriented thread-shaped structures are observed, obviously corresponding to mitochondria. In the control conditions, lysosomes (stained with Acridine orange) in spreading cells are dispersed chaotically, all over the cytoplasm, or are localized in the perinuclear area. In the presence of sodium aside lysosomes are observed only in the perinuclear area. Under DCCD treatment lysosomes do not accumulate the dye. Thus, the cytoskeleton modification and changes in the properties of membrane organelles, induced by ATP-synthesis inhibitors, do not prevent attachment, spreading or cell polarization.     

Induction in primary culture of 'gluconeogenic' and 'glycolytic' hepatocytes resembling periportal and perivenous cells

Adult rat hepatocytes were kept in primary culture for 48 h under different hormonal conditions to induce an enzyme pattern which with respect to carbohydrate metabolism approximated that of periportal and perivenous hepatocytes in vivo. 1. Glucagon-treated cells compared with control cells possessed a lower activity of glucokinase, a 4.5-fold higher activity of phosphoenolpyruvate carboxykinase and unchanged levels of glucose-6-phosphatase, phosphofructokinase, fructose-bisphosphatase and pyruvate kinase; they resembled in a first approximation the periportal cell type and are called for simplicity 'periportal'. Inversely, insulin-treated cells compared with control cells contained a 2.2-fold higher activity of glucokinase, a slightly decreased activity of phosphoenolpyruvate carboxykinase, increased activities of phosphofructokinase and pyruvate kinase and unaltered levels of glucose-6-phosphatase and fructose-bisphosphatase; they resembled perivenous cells and are called simply 'perivenous'. Gluconeogenesis and glycolysis were studied under various substrate and hormone concentrations. 2. Physiological concentrations of glucose (5 mM) and lactate (2 mM) gave about 80% saturation of gluconeogenesis from lactate and less than 15% saturation of glycolysis at a simultaneous 40% inhibition of the glycolytic rate by lactate. 3. Comparison of the two cell types showed that under identical assay conditions (5 mM glucose, 2 mM lactate, 0.5 nM insulin, 0.1 muM dexamethasone) gluconeogenesis was 1.5-fold faster in the 'periportal' cells and glycolysis was 2.4-fold faster in the 'perivenous' cells. 4. Metabolic rates were under short-term hormonal control. Insulin increased glycolysis three fold in both cell types with a half-maximal effect at about 0.4 nM, but did not influence the gluconeogenic rate. Glucagon inhibited glycolysis by 70% with a half-maximal effect at about 0.1 nM. Gluconeogenesis was stimulated by glucagon (half-maximal dose: 0.5 nM) 1.8-fold only in 'periportal' cells containing high phosphoenolpyruvate carboxykinase activity, not in the 'perivenous' cells with a low level of this enzyme. 5. A comparison of the two cell types showed that with maximally stimulating hormone concentrations gluconeogenesis was threefold faster in 'periportal' cells and glycolysis was eightfold faster in 'perivenous' cells. The results support the view that periportal and perivenous hepatocytes in vivo catalyse gluconeogenesis and glycolysis at inverse rates.     

Non-proton-motive-force-dependent sodium efflux from the ruminal bacterium Streptococcus bovis: bound versus free pools.

Growing cells of Streptococcus bovis JB1 had a sodium content of 1,125 nmol/mg of protein and, based on a ratio of cell volume to protein of 4.3 microliters/mg, the apparent intracellular sodium concentration was more than 240 mM. Much of this sodium could not be removed by water washing even if cells were boiled or treated with the pore-forming ionophore, gramicidin, but it could be exchanged for potassium. Stationary cultures had a 2.6-microliters volume per milligram of protein and a total sodium content of 410 mM. When stationary cultures were energized with glucose at pH 6 to 8, sodium (more than 200 mM) was expelled within 2 min, and it appeared that growing cells had a very small pool of free intracellular sodium. Sodium-proton antiport activity could not be demonstrated with a sodium pulse, and the protonophore SF6847, valinomycin, and the H+-ATPase inhibitor dicyclohexylcarbodiimide (DCCD) had little effect on sodium efflux, even though these inhibitors greatly reduced the proton-motive force. SF6847, valinomycin, and DCCD had little effect on intracellular ATP, but iodoacetate, an inhibitor of glycolysis, decreased ATP as well as sodium efflux. Stationary cells from sodium-deficient medium expelled little sodium after glucose addition and had 35% more ATP than stationary cells which were grown in sodium medium and expelled sodium. An artificial electrochemical gradient of sodium was able to drive ATP synthesis in stationary cells, and this ATP formation was not sensitive to DCCD. These results indicated that bacteria could have a significant pool of bound sodium and that sodium expulsion from S. bovis was directly coupled to ATP hydrolysis.     

Phosphorus-31 nuclear magnetic resonance studies of bioenergetics in wild-type and adenosinetriphosphatase(1-) Escherichia coli cells

By use of 31P NMR, the transmembrane pH gradient (delta pH) and the intracellular levels of phosphorylated metabolites were measured in aerobic suspensions of wild-type Escherichia coli cells in the presence and absence of the adenosinetriphosphatase (ATPase) inhibitor dicyclohexylcarbodiimide (DCCD); the same parameters were also determined in E. coli mutants deficient in ATPase activity under both anaerobic and aerobic conditions. A method is described by which dense suspensions of E. coli cells (approximately 3 X 10(11) cells/mL) were oxygenated so that steady-state O2 levels in the suspensions were far greater than the Km for O2 consumption. Under these conditions, in wild-type MRE600 cells, the intracellular concentrations of PI, NTP, and NDP were measured to be 3.0 +/- 1.5, 8 +/- 1, and 1.2 +/- 1 mM, respectively, while the intracellular pH was approximately 7.5 over the external pH range studied (6 to approximately 7.0). Upon treatment with DCCD, the intracellular NTP level was drastically reduced and intracellular Pi concentration increased in respiring wild-type cells; in the same cells, however, DCCD did not affect the intracellular pH and the delta pH. During respiration in the presence of lactate, ATPase- cells established a delta pH but failed to synthesize any detectable levels of NTP. Conversely, ATPase- cells accumulated high levels of NTP but did not generate a delta pH during glycolysis under anaerobic conditions. These results are in complete agreement with the generally accepted chemiosmotic hypothesis. 31P NMR data on intact ATPase- NR70 cells were in agreement with the previously proposed [Rosen, B. P., Brey, R., & Hasan, S. (1978) J. Bacteriol. 134, 1030] existence of a proton leak in this strain which is sealed by DCCD or by spontaneous mutation into strain NR71. However, the NMR data also indicated that other major differences exist between NR71 and NR70 cells.     

Non-proton-motive-force-dependent sodium efflux from the ruminal bacterium Streptococcus bovis: bound versus free pools

Growing cells of Streptococcus bovis JB1 had a sodium content of 1,125 nmol/mg of protein and, based on a ratio of cell volume to protein of 4.3 microliters/mg, the apparent intracellular sodium concentration was more than 240 mM. Much of this sodium could not be removed by water washing even if cells were boiled or treated with the pore-forming ionophore, gramicidin, but it could be exchanged for potassium. Stationary cultures had a 2.6-microliters volume per milligram of protein and a total sodium content of 410 mM. When stationary cultures were energized with glucose at pH 6 to 8, sodium (more than 200 mM) was expelled within 2 min, and it appeared that growing cells had a very small pool of free intracellular sodium. Sodium-proton antiport activity could not be demonstrated with a sodium pulse, and the protonophore SF6847, valinomycin, and the H+-ATPase inhibitor dicyclohexylcarbodiimide (DCCD) had little effect on sodium efflux, even though these inhibitors greatly reduced the proton-motive force. SF6847, valinomycin, and DCCD had little effect on intracellular ATP, but iodoacetate, an inhibitor of glycolysis, decreased ATP as well as sodium efflux. Stationary cells from sodium-deficient medium expelled little sodium after glucose addition and had 35% more ATP than stationary cells which were grown in sodium medium and expelled sodium. An artificial electrochemical gradient of sodium was able to drive ATP synthesis in stationary cells, and this ATP formation was not sensitive to DCCD. These results indicated that bacteria could have a significant pool of bound sodium and that sodium expulsion from S. bovis was directly coupled to ATP hydrolysis.     

Spreading of HeLa cells on a collagen substratum requires a second messenger formed by the lipoxygenase metabolism of arachidonic acid released by collagen receptor clustering.

HeLa cells attach to a variety of substrata but spread only on collagen or gelatin. Spreading is dependent on collagen-receptor upregulation, clustering, and binding to the cytoskeleton. This study examines whether second messengers are involved in initiating the spreading process on gelatin. The levels of cytosolic free calcium ([Ca++]i), cAMP, and cytoplasmic pH (pHi) do not change during cell attachment and spreading. However, a basal level of [Ca++]i and an alkaline pH(i) are required for spreading. There is an activation of protein kinase C (PKC) and a release of arachidonic acid (AA) on attachment and before cell spreading. Inhibition of PKC does not block cell spreading, indicating that PKC activation is not essential for spreading. Inhibition of phospholipase A2 blocks cell spreading, whereas addition of exogeneous AA overcomes this inhibitory effect. Among AA metabolic pathways, inhibitors of lipoxygenase (LOX) block cell spreading, suggesting that a LOX product(s) formed from AA initiates spreading. Clustering receptors for collagen with polyclonal antibodies, or with anti-collagen-receptor antigen-binding fragments (Fab) in combination with a secondary antibody, induce AA release. Also, AA is released when cells attach to either immobilized gelatin or immobilized Arg-Gly-Asp (RGD) peptide. Thus, AA is released whenever receptor clustering is observed. Receptor occupancy is not sufficient to release AA; when cells are treated with gelatin or RGD peptide in solution or anti-collagen-receptor Fab fragments without secondary antibody, conditions where receptor clustering is not observed, AA is not released. Thus, a LOX metabolite(s) of AA formed by collagen-receptor clustering is a second messenger(s) that initiates HeLa cell spreading. LOX inhibitors also block the spreading of bovine aortic endothelial cells, chicken embryo fibroblasts, and CV-1 fibroblasts on gelatin or fibronectin, indicating that other cells might use the same second messenger system in initiating cell-substratum adhesion.     

Energy coupling for membrane hyperpolarization in Lemna: Evidence against an ATP-fueled electrogenic pump as the exclusive mechanism

The vacuolar electrical potential of Lemna paucicostata 6746 has an active component of about-130 mV. This hyperpolarization above the diffusion potential was maintained when dicyclohexyl carbodiimide (DCCD) or arsenate (0.1 mM or 5 mM final concentrations, respectively) were added in the light or after the plants had been kept in darkness for 1 h. The ATP level was reduced to 11±3% by DCCD and to 56±6% by arsenate under conditions identical to those during the potential measurements. In this report, it is discussed whether these results could be interpreted in terms of a putative electrogenic ATPase in the plasma membrane of Lemna. Rb⁺-influx in illuminated plants was 12.5% or 52% of the control when ATP generation was inhibited by DCCD or arsenate. This finding is regarded as justifying the assumption that the availability of ATP at plasmalemma-located transport sites is drastically decreased by these inhibitors.A passive proton-permeability in the cell membrane was induced with different concentrations of carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The potential decrease, caused by the current through this shunt, was not affected by DCCD. It therefore seems less conceivable that the cell membrane remains hyperpolarized because of an increase of membrane resistance concomitant to the inhibition of the pump.The significance of respiratory processes for membrane hyperpolarization is displayed by the depolarizing action of anoxia or KCN. As ATP was found to be non-limiting under these conditions, the inhibition of the electrogenic pump is regarded as being in discord with the concept of an electrogenic ATPase, which is solely responsible for membrane hyperpolarization.Abbreviations CCCP carbonyl cyanide m-chlorophenyl hydrazone - DCCD N, N-dicyclohexyl carbodiimide - DES diethylstilbestro - DNP 2,4-dinitrophenol - POPOP 1,4-bis (2-(5-phenyloxazolyl))-benzene - PPO 2,5-diphenyloxazole     

Properties of a transplasma membrane electron transport system in HeLa cells

A transmembrane electron transport system has been studied in HeLa cells using an external impermeable oxidant, ferricyanide. Reduction of ferricyanide by HeLa cells shows biphasic kinetics with a rate up to 500 nmoles/min/g w.w. (wet weight) for the fast phase and half of this rate for the slow phase. The apparentK _m is 0.125 mM for the fast rate and 0.24 mM for the slow rate. The rate of reduction is proportional to cell concentration. Inhibition of the rate by glycolysis inhibitors indicates the reduction is dependent on glycolysis, which contributes the cytoplasmic electron donor NADH. Ferricyanide reduction is shown to take place on the outside of cells for it is affected by external pH and agents which react with the external surface. Ferricyanide reduction is accompanied by proton release from the cells. For each mole of ferricyanide reduced, 2.3 moles of protons are released. It is, therefore, concluded that a transmembrane redox system in HeLa cells is coupled to proton gradient generation across the membrane. We propose that this redox system may be an energy source for control of membrane function in HeLa cells. The promotion of cell growth by ferricyanide (0.33–0.1 mM), which can partially replace serum as a growth factor, strongly supports this hypothesis.     

Cytometric assessment of DNA damage by exogenous and endogenous oxidants reports aging-related processes.

The ongoing DNA damage caused by reactive oxygen species generated during oxidative metabolism is considered a key factor contributing to cell aging as well as preconditioning cells to neoplastic transformation. We postulated before that a significant fraction of constitutive histone H2AX phosphorylation (CHP) and constitutive activation of ATM (CAA) seen in untreated normal and tumor cells occurs in response to such DNA damage. In the present study, we provide further evidence in support of this postulate. The level of ATM activation and H2AX phosphorylation, detected immunocytochemically, has been monitored in WI-38, A549, and TK6 cells treated with H2O2 as well as growing under conditions known or suspected to affect the level of endogenous oxidants. Thirty- to 60-min exposure of cells to 100 or 200 microM H2O2 led to an increase in the level of H2AX phosphorylation and ATM activation, particularly pronounced (nearly fivefold) in S-phase cells. Cell growth for 24-48 h under hypoxic conditions (3% O2) distinctly lowered the level of CHP and CAA while it had minor effect on cell cycle progression. Treatment (4 h) with 0.1 or 0.3 mM 3-bromopyruvate, an inhibitor of glycolysis and mitochondrial oxidative phosphorylation, reduced the level of CHP (up to fourfold) and also decreased the level of CAA. Growth of WI-38 cells in 2% serum concentration for 48 h led to a 25 and 30% reduction in CHP and CHA, respectively, compared with cells growing in 10% serum. The antioxidant vitamin C (2 mM) reduced CHP and CAA by 20-30% after 24 h of treatment, while the COX-2 inhibitor celecoxib (5 microM) had a minor effect on CHP and CAA, though it decreased the level of H2O2-induced H2AX phosphorylation and ATM activation. In contrast, dichloroacetate known to shift metabolism from anaerobic to oxidative glycolysis through its effect on pyruvate dehydrogenase kinase enhanced the level of CHP and CAA. Our present data and earlier observations strongly support the postulate that a large fraction of CHP and CAA occurs in response to DNA damage caused by metabolically generated oxidants. Cytometric analysis of CHP and CAA provides the means to measure the effectiveness of exogenous factors, which either through lowering aerobic metabolism or neutralizing radicals may protect DNA from such damage.     

Calcium-mediated cell surface changes in Chinese hamster cells.

Scanning electron microscopy (SEM) revealed quantitative differences in both surface characteristics and spreading behavior of young and senescent WI38 human diploid fibroblasts. Measurement of spreading behavior involved the determination of the rate of cell elongation beyond an axial ratio of 1.5 after time intervals of 1.5, 3, 6, 9, and 12 h following subculture. Early passage cells exhibited a 44–52% increased spreading rate compared with later passage cells, both obeying zero-order kinetics. Surface microvilli and blebs were also found to vary in number and size after those same time intervals following subculture. In young cells, microvilli density was found initially to be very low but then increased steadily with time. Later passage cells exhibited an initial high density of microvilli, followed by a gradual disappearance with time. The density of cellular blebs did not, however, vary significantly with time following subculture. After approx. 24 h, both early and late passage cells resembled each other. These studies do show that physical membrane characteristics of young and senescent cells differ under certain conditions, and suggest the importance of that structure in the phenomena of senescence.     

An antiglycolipid antibody inhibits Madin-Darby canine kidney cell adhesion to laminin and interferes with basolateral polarization and tight junction formation

Epithelial cells polarize not only in response to cell-cell contacts, but also to contacts with a substratum composed of extracellular matrix molecules. To probe the role of specific matrix constituents in epithelial cell polarization, we investigated the effects of an adhesion-blocking mAb, 12B12, on initial polarization of MDCK cells. The 12B12 antibody, raised against whole MDCK cells, blocks adhesion to laminin by 65% but has no effect on adhesion of cells to collagen type I. Taking advantage of this antibody's function-blocking activity, as well as the fact that MDCK cells secrete laminin, the role of endogenous laminin in polarization was examined by plating cells on collagen-coated substrata in the presence of the antibody. Under these conditions, cell spreading was reduced 1.5h after plating, and cells were flatter and had fewer microvilli after 24 h. Even though lateral cell membranes were closely apposed, transepithelial resistance in the presence of the antibody was significantly reduced relative to controls. When the polarization of specific apical and basolateral markers was examined both biochemically and immunocytochemically in the presence of the antibody, we observed that the apical marker polarized at normal rates while basolateral markers did not. Surprisingly, the 12B12 antibody was not directed against any known cell adhesion protein but reacted specifically with Forssman antigen, a glycosphingolipid. These results suggest that glycolipids may play a significant role in cell adhesion via laminin and in epithelial cell polarization.     

The Relationship between Cell Membrane Potassium Ion Transport and Glycolysis : The effect of ethacrynic acid

Cell membrane transport of K⁺ stimulates the rate of glycolysis in Ehrlich ascites tumor cells. A study of the characteristics of this relationship indicates that the stimulation occurs under anaerobic as well as under aerobic conditions. The data suggest that glycolysis is stimulated by a K⁺ transport mechanism that is coupled to Na⁺ transport because the effect is blunted or abolished when the principal intracellular ion is lithium or choline. This stimulus to glycolysis is blocked by ouabain and ethacrynic acid, agents that have been shown to inhibit monovalent cation transport in erythrocytes. In contrast to the action of ouabain, glycolysis is inhibited by ethacrynic acid in Ehrlich ascites tumor cells in the absence of cell membrane K⁺ transport. In studies with ghost-free hemolysates of human erythrocytes and with cytosol prepared from Ehrlich ascites tumor cells, ethacrynic acid significantly blocks lactate formation from fructose diphosphate demonstrating the direct inhibitory effect of this agent on one or more enzymes of the Embden-Meyerhof pathway. Since ethacrynic acid has no influence on lactate formation in intact erythrocytes utilizing an endogenous substrate, the presumptive site of inhibition is proximal to the 3-phosphoglycerate level.     

Involvement of glycolytic metabolism in developmental inhibition of rat two-cell embryos by phosphate

To elucidate the mechanism by which phosphate induces developmental inhibition of rat 2-cell embryos, we examined the mutual effects of glucose and other glycolytic and non-glycolytic sugars, the non-metabolizable glucose analogue, and glycolytic inhibitors on the inhibitory effect of phosphate. In the absence of glucose, 30-49% of embryos treated with 10-500 microM phosphate were able to develop to morula and blastocysts. On the other hand, in the presence of 5 mM glucose, 10 microM phosphate decreased the developmental rate of 2-cell embryos to the 4-cell stage and completely inhibited the development beyond the 4-cell stage. In contrast, glucose showed no influence on development in phosphate-free medium. Similarly to glucose, the other glycolytic sugars fructose (5 mM) and mannose (5 mM) enhanced the inhibitory effect of 10 microM phosphate but had no influence in the absence of phosphate. In contrast, the non-glycolytic sugar and non-metabolizable glucose analogue N-acetylglucosamine and 3-O-methylglucose (3-O-MGlc), respectively, did not enhance the effects of phosphate. 2-Deoxyglucose (2DGlc), another glucose analogue that is non-metabolizable but is converted by hexokinase to 2DGlc 6-phosphate, at concentrations as low as 0.1 mM completely inhibited cell cycle progression of 2-cell embryos cultured in glucose-free (Glc(-)) medium with 10 microM phosphate. In contrast, in the absence of phosphate, 2DGlc at the same concentration allowed 55% of 2-cell embryos to develop to morula and blastocyst stages. Addition of an inhibitor of enolase in glycolysis, sodium fluoride (NaF), at 1 mM to the Glc(-) medium also enhanced the inhibitory effects of 10 microM phosphate, whereas 1 mM NaF in the absence of phosphate showed no inhibitory effects on the development of 2-cell embryos to morula and blastocyst stages. From these results, disturbance of glycolysis is a critical reason for the developmental inhibition caused by phosphate in early rat embryos in culture.     

Kinetics of inhibition of the plasma membrane calcium pump by vanadate in intact human red cells.

The lack of specific inhibitors of the plasma membrane Ca2+ pump (PMCA) has made vanadate (VO3-), a non-specific inhibitor, an invaluable tool in the study of PMCA function. However, three important properties of vanadate as an inhibitor of the PMCA in intact cells, namely its speed of action in different experimental conditions, the reversibility of its inhibitory effects at different doses, and its dose-response, had never been characterized, despite extensive use. We report here the speed, reversibility and dose-response of PMCA inhibition by vanadate in intact human red cells. Near maximal inhibitory concentrations (1mM) in the red cell suspension blocked almost instantly the uphill Ca2+ extrusion by the PMCA, regardless of the intracellular Ca2+ concentration, cation composition of the external media, membrane potential or volume-stability of the cell. PMCA inhibition by vanadate, at concentrations of 10mM and 1mM, was not reversed by washing, resuspending, and incubating the cells for up to 2h in vanadate-free media. Vanadate inhibited PMCA-mediated Ca2+ efflux in intact red cells with a K1/2 of approximately 3 microM, a value similar to that described for the Ca2+-ATPase in isolated red cell membranes.     

Adenine Nucleotide Levels in Neurospora, as Influenced by Conditions of Growth and by Metabolic Inhibitors

By a combination of luciferase and fluorescence methods adenine nucleotide pools in Neurospora crassa have been examined under various conditions of growth and metabolic inhibition. During sustained exponential growth (25 C, shaking liquid cultures), the intracellular adenosine 5'-triphosphate (ATP) concentration, [ATP](i), rises slowly from the conidial level near 1 mM (1 mmol/kg of cell water) to a maximum of 2.0 to 2.5 mM at 14 h, after which it slowly declines. The adenosine 5'-diphosphate and adenosine 5'-monophosphate (AMP) curves show two peaks, at 8 and 20 h, with a minimum at 16 h. The "energy charge" function varies around a mean of 0.72 throughout the period of exponential growth. Transferral of growing cells to buffer lacking a nitrogen source stabilizes the [ATP](i) near 2.5 mM, apparently independent of the cell age, and most studies of metabolic inhibitors were carried out on cells grown 14 to 16 h and then shifted to N-free buffer. Under these conditions sudden respiratory blockade (cyanide) produces exponential decay of ATP with a time constant of about 5.7 s (half-time of 3.9 s), and at a rate which implies a minimal ATP turnover of 0.44 mM/min. This figure is about one-third the rate (1.17 mM/min) which would be calculated from steady-state respiration, a discrepancy which may partly be accounted for by transphosphorylation from appreciable amounts of non-adenine nucleoside di- and triphosphates present in Neurospora. For all three adenine nucleotides, the transients associated with sudden respiratory blockade include overshoots or undershoots of several minutes duration, which are consistent with feedback regulation of glycolysis by the AMP/ATP ratio.     

Cell proliferation and polyamine metabolism in 9L cells treated with (2R,5R)-6-heptyne-2,5-diamine or alpha-difluoromethylornithine

We studied the effects of the ornithine decarboxylase inhibitors (2R,5R)-6-heptyne-2,5-diamine (R,R,-MAP) and alpha-difluoromethylornithine (DFMO) on cell proliferation and polyamine metabolism in 9L rat brain tumour cells. Treatment with 5 microM R,R-MAP inhibited cell proliferation to the same extent as did treatment with 1 mM DFMO. Both inhibitors depleted putrescine and spermidine concentrations to less than detectable levels within 24 h and 48 h of drug treatment, respectively; spermine levels were not affected significantly by either inhibitor. The effects of DFMO on 9L cell cycle kinetics were similar to those of R,R-MAP. During the first 3 days of treatment, both drugs caused an accumulation of cells in G1 and a reduction of cells in S phase, as compared with control cells with a slowing in the rate of cell cycle traverse. In cultures seeded at low (1 x 10(5)), medium (5 x 10(5)), or high (2 x 10(6)) cell densities in a 25 cm2 flask, inhibition of cell proliferation and polyamine depletion by both R,R-MAP and DFMO was more pronounced at the lower densities relative to the density-matched control cells. Thus, R,R-MAP was a more potent inhibitor of ornithine decarboxylase than was DFMO in 9L cells, and the inhibitory effects of both compounds on cell proliferation and polyamine biosynthesis were greater in actively proliferating cells.     

Inhibition of ATP dephosphorylation by acaricides with emphasis on the anti-ATPase activity of the carbodiimide metabolite of diafenthiuron.

3-(2,6-Diisopropyl-4-phenoxyphenyl)-1-tert-butylcarbodiimide (DFCD), a toxic metabolite and photodegradation product of the propesticide diafenthiuron, and dicyclohexylcarbodiimide (DCCD), a commonly used biochemical inhibitory probe, inhibited Mg(2+)-, Na+K(+)-, and Ca2+Mg(2+)-ATPase activities in preparations from bulb mites (Rhizoglyphus echinopus (Fumouze and Robin)), twospotted spider mites (Tetranychus urticae Koch), and bluegill (Lepomis macrochirus Rafinesque) brain. DCCD was more active than DFCD, but neither carbodiimide was very potent. A possible exception occurred with DCCD, which caused 100% inhibition of bulb mite oligomycin-sensitive Mg(2+)-ATPase activity at a concentration of 0.1 mM. Using house fly, Musca domestica L., thorax mitochondrial Mg(2+)-ATPase, we showed that the binding domain for both carbodiimides was in the F0 portion of the enzyme, probably the transmembrane proton channel which is the known site of DCCD binding in proton-translocating ATPases. Certain other specific acaricides were inhibitors (greater than 50% at 0.1 mM) of ATPase preparations from bulb mites. These acaricides included chloropropylate, bromopropylate, oxythioquinox, cyhexatin, and flubenzimine (Mg2+ and Na+K(+)-ATPase), and ovex, chlorbenside, and propargite (Mg(2+)-ATPase). The role of ATPase inhibition in the modes of acaricidal and insecticidal actions of diafenthiuron, the two carbodiimides, and the other compounds is discussed.     

Comparative study of inhibiting photophosphorylation and light-activated ATP hydrolysis in pea chloroplasts by N,N'-dicyclohexylcarbodiimide

Inhibition kinetics of photophosporylation in chloroplast preparations preincubated by N,N'-dicyclohexylcarbodiimide (DCCD) in darkness has been studied. It was found that the higher membrane concentration the lower DCCD/chlorophyll relation sufficient for blocking of ATP synthesis and light-activated hydrolysis. Comparative studies of DCCD-inhibition of the ATP synthesis and light-activated hydrolysis showed that the latter process was more sensitive to DCCD. In the thylakoid suspensions with concentration Chlorophyll 4 mg/ml 50% inhibition of ATP hydrolysis was observed at the DCCD/Chlorophyll ratio of 0.012, and 50% inhibition of ATP--at 0.02. Inhibition kinetics of light-activated hydrolysis and synthesis corresponded to Hill equation with Hill coefficients 9.1 and 5.8 correspondingly. Different mechanisms of participation of DCCD-binding subunits in ATP-synthesis and ATP-hydrolysis processes have been discussed.     

Role of Src in angiopoietin 1-induced capillary morphogenesis of endothelial cells: Effect of chronic hypoxia on Src inhibition by PP2

Signal transduction pathways leading to angiopoietin 1 (Ang1)-induced capillary morphogenesis by endothelial cells remain poorly defined. Angiogenic cellular responses by endothelial cells may be modulated in vivo by chronic hypoxia, such as that induced by tumors. Here, we studied Ang1-induced capillary morphogenesis in human umbilical-vein endothelial cells (HUVECs) cultured chronically under normoxic (21% oxygen) or hypoxic (1.5% oxygen) conditions. Downregulation of Src using a small interfering RNA (siRNA) inhibited Ang1-induced capillary morphogenesis of HUVECs cultured under both conditions by blocking cell spreading and protrusion. Ang1 upregulated the Src-dependent secretion of vascular endothelial growth factor-A (VEGF-A). Blockade of endogenous VEGF-A also inhibited Ang1-induced capillary morphogenesis. Addition of exogenous VEGF-A restored cell spreading and protrusion, leading to Ang1-induced capillary morphogenesis of Src siRNA-treated HUVECs, suggesting that Ang1-induced VEGF-A secretion through Src was required for capillary morphogenesis. PP2 inhibited both Ang1-induced capillary morphogenesis and Src activation in HUVECs cultured under normoxic conditions, but the PP2 activity was significantly impaired in HUVECs cultured under hypoxic conditions. Expression of multidrug resistance-associated protein 1 (MRP 1) was upregulated in hypoxic HUVECs, and treatment with MRP 1 siRNA restored the inhibitory action of PP2. Taken together, our results suggest that Ang1 induces capillary morphogenesis in HUVECs through Src-dependent upregulation of endogenous VEGF-A. Conditions of chronic hypoxia impaired the effect of PP2, possibly via MRP 1.