Hypoxia as a risk factor for doxorubicin-induced cardiotoxicity: a NMR evaluation

Previous studies suggested that one possible mechanism of doxorubicin (DXR)-induced cardiomyopathy involves the depletion of high-energy phosphate stores. In this study, we used 31P nuclear magnetic resonance to assess the high-energy phosphate content in Langendorff perfused rat hearts. Hearts were perfused in normoxic conditions (spontaneous flow) or in partially hypoxic conditions obtained by perfusing at 50% of the spontaneous flow. DXR was used at the subtoxic conditions of 50 mg/l for 15 min and at the cardiotoxic concentration of 100 mg/l for 60 min. Left ventricular pressure (dP/dt), heart rate, myocardial ATP and PCr levels and PCr/ATP ratio were measured. We found that, in normoxic conditions, DXR (50 mg/l, 15 min) does not impair cellular high-energy phosphate metabolism. However, in mild hypoxic conditions, DXR induces a significant decrease in PCr/ATP ratio, due to a decrease in PCr and to a simultaneous increase in ATP. Similar results are obtained after 60 min perfusion with the cardiotoxic dose of DXR. This study suggests that hypoxia may represent a risk factor for the development of DXR-induced acute cardiotoxicity.     

Optimization of adenosine 5′-triphosphate extraction for the measurement of␣acidogenic biomass utilizing whey wastewater

A set of experiments was carried out to maximize adenosine 5′-triphosphate (ATP) extraction efficiency from acidogenic culture using whey wastewater. ATP concentrations at different microbial concentrations increased linearly as microbial concentration decreased. More than 50% of ATP was extracted from the sample of 39 mg volatile suspended solids (VSS)/l compared to the sample of 2.8 g VSS/l. The ATP concentrations of the corresponding samples were 0.74±0.06 and 0.49±0.05 mg/l, respectively. For low VSS concentrations ranging from 39 to 92 mg/l, the extracted ATP concentration did not vary significantly at 0.73±0.01 mg ATP/l. Response surface methodology with a central composite in cube design for the experiments was used to locate the optimum for maximal ATP extraction with respect to boiling and bead beating treatments. The overall designed intervals were from 0 to 15 min and from 0 to 3 min for boiling and bead beating, respectively. The extracted ATP concentration ranged from 0.01 to 0.74 mg/l within the design boundary. The following is a partial cubic model where η is the concentration of ATP and x _k is the corresponding variable term (k=boiling time and bead beating time in order): η=0.629+0.035x ₁–0.818x ₂–0.002x ₁ x ₂–0.003x ₁ ² +0.254x ₂ ² +0.002x ₁ ² x ₂. This model successfully approximates the response of ATP concentration with respect to the boiling- and bead beating-time. The condition for maximal ATP extraction was 5.6 min boiling without bead beating. The maximal ATP concentration using the model was 0.74 mg/l, which was identical to the experimental value at optimum condition for ATP extraction.     

Oxygen consumption in fathead minnows (Pimephales promelas) following acute exposure to water-borne selenium

Following determination of a concentration of selenate-Se lethal during 24-hr exposures at 22 degrees C, routine oxygen consumption was measured in fathead minnows (Pimephales promelas) exposed to various sublethal concentrations of Se for 24 hr. A median lethal concentration for 24-hr exposures (24-hr LC50) of fathead minnows equalled 82 mg Se/l with 95% fiducial limits of 76-89 mg Se/l. Mean weight-specific oxygen consumption rates of minnows exposed to 0, 15, 40 and 60 mg Se/l for 24-hr ranged from 0.43 to 0.49 mg O2/g-1/hr-1 and were not significantly different. These results indicate that acute exposure to Se does not affect oxygen consumption; however, observations during respirometry trials suggest that minnows at the highest Se concentration may have physiologically compensated to maintain oxygen consumption rates.     

Compartmentation of high-energy phosphates in resting and beating heart cells

The subcellular distribution of ATP, ADP, creatine phosphate and creatine has been analyzed by fast detergent fractionation of isolated frog heart cells. Digitonin fractionation (0.5 mg/ml, 10 s at 2 degrees C in 20 mM 4-morpholinepropanesulfonic acid/3 mM EDTA/230 mM mannitol medium) was used to separate mitochondria and myofilaments from cytosol. To separate myofilaments from the other cellular compartments. Triton X-100 was used (2%, 15 s in the same medium as digitonin). For either resting or beating cells the total cellular contents of ATP, ADP, creatine phosphate and creatine was similar, nevertheless the O2 consumption was 6-times higher. The compartmentation of these metabolites was also identical. Myofilaments contain 1.1 nmol ADP per mg total cellular proteins. In the cytosolic compartment the metabolite concentrations, all measured in nmol per mg total cellular proteins, were: ATP, 13; ADP, 0.25-0.05; creatine phosphate, 18.5 and creatine, 14. This indicated that the reaction catalyzed by creatine kinase was in a state of (or near) equilibrium.     

Correlation of microbial mass with ATP and DNA concentrations in acidogenesis of whey permeate

In this paper, we examine variations in the contents of ATP and DNA per unit microbial mass in an acidogenesis of whey permeate. We also introduce a novel approach to estimate microbial mass by measuring ATP and DNA when the ratios of ATP and DNA to microbial mass vary. Acidogenic experiments were performed at 35°C and pH 6.0 in batch mode. The amounts of ATP and DNA per unit microbial mass were not consistent during the incubation except during the post-decay phase. Especially within the exponential phase, each showed a 10-fold difference between maximal and minimal values. In this case, the conventional method which converts ATP or DNA concentration into microbial mass using a fixed conversion factor can give inaccurate results. While the constant ratios of 0.74 mg ATP/g VSS and 1.96 mg DNA/g VSS were determined for the post-decay phase, the ATP and DNA concentrations showed strong linear relationships with the microbial mass (r ² = 0.99) within the ranges of 0.039–1.078 mg ATP/l and 0.075–2.080 mg DNA/l, respectively. The linear regression equations are as follows: (1) microbial mass concentration (mg/l) = 478.5 × ATP concentration (mg/l) + 293.5, (2) microbial mass concentration (mg/l) = 257.2 × DNA concentration (mg/l) + 250.4. Therefore, changes in the mass of the acidogenic population should be monitored by the combined use of the regression equations obtained in the exponential phase and the constant ratios determined in the post-decay phase. This procedure should be widely applicable to the acidogenesis of dairy processing wastewaters, especially of a highly suspended organic wastewater such as whey.     

Ascorbic acid supplementation to primary culture of chicken hepatocytes with non-serum medium

Chicken liver is lack of ascorbic acid biosynthesis system, different from mammals and highly evoluted birds. Chicken hepatocytes cultured without ascorbate was expected to have lower ascorbate amounts than physiological levels. Intracellular was decreased as compared with intact liver by cell preparation performed with in situ collagenase perfusion. We added ascorbate to a primary culture of chicken hepatocytes in order to restore the amount of ascorbate. Serum-free Leivobitz's L-15 medium which do not contain ascorbate was used for control medium. Cells were cultured with several concentrations of ascorbate for 24 or 48 h. After ascorbate supplementation for 24 to 48 h, cellular ascorbate concentration increased depending on the dose of medium ascorbate. Medium lactate dehydrogenase activity derived from hepatocytes, an index of cell injury, decreased upon 5-100 mg/l of ascorbate supplementation for 48 h. Tyrosine aminotransferase activity, an index of liver function, increased following culture with 50 and 100 mg/l ascorbate for 48 h. The activities, however, decreased by supplementation with 1000 mg/l of ascorbate. In conclusion hepatocytes lost intracellular ascorbate during preparation by in situ collagenase perfusion. Supplementation of ascorbate restored cellular ascorbate concentration, lowered cell injury and raised tyrosine aminotransferase activitv in primary cultured chicken hepatocytes. Ascorbate treatment for 48 h at 50 mg/l was the best combination in this study for primary culture of chicken hepatpcyte with non-serum L-15 medium     

Mitochondrial respiratory chain adjustment to cellular energy demand

Because adaptation to physiological changes in cellular energy demand is a crucial imperative for life, mitochondrial oxidative phosphorylation is tightly controlled by ATP consumption. Nevertheless, the mechanisms permitting such large variations in ATP synthesis capacity, as well as the consequence on the overall efficiency of oxidative phosphorylation, are not known. By investigating several physiological models in vivo in rats (hyper- and hypothyroidism, polyunsaturated fatty acid deficiency, and chronic ethanol intoxication) we found that the increase in hepatocyte respiration (from 9.8 to 22.7 nmol of O(2)/min/mg dry cells) was tightly correlated with total mitochondrial cytochrome content, expressed both per mg dry cells or per mg mitochondrial protein. Moreover, this increase in total cytochrome content was accompanied by an increase in the respective proportion of cytochrome oxidase; while total cytochrome content increased 2-fold (from 0.341 +/- 0.021 to 0.821 +/- 0.024 nmol/mg protein), cytochrome oxidase increased 10-fold (from 0.020 +/- 0.002 to 0.224 +/- 0.006 nmol/mg protein). This modification was associated with a decrease in the overall efficiency of the respiratory chain. Since cytochrome oxidase is well recognized for slippage between redox reactions and proton pumping, we suggest that this dramatic increase in cytochrome oxidase is responsible for the decrease in the overall efficiency of respiratory chain and, in turn, of ATP synthesis yield, linked to the adaptive increase in oxidative phosphorylation capacity.     

Effects of nitroglycerin on energy metabolism of rat reticulocytes.

Nitric oxide (NO) in many cells inactivates aconitase and mitochondrial respiratory chain, and influenced glyceraldehyde 3-phosphate dehydrogenase activity. The aim of this study was to evaluate role of nitroglycerin (NTG), a widely used NO donor, on energy metabolism of rat reticulocytes. Rat reticulocyte rich red blood cell suspensions containing 70-100% of reticulocytes, were aerobically incubated without (control) or in the presence of different concentrations of (a) NTG (0.1, 0.25, 0.5, 1.0, 1.5 mmol/l), (b) 8-Br-cGMP (0.1, 0.5, 1.0 mmol/l) and (c) NaNO2 and NaNO3 (1 mmol/l). NTG in dose- and time-dependent manner decreased total (p>0.05; EC50 = 0.78+/-0.05 mmol/l) and coupled (p<0.05; EC50 = 0.50+/-0.04 mmol/l) and increased uncoupled oxygen consumption (p<0.05: EC50 = 0.36+/-0.01 mmol/l). They were accompanied by stimulation of glycolysis, as measured by increased glucose consumption and lactate accumulation (p<0.001 EC50 = 0.53 and 0.53 mmol/l, respectively). Levels of all glycolytic intermediates in the presence of NTG indicate stimulation of HK-PFK, GA3PDH and PK activity. NTG significantly decreased ATP level, which accompanied by increased ADP and AMP levels. However, level of total adenine nucleotides (TAN) was significantly lower, which was consequence of increased catabolism of adenine nucleotides (increased hypoxanthine level; p<0.05). Stimulation of glycolysis accompanied with inhibition of the OxP, activation of HK-PFK, decrease of ATP and simultaneous rise of ADP and AMP levels, all together represent an example of Pasteur effect occurring in NTG-treated reticulocytes. In rat reticulocytes under steady state conditions 93% of overall energy was produced by OxP, but only 7% by glycolysis. Due to decrease of coupled oxygen consumption in the presence of NTG, ATP production via OxP was significantly diminished. Simultaneous increase of glycolytic ATP production is not enough to provide constant either ATP production or concentration. Calculated mean ATP-turnover time was prolonged even for 45% in the presence of 1.5 mmol/l NTG. Metabolic effects of NTG were not mimic by exogenous 8-Br-cGMP, NaNO2 or NaNO3, which indicate that NTG induced a) inhibition of coupled respiration and b) stimulation of glycolysis in rat reticulocytes are mediated by NO as an effector molecule.     

Nitrogenase activity and regeneration of the cellular ATP pool in Azotobacter vinelandii adapted to different oxygen concentrations.

The in vivo activity of nitrogenase under aerobiosis was studied with diazotrophic chemostat cultures of Azotobacter vinelandii grown under glucose- or phosphate-limited conditions at different dilution rates (Ds, representing the growth rate mu) and different dissolved oxygen concentrations. Under steady-state conditions, the concentration as well as the cellular level of ATP increased in glucose-limited cultures when D was increased. Irrespective of the type of growth limitation or the dissolved oxygen concentration, the steady-state concentrations of ATP and of dinitrogen fixed by nitrogenase increased in direct proportion to each other. Specific rates of dinitrogen fixation as well as of the regeneration of the cellular ATP pool were compared with specific rates of cellular respiration. With glucose-limited cultures, the rate of regeneration of the ATP pool and the rate of respiration varied in direct proportion to each other. This relationship, however, was dependent on the dissolved oxygen concentration. As compared to the phosphate-sufficient control, phosphate-limited cultures exhibited the same nitrogenase activity but significantly increased respiratory activities. Rates of ATP regeneration and of cellular respiration of phosphate-limited cultures did not fit into the relationship characteristic of glucose-limited cultures. However, a linear relationship between the rates of dinitrogen fixation and ATP regeneration was identified irrespective of the type of growth limitation and the dissolved oxygen concentration. The results suggest that the ATP supply rather than cellular oxygen consumption is of primary importance in keeping nitrogenase activity in aerobic cultures of A. vinelandii.     

Interdependence of mitochondrial ATP production and extramitochondrial ATP utilization in intact spermatozoa

The dependence of both respiration and total activity of ATP-consuming reactions on the cellular adenine nucleotide pattern was investigated in intact bovine spermatozoa. ATP consumption was manipulated by inhibition with vanadate and activation with caffeine, leading to a decrease or increase in the rate of respiration up to 70% or 20%, respectively. Oligomycin blocked the respiration to the same extent as did vanadate, suggesting that the total extramitochondrial ATP-consuming activity is vanadate-sensitive. The major part of ATP utilization must be linked to dynein ATPase, since inhibition of (Na⁺, K⁺) ATPase by ouabain showed only a small effect on respiration (−17%). Being a potent inhibitor of dynein ATPase, vanadate drastically reduced the amount of motile cells, whereas caffeine tended to increase the intensity of motion. The effects of vanadate or caffeine on respiration were paralleled by changes in cellular ATP, reflecting the response of mitochondrial respiration on the cellular ATP/ADP ratio. Respiration was found to depend on changes in the ATP/ADP ratio in the range from about 3 (+ caffeine) to 9 (+ vanadate). The range of response of ATP consumption to the ATP/ADP ratio was determined by varying the mitochondrial ATP production via the concentration of lactate which was used as substrate. The measured effects on both respiratory rate and ATP/ADP ratio suggested that ATP consumption was markedly dependent on ATP/ADP ratios below 5. It is concluded that lactate concentrations above 1 mM sufficiently supply bovine spermatozoa with substrate and the energy turnover is mainly limited by the activity of dynein ATPase rather than by the capacity of mitochondrial oxidative phosphorylation.     

Oxygen consumption of the lobster,Homarus americanus milne-edwards

Summary 1. Oxygen consumption by a group of 25 lobsters was essentially constant over a range of ambient oxygen concentrations from 1.0 to 8.5 mg/l. Consumption by groups of 35 and 50 lobsters at 15° C decreased as the concentration decreased.2. Oxygen consumption by individuals at 10° and 15° C increased as the oxygen concentration increased.3. Oxygen consumption increased as activity increased with crowding.4. Oxygen consumption almost doubled after feeding.5. Oxygen consumption per unit weight decreased with increasing size.6. The average rate of oxygen consumption by individuals doubled over the temperature range 12° to 25° C.7. Oxygen consumption in air at 6° to 25° C was much less than in water.

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Sauerstoffverbrauch des HummersHomarus americanus Milne-Edwards

Kurzfassung Bei einer Gruppe von 25 Hummern wurde der Sauerstoffverbrauch in Dauerfluß-Respirometern gemessen. Bei 10° C erwies er sich als im wesentlichen konstant über einen Bereich der Sauerstoffkonzentration im umgebenden Wasser von 1,0 bis 8,5 mg/l. Bei Gruppen von 35 und 50 Hummern, welche bei 15° C getestet wurden, nahm der Sauerstoffverbrauch jedoch mit fallender Sauerstoffkonzentration etwas ab. Anstieg der Individuenzahl pro Raumeinheit (crowding) führte zu steigender Bewegungsaktivität und zu erhöhtem Sauerstoffverbrauch. Nahrungsaufnahme verursachte fast eine Verdoppelung des Sauerstoffverbrauchs. Kleine Individuen verbrauchen pro Gewichtseinheit mehr Sauerstoff als große. In manometrischen Respirometern stieg der Sauerstoffverbrauch bei hohen Sauerstoffkonzentrationen im umgebenden Wasser mit der Temperatur. Bei 6° bis 25° C war der Sauerstoffverbrauch in der Luft wesentlich geringer als im Wasser.

     

Bongkrekic acid facilitates glycolysis in cultured cells and induces cell death under low glucose conditions

Bongkrekic acid (BKA) inhibits adenine nucleotide translocator (ANT) and suppresses ADP/ATP exchange in the mitochondrial inner membrane. Previously, we demonstrated that BKA exhibited cytotoxic effects on 4T1 tumor cells, depending on the cell number in the culture, but not on NIH3T3 cells. However, the cause of this differential sensitivity was unelucidated. Here we demonstrate that BKA reduced the O₂ consumption in both cell lines and increased the mitochondrial membrane potential, thereby facilitating glucose consumption. BKA reduced cellular ATP in 4T1 cells in a dose-dependent manner but not in NIH3T3 cells. The cellular ATP of 4T1 cells was decreased with a reduced glucose concentration in the media, but that of NIH3T3 cells remained constant. We also demonstrated that BKA-induced cell death in both cell lines in low glucose media; however, the susceptibility to the reduced glucose concentration was slightly higher in 4T1 cells, which may be attributed to the difference in the dependency on glycolysis as their energy source. These results indicate that 4T1 tumor cells rely heavily on glucose for energy production. Our data demonstrate that BKA disturbs ATP production in mitochondria and increases the susceptibility to a low glucose condition.     

Effects of nitric oxide donor, isosorbide dinitrate, on energy metabolism of rat reticulocytes

Since nitric oxide (NO) in many cells is involved in energy metabolism, the aim of this study was to evaluate the role of isosorbide dinitrate (ISDN), a NO donor, in energy metabolism of rat reticulocytes, particularly due to their high content of hemoglobin--an effective scavenger of NO. Rat reticulocyte-rich red blood cell suspensions were aerobically incubated in the absence (control) or in the presence of different concentrations of ISDN. ISDN decreased total and coupled oxygen consumption (p<0.05) while increased uncoupled oxygen consumption (p<0.05) in a dose- and time-dependent manner. This was followed by enhancement of glycolysis, as measured by increased glucose consumption and lactate accumulation (p<0.05). Levels of all glycolytic intermediates in the presence of ISDN indicate only stimulation of pyruvate kinase activity. ISDN did not alter the concentration of ATP, while increased ADP and AMP levels (p>0.05). In rat reticulocytes under steady-state conditions, 95.4% of overall energy was produced by oxidative phosphorylation but only 4.6% by glycolysis. Due to a reduced coupled oxygen consumption in the presence of ISDN, ATP production via oxidative phosphorylation was significantly diminished. A simultaneous increase of glycolytic ATP production is not enough to ensure constant ATP production. The calculated mean ATP turnover time was prolonged by 199% in the presence of 1.5 mmol/l ISDN. In conclusion, ISDN a) inhibited total and coupled respiration but enhanced uncoupled respiration, b) stimulated glycolysis, c) decreased ATP production and d) prolonged ATP turnover time in rat reticulocytes. These effects were mediated by NO as the effector molecule.     

Nucleotides and the adenylate energy charge as indicators of stress in rainbow trout (Oncorhynchus mykiss) subjected to a range of dissolved oxygen concentrations

Liver nucleotides (ATP, ADP, AMP, IMP), the adenylate energy charge (AEC), total adenylate concentration (TA), and IMP-load were used as measures of stress in rainbow trout (Oncorhynchus mykiss) acclimated to normoxic (10.0 mg/l), hypoxic (6.5 mg/l), and supersaturated (13.0 mg/l) dissolved oxygen concentrations and subjected to a challenge by confinement. Liver ATP (783.0 nmol/g) was significantly different in the normoxic fish compared to either hyperoxic (447.7 nmol/g) or hypoxic (402.0 nmol/g) fish at the end of the confinement. Within 6.0 hr in the confinement, liver AEC in the normoxic fish increased significantly (0.58) compared to hypoxic (0.42) and hyperoxic fish (0.42). Similarly, the IMP-load in normoxic fish (0.16) decreased to near prestress levels by 6.0 hr in confinement compared to either the hypoxic (0.31) or hyperoxic (0.30) fish. Nucleotides in liver were significantly affected by the dissolved oxygen treatments and the confinement stress in contrast to the muscle nucleotides which were not.     

K(ATP)(+) channels, nitric oxide, and adenosine are not required for local metabolic coronary vasodilation

The role of ATP-sensitive K(+) (K(ATP)(+)) channels, nitric oxide, and adenosine in coronary exercise hyperemia was investigated. Dogs (n = 10) were chronically instrumented with catheters in the aorta and coronary sinus and instrumented with a flow transducer on the circumflex coronary artery. Cardiac interstitial adenosine concentration was estimated from arterial and coronary venous plasma concentrations using a previously tested mathematical model. Experiments were conducted at rest and during graded treadmill exercise with and without combined inhibition of K(ATP)(+) channels (glibenclamide, 1 mg/kg iv), nitric oxide synthesis (N(omega)-nitro-L-arginine, 35 mg/kg iv), and adenosine receptors (8-phenyltheophylline, 3 mg/kg iv). During control exercise, myocardial oxygen consumption increased ~2.9-fold, coronary blood flow increased ~2.6-fold, and coronary venous oxygen tension decreased from 19.9 +/- 0.4 to 13.7 +/- 0.6 mmHg. Triple blockade did not significantly change the myocardial oxygen consumption or coronary blood flow response during exercise but lowered the resting coronary venous oxygen tension to 10.0 +/- 0.4 mmHg and during exercise to 6.2 +/- 0.5 mmHg. Cardiac adenosine levels did not increase sufficiently to overcome the adenosine receptor blockade. These results indicate that combined inhibition of K(ATP)(+) channels, nitric oxide synthesis, and adenosine receptors lowers the balance between total oxygen supply and consumption at rest but that these factors are not required for local metabolic coronary vasodilation during exercise.     

Effects of hypoxia, anoxia, and metabolic inhibitors on KATP channels in rat femoral artery myocytes

Vascular ATP-sensitive potassium (KATP) channels have an important role in hypoxic vasodilation. Because KATP channel activity depends on intracellular nucleotide concentration, one hypothesis is that hypoxia activates channels by reducing cellular ATP production. However, this has not been rigorously tested. In this study we measured KATP current in response to hypoxia and modulators of cellular metabolism in single smooth muscle cells from the rat femoral artery by using the whole cell patch-clamp technique. KATP current was not activated by exposure of cells to hypoxic solutions (Po2 approximately 35 mmHg). In contrast, voltage-dependent calcium current and the depolarization-induced rise in intracellular calcium concentration ([Ca2+]i) was inhibited by hypoxia. Blocking mitochondrial ATP production by using the ATP synthase inhibitor oligomycin B (3 microM) did not activate current. Blocking glycolytic ATP production by using 2-deoxy-D-glucose (5 mM) also did not activate current. The protonophore carbonyl cyanide m-chlorophenylhydrazone (1 microM) depolarized the mitochondrial membrane potential and activated KATP current. This activation was reversed by oligomycin B, suggesting it occurred as a consequence of mitochondrial ATP consumption by ATP synthase working in reverse mode. Finally, anoxia induced by dithionite (0.5 mM) also depolarized the mitochondrial membrane potential and activated KATP current. Our data show that: 1) anoxia but not hypoxia activates KATP current in femoral artery myocytes; and 2) inhibition of cellular energy production is insufficient to activate KATP current and that energy consumption is required for current activation. These results suggest that vascular KATP channels are not activated during hypoxia via changes in cell metabolism. Furthermore, part of the relaxant effect of hypoxia on rat femoral artery may be mediated by changes in [Ca2+]i through modulation of calcium channel activity.     

Lactate stimulates insulin secretion without blocking the K+ channels in HIT-T15 insulinoma cells.

To clarify the mechanism by which lactate affects insulin secretion, we investigated the effect of lactate on insulin secretion, cytosolic free Ca2+ ([Ca2+](i), the ATP sensitive K+ channel (K(ATP)) and the Ca2+-activated K+ channel (K(Ca)) in HIT-T15 cells, and the results were compared with those of glucose and glibenclamide. All three agents caused insulin secretion and increased [Ca2+](i), but the effects on the K+ channels were different. In cell-attached patch configurations, 10 mmol/l glucose blocked both the K(ATP) and KCa channels, while 100 nmol/l glibenclamide had no effect on KCa channels, but blocked K(ATP) channels. Lactate at a concentration of 10 mmol/l activated both the K(ATP) and KCa channels, not only in cell-attached, but also in inside-out patch configurations, indicating that the increase in [Ca2+](i) and secretion of insulin by lactate cannot be explained by the blocking of the K+ channels. Lactate, at concentrations of 10 mmol/l and 50 mmol/l decreased 45Ca2+ efflux, while glibenclamide increased the efflux. These results suggest that the lactate-induced Ca2+ increase is not due to the closing of K+ channels, but at least in part, to the suppression of Ca2+ efflux from HIT cells.     

Adaptive respiratory significance of organophosphates (ATP & GTP) in air-breathing fishes

The ATP and GTP contents of erythrocytes of Protopterus annectens, Polypterus senegalus and Clarias lazera were assessed in relation to maturation and hypoxia. The steady state normal concentrations of ATP were: 446, 316 and 475 µM/100 ml and those of GTP were 286, 93 and 85 µM/100 ml in Protopterus, Polypterus and Clarias, respectively. The level of ATP decreased and that of GTP increased with increments in body growth, especially during development of the pulmonary organs. The concentration of GTP also increased progressively with hypoxia (O₂: 7.4 to 3.6 mg/l); the level ATP remained almost unaltered. The ratio of GTP increased markedly with increased hypoxia particularly in the adaptive respiratory range of aquatic oxygen concentrations (6.5–4.8 mg/l) when the fish were forced to depart from branchial respiration and became reliant on air-breathing.     

Demonstration of hormonal actions on Escherichia coli K 12 by potentiometry in the presence of lipoic acid; correlation with measurements of microbial growth and glucose consumption

The action of hormones on Escherichia coli K 12 was followed by potential-time measurements in the presence of lipoic acid (LA), photometric determinations of growth and glucose titrations. Bovine growth hormone (5 to 60 IU/1) increased O2 consumption. The (10 IU/1) insulin-facilitated transport of LA was stimulated by glucose. Adrenaline (0.5 to 15 mg/l) increased more glucose and O2 consumption than growth.     

Kinetics and compartmentation of energy metabolism in intact skeletal muscle determined from 18O labeling of metabolite phosphoryls

Analyses of isolated intact diaphragm muscle show that at rest only about 30% of the total cellular Pi is metabolically reactive as indicated by 18O incorporation from [18O]water, whereas up to 90% becomes metabolically active incrementally with contractile frequency. Kinetics of [gamma-18O]ATP appearance show that about 90% of the cellular ATP is metabolically active and suggest slowly and rapidly metabolizing compartments of ATP in resting muscle and only rapidly metabolizing compartments in contracting muscle. Rates of [18O]creatine phosphate [( 18O]CrP) appearance are consistent with creatine kinase-catalyzed phosphoryl exchange functioning in an obligatory phosphoryl shuttle system. In noncontracting muscle, ATP turnover rate was 83 nmol.mg protein-1.min-1, and the P/O ratio was determined to be 3.2. ATP utilization increases in direct proportion to contractile frequency with each contracture consuming the equivalent of 0.96 nmol of ATP.mg protein-1 or 2.5-3.5 molecules of ATP/myosin active site. Basal concentrations of nucleotide polyphosphates are not altered when ATP utilization rates increase during contraction. At high contractile frequencies, decreases in CrP concentration occur, but this accounts for less than 4% of total high energy phosphoryls consumed. If metabolic intermediates are free in the aqueous cellular cytosol, each twitch contracture would result in a decrease in ATP concentration of no more than 2% and increases in ADP and AMP concentrations of less than 20 and 7%, respectively. Thus, changes in metabolite concentration must be highly localized or metabolic regulation can be accomplished by a nonallosteric mechanism.