Decrease In ATP Content Research Articles

Pentachlorophenol and its derivatives induce oxidative damage and morphological changes in human lymphocytes (in vitro)

In this study, the effect of environmental toxins such as pentachlorophenol (PCP), tetrachlorocatechol (TeCC) and tetrachloroguaiacol (TeCG) on human peripheral blood lymphocytes was investigated. All the compounds studied increased the size and granularity of the lymphocytes in the concentrations range from 5 to 600 ppm. The PCP caused the strongest increase in the size of the cells, whereas lymphocytes granularity was more strongly increased by TeCC and PCP than by TeCG. The PCP and its derivatives in the concentrations range from 1 to 125 ppm significantly depleted ATP level. It was also observed that PCP most strongly decreased ATP content at its highest concentration of 125 ppm. Moreover, PCP caused the highest loss of lymphocytes viability in the concentrations range from 125 to 600 ppm. The TeCC in the concentrations of 1 and 5 ppm significantly increased the level of strand breaks in DNA, whereas lower damage was noted for PCP, and particularly for TeCG. The increase in carbonyl groups content was more strongly induced by TeCG and TeCC than by PCP in the concentrations range from 0.04 to 1 ppm; however, in a concentration of 5 ppm, all the compounds studied increased this parameter to a similar degree. DNA and protein damage was the most probably induced by free radical formation, as it was observed that all the compounds examined, and TeCC, in particular, were able of oxidize a fluorescent probe 6-carboxy-2',7'-dichlorodihydrofluorescein in the concentrations range from 0.01 to 1 ppm.

Simulation of the Impact of Elevated Cytosolic Na+ on Ca2+ Handling, Mitochondrial Energetics and Cellular Electrophysiology in Guinea Pig Myocytes

Chronic heart failure is one of the leading causes of morbidity and mortality in the United States. One of the classical strategies for treating heart failure is to inhibit sarcolemmal Na+/K+-ATPase (NKA). Blocking NKA can result in dramatic elevation of [Na+]i, increasing the sarcoplasmic reticulum (SR) Ca2+ load by acting on the plasmalemmal Na+/Ca2+ exchanger (NCX). Whether and how change of [Na+]i affects mitochondrial Ca2+ dynamics and energetics is still under investigation. Since intracellular Na+ is regulated by a complex system involving multiple ions, channels, exchangers and membrane potentials, unraveling its effect on cell physiology and function requires an integrative view of cardiomyocyte physiology. In the present study we developed a mathematical model of cardiomyocyte that incorporates mitochondrial energetics, ion channels and exchangers, and E-C coupling. Using this model, we simulated the effect of elevated cytosolic Na+ on Ca2+ handling, mitochondrial energetics and reactive oxygen species (ROS) generation. Model simulations show that inhibition of NKA (50%) dramatically increased [Na+] in both the cytosol and mitochondria, which consequently caused Ca2+ overload in the cytoplasm during increased workload. Elevated Na+ also decreased ATP concentration and increased mitochondrial ROS production. Concomitant inhibition of mitochondrial Na+/Ca2+ exchanger (mNCE) ameliorated these effects by attenuating cellular Ca2+ overload and increasing [Ca2+]m. Furthermore, inhibiting mNCE also prevented the [ATP]i drop and decreased ROS production. The findings indicate that increasing cytosolic Na+ has an adverse effect on mitochondrial energetics that can be attenuated by simultaneous inhibition of mNCE.

Stimulation of Suicidal Erythrocyte Death by α-Lipoic Acid

Α-lipoic acid, a nutrient with both, antioxidant and oxidant activity induces apoptosis in a variety of cells. Owing to its proapoptotic potency Α-lipoic acid has been suggested for the therapy of cancer. Α-Lipoic acid stimulates apoptosis by induction of oxidative stress and subsequent activation of caspases. Oxidative stress could similarly trigger caspase activation and suicidal erythrocyte death or eryptosis, which is characterized by cell membrane scrambling and cell shrinkage. Eryptosis is triggered by increase of cytosolic Ca²⁺ concentration and/or ceramide formation. The present study explored whether Α -lipoic acid influences eryptosis. Cell membrane scrambling was estimated from binding of annexin V to phosphatidylserine at the erythrocyte surface, cell volume from forward scatter in FACS analysis, cytosolic Ca²⁺ concentration from Fluo3 fluorescence, caspase activation and ceramide formation utilizing respective antibodies, cytosolic ATP concentration from a luciferase-assay. Within 48 hours, exposure to Α-lipoic acid (10 - 75 mM) significantly decreased forward scatter, increased cytosolic Ca²⁺ concentration, decreased ATP concentration, activated caspase 3, stimulated formation of ceramide and triggered annexin V-binding. Glucose depletion (48 h) was followed by decrease of forward scatter and increase of annexin V-binding, effects significantly augmented in the presence of Α-lipoic acid (20 mM). Oxidative stress (30 min 0.3 mM tert-butylhydroperoxide) similarly triggered annexin binding, an effect slightly but significantly blunted by Α-lipoic acid. In conclusion, Α-lipoic acid triggers eryptosis but by the same token counteracts eryptosis during oxidative stress. Α-lipoic acid sensitive eryptosis may lead to anemia and derangements of microcirculation.

Salicylic acid-induced hepatotoxicity triggered by oxidative stress

Salicylic acid is a widely used nonsteroidal anti-inflammatory drug (NSAID). But it is known to cause serious liver damage occasionally. Mitochondrial dysfunction and oxidative stress are predicted to be the major factors of salicylic acid-induced liver injury. We investigated the influence of salicylic acid on ATP contents, oxygen consumption and lipid peroxidation in the presence of the same concentration of salicylic acid. Leakage of lactate dehydrogenase (LDH) was significantly higher in the presence of 5 mM salicylic acid than in its absence. Salicylic acid-induced thiobarbituric acid-reactive substance (TBARS) formation and spontaneous chemiluminescence (CL) in rat hepatocytes, whereas antioxidants, such as promethazine (PMZ) and N,N-diphenylphenylenediamine (DPPD), suppressed both TBARS formation and LDH leakage. TBARS formation in rat liver microsomes was suppressed by diethyldithiocarbamate (a specific inhibitor of cytochrome P450 (CYP)2E1) and diclofenac (a specific inhibitor of CYP2C11). These results suggest that salicylic acid-induced lipid peroxidation was related to oxidative metabolism mediated by CYP2E1 and CYP2C11. On the other hand, 5 mM salicylic acid induced a drastic decrease of ATP contents in rat isolated hepatocytes. Furthermore, mitochondrial respiration control ratio (RC ratio), calculated by State 3/State 4 also decreased with the increase of salicylic acid concentration. These findings suggest that salicylic acid would trigger mitochondrial dysfunction and cause ATP decrease, leading to lethal liver cell injury by lipid peroxidation, although this hypothesis remains to be elucidated in vivo.

Keratins modulate the shape and function of hepatocyte mitochondria: a mechanism for protection from apoptosis

Absence or mutation of keratins 8 (K8) or 18 (K18) cause predisposition to liver injury and apoptosis. We assessed the mechanisms of hepatocyte keratin-mediated cytoprotection by comparing the protein expression profiles of livers from wild-type and K8-null mice using two-dimensional differential-in-gel-electrophoresis (2D-DIGE) and mass spectrometry. Prominent among the alterations were those of mitochondrial proteins, which were confirmed using 2D-DIGE of purified mitochondria. Ultrastructural analysis showed that mitochondria of livers that lack or have disrupted keratins are significantly smaller than mitochondria of wild-type livers. Immunofluorescence staining showed irregular distribution of mitochondria in keratin-absent or keratin-mutant livers. K8-null livers have decreased ATP content; and K8-null mitochondria have less cytochrome c, increased release of cytochrome c after exposure to Ca(2+) and oxidative stimulation, and a higher sensitivity to Ca(2+)-induced permeability transition. Therefore, keratins play a direct or indirect role in regulating the shape and function of mitochondria. The effects of keratin mutation on mitochondria are likely to contribute to hepatocyte predisposition to apoptosis and oxidative injury, and to play a pathogenic role in keratin-mutation-related human liver disease.

ATP modulates Ca2+uptake by TRPV6 and is counteracted by isoform‐specific phosphorylation

Ca(2+) homeostasis requires balanced uptake and extrusion, and dysregulation leads to disease. TRPV6 channels are homeostasis regulators, are upregulated in certain cancers, and show an unusual allele-specific evolution in humans. To understand how Ca(2+) uptake can be adapted to changes in metabolic status, we investigate regulation of Ca(2+)-influx by ATP and phosphorylation. We show that ATP binds to TRPV6, reduces whole-cell current increments, and prevents channel rundown with an EC(50) of 380 microM. By using both biochemical binding studies and patch-clamp analyses of wild-type and mutant channels, we have mapped one relevant site for regulation by ATP to residues within the ankyrin repeat domain (ARD) and identify an additional C-terminal binding region. Stimulation of PKC largely prevented the effects of ATP. This regulation requires PKC(betaII) and defined phosphorylation sites within the ARD and the C-terminus. Both regulatory sites act synergistically to constitute a novel mechanism by which ATP stabilizes channel activity and acts as a metabolic switch for Ca(2+) influx. Decreases in ATP concentration or activation of PKC(betaII) disable regulation of the channels by ATP, rendering them more susceptible to inactivation and rundown and preventing Ca(2+) overload.

Mitochondrial dysfunction enhances the migration of vascular smooth muscles cells via suppression of Akt phosphorylation

Background Atherosclerosis is one of the major complications of diabetes, which may result from insulin resistance via mitochondrial dysfunction. Although a strong association between insulin resistance and cardiovascular disease has been suggested, it is not clear yet whether stress-inducing factors damage mitochondria and insulin signaling pathway in cardiovascular tissues. Methods We investigated whether stress-induced mitochondrial dysfunction might alter the insulin/Akt signaling pathway in A10 rat vascular smooth muscle cells (VSMC). Results The treatment of oxidized low density lipoprotein (oxLDL) decreased ATP contents, mitochondrial respiration activity, mRNA expressions of OXPHOS subunits and IRS-1/2 and insulin-mediated phosphorylations of Akt and AMP-activated protein kinase (AMPK). Similarly, dideoxycytidine (ddC), the mtDNA replication inhibitor, or rotenone, OXPHOS complex I inhibitor, inhibited the insulin-mediated pAkt while increased pAMPK regardless of insulin. Reciprocally, an inhibitor of Akt, triciribine (TCN), decreased cellular ATP contents. Overexpression of Akt dominant positive reversed the oxLDL- or ddC-mediated ATP decrease but AMPK activator did not. Akt activation also normalized the aberrant VSMC migration induced by ddC. Conclusions Defective insulin signaling and mitochondrial function may collectively contribute to developing cardiovascular disease. General significance Akt may be a possible therapeutic target for treating insulin resistance-associated atherosclerosis.

CO2 assimilation, ribulose-1,5-bisphosphate carboxylase/oxygenase, carbohydrates and photosynthetic electron transport probed by the JIP-test, of tea leaves in response to phosphorus supply.

BackgroundAlthough the effects of P deficiency on tea (Camellia sinensis (L.) O. Kuntze) growth, P uptake and utilization as well as leaf gas exchange and Chl a fluorescence have been investigated, very little is known about the effects of P deficiency on photosynthetic electron transport, photosynthetic enzymes and carbohydrates of tea leaves. In this study, own-rooted 10-month-old tea trees were supplied three times weekly for 17 weeks with 500 mL of nutrient solution at a P concentration of 0, 40, 80, 160, 400 or 1000 μM. This objective of this study was to determine how P deficiency affects CO2 assimilation, Rubisco, carbohydrates and photosynthetic electron transport in tea leaves to understand the mechanism by which P deficiency leads to a decrease in CO2 assimilation.ResultsBoth root and shoot dry weight increased as P supply increased from 0 to 160 μM, then remained unchanged. P-deficient leaves from 0 to 80 μM P-treated trees showed decreased CO2 assimilation and stomatal conductance, but increased intercellular CO2 concentration. Both initial and total Rubisco activity, contents of Chl and total soluble protein in P-deficient leaves decreased to a lesser extent than CO2 assimilation. Contents of sucrose and starch were decreased in P-deficient leaves, whereas contents of glucose and fructose did not change significantly except for a significant increase in the lowest P leaves. OJIP transients from P-deficient leaves displayed a rise at the O-step and a depression at the P-step, accompanied by two new steps at about 150 μs (L-step) and at about 300 μs (K-step). RC/CSo, TRo/ABS (or Fv/Fm), ETo/ABS, REo/ABS, maximum amplitude of IP phase, PIabs and PItot, abs were decreased in P-deficient leaves, while VJ, VI and dissipated energy were increased.ConclusionP deficiency decreased photosynthetic electron transport capacity by impairing the whole electron transport chain from the PSII donor side up to the PSI, thus decreasing ATP content which limits RuBP regeneration, and hence, the rate of CO2 assimilation. Energy dissipation is enhanced to protect P-deficient leaves from photo-oxidative damage in high light.

Disturbances of energetic metabolism in rat epididymal epithelial cells as a consequence of chronic lead intoxication

Adult male Wistar rats were intoxicated with 1% lead acetate (PbAc) administered in drinking water for nine months, which amounts to a period five times longer than the duration of one spermatogenesis. There were mitochondrial ultrastructure disorders of epididymal epithelial cells observed in PbAc-treated rats; also a significant lead-induced decrease in ATP concentration in epididymal epithelial cells (by 32%, P < 0.05), Adenylate Energy Charge value (AEC) (by 8%, P < 0.05) and an increase in ADP (28.5%, P < 0.05), AMP (27%, P < 0.05) and adenosine (by 56%, P < 0.05). The results were measured using high performance liquid chromatography (HPLC) and detected even at low lead concentrations in whole blood (M:7.03 μg/dL; Q1-Q3: 2.99-7.65). The function of mitochondria in cultured epididymal epithelial cells of control and PbAc-treated animals were evaluated using fluorophores: Mitotracker Green FM and JC-1. After incubation with Mitotracker Green FM, we observed active mitochondria producing bright green fluorescence in the cytoplasm of cultured epididymal epithelial cells, both in the control group and the Pb-treated animals. Incubation of cultured epididymal epithelial cells of animals from both groups produced red-orange fluorescence with the mitochondrial JC-1 probe indicating mitochondria with high membrane potential (ΔΨm > 80-100 mV) and green fluorescence in the mitochondria with low membrane potential (ΔΨm < 80 mV). The results showed that a chronic low-level exposure to lead, even without severe clinical symptoms of contamination, disrupted the ultrastructure and energy metabolism of mitochondria in epididymal epithelial cells.

Bioenergetics of fish spermatozoa during semen storage

This mini-review focuses on changes in ATP and creatine phosphate concentrations in fish sperm under storage conditions. The storage of catfish sperm at 4 degrees C leads to ATP depletion and decreased sperm motility. The rate of intracellular ATP depletion can be diminished through the addition of energetic substrates to the sperm storage medium, with lactate + pyruvate being the most efficient substrates for maintaining ATP concentrations in catfish sperm. The decrease in ATP concentration is closely associated with increases in AMP and hypoxanthine content. In contrast to catfish sperm, carp sperm is able to maintain intracellular ATP concentration close to the physiological level during storage. Collectively, these results suggest that fish species differ in terms of the energy metabolism of their spermatozoa and that the semen storage medium must be carefully selected for a particular fish species so as to maintain the ATP concentration and adenylate energy charge close to physiological values as long as possible.

Inhibition of erythrocyte phosphoribosyltransferases (APRT and HPRT) by Pb 2+: A potential mechanism of lead toxicity

Many reports show that red blood cells of people exposed to lead have a decreased ATP concentration, decreased adenylate energy charge value and many metabolic and morphological abnormalities. Since the synthesis of nucleotides in erythrocytes occurs only through salvage pathways, we hypothesized that a decrease in nucleotide concentrations may be caused by lead-induced inhibition of erythrocyte phosphoribosyltransferases: adenine APRT (EC 2.4.2.7) and hypoxanthine-guanine HPRT (EC 2.4.2.8). These enzymes enable the reutilization of purine bases (adenine, guanine, hypoxanthine) converting them to mononucleotides (AMP, GMP, IMP), substrates for the synthesis of high-energy nucleotides. To confirm the hypothesis two experiments were performed: (i) in vitro, using a lysate of human erythrocytes incubated (5, 10, 30 min) with lead ions (100 μM, 10 μM, 1 μM, 500 nM, 100 nM lead acetate) and 100 μM sodium acetate for the control, (ii) in vivo, using a lysate of rat erythrocytes taken from rats chronically exposed to lead (0.1% lead acetate in drinking water for 9 months, resulting in whole blood lead concentration 7 μg/dL). The activities of APRT and HPRT were determined using HPLC method, which allowed concurrent determination of the activity of both enzymes in erythrocyte lysates. We have shown that, lead ions: (i) moderately inhibit both phosphoribosyltransferases in erythrocytes, this influence being detectable even at very low concentrations (ii) participate in hemolysis, the intensity of which negatively correlates with the activity of phosphoribosyltransferases. Our results indicate the necessity of further research on the role of lead-induced APRT and HPRT inhibition as one of the mechanisms of lead toxicity.

Intracellular Water Lifetime Depends on Cellular Energetic State

Intra- and extracellular water undergoes equilibrium exchange via mechanisms that include passive diffusion across the membrane and movement through channels, like aquaporin. Longitudinal 1H2O NMR relaxography can employ extracellular relaxation agent GdDTPA2- to distinguish intra- and extracellular 1H2O signals by creating a difference in their relaxation time constant (T1) values. Inverse Laplace transform of relaxation decay produces the relaxogram, the T1 distribution, which reports apparent water populations, pi′ and pe′. The true water fractions, pi and pe - measures of the volume fractions vi and ve, are determined if the kinetics of transcytolemmal water exchange (the mean intracellular water life time τi) are quantified using two-site-exchange analysis. The “well-mixed” kinetic expression is: τi-1 = PW(A/V), where PW is the water permeability coefficient; A and V are the individual cell surface area and volume, respectively. Furthermore: vi/τi = PWS, where S is the total cell surface area.We determined the relationship between τi and cellular energetics in suspensions of Saccharomyces cerevisiae. Under aerobic conditions τi was 333 (± 4) ms; under anaerobic conditions τi was 670 (± 62) ms. Changing from aerobic to anaerobic conditions also resulted in a decrease of ATP content. In the yeast strain MR6, which requires adenine supplementation to grow, ATP content increased with medium adenine concentration. As ATP was thus increased, the τi was found to inversely correlate with ATP content (R2 = 0.96). Therefore, τi is a sensitive indicator of the cellular energetic state and energetics affects the kinetics of transcytolemmal water exchange. The aerobic PWS was greater than the anaerobic PWS. The underlying mechanism(s) are not yet clear. Experiments with baker's yeast stored in an anaerobic state found that inhibition of protein synthesis prevented the anaerobic-to-aerobic τi decrease and that aquaporin is not responsible.1

The relationship between diphenylamine structure and NSAIDs-induced hepatocytes injury

Many nonsteroidal anti-inflammatory drugs (NSAIDs) with diphenylamine structure induce severe hepatotoxicities. We evaluated the role of diphenylamine structure in liver injuries induced by these NSAIDs. Effects of diphenylamine, diclofenac and tolfenamic acid on mitochondrial permeability transition (MPT) and efflux of calcium in isolated liver mitochondria as well as on cellular ATP content and mitochondrial membrane depolarization in rat primary hepatocyte cultures were examined. Diclofenac and tolfenamic acid induced cyclosporine A (CsA)-sensitive mitochondrial swelling and membrane depolarization in isolated liver mitochondria. Only diclofenac caused the release of calcium in isolated liver mitochondria. Diphenylamine had no effects on isolated liver mitochondria. All three compounds decreased ATP content and induced mitochondrial membrane depolarization. CsA attenuated these effects, suggesting MPT might be involved in the hepatotoxicities caused by diphenylamine, diclofenac and tolfenamic acid. SKF-525A, a general inhibitor of CYP450, markedly inhibited the injury induced by diphenylamine, but not diclofenac or tolfenamic acid. The hepatotoxicities caused by diclofenac and tolfenamic acid may be attributed to the mitochondrial dysfunction induced by these drugs instead of the diphenylamine structure per se.

Abnormal changes in mitochondria, lipid droplets, ATP and glutathione content, and Ca2+ release after electro-activation contribute to poor developmental competence of porcine oocyte during in vitro ageing

The present study aims to investigate major changes in porcine oocytes during ageing in vitro. After the oocytes were cultured for 44, 56, 68 and 80 h, changes to porcine oocytes in ultrastructure, mitochondrial distribution, glutathione (GSH) and ATP content, Ca(2+) release patterns and developmental competence after electro-activation were observed. Mitochondria were evenly distributed in oocytes at 44 h, aggregated in clusters or in peripheral cytoplasm at 68 h and dimly dispersed throughout ooplasm at 80 h. Mitochondrial shape during ageing was also observed by transmission electron microscopy (TEM) at the same time intervals. Most mitochondria were spherical at 44 h, and became elongated when the culture time was extended to 68 h and 80 h. Moreover, mitochondrial clustering became increasingly loose from 56 h. Lipid droplets in oocytes appeared prominent and electron-dense at 44 h, but electron density was lost at 56 h. Lipid droplets were solidified as of 68 h. There was an age-dependent decrease in ATP content per oocyte. Glutathione content per oocyte decreased significantly and remained lower after 56 h. Amplitudes of [Ca(2+)] rise decreased dramatically following 56 h, and the time required for [Ca(2+)] to plateau became shorter after electro-activation with prolonged culture time. Cleavage and blastocyst rates of aged oocytes progressively decreased, while the fragmentation rate gradually increased after electro-activation. It is concluded that abnormal changes in mitochondria, lipid droplets, Ca(2+) release after electro-activation, and ATP and GSH content in oocytes during ageing may result in poor developmental competence of parthenotes.

Decreased energy synthesis is partially compensated by a switch to sucrose synthase pathway of sucrose degradation in restricted root of tomato plants

Tomato (Solanum lycopersium L.) plants were grown hydroponically to investigate the changes of energy metabolism and adaptive mechanism in response to root restriction. Root restriction resulted in a significant increase in root lipid peroxidation and reduction in leaf net CO(2) assimilation rate, which was accompanied by increase of alcohol dehydrogenase (ADH; EC 1.1.1.1) and lactate dehydrogenase (LDH; EC 1.1.1.27) activities. Total, cytochrome pathway, and alternative pathway respirations were all decreased in the roots after 15 days of root restriction treatment. Accompanied with the decrease of ATP content, ratio of invertase/sucrose synthase activity was increased in the restricted roots together with a decrease in glucose content and an increase in fructose content. We concluded that the decreased energy synthesis under root restriction condition was partially compensated by the energy-conserving sucrose synthase pathway of sucrose metabolism.

Src activation generates reactive oxygen species and impairs metabolism–secretion coupling in diabetic Goto–Kakizaki and ouabain-treated rat pancreatic islets

Na(+)/K(+)-ATPase inhibition by ouabain suppresses ATP production by generating reactive oxygen species (ROS) and impairs glucose-induced insulin secretion from pancreatic islets. To clarify the signal-transducing function of Na(+)/K(+)-ATPase in decreasing ATP production by the generation of ROS in pancreatic islets, the involvement of Src was examined. In addition, the significance of Src activation in diabetic islets was examined. Isolated islets from Wistar rats and diabetic Goto-Kakizaki (GK) rats (a model for diabetes) were used. ROS was measured by 5-(and 6)-chloromethyl-2',7'-dichlorofluorescein fluorescence using dispersed islet cells. After lysates were immunoprecipitated by anti-Src antibody, immunoblotting was performed. Ouabain caused a rapid Tyr(418) phosphorylation, indicating activation of Src in the presence of high glucose. The specific Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) restored the ouabain-induced decrease in ATP content and the increase in ROS production. Both PP2 and ROS scavenger restored the impaired insulin release and impaired ATP elevation in GK islets, but had no such effect in control islets. PP2 reduced the high glucose-induced increase in ROS generation in GK islet cells but had no effect on that in control islet cells. Moreover, ouabain had no effect on ATP content and ROS production in the presence of high glucose in GK islets. These results indicate that Src plays a role in the signal-transducing function of Na(+)/K(+)-ATPase, in which ROS generation decreases ATP production in control islets. Moreover, ROS generated by Src activation plays an important role in impaired glucose-induced insulin secretion in GK islets, in which Src is endogenously activated independently of ouabain.

Partitioning of photosynthetic electron flow between CO&lt;sub&gt;2&lt;/sub&gt; assimilation and O&lt;sub&gt;2&lt;/sub&gt; reduction in sunflower plants under water deficit

In sunflower (Helianthus annuus L.) grown under controlled conditions and subjected to drought by withholding watering, net photosynthetic rate (P N) and stomatal conductance (g s) of attached leaves decreased as leaf water potential (Ψw) declined from -0.3 to -2.9 MPa. Although g s decreased over the whole range of Ψw, nearly constant values in the intercellular CO2 concentrations (C i) were observed as Ψw decreased to -1.8 MPa, but C i increased as Ψw decreased further. Relative quantum yield, photochemical quenching, and the apparent quantum yield of photosynthesis decreased with water deficit, whereas non-photochemical quenching (qNP) increased progressively. A highly significant negative relationship between qNP and ATP content was observed. Water deficit did not alter the pyridine nucleotide concentration but decreased ATP content suggesting metabolic impairment. At a photon flux density of 550 µmol m-2 s-1, the allocation of electrons from photosystem (PS) 2 to O2 reduction was increased by 51 %, while the allocation to CO2 assimilation was diminished by 32 %, as Ψw declined from -0.3 to -2.9 MPa. A significant linear relationship between mean P N and the rate of total linear electron transport was observed in well watered plants, the correlation becoming curvilinear when water deficit increased. The maximum quantum yield of PS2 was not affected by water deficit, whereas qP declined only at very severe stress and the excess photon energy was dissipated by increasing qNP indicating that a greater proportion of the energy was thermally dissipated. This accounted for the apparent down-regulation of PS2 and supported the protective role of qNP against photoinhibition in sunflower.

Modulation of the activities of AMP‐activated protein kinase, protein kinase B, and mammalian target of rapamycin by limiting energy availability with 2‐deoxyglucose

2-Deoxyglucose (2-DG), which has been shown to inhibit mammary carcinogenesis, was used as a metabolic probe to investigate effects of limiting energy availability (reduced cellular ATP) on patterns of proteins' phosphorylation that play a role in the development of cancer. Experiments were conducted using a human breast cancer cell line, MDA-MB-468, and 1-methyl-1-nitrosourea-induced rat model for mammary carcinogenesis. Under in vitro conditions in which cellular ATP concentration decreased rapidly with increasing 2-DG in a dose and time dependent manner, levels of phosphorylated mammalian target of rapamycin (P-mTOR) decreased in parallel to decreases in ATP concentration. Concomitantly, phosphorylation of two upstream regulators of mTOR, AMP-activated protein kinase (AMPK) and Akt/protein kinase B were increased and decreased, respectively, with increased levels of phosphorylated acetyl-CoA carboxylase as an indicator of AMPK activation. Levels of insulin like growth factor 1-receptor and phosphoinositide-3 kinase p110 alpha were also reduced. Similar effects were observed in mammary carcinomas in vivo at concentration of 0.03% (w/w) dietary 2-DG that inhibited carcinogenesis. In vitro, downregulation of mTOR was accompanied by decreases in phosphorylation of two of mTOR's targets, 70-kDa ribosomal protein S6 kinase and eukaryote initiation factor 4E binding protein 1. Glucose treatment reversed 2-DG effects. When cells were transfected with dominant-negative AMPK alpha 2, effects of 2-DG on mTOR and its downstream effectors were diminished, providing evidence of a link between AMPK and mTOR when energy availability was limited. This work indicates that AMPK, Akt, and mTOR are candidate targets for efforts to inhibit the carcinogenic process by limiting energy availability.

Comparison of the Mechanisms of Cataract Development Involving Differences in Ca2+ Regulation in Lenses among Three Hereditary Cataract Model Rats

We previously found that the increases in Ca2+ content in the lenses of three hereditary cataract model rats, UPL rat (UPLR), Shumiya cataract rat (SCR) and Ihara cataract rat (ICR), are inhibited by aminoguanidine, a selective inhibitor of inducible nitric oxide synthase, and that the mechanisms of Ca2+ enhancement in these rat models differ. In this study, we compare the mechanisms for dysfunction in Ca2+ regulation in UPLR, SCR and ICR. Decreases in the activity of Ca2+-ATPase were found in the lenses of SCR and ICR concurrent with cataract development. In contrast, the Ca2+-ATPase activity in UPLR with opaque lenses was higher than in those with transparent lenses. On the other hand, ATP levels were markedly decreased in UPLR with opaque lenses. The expression of cytochrome c oxidase (CCO)-1 mRNA and CCO activity in UPLR lenses was found to decrease during cataract development. The nitric oxide (NO) and lipid peroxide levels were also increased in the lenses of UPLR, SCR and ICR with opaque lenses. In UPLR, excessive NO may cause damage to the mitochondrial genome, resulting in a decrease in ATP production and increase in Ca2+-ATPase activity. The decrease in ATP content may cause the decrease in Ca2+-ATPase function resulting in the elevation in lens Ca2+. In SCR and ICR, excessive NO may cause an enhancement of lipid peroxidation resulting in the oxidative inhibition of Ca2+-ATPase. The decrease in Ca2+-ATPase activity may cause the elevation in the level of lens Ca2+, thus leading to lens opacification. Our findings show that the Ca2+ contents in the cataractous lenses of all three model rats are increased, the mechanisms for this Ca2+ enhancement is different in each rat model.

Na–Picotransporter type I activity causes a transient intracellular alkalinization during ATP depletion in rabbit medullary thick ascending limb cells

The cellular pathophysiology of renal ischemia-reperfusion injury was investigated in primary cell cultures from rabbit medullary thick ascending limb (MTAL). Metabolic inhibition (MI) was achieved with cyanide and 2-deoxyglucose. Sixty minutes of MI caused a profound but reversible decrease in intracellular concentration of ATP ([ATP]i). Intracellular pH (pHi) first decreased after initiation of MI, followed by a transient alkalinization. When [ATP]i reached its lowest value (<1% of control), the cells slowly acidified to reach a stable pHi of 6.92 after 50 min of MI. In the presence of EIPA (10 micromol/L), the pattern of changes in pHi was unchanged and acidification was not increased, indicating that the Na+/H+ exchangers were inactive during ATP depletion. When inorganic phosphate (P(i)) or Na+ was omitted from the apical solutions during MI, the transient alkalinization was no longer observed and the cytosol slowly acidified. Experiments on Na+-dependent alkalinizations revealed the presence of a Na-P(i) cotransporter in the apical cell membrane. With indirect immunofluorescence, the Na-P(i) cotransporter expressed in these primary cell cultures could be identified as Na-P(i) type I. Although the exact physiological role of Na-P(i) type I still is unresolved, these experiments demonstrate that apical Na-P(i) type I activity is increased at the onset of ATP depletion in MTAL cells.