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Characterization of lactic acid formation and adenosine triphosphate consumption in calcium-loaded erythrocytes of broiler chickens.

The formation of lactic acid and consumption of adenosine triphosphate (ATP) were measured in erythrocytes from broiler and White Leghorn chickens with the goal of providing further evidence that lactic acid, a metabolite from glycolysis, is an etiological factor for sudden death syndrome (SDS) in broiler chickens. When loaded with Ca2+, erythrocytes exhibited increased lactic acid concentrations. The effect of Ca2+ loading was significantly faster in broiler erythrocytes. In the absence of adenosine used as an energy-yielding substrate, Ca2+ loading was followed by a reduction in ATP concentrations. The effect was also significantly faster in broiler erythrocytes. Intravenous injection of a 20% lactate solution at 0.1 mL/kg produced SDS-like death of broilers but not White Leghorns. The results obtained in erythrocytes indicate that, in broilers, a greater amount of energy is required for regulation of intracellular Ca2+ concentrations, and, thereby, formation of lactic acid is more accelerated. In addition to the result that intravenous lactate injection caused death in broilers, an elevation of lactic acid concentrations in blood, arising from operation of Ca2+ regulation mechanisms, may predispose broilers to incidence of SDS.

Adenosine Triphosphate↗

Effect of blood purification on the kidney function of the jaundiced rat.

This paper is aimed at evaluating the effect of blood purification on the mitochondrial functions of the kidney in jaundiced rats. At 1, 2, or 3 weeks after bile duct ligation and dissection, obstructive jaundiced rats were utilized for experiment. Cross circulation in 1 ml/min between jaundiced rats and normal rats was performed for 1 or 3 h. Immediately and 24 h after cross circulation, the rats were killed and mitochondrial respiratory functions of the kidney were assessed by measuring oxygen consumption. Mitochondrial function, especially ATP synthesis, showed significant improvement in jaundiced rat with 2 or 3 weeks of biliary obstruction. Cross circulation for 3 h was more effective than that for 1 h. These data suggest that the renal energy production can be increased significantly in obstructive jaundice by blood purification treatment.

Animals↗

Compensatory responses of protein import and transcription factor expression in mitochondrial DNA defects.

Defects in mitochondrial DNA (mtDNA) evoke distinctive responses in the nuclear genome, leading to altered mitochondrial biogenesis. We used C(2)C(12) cells depleted of mtDNA (rho(-) cells) and fibroblasts from a mitochondrial encephalopathy, lactic acidosis, and strokelike episodes (MELAS) patient to examine adaptations of the protein import machinery and transcription factors involved in mitochondrial biogenesis. In rho(-) cells, Tom20 and Tim23 protein levels were reduced by 25% and 59%, whereas mtHSP70 was induced by twofold relative to control cells. These changes were accompanied by a 21% increase in enhanced yellow fluorescent protein (EYFP) import into mitochondria in rho(-) cells (P < 0.05). In contrast, in MELAS cells mtHSP70 was elevated by 70%, whereas Tom20 and Tom34 protein levels were increased by 45% and 112% relative to control values. EYFP import was not altered in MELAS cells. In rho(-) cells, protein levels of the transcription factors nuclear respiratory factor-1 (NRF-1) and transcription factor A (Tfam) declined by 33% and 54%, whereas no change was observed for the coactivator peroxisome proliferator receptor-gamma coactivator-1alpha (PGC-1alpha). In contrast, Tfam was increased by 40% in MELAS cells. Rho(-) cells displayed reduced oxygen consumption (Vo(2)) and ATP levels, along with a twofold increase in lactate levels (P < 0.05). In electrically stimulated C(2)C(12) cells, 109%, 78%, 60%, and 67% increases were observed in mtDNA, Vo(2), cytochrome-c oxidase (COX) activity, and Tom34 levels, respectively (P < 0.05). Our findings suggest that compensatory adaptations occurred to maintain normal rates of protein import in response to mtDNA defects and support a role for contractile activity in reducing pathophysiology associated with mtDNA depletion. Because the expression of nuclear-encoded transcription factors and protein import machinery components was dependent on the type of mtDNA defect, these findings suggest involvement of distinct signaling cascades, each dependent on the type of mitochondrial defect, resulting in divergent changes in nuclear gene expression patterns.

Animals↗

Effects of taxol on isolated rat hepatocyte metabolism.

Taxol is a natural product, isolated from Taxus brevifolia, with increasing clinical applications because of its potent antitumor activity. Although it is mainly metabolized in the liver, its effect on hepatocyte metabolism has been scarcely investigated. In this study, the response of isolated rat hepatocytes to taxol was evaluated by correlating the changes observed in global indexes such as viability and heat dissipation with those produced in oxygen consumption and intracellular metabolites (ATP, fructose 2,6-bisphosphate, and lactate). The results indicate that taxol reduces aerobic metabolism, which induces an insufficient increase of the ATP via anaerobic glycolysis. Moreover, incubations of isolated mitochondria with taxol indicate that the respiratory chain is directly affected by this drug.

Animals↗

Bicarbonate conservation during incomplete cerebral ischemia with superimposed hypercapnia.

We superimposed extreme hypercapnia (arterial Pco2 400-450 mmHg) immediately before and during incomplete cerebral ischemia to distinguish the role of intracellular pH (pHi) and bicarbonate [( HCO3-]i) in postischemic metabolic and electrophysiological recovery. Incomplete global ischemia was produced in seven anesthetized dogs by 30 min of intracranial hypertension followed by 4 h of reperfusion. ATP, phosphocreatine (PCr), and pHi were measured with 31P magnetic resonance spectroscopy, and [HCO3-]i was calculated from the Henderson-Hasselbalch equation using the measured pHi and sagittal sinus Pco2. Cerebral blood flow was reduced to 7 +/- 1 ml.min-1.100 g-1 (+/- SE) during ischemia with extreme hypercapnia, and pHi decreased to 5.72 +/- 0.09. During normocapnic reperfusion, pHi rapidly returned to near baseline values by 14 min. [HCO3-]i fell from 12.1 +/- 0.9 to 6.0 +/- 1.2 mM by the midpoint of ischemia and recovered by 30 min of reperfusion. ATP, PCr, and O2 consumption also recovered rapidly and completely. Somatosensory-evoked potentials (SEP) recovered to 43 +/- 10% of control amplitude. These results are in marked contrast to the poor metabolic and SEP recovery previously observed in hyperglycemic dogs in which pHi decreased to the same range as with hypercapnic ischemia, but in which [HCO3-]i was much lower (1.1 +/- 0.5 mM). Therefore, [HCO3-]i depletion during hyperglycemic ischemia may be a more important factor in recovery than end-ischemic pHi per se. We speculate that higher [HCO3-]i may improve glial cell buffering capacity or decrease iron availability for hydroxyl radical production.

Adenosine Triphosphate↗

Effects of ouabain and isoproterenol on left ventricular diastolic function during low-flow ischemia in isolated, blood-perfused rabbit hearts.

Myocardial ischemia causes both systolic and diastolic dysfunction. A variety of positive inotropic agents with different subcellular mechanisms may be used clinically in an attempt to reverse ischemic contractile failure. We tested the hypothesis that two inotropic agents, isoproterenol (a beta-adrenergic agonist) and ouabain (a sodium pump inhibitor), might have different effects on left ventricular (LV) diastolic function during ischemic failure despite an equivalent inotropic effect. Isolated isovolumic (balloon-in-LV) blood perfused rabbit hearts were paced at constant physiological heart rate (4 Hz), given either no drug (controls, n = 7), isoproterenol (n = 7), or ouabain (n = 7), and then subjected to 6 minutes of low flow ischemia (75% reduction of baseline coronary flow). The doses of isoproterenol and ouabain were selected to produce equivalent modest inotropic effects (15% increase in LV + dP/dt) in each heart during baseline perfusion conditions. During the ischemic period, there was a marked decrease in contractility, and neither isoproterenol nor ouabain demonstrated a positive inotropic effect relative to the control group. However, these agents had markedly different effects on diastolic chamber distensibility (assessed by end-diastolic pressure at constant LV volume) during ischemia. In the control and isoproterenol groups, diastolic chamber distensibility did not change during the ischemic period. In contrast, ouabain treatment resulted in a marked decrease in diastolic chamber distensibility during ischemia; this change was not completely reversible during the 10-minute reperfusion period. The mechanism by which ouabain decreased diastolic chamber distensibility relative to isoproterenol was assessed indirectly. The ouabain and isoproterenol groups were subjected to equivalent degrees of ischemia as assessed by oxygen supply/demand imbalance; during ischemia, each drug group did not differ with regard to myocardial perfusion rates, determinants of myocardial oxygen demand (heart rate, LV developed pressure, LV + dP/dt), myocardial oxygen consumption, lactate production, and ATP and creatine phosphate content. We therefore inferred that the greater decrease in diastolic distensibility in the ouabain group was not due to a greater metabolic severity of ischemia. These observations are consistent with a mechanism of cytosolic calcium overload induced by ouabain, resulting in persistent active myofilament tension development throughout diastole, to cause the observed decrease in diastolic chamber distensibility during ischemia in the ouabain group.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Three mechanistically distinct kinase assays compared: Measurement of intrinsic ATPase activity identified the most comprehensive set of ITK inhibitors.

Numerous assay methods have been developed to identify small-molecule effectors of protein kinases, but no single method can be applied to all isolated kinases. The authors developed a set of 3 high-throughput screening (HTS)-compatible biochemical assays that can measure 3 mechanistically distinct properties of a kinase active site, with the goal that at least 1 of the 3 would be applicable to any kinase selected as a target for drug discovery efforts. Two assays measure catalytically active enzyme: A dissociation-enhanced lanthanide fluoroimmuno assay (DELFIA) uses an antibody to quantitate the generation of phosphorylated substrate; a second assay uses luciferase to measure the consumption of adenosine triphosphate (ATP) during either phosphoryl-transfer to a peptide substrate or to water (intrinsic ATPase activity). A third assay, which is not dependent on a catalytically active enzyme, measures the competition for binding to kinase between an inhibitor and a fluorescent ATP binding site probe. To evaluate the suitability of these assays for drug discovery, the authors compared their ability to identify inhibitors of a nonreceptor protein tyrosine kinase from the Tec family, interleukin-2-inducible T cell kinase (ITK). The 3 assays agreed on 57% of the combined confirmed hit set identified from screening a 10,208-compound library enriched with known kinase inhibitors and molecules that were structurally similar. Among the 3 assays, the one measuring intrinsic ATPase activity produced the largest number of unique hits, the fewest unique misses, and the most comprehensive hit set, missing only 2.7% of the confirmed inhibitors identified by the other 2 assays combined. Based on these data, all 3 assay formats are viable for screening and together provide greater options for assay design depending on the targeted kinase.

Adenosine Triphosphatases↗

Uncoupling protein-2 is critical for nigral dopamine cell survival in a mouse model of Parkinson's disease.

Mitochondrial uncoupling proteins dissociate ATP synthesis from oxygen consumption in mitochondria and suppress free-radical production. We show that genetic manipulation of uncoupling protein-2 (UCP2) directly affects substantia nigra dopamine cell function. Overexpression of UCP2 increases mitochondrial uncoupling, whereas deletion of UCP2 reduces uncoupling in the substantia nigra-ventral tegmental area. Overexpression of UCP2 decreased reactive oxygen species (ROS) production, which was measured using dihydroethidium because it is specifically oxidized to fluorescent ethidium by the superoxide anion, whereas mice lacking UCP2 exhibited increased ROS relative to wild-type controls. Unbiased electron microscopic analysis revealed that the elevation of in situ mitochondrial ROS production in UCP2 knock-out mice was inversely correlated with mitochondria number in dopamine neurons. Lack of UCP2 increased the sensitivity of dopamine neurons to 1-methyl-4-phenyl-1,2,5,6 tetrahydropyridine (MPTP), whereas UCP2 overexpression decreased MPTP-induced nigral dopamine cell loss. The present results expose the critical importance of UCP2 in normal nigral dopamine cell metabolism and offer a novel therapeutic target, UCP2, for the prevention/treatment of Parkinson's disease.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

Preservation of resuspended red cell concentrate. Analysis of purines, nucleosides and nucleotides in stored red cells.

Purine nucleotides of red blood cells (RBC) during storage in two different media with addition of adenine/nicotinamide (NAP) or adenine/guanosine (CDS-AG) were estimated by HPLC. Synthesis of guanine nucleotides reached a maximum after 14 days in RBC stored with adenine/guanosine. The higher adenine concentration in the NAP solution (3 mmol/l) did not increase adenine consumption and the ATP-level of the erythrocytes. The adenylate energy charge (AEC) of RBC decreased from 0.91 to 0.63 during 42 days of storage in CDS-AG solution.

Adenine Nucleotides↗

The effect of cell K+ on the volume regulation in rat kidney cortex cells.

Incubation of outermost rat kidney cortex slices (rich in proximal tubules) at 0 degrees C in a medium without K+ produces an increase in their cell water, Na+ and Cl- contents and a diminution in their cell K+ content. When these slices are rewarmed at 25 degrees C in a medium without K+, they can regulate their cell volume and their cell Na+ and Cl- contents. When the rewarming period lasts more than 30 min, there is again a cellular swelling, which seems to be correlated with the diminution of the cell K+ concentration. This effect of cellular K+ appears to be related to severe changes in cellular permeability to ions and water. The oxygen consumption and the cell ATP concentrations are similar to those in control conditions. The ouabain-insensitive Na-pump activity is not affected by different concentrations of K+.

Animals↗

Interaction of aspartate and aspartate-derived antimetabolites with the enzymes of the threonine biosynthetic pathway of Escherichia coli.

The five enzymes responsible for the conversion of L-aspartate to L-threonine in Escherichia coli were purified to homogeneity and subsequently reconstituted in vitro in ratios approximating those found in vivo. 31P NMR was used to conveniently monitor the rates of consumption of the substrates ATP and NADPH, the accumulation of the intermediates beta-aspartyl phosphate and homoserine phosphate, and the formation of the products ADP, NADP+, and Pi in a single experiment. By this method, the flux of aspartic acid through the enzymes of the pathway was monitored in the absence and in the presence of several alternative substrates and inhibitors. Several known antimetabolites were found to be alternative substrates that ultimately became inhibitors of pathway flux. L-threo-3-Hydroxyaspartic acid was converted to 3-hydroxyhomoserine phosphate by the first four enzymes of the pathway. The antimetabolite L-threo-3-hydroxyhomoserine was found to bind to and inhibit aspartokinase-homoserine dehydrogenase I in a cooperative fashion (I 0.5 = 3 mM, nH = 2.5), similar to the action of the allosteric end product inhibitor L-threonine (I 0.5 = 0.36 mM, nH = 2.4). In the presence of the remaining enzymes of the pathway, however, L-threo-3-hydroxyhomoserine was phosphorylated to the apparent ultimate antimetabolite L-threo-3-hydroxyhomoserine phosphate that was a potent inhibitor of threonine synthase and consequently of L-threonine biosynthesis. When aspartic acid alone was examined as a substrate of the enzymes of the pathway, no accumulation of the beta-aspartyl phosphate and homoserine phosphate intermediates was observed. However, in the presence of either 5 mM L-threo-3-hydroxyhomoserine or 5 mM L-threo-3-hydroxyhomoserine phosphate, homoserine phosphate was found to accumulate. In contrast to the homoserine phosphate and 3-hydroxyhomoserine phosphate intermediates, both of which were very stable, the acylphosphate intermediates beta-aspartyl phosphate and beta-3-hydroxyaspartyl phosphate were highly susceptible to hydrolysis, with first-order rate constants of 4.6 X 10(-3) min-1 and 4.5 X 10(-2) min-1 (pH 7.8, 25 degrees C), respectively.

Aspartate Kinase↗

[Department of the Biochemistry of Muscles].

The basic scientific achievements of the Department of Biochemistry of Muscles organized at the Academy of Sciences of Ukrainian SSR in 1944 are presented in this short historical overview. The basic guidelines for activities in the scientific field are as follows: study of biochemical processes in the working muscles as well as during misfunctions and disabilities, processes of adenine nucleotides exchange and ammonia creation, biochemical characterization of Ca2+ and H+ transport through the plasma and sarcoplasmic reticulum membranes. It is shown that creatine and creatine phosphate as well as adenine nucleotide content and metabolism affect the muscle functioning, glycogen metabolism proceeds simultaneously with the lowering of content of inorganic phosphate. The facts of glucose phosphorylation and its conversion via glycolytic pathways and the backward reaction of glycolysis (the aerobic synthesis of phosphopyruvate, glycogen synthesis from glucose in the presence of phosphorylase) were determined. After the muscle work up to tiredness adenine nucleotide depletion is not limited by its dephosphorylation, but goes up to formation of inosine acid and ammonia. Deamidation is shown to be in myofibrillar fraction and in sarcoplasmic reticulum of the skeletal muscle. Deamidation activity is not registered in myocardium myofibrillar fraction but it is registered in sarcoplasmic reticulum. AMP-phosphohydrolase and adenosine desaminase were found in membranes of the sarcoplasmic reticulum. The decrease in activity of all enzymes mentioned above is registered during myocardium hypertrophy, because of aorta narrowing. These data permit creating the methods for obtaining substance "adenosine phosphate" for treatment of cardiac pathologies. Glutaminase was found to be active in the muscles. This activity depended on the organism functioning. The ammonia usage by the muscle cells goes with glutamine synthesis and consumption of energy of ATP, e.g. protein amidation. The later is of all-biological significance and is used in the fields of medicine actualls concerned with the following fact: the velocity of hydrolysis of amidated protein is different for such pathology as epilepsia, tuberculosis, poisoning with manganese oxides. The methods for diagnostics of the above pathological states were developed on this basis. It is proved that glutamine nitrogen can be also used in the reaction of transamination, particularly during synthesis of purines, inosine acid and it is stored in a form of glutaminic acid. Changes in carbohydrate and phosphorus metabolism, in nitrogen and energetic exchanges and mitochondria overfilling with calcium were determined under E-avitaminosis dystrophy.(ABSTRACT TRUNCATED)

Academies and Institutes↗

Alterations of oxygen uptake and the redox state of ubiquinone in rabbit sperm exposed to a variety of physiologic treatments.

The rate of TEMPONE reduction by electrons originating from ubiquinone in intact rabbit spermatozoa was observed for control, high ionic strength (HIS) medium-treated, and HIS-seminal plasma-treated (HIS-SP) samples. The presence of TEMPONE in the incubation medium had no effect on oxygen consumption, demonstrating the utility of TEMPONE as a nonperturbing probe of the ubiquinol redox state. The rate of TEMPONE reduction was significantly increased over control levels for sperm incubated in hypertonic medium and was correlated to a decrease in oxygen consumption and a relative increase in ATP in the total adenine nucleotide pool. This increase in TEMPONE reduction in HIS sperm was reversed by treatment of sperm with seminal plasma, but seminal plasma had no effect on oxygen consumption or relative amounts of ATP in the adenine nucleotide pool. These observations are consistent with state 3 respiration in control sperm and state 4 respiration in HIS- and HIS-SP-treated sperm. Arrhenius data were obtained for ejaculated and epididymal sperm subjected to a variety of treatments. Lines fitted to plots of Arrhenius data revealed that each treatment affected the activation energy and intercept relative to controls. Evidence is presented for a phase transition occurring at 13 degrees C based on changes in the rate of TEMPONE reduction by ubiquinol. It was noted that, above the phase transition, rate constants for the reaction were dependent upon both treatment and temperature, but below the transition the differential effects of treatment were no longer apparent. The present study has demonstrated that events taking place in the respiratory chain can be closely monitored by measuring oxygen uptake and TEMPONE reduction, and that these events are affected by alterations in the sperm environment.

Animals↗

A relationship between adenosine 5'-triphosphate and pregnenolone syntheses by ovarian mitochondria as demonstrated with aminoglutethimide.

Aminoglutethimide, an inhibitor of pregnenolone synthesis, was used to study the relationship between oxidative phosphorylation and steroidogenesis in ovarian mitochondria. In vitro addition of aminoglutethimide to mitochondria prepared from porcine corpora lutea resulted in a decrease in oxygen uptake and an increase in the ADP to oxygen ratio, with maximal changes occurring at a concentration of 300 microM. Similar decreases in oxygen uptake were seen when aminoglutethimide was added to ovarian mitochondria from untreated and PMS gonadotropin-treated immature rats and intact and hypophysectomized mature rats. Also, aminoglutethimide increased the ADP to oxygen ratio in ovarian mitochondria from PMS gonadotropin-treated immature rats and intact mature rats. The addition of aminoglutethimide to luteal mitochondria inhibited their [4-14C]cholesterol conversion activity but increased their ATP synthesis rate by 25%. It is concluded that inhibition of ovarian mitochondrial steroidogenesis by aminoglutethimide is accompanied by both a decrease in oxygen consumption and an increase in ATP synthesis.

Adenosine Triphosphate↗

Myocardial force production and energy turnover in anoxia.

ATP turnover of isolated rabbit papillary muscles, contracting isometrically at 20 degrees C, was determined in oxygen and during 40 min of exposure to nitrogen (anoxia). Stimulus frequency was 0.2 hertz (Hz) in oxygen and 0.2 or 1.0 Hz in nitrogen. In oxygen, ATP turnover was determined from oxygen consumption using a P/O2 ratio of 6.3. The time-dependent rate of ATP turnover in nitrogen was found from the production of lactate, and the changes in adenine nucleotides and phosphocreatine, measured in rapidly frozen preparations at different time-points during the anoxic period. A P/lactate ratio of 1.5 was used. In muscles stimulated at 0.2 Hz, twitch force dropped during the anoxic period to 33% while force production of muscles stimulated at 1.0 Hz stopped completely. However, in the latter muscles, resting force rose to 19% of the twitch force in oxygen. The rate of ATP hydrolysis in anoxia depended strongly on stimulus frequency, indicating that it is not solely determined by the glycolytic capacity. In the 0.2 Hz-stimulated muscles the decrease in energy turnover occurred in parallel with the drop in force. However, the rise in resting force in muscles stimulated at 1.0 Hz occurred when ATP turnover was close to zero. It was concluded that anoxia hardly affects the energy required for twitch force production, but that the rise of resting force measured when twitch force had disappeared occurred when the rates of cross-bridge cycling and calcium turnover were very low.

Adenine Nucleotides↗

A novel technique for rapid determination of energy consumption in platelets. Demonstration of different energy consumption associated with three secretory responses.

A novel method has been developed for rapid and quantitative determination of the rate of energy consumption in platelets. In platelets suspended in a cyanide-containing medium. ATP resynthesis is abruptly blocked by addition of 2-deoxyglucose and D-glucono-1,5-lactone. We demonstrate that the subsequent changes in the levels of cytoplasmic ATP and ADP reflect the velocity of energy consumption in the platelets immediately before addition of the inhibitors. Despite the arrest in ATP resynthesis the platelets remain responsive to stimulation by thrombin (5 units x ml-1) which triggers the secretion of the contents of dense, alpha- and acid hydrolase granules. Unstimulated platelets were found to consume about 3.5 and 0.5 mumol of ATP equivalents x min-1 x (10(11) cells)-1 at 37 degrees C and 15 degrees C, respectively; the thrombin-treated platelets consumed respectively 16 and 2 mumol of ATP equivalents x min-1 x (10(11) cells)-1 at these temperatures. When the velocity of energy consumption was varied by (a) changing the temperature and (b) preincubation with glyco(geno)lytic inhibitors, it was found to be linearly related to the initial rate of secretion from the three types of granules. The precise nature of this relationship differed between the three types of secretion responses and indicated an increasing requirement for metabolic energy for secretion from the three types of granules in the order: dense granule less than alpha-granule less than acid hydrolase granule. The results obtained with changes in temperature were superimposable on those obtained with the glyco(geno)lytic inhibitors for dense granule secretion and alpha-granule secretion, suggesting an apparent coupling between energy consumption and the rate of these secretion responses. The rate of secretion of acid hydrolase was always higher when energy consumption was varied by temperature changes than when glyco(geno)lytic inhibitors were used, probably as a result of metabolic changes prior to induction of secretion. On the basis of these experiments, we calculated an incremental energy consumption during complete secretion of dense, alpha- and acid hydrolase granule contents of 2.5, 4.2 and 6.7 mumol of ATP equivalents x (10(11) platelets)-1, respectively.

Adenosine Triphosphate↗

Effects of Lewisite on cell membrane integrity and energy metabolism in human keratinocytes and SCL II cells.

Lewisite is a highly toxic arsenic compound which can cause skin damage. In the present study effects of Lewisite on cell membrane integrity and energy metabolism as well as antidotal effects of DL-2,3-dimercaptopropanesulfonate (DMPS), and meso-2,3-dimercaptosuccinic acid (m-DMSA) were investigated in a keratinocyte derived cell line (SCL II) and primary human keratinocytes (HK). Cells were incubated in Lewisite (60 microM) containing medium for 5 min. During the following 6 h lactate dehydrogenase (LDH) activity in the supernatant, intracellular ATP content, tetrazolium reduction, glucose consumption and lactate formation were measured. Glucose consumption and lactate production were decreased in both cell lines after Lewisite exposure. In SCL II cells an increase of LDH activity in the supernatant, a decrease of ATP content, and an impaired ability to reduce tetrazolium was found 3 h after Lewisite exposure. In HK cultures tetrazolium reduction was significantly decreased already after 2 h, whereas LDH increase in the supernatant and ATP content decrease occurred only at 6 h after Lewisite exposure. When DMPS or m-DMSA was added directly after Lewisite exposure to SCL II cells, glucose consumption and lactate formation were restored and LDH leakage was prevented. SCL II cells might be more prone to membrane damage whereas in keratinocytes mitochondrial impairment seems to be the predominant effect of Lewisite.

Adenosine Triphosphate↗

Role of K(ATP)(+) channels and adenosine in the control of coronary blood flow during exercise.

The present study was designed to examine the role of ATP-sensitive potassium (K(ATP)(+)) channels during exercise and to test the hypothesis that adenosine increases to compensate for the loss of K(ATP)(+) channel function and adenosine inhibition produced by glibenclamide. Graded treadmill exercise was used to increase myocardial O(2) consumption in dogs before and during K(ATP)(+) channel blockade with glibenclamide (1 mg/kg iv), which also blocks adenosine mediated coronary vasodilation. Cardiac interstitial adenosine concentration was estimated from arterial and coronary venous values by using a previously tested mathematical model (Kroll K and Stepp DW. Am J Physiol Heart Circ Physiol 270: H1469-H1483, 1996). Coronary venous O(2) tension was used as an index of the balance between O(2) delivery and myocardial O(2) consumption. During control exercise, myocardial O(2) consumption increased approximately 4-fold, and coronary venous O(2) tension fell from 19 to 14 Torr. After K(ATP)(+) channel blockade, coronary venous O(2) tension was decreased below control vehicle values at rest and during exercise. However, during exercise with glibenclamide, the slope of the line of coronary venous O(2) tension vs. myocardial O(2) consumption was the same as during control exercise. Estimated interstitial adenosine concentration with glibenclamide was not different from control vehicle and was well below the level necessary to overcome the 10-fold shift in the adenosine dose-response curve due to glibenclamide. In conclusion, K(ATP)(+) channel blockade decreases the balance between resting coronary O(2) delivery and myocardial O(2) consumption, but K(ATP)(+) channels are not required for the increase in coronary blood flow during exercise. Furthermore, interstitial adenosine concentration does not increase to compensate for the loss of K(ATP)(+) channel function.

ATP-Binding Cassette Transporters↗