Increase In ATP Hydrolysis Research Articles

Increased Graft Survival through Ectonucleotidases Modulation in Platelets and Lymphocytes of Kidney Transplanted Patients

Background: despite limited long-term survival, kidney transplantation is the best form of renal replacement therapy for terminal disease patients. Components of extracellular purinergic signaling plays a fundamental role on inflammation and immune response related to organ transplantation. They could be alternative targets to avoid graft rejection.
 Materials and Methods: The hydrolysis of ATP, ADP and AMP nucleotides was analyzed in both lymphocytes and platelets, as well as the quantification of ATP and ADA activity. A sample of 30 patients who underwent kidney transplants was obtained, of which 15 had a transplant time of less than one year (acute response) and 15 had a transplant time between one and three years (chronic response).
 Results: In the group with transplantation time between one and three years, it was possible to identify a significant decrease in the amount of ATP, increase in ATP hydrolysis in platelets, decrease in AMP hydrolysis and increase in ADA activity, also in platelets. In the lymphocyte sample, there was a significant reduction in ADA activity as well as a decrease in the amount of ATP.
 Conclusions: From the data obtained in the study, it can be inferred that adenosine can reduce pro-inflammatory cytokines, providing greater graft survival and reducing the intensity of graft-versus-host disease. ATP signaling exerts inflammatory effects and modulates the purinergic signaling cascade, offering new avenues for drug therapies to combat chronic graft rejection.

TNFα Induces Mitochondrial Fragmentation and Mitochondrial Biogenesis in Human Airway Smooth Muscle Cells

Airway inflammation is a key aspect of asthma and is associated with airway smooth muscle (ASM) hyperreactivity (airway narrowing due to increased Ca2+ and force responses to agonist stimulation) as well as airway remodeling (ASM cell proliferation and altering extracellular matrix composition). Proinflammatory cytokines, such as TNF‐α mediate this airway inflammatory response. Previously, we found that the TNF‐α induces an increase ASM force, which is associated with an increase in ATP hydrolysis, imposing a metabolic stress on ASM. Mitochondria play an important role in providing ATP to support contraction in ASM. Mitochondria are dynamically remodelling within ASM cells displaying changing filamentous or fragmented tubular networks. Changing expression of Mitofusin 2 (Mfn2), which is associated with mitochondria fusion, or Dynamin Related Protein 1 (Drp1), which is associated with fission mediate this mitochondrial remodelling. The stressors that induce mitochondrial remodelling in ASM are unknown, but mitochondrial fragmentation may be an initial step in mitochondrial biogenesis to meet increasing energy demand. We hypothesized that human ASM (hASM) cells exposed to TNF‐α will display reduced Mfn2 and increased Drp1 expression resulting in mitochondrial fragmentation and mitochondrial biogenesis. To test our hypothesis, serum‐deprived primary hASM cells were exposed to 20 ng/ml TNF‐α for 24 h after which they were loaded with 500 μM MitoTracker Green to visualize mitochondria and imaged using confocal microscopy. Images were analyzed using MATLAB Image J software to quantify mitochondrial morphometry: form factor and aspect ratio. An increase in form factor and aspect ratio indicates greater mitochondrial filamentous networks. Expression of Mfn2 and Drp1 were determined by Western blot analysis. In hASM exposed to TNF‐α, Mfn2 expression decreased by ~40%, while Drp1 expression increased by ~75% compared to controls. These changes in fusion/fission proteins were associated with mitochondrial fragmentation (reduced form factor and aspect ratio) and an increase in mitochondria number. Our results indicate that hASM cells exposed to TNF‐α display mitochondrial fragmentation due to changes in Mfn2 and Drp1 expression. Mitochondrial fragmentation may be involved in mitochondrial biogenesis in response to increased metabolic demands imposed by airway hyperreactivity.Support or Funding InformationAPS fellowship (EK) and NIH grant HL126451 (GCS)

Data showing the circumvention of oxaliplatin resistance by vatalanib in colon cancer.

We have recently reported that vatalanib, an orally active small molecule multi-tyrosine kinase inhibitor (Hess-Stumpp et al., 2005 [1]), can sensitize multidrug resistant (MDR) colon cancer cells to chemotherapy under hypoxia by inhibiting two MDR transporters ABCB1 and ABCG2 (To et al., 2015 [2]). This data article describes the possible circumvention of resistance to specifically platinum (Pt)-based anticancer drugs by vatalanib via inhibition of two other efflux transporters ABCC2 and ATP7A. Data from the flow cytometric transporter efflux assay showed specific inhibition of ABCC2 activity by vatalanib in stable transfected cells and ABCC2-overexpressing oxaliplatin-resistant colon cancer cells HCT116/Oxa. We also performed the transporter ABCC2 ATPase assay and showed an increase in ATP hydrolysis by ABCC2 in the presence of vatalanib. ATP7A mRNA expression was also shown to be upregulated in HCT116/Oxa cells. Vatalanib was shown to suppress this upregulated ATP7A expression. Data from the cellular Pt accumulation assay showed a lower Pt accumulation in HCT116/Oxa cells than the parental sensitive HCT116 cells. Vatalanib was shown to increase cellular Pt accumulation in a concentration-dependent manner. Combination of oxaliplatin and vatalanib was shown to restore the suppressed apoptosis in HCT116/Oxa cells.

The R403Q mutation alters isometric and energetic properties in 2 month mice (1081.1)

Familial hypertrophic cardiomyopathy is a primary disease of the sarcomere. The R403Q mutation resides at the actin‐interaction site on myosin and leads to progressive hypertrophic cardiomyopathy that can lead to sudden cardiac death or congestive heart failure. We hypothesized that the R403Q mutation intrinsically increases the energetic cost of contraction.To do this, we assessed Ca2+‐sensitivity of tension and cross‐bridge kinetics in de‐membranated cardiac trabeculae from wild‐type (WT) and R403Q hearts at 2 months of age. We measured cross‐bridge kinetics using 2 methods: (1) the rate of ATP hydrolysis (off‐rate) by NADH oxidation, and (2) the rate of force redevelopment (ktr) by a rapid release‐restretch protocol.Male R403Q mice display a decrease in Ca2+ sensitivity compared to WT males and females at SL 2.0μm. R403Q males display increased tension cost at SL 2.0μm when compared to WT counterparts. At maximal activation, the rate of force redevelopment is not significantly different between R403Q and WT males. Myosin heavy chain isoform gel analysis reveals no difference in β‐myosin content between R403Q mice and WT controls.Although maximal ktr is not significantly different between WT and R403Q males, an increase in ATP hydrolysis suggests an alteration in binding kinetics. Along with no overt pathology at 2 months, the R403Q mutation alters the energetic cost of contraction and isometric properties.Grant Funding Source: Supported by R01 HL098256A and T32HL007955

The R403Q Mutation Alters Isometric and Energetic Properties in 2 Month Mice

Introduction: Familial hypertrophic cardiomyopathy (FHC) is a primary disease of the sarcomere. The R403Q mutation resides at the actin-interaction site on myosin and leads to progressive hypertrophic cardiomyopathy that can result in sudden cardiac death or congestive heart failure. Previous studies examining the functional impact of the R403Q mutation give inconsistent results such as either reduced or enhanced actin-activated ATPase. We hypothesized that the R403Q mutation intrinsically increases the energetic cost of contraction.Methods: To do this, we assessed Ca2+-sensitivity of tension and cross-bridge kinetics in de-membranated cardiac trabeculae from wild-type (WT) and R403Q hearts at 2 months of age. Assuming a two-state model of cross-bridge binding and un-binding, we measured cross-bridge kinetics using 2 methods: (1) the rate of ATP hydrolysis (off-rate) by NADH oxidation that is enzymatically and stoichiometrically coupled to ATP consumption, and (2) the rate of force redevelopment (ktr; on-rate plus off-rate) by a rapid release-restretch protocol.Results: Male R403Q mice display a decrease in Ca2+ sensitivity (2.461μM ± 0.072) compared to WT males and females (2.189 μM ± 0.059 and 2.166 μM ±0.044, respectively) at SL 2.0μm. R403Q males display increased tension cost at SL 2.0μm when compared to WT counterparts (9.56 ± 0.68 and 6.82 ±1.07 pmol s-1 mm-1 mN-1, respectively). At maximal activation, the rate of force redevelopment (ktr) is not significantly different between R403Q and WT males (40.26±6.86 and 30.26±3.95 s-1).Conclusions: Although maximal ktr is not significantly different between WT and R403Q males, an increase in ATP hydrolysis (off rate) suggests an alteration in cross-bridge kinetics. Importantly, future studies will explore the relationship between submaximal activation and ktr. Along with no overt pathology at 2 months, the R403Q mutation alters the energetic cost of contraction and isometric properties.

Mechanisms of respiration intensification of rat pancreatic acini upon carbachol‐induced Ca2+ release

Acetylcholine as one of the main secretagogues modulates mitochondrial functions in acinar pancreacytes, presumably due to increase in ATP hydrolysis or Ca(2+) transport into mitochondria. The aim of this work was to investigate the mechanisms of carbachol (CCh) action on respiration and oxidative phosphorylation of isolated pancreatic acini. Respiration of intact or permeabilized rat pancreatic acini was studied at 37 °C using a Clark oxygen electrode. Respiration rate of isolated acini in rest was 0.27 ± 0.01 nmol O2 s(-1) 10(-6) cells. Addition of 10 μM CCh into respiration chamber evoked biphasic stimulation of respiration. Rapid increase of respiration by 20.1% lasted for approx. 1 min, followed by decrease to level by 11.5% higher than control. Addition of 1 μm CCh caused monophasic increase by 11.5%. Preincubation (5 min) with 1 or 10 μm CCh elevated respiration rate by 12.5 or 11.2% respectively. FCCP prevented the effect of CCh. Preincubation with 1 (but not 10) μm CCh increased FCCP-uncoupled respiration rate. Thapsigargin slightly elevated respiration, but ryanodine did not. Application of 2-aminoethoxydiphenyl borate or ruthenium red prevented the effects of CCh on respiration, while oligomycin abolished them. Preincubation with 1 μm CCh prior to cell permeabilization increased respiration rate at pyruvate+malate oxidation, but not at succinate oxidation. In contrast, preincubation with 10 μm CCh decreased pyruvate+malate oxidation. Medium CCh dose (1 μm) intensifies respiration and oxidative phosphorylation of acinar pancreacytes by feedforward mechanism via Ca(2+) transport into mitochondria and activation of Ca(2+) /ADP-sensitive mitochondrial dehydrogenases. Prolonged action of high CCh dose (10 μm) might impair mitochondrial functions.

Gingerol discharges NG115–401L neural cell Ca2+ stores, activates Ca2+ influx and promotes cell death

Gingerol's effect on sarco/endoplasmic reticulum Ca2+‐ ATPase (SERCA) and cytosolic free Ca2+ ([Ca2+]i) was tested on sarcoplasmic reticulum (SR) microsomes and NG115–401L, a neural cell line with diminished level of stromal interacting molecule 1 (STIM1) and deficient endoplasmic reticulum (ER)− regulated Ca2+ signaling pathways. Cells were loaded with the Ca2+ probe fura‐2 to detect [Ca2+]i changes and SR microsomes were prepared to measure Ca2+‐ATPase activity. Gingerol stimulates an approximately two‐fold increase in ATP hydrolysis in skeletal muscle SR microsomes. Moreover, gingerol incubation nearly completely reverses SERCA enzymatic function against SERCA blocker cyclopiazonic acid (CPA). However, in 401L cells, gingerol fails to enhance ER Ca2+ uptake, but induces a transient [Ca2+]i elevation in the absence of external Ca2+ much like thapsigargin (TG) and CPA. Additionally, gingerol is unable to protect 401L cells against CPA‐induced apoptosis, confirming that the compound has no ER Ca2+ enhancing effect in 401L cells. Surprisingly, gingerol's apparent ER Ca2+ depleting action couples to long‐lived Ca2+ influx pathways, whereas those of TG and CPA do not. These results argue for a finer regulatory control on SERCA function with gingerol actions revealing potentially novel routes of coupling altered pump regulation to the assembly of functional Ca2+ influx units. Research supported by University of the Pacific

Profile of Nucleotide Catabolism and Ectonucleotidase Expression from the Hippocampi of Neonatal Rats After Caffeine Exposure

Nucleotides and nucleosides play an important role in neurodevelopment acting through specific receptors. Ectonucleotidases are the major enzymes involved in controlling the availability of purinergic receptors ligands. ATP is co-released with several neurotransmitters and is the most important source of extracellular adenosine by catabolism exerted by ectonucleotidases. The main ectonucleotidases are named NTPDases (1-8) and 5'-nucleotidase. Adenosine is a powerful modulator of neurotransmitter release. Caffeine blocks adenosine receptor activity as well as adenosine-mediated neuromodulation. Considering the susceptibility of the immature brain to caffeine and the need for correct purinergic signaling during fetal development, we have analyzed the effects of caffeine exposure during gestational and lactational periods on nucleotide degradation and ectonucleotidase expression from the hippocampi of 7-, 14- and 21-days-old rats. Nucleotides hydrolysis was assessed by colorimetric determination of inorganic phosphate released. Ectonucleotidases expression was performed by RT-PCR. ATP and ADP hydrolysis displayed parallel age-dependent decreases in both control and caffeine-treated groups. AMP hydrolysis increased with caffeine treatment in 7-days-old rats (75%); although there was no significant difference in AMP hydrolysis between control (non caffeine-treated) rats and 14- or 21-days caffeine-treated rats. ADP hydrolysis was not affected by caffeine treatment. Caffeine treatment in 7- and 14-days-old rats decreased ATP hydrolysis when compared to the control group (19% and 60% decrease, respectively), but 21-days-treated rats showed an increase in ATP hydrolysis (39%). Expression levels of NTPDase 1 and 5 decreased in hippocampi of caffeine-treated rats. The expression of 5'-nucleotidase was not affected after caffeine exposure. The changes observed in nucleotide hydrolysis and ectonucleotidases expression could promote subtle effects on normal neural development considering the neuromodulatory role of adenosine.

Single Molecule Observations of Rad51 Nucleoprotein Filament Formation and Removal via Yeast Srs2

The Rad51 in Saccharomyces cerevisiae plays a vital role in promoting homologous recombination (HR) by self-assembling into elongated nucleoprotein filaments on DNA and repair double-stranded DNA breaks (DSBs). However, mutations can arise from undesirable HR that lead to debilitating diseases such as rapid aging or breast cancer. The Srs2 helicase (a member of the SF1 helicase family) is an ATP dependent antirecombinase that disrupts the Rad51 presynaptic filament through physical interaction with the Rad51 recombinase.We are investigating the dynamics of individual Rad51 nucleoprotein filament on DNA and their interactions with Srs2 using ensemble and single molecule fluorescence microscopy. Our ensemble ATPase measurement shows a substantial increase in ATP hydrolysis when Srs2 is introduced in the presence of preformed Rad51 filament while fluorescence studies indicate that Srs2 efficiently removed Rad51 filaments. Unexpectedly, the burst of ATPase activity was not dependent on Srs2 concentrations applied, revealing a substoichiometric relationship between Rad51 and Srs2. Using single-molecule FRET assay, we demonstrate real time dynamics of individual Rad51 nucleoprotein filament and its interaction with Srs2. Our preliminary data suggests a possible mechanism by which Srs2 removes Rad51 nucleoprotein filaments.

Characterization of mitochondria isolated from normal and ischemic hearts in rats utilizing atomic force microscopy

Mitochondria play critical roles in both the life and the death of cardiac myocytes. Various factors, such as the loss of ATP synthesis and increase of ATP hydrolysis, impairment in ionic homeostasis, formation of reactive oxygen species (ROS), and release of proapoptotic proteins are related to the generation of irreversible damage. It has been proposed that the release of cytochrome c is caused by a swelling of the mitochondrial matrix triggered by the apoptotic stimuli. However, there is a controversy about whether or not the mitochondria, indeed, swell during apoptosis. The major advantages of atomic force microscopy (AFM) over conventional optical and electron microscopes for bio-imaging include the fact that no special coating and vacuum are required and imaging can be done in all environments—air, vacuum or aqueous conditions. In addition, AFM force–distance curve measurements have become a fundamental tool in the fields of surface chemistry, biochemistry, and material science. In this study, we used AFM to observe the morphological and property changes in heart mitochondria that were isolated from a rat myocardial infarction model. From the shape parameters of the mitochondria in the AFM topographic image, it seemed that myocardial infarction caused the mitochondrial swelling. Also, the results of force–distance measurements showed that the adhesion force of heart mitochondria was significantly decreased by myocardial in infarction. Therefore, we suggested that myocardial infarction might be the cause of mitochondrial swelling and the changes in outer membrane of heart mitochondria.

L-NAME-treatment alters ectonucleotidase activities in kidney membranes of rats

Aims To investigate the effect of Nω-Nitro- l-arginine methyl ester ( l-NAME) treatment, known to induce a sustained elevation of blood pressure, on ectonucleotidase activities in kidney membranes of rats. Main methods l-NAME (30 mg/kg/day) was administered to Wistar rats for 14 days in the drinking water. Enzyme activities were determined colorimetrically and their gene expression patterns were analyzed by semi-quantitative RT-PCR. The metabolism of ATP and the accumulation of adenosine were evaluated by HPLC in kidney membranes from control and hypertensive rats. PKC phosphorylation state was investigated by Western blot. Key findings We observed an increase in systolic blood pressure from 115 ± 12 mmHg (control group) to 152 ± 18 mmHg ( l-NAME-treated group). Furthermore, the hydrolysis of ATP, ADP, AMP, and p-Nph-5′TMP was also increased (17%, 35%, 27%, 20%, respectively) as was the gene expression of NTPDase2, NTPDase3 and NPP3 in kidneys of hypertensive animals. Phospho-PKC was increased in hypertensive rats. Significance The general increase in ATP hydrolysis and in ecto-5′-nucleotidase activity suggests a rise in renal adenosine levels and in renal autoregulatory responses in order to protect the kidney against the threat presented by hypertension.

Neonatal morphine exposure alters E-NTPDase activity and gene expression pattern in spinal cord and cerebral cortex of rats

The neonate opioid system has been frequently investigated, and studies have shown that exposure to drugs in early life can have implications for nervous system development. It has been proposed that adenosine is involved in opioid antinociception, and ATP is involved in central and peripheral mechanisms of nociception. Extracellular nucleotides can be hydrolyzed by E-NTPDases and ecto-5′nucleotidase, which present the functions of removing ATP and generating adenosine. In this study, we evaluated ATP, ADP, and AMP hydrolysis in synaptosomes from spinal cord and cerebral cortex of rats at postnatal day 16 after repeated morphine exposure in early life (postnatal day 8 to 14). Additionally, we evaluated E-NTPDase (1, 2 and 3) and ecto-5′nucleotidase gene expression by semi-quantitative RT-PCR analysis. We observed an increase in ATP hydrolysis in the cerebral cortex, and a decrease in ADP hydrolysis in spinal cord. Expression levels of E-NTPDase 1 decreased in cerebral cortex and increased in spinal cord. Our findings highlight the importance of the purinergic system in young rats submitted to repeated morphine exposure by showing that in the neonatal period such exposure is capable of affecting the control system for nucleotide levels, which can promote changes in modulation or transmission of painful stimuli.

Effect of ketogenic diet on nucleotide hydrolysis and hepatic enzymes in blood serum of rats in a lithium-pilocarpine-induced status epilepticus

The ketogenic diet (KD) is a high-fat and low-carbohydrate diet, used for treating refractory epilepsy in children. We have previously shown alterations in nucleotidase activities from the central nervous system and blood serum of rats submitted to different models of epilepsy. In this study we investigated the effect of KD on nucleotidase activities in the blood serum, as well if KD has any influence in the activity of liver enzymes such as alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase activities in Wistar rats submitted to the lithium-pilocarpine model of epilepsy. At 21 days of age, rats received an injection of lithium chloride and, 18-19 h later, they received an injection of pilocarpine hydrochloride for status epilepticus induction. The results reported herein show that seizures induced by lithium-pilocarpine elicit a significant increase in ATP hydrolysis and alkaline phosphatase activity, as well as a decrease in ADP hydrolysis and aspartate aminotransferase activity. The KD is a rigorous regimen that can be associated with hepatic damage, as shown herein by the elevated activities of liver enzymes and 5'-nucleotidase in blood serum. Further studies are necessary to investigate the mechanism of inhibition of lithium on nucleotidases in blood serum.

GP10 of Bacteriophage Phi29 Exhibits ATPase Activity

Previously thought to function only as a passive portal for the phi29 DNA packaging motor, we demonstrate that the connector protein gene product 10 (gp10) exhibits significant ATPase activity. Bioluminescence assays have shown a concentration dependent decrease in available ATP after the addition of gp10. Also, enzyme-linked inorganic phosphate assays (ELIPA) have confirmed a time-dependent increase of phosphate in the presence of gp10 and ATP. Alone, gp10 acts as a slow ATPase with optimal hydrolysis of ATP occurring above room temperature at conditions native to the phi29 DNA motor. Overall increase of ATP hydrolysis in the system has been observed upon the addition of other phi29 motor components to gp10. Compared to gp16, a known ATPase, gp10 consumes ATP at a notably slower rate. Moreover, pRNA has shown no measurable ATPase activity. Combined, the phi29 motor assembly (i.e. gp10, gp16, and pRNA) has exhibited greater rates of ATP hydrolysis than any of these components independently.

Binding of MG132 or Deletion of the Thr Active Sites in HslV Subunits Increases the Affinity of HslV Protease for HslU ATPase and Makes This Interaction Nucleotide-independent

HslVU is an ATP-dependent protease in bacteria consisting of HslV dodecamer and HslU hexamer. Upon ATP binding, HslU ATPase allosterically activates the catalytic function of HslV protease by 1-2 orders of magnitude. However, relatively little is known about the role of HslV in the control of HslU function. Here we describe the involvement of the N-terminal Thr active sites (Thr-1) of HslV in the communication between HslV and HslU. Binding of proteasome inhibitors to Thr-1 led to a dramatic increase in the interaction between HslV and HslU with a marked increase in ATP hydrolysis by HslU. Moreover, carbobenzoxy-leucyl-leucyl-leucinal (MG132) could bind to Thr-1 of free HslV, and this binding induced a tight interaction between HslV and HslU with the activation of HslU ATPase, suggesting that substrate-bound HslV can allosterically regulate HslU function. Unexpectedly, the deletion of Thr-1 also caused a dramatic increase in the affinity between HslV and HslU even in the absence of ATP. Furthermore, the increase in the number of the Thr-1 deletion mutant subunit in place of HslV subunit in a dodecamer led to a proportional increase in the affinity between HslV and HslU with gradual activation of HslU ATPase. Although the molecular mechanism elucidating how the Thr-1 deletion influences the interaction between HslV and HslU remains unknown, these results suggest an additional allosteric mechanism for the control of HslU function by HslV. Taken together, our findings indicate a critical involvement of Thr-1 of HslV in the reciprocal control of HslU function and, thus, for their communication.

Metabolic and Developmental Adaptations of Growing Potato Tubers in Response to Specific Manipulations of the Adenylate Energy Status

Heterotrophic carbon metabolism has been demonstrated to be limited by oxygen availability in a variety of plant tissues, which in turn inevitably affects the adenylate status. To study the effect of altering adenylate energy metabolism, without changing the oxygen supply, we expressed a plastidially targeted ATP/ADP hydrolyzing phosphatase (apyrase) in tubers of growing potato (Solanum tuberosum) plants under the control of either inducible or constitutive promoters. Inducible apyrase expression in potato tubers, for a period of 24 h, resulted in a decrease in the ATP-content and the ATP-ADP ratio in the tubers. As revealed by metabolic profiling, this was accompanied by a decrease in the intermediates of sucrose to starch conversion and several plastidially synthesized amino acids, indicating a general depression of tuber metabolism. Constitutive tuber-specific apyrase expression did not lead to a reduction of ATP, but rather a decrease in ADP and an increase in AMP levels. Starch accumulation was strongly inhibited and shifted to the production of amylopectin instead of amylose in these tubers. Furthermore, the levels of almost all amino acids were decreased, although soluble sugars and hexose-Ps were highly abundant. Respiration was elevated in the constitutively expressing lines indicating a compensation for the dramatic increase in ATP hydrolysis. The increase in respiration did not affect the internal oxygen tensions in the tubers. However, the tubers developed a ginger-like phenotype having an elevated surface-volume ratio and a reduced mass per tuber. Decreased posttranslational redox activation of ADP-glucose pyrophosphorylase and a shift in the ratio of soluble starch synthase activity to granule-bound starch synthase activity were found to be partially responsible for the alterations in starch structure and abundance. The activity of alcohol dehydrogenase was decreased and pyruvate decarboxylase was induced, but this was neither reflected by an increase in fermentation products nor in the cellular redox state, indicating that fermentation was not yet induced in the transgenic lines. When taken together the combined results of these studies allow the identification of both short- and long-term adaptation of plant metabolism and development to direct changes in the adenylate status.

E-NTPDase 3 (ATP diphosphohydrolase) from cardiomyocytes, activity and expression are modulated by thyroid hormone

Degradation of adenine nucleotides by myocardial cells occurs, in part, by a cascade of surface-located enzymes converting ATP into adenosine that has important implications for the regulation of the nucleotide/nucleoside ratio modulating the cardiac functions. Thyroid hormones have profound effects on cardiovascular system, as observed in hypo- and hyperthyroidism. Combined biochemical parameters and gene expression analysis approaches were used to investigate the influence of tri-iodothyronine (T3) on ATP and ADP hydrolysis by isolated myocytes. Cultures of cardiomyocytes were submitted to increasing doses of T3 for 24 h. Enzymatic activity and expression were evaluated. T3 (0.1 nM) caused an increase in ATP and ADP hydrolysis. Experiments with specific inhibitors suggest the involvement of an NTPDase, which was confirmed by an increase in NTPDase 3 messenger RNA (mRNA) levels. Since T3 promotes an increase in the contractile protein, leading to cardiac hypertrophy, it is tempting to postulate that the increase in ATP hydrolysis and the decrease in the extracellular levels signify an important factor for prevention of excessive contractility.

Hindering the Strand Passage Reaction of Human Topoisomerase IIα without Disturbing DNA Cleavage, ATP Hydrolysis, or the Operation of the N-terminal Clamp

DNA topoisomerase II is an essential enzyme that releases a topological strain in DNA by introduction of transient breaks in one DNA helix through which another helix is passed. While changing DNA topology, ATP is required to drive the enzyme through a series of conformational changes dependent on interdomain communication. We have characterized a human topoisomerase IIalpha enzyme with a two-amino acid insertion at position 351 in the transducer domain. The mutation specifically abolishes the DNA strand passage event of the enzyme, probably because of a sterical hindrance of T-segment transport. Thus, the enzyme fails to decatenate and relax DNA, even though it is fully capable of ATP hydrolysis, closure of the N-terminal clamp, and DNA cleavage. The cleavage activity is increased, suggesting that the transducer domain has a role in regulating DNA cleavage. Furthermore, the enzyme has retained a tendency to increase DNA cleavage upon nucleotide binding and also responds to DNA with elevated ATP hydrolysis. However, the DNA-mediated increase in ATP hydrolysis is lower than that obtained with the wild-type enzyme but similar to that of a cleavage-deficient topoisomerase IIalpha enzyme. Our results strongly suggest that the strand passage event is required for efficient DNA stimulation of topoisomerase II-mediated ATP hydrolysis, whereas the stimulation occurs independent of the DNA cleavage reaction per se. A comparison of the strand passage deficient-enzyme described here and the cleavage-deficient enzyme may have applications in other studies where a clear distinction between strand passage and topoisomerase II-mediated DNA cleavage is desirable.

Evidence for active CO2 uptake by a CO2-ATPase in the acidophilic green alga Eremosphaera viridis

We examined the mechanism(s) responsible for active uptake of dissolved inorganic carbon (DIC) during photosynthesis in the green alga Eremosphaera viridis De Bary. O2 electrode measurements of algal oxygen evolution and CO2 fluxes as a function of DIC availability indicate that E. viridis actively imports only CO2 during photosynthesis, and does not possess external carbonic anhydrase (CA). The K0.5[CO2] was 14.2 and 10.1 µM at pH 5.0 and 8.0, respectively. Both membrane potential and cellular resistance were measured under controlled conditions of [CO2] at either 2 or 15 µM. Active CO2 uptake was electrically silent, suggesting that CO2 uptake might be mediated by a CO2-ATPase. Comparison of ATPase activity in microsomal preparations at low (0 µM) and high (15 µM) [CO2] indicated a 1.25-fold increase in ATP hydrolysis in high [CO2]. The CO2-ATPase activity was inhibited by the broad-acting inhibitors diethylstilbestrol (DES) and N',N'-dicyclohexylcarbodiimide (DCCD) but unaffected by vanadate, fluoride, and nitrate. The K0.5[CO2] of the ATPase activity was 22.5 µM, a value very similar to the K0.5[CO2] for CO2 uptake by whole algal cells. These results suggest the existence of a CO2-ATPase as the major importer of DIC for photosynthesis in the microalga E. viridis.Key words: chlorophyte, CO2 transport, CO2-ATPase, photosynthesis, electrical potential, mass spectrometry.

Altered ATP hydrolysis induced by pentylenetetrazol kindling in rat brain synaptosomes.

The ectonucleotidase pathway is an important metabolic source of extracellular adenosine. Adenosine has potent anticonvulsant effects on various models of epilepsy. One of these models is pentylenetetrazol (PTZ) kindling, in which repeated administration of subconvulsive doses of this drug induces progressive intensification of seizure activity. In this study, we examine the effect of a single convulsive injection (60 mg/kg, i.p.) or 10 successive (35 mg/kg, i.p.) injections of PTZ on synaptosomal ectonucleotidases. Our results have shown that no changes in ectonucleotidase activities were seen at 0, 1, and 24 h or at 5 days after a single convulsive PTZ injection. However, after PTZ-kindling, rats which were more resistant to seizure development presented an increase in ATP hydrolysis in synaptosomes from hippocampus and cerebral cortex (44% and 28%, respectively). These results suggest that changes in nucleotide hydrolysis may represent an important mechanism in the modulation of chronic epileptic activity in this model.