Total Adenylate Research Articles

Metabolic Effects of Cold Storage on Livers from Euthermic and Hibernating Columbian Ground Squirrels

The current study was undertaken to investigate energy metabolism during hypoxia in the cold in livers from euthermic and hibernating Columbian ground squirrels. We hypothesized that the hibernating Columbian ground squirrel would be able to maintain liver energetics for a considerably longer time than euthermic animals. Particular reference was made to the function of glycolysis, which is the only mechanism for energy production under hypothermic ischemia. The transition from aerobic to anaerobic metabolism was apparent in both euthermic and hibernating animals as lactate levels rose within 1–3 h; total lactate accumulation was 2.5 μmol/g in both groups. In euthermic squirrels, liver ATP and ADP decreased considerably over the first 3-h storage; values dropped by 55% and 34%, respectively. Conversely, as the drain on high energy phosphate pools progressed, there was an increase in low energy adenylate, AMP. Between 10 and 24 h of storage, increases in AMP accounted for approximately 25–30% of total ATP + ADP decrease. The remainder of the drop in adenylates was accounted for by considerable decreases in total adenylate (TA) contents; by 24 h TA contents had decreased by 2.0 μmol/g. Livers from hibernating squirrels exhibited similar patterns of adenylate change and were not significantly higher than their euthermic counterparts. With respect to regulatory control of glycolysis, livers from euthermic squirrels exhibited no regulatory control at phosphofructokinase (PFK) or pyruvate kinase (PK). Livers from hibernating animals, however, showed an activation at PFK by 10 h of cold storage; levels of hexose phosphates, glucose-6-phosphate + fructose 6-phosphate (G6P + F6P), dropped and fructose 1,6-biphosphate (F1,6P2), increased. Changes in metabolite levels (phosphoenolpyruvate and pyruvate) associated with another key suspect regulatory enzyme, PK, indicated no role in regulatory control of glycolysis during the 24-h period. The apparent increase in PFK responsiveness to declining energy stores may be a futile activation since there was no accompanying increase in anaerobic end product, lactate, and no maintenance of energetics.

Ontogenetic differences in energy metabolism and inhibition of protein synthesis in hippocampal slices during in vitro ischemia and 24 h of recovery

The present study was designed to clarify whether ontogenetic differences in the vulnerability of the brain towards hypoxic-ischemic insults are only caused by the low cerebral energy demand of immature animals or whether there are additional mechanisms, such as protein synthesis (PSR), that may be involved in this phenomenon. We therefore measured tissue levels of adenylates and PSR in hippocampal slices from immature (1340) and mature (1360) guinea pigs fetuses and from adult guinea pigs during in vitro ischemia and 24 h of recovery using a recently modified method. Hippocampal slices were incubated in a temperature controlled flow-through chamber, gassed with 95% 02/5% C02. In vitro ischemia was induced by transferring slices to a glucose-free artificial cerebrospinal fluid (aCSF) eqiilibrated with 95% N2/5% C02. The duration of ischemia ranged from 10 to 40 min. Adenylates were measured by HPLC after extraction with perchloric acid. PSR was evaluated as the incorporation rate of (t4C)leucine into proteins. Under control conditions, tissue levels in adenylates did not change, whereas PSR increased slightly in hippocampal slices from mature fetuses and adult animals during a 24-h control incubation period. In slices from immature fetuses ATP levels were only maintained for 2 h. During in vitro ischemia the decline in ATP, total adenylate pool, and adenylate energy charge was much slower in slices from immature fetuses than in slices from mature fetuses or adults. After in vitro ischemia, ATP and the total adenylate pool did not completely recover in mature fetuses and adults, whereas adenylate energy charge almost returned to control values independently of the developmental stage. Two hours after in vitro ischemia PSR was undisturbed in slices from immature fetuses, but severely inhibited in slices from mature fetuses and adults. With ongoing recovery, PSR in mature fetuses returned to control values, while in adults it was still inhibited even 24 h after in vitro ischemia. From these results we conclude that hippocampal slices prepared from mature guinea pig fetuses as well as from adult guinea pigs can be held metabolically stable during long-term incubation using a recently modified technique. However, in slices from immature fetuses a stable energy state could not be maintained for more than 2 h. We further conclude that postischemic disturbances in PSR closely reflect the ontogenetic changes in the vulnerability of the brain to ischemia and that low energy metabolism is certainly not the only cause of the increased vulnerability of the fetal brain to ischemia.

Purine metabolism in cells of a mangrove plant, Sonneratia alba, in tissue culture

Summary A tissue culture system was established from pistils of flower buds of a mangrove plant, Sonneratia alba . The optimum growth rate of callus was observed in Murashige and Skoog's medium that contained 50 mM NaCl. In this tissue culture system, we examined the effects of short-term salt stress on the size of the pool of purine nucleotides, as well as the capacity for the salvage and the degradation of purines. At 100 mM NaCl, the size of the total adenylate pool was reduced, but the «adenylate energy charge» was, by contrast, increased. No marked change in the size of guanylate pool occurred in response to NaCl. The rate of conversion of [8- 14 C] adenosine to adenylate was decreased by NaCl, but that to nucleic acids was not significandy affected. In contrast to the results obtained with maize root tips (Peterson et al., 1988), salt stress did not increase the catabolism of [8- 14 C] hypoxanthine. Most of the radioactivity from [8- 14 C] adenosine was accumulated in adenine while that in [8- 14 C] hypoxanthine remained unaffected in Sonneratia alba . The low capacity of the purine-degradation system might ensure a supply of purine bases and nucleosides that can be utilized as substrates for the rapid and efficient biosynthesis of nucleotides by the salvage pathways.

Purine and carnitine metabolism in muscle of patients with Duchenne muscular dystrophy

We determined levels of purines, purine metabolites, related enzymes and carnitine in muscle of 8 untreated Duchenne muscular dystrophy (DMD) patients, 12 allopurinol-treated DMD patients and 12 age-matched controls. Muscle of DMD patients was found to be deficient in ATP, ADP, adenylsuccinate, hypoxanthine, guanine and adenylsuccinate synthetase. In allopurinol-treated DMD patients, mean total adenylate level was only three times less than in controls (versus 14 times less in untreated DMD patients). Mean inosine monophosphate (IMP), adenine, adenosine, inosine, xanthine, guanine, guanosine and uric acid levels were higher in allopurinol-treated patients than in controls, while mean adenylsuccinate levels were higher than in untreated patients. Allopurinol also restored acylcarnitine levels to normal and significantly increased free carnitine levels. These findings strongly support the hypothesis that Duchenne muscular dystrophy involves alterations leading to blockage of the IMP → purine pathway and that allopurinol treatment favours restoration of purine levels by this route. Furthermore, our results suggest that the observed deficiencies in cell components unrelated to purine metabolism are long-term secondary effects.

Protective properties of amino acids in liver preservation: effects of glycine and a combination of amino acids on anaerobic metabolism and energetics

Background/Aims/Methods: In this study, we investigated the hepatoprotective effects of three storage solutions containing glycine (180 mM), glycylglycine (180 mM), and a mixture of 20 amino acids (combined concentration of 180 mM) on energy metabolism and levels of glucose and lactate (as an index of glycolytic flux) in rat livers. All effects were compared to those of livers flushed/stored with a modified University of Wisconsin solution. Results: Glycine-treatment showed no improvement in liver energetics (ATP, ADP, AMP) and lactate accumulation; this solution had the lowest buffering capacity of the four tested (approximately 30% of the University of Wisconsin solution). The glycylglycine solution had the highest buffering capacity of the four solutions tested (including University of Wisconsin solution). Complete titration of the glycine-, combined amino acids-, and University of Wisconsin solutions (from 8.0 to pH=6.0) resulted in a minor decrease in glycylglycine buffer pH; pH dropped by 0.2 pH units. In glycylglycine-treated livers, energetics showed an improvement over the first 1 h cold storage; ATP and ‘energy charge’ values remained high and ADP levels (and consequently total adenylate contents) were 0.7–2.4 μmol/g greater than livers stored in University of Wisconsin solution. A 2-fold increase in lactate accumulation suggested that the improvement in liver energetics for the glycylglycine buffer was due to maintained flux through glycolysis brought about by enhanced buffering capacity. The solution containing a combination of amino acids exhibited maximum maintenance of liver energetics via increased glycolytic flux, despite its slightly inferior buffering capacity (85% of University of Wisconsin solution). ATP levels were maintained over the first 2 h storage and ADP levles (and consequently, total adenylate contents) were 1.2–2.1 μmol/g greater than University of Wisconsin solution-treated livers during the entire 24 h storage period. Energy charge values for livers treated with the combination of amino acids were also significantly higher than with glycine-, glycylglycine- and University of Wisconsin solution-treatment; even at 24 h, energy charge was 0.36 (comparable to only 4 h storage in University of Wisconsin solution). Conclusions: Our data suggest that a combination of amino acids may be required for maximum protection of the liver, and furthermore there may be several independent mechanisms, including buffering capacity, responsible for cytoprotection of the liver during cold storage.

The Importance of Calcium-Related Effects on Energetics at Hypothermia: Effects of Membrane-Channel Antagonists on Energy Metabolism of Rat Liver

During normothermic metabolism, the active pumping of Ca2+ across the cell membrane, mitochondria, and specialized sequestration organelles accounts for a large proportion of total energy expenditure in the cell. This study was designed to determine the effects of Ca2+ channel antagonists (chlorpromazine, verapamil, nifedipine, prenylamine, and nisoldipine) on energy metabolism and levels of glycolytic substrate (glucose) and anaerobic endproduct (lactate) during cold ischemia in rat livers. We hypothesized that if the passive channels were blocked during cold ischemia, then the ATP requirement of active ion pumping would be reduced and ATP levels and energy charge ratios would remain higher throughout the ischemic period; thus, viability of the liver would also be increased after prolonged ischemia. The most positive effect on energy metabolism was observed in the chlorpromazine-treated livers, followed by verapamil treatment. In the chlorpromazine treatment, total adenylate (TA) contents were 0.5-1.0 μmol/g (P < 0.05) higher than the sham group for most of the 24-h time course. Energy charge (EC) ratios were 0.05-0.07 higher than sham values up to 4-10 h ischemia. Verapamil treatment was less effective, but still exhibited positive effects on TA levels at several time points (20 min, 10 h, and 24 h) throughout the entire 24-h period. In both of these groups, TA values by 24 h ischemia were similar to levels at 10 h in the sham group (3.1 μmol/g), thus showing a considerable effect in maintaining adenylate levels. Despite similar pharmacological antagonist activities, ATP levels in the nifedipine, prenylamine, and nisoldipine treatment groups were 1.0-1.5 μmol/g (P < 0.05) less than the corresponding sham group (without Ca2+ antagonists) over the first 1 h ischemia. The decreases in high energy adenylate levels were reflected in lower EC ratios in these three groups; values were 0.06-0.17 (P < 0.05) lower than corresponding sham values. Finally, it was an unexpected finding that the sham injection (0.5 μmg/kg ethanol + PEG400) resulted in the sustained elevation of ATP, total adenylates, and EC values over the first h; EC ratios remained at initial (t = 0) values (EC = 0.71 ± 0.01) up to 1 h.

The dephosphorylation of AMP and IMP by a soluble low Km 5′nucleotidase from human seminal plasma: Some regulatory aspects

In this study, a soluble low K m 5′nucleotidase, dephosphorylating IMP with a V max/ K m ratio 10-times higher than that of AMP, has been purified from human seminal plasma. The effect of inorganic phosphate (P i) and adenylate energy charge variations on the activity of this enzyme has also been investigated. In the physiological range, with IMP as substrate, the activity of the enzyme does not change whereas the hydrolysis of AMP increases with decreasing energy charge values. In the presence of both the substrates, phosphate exerts an inhibitory effect on the enzyme activity with a similar concentration dependence pattern. The results show that AMP-hydrolysing activity responds to variations of energy charge by increasing the AMP degradation thus protecting the value of energy charge at the expense of a decrease in the total adenylate pool. In contrast, the dephosphorylation of IMP is not regulated by changes in energy charge. This data suggests that the degradation of IMP and AMP, although carried out by the same enzyme, is controlled by different regulatory mechanisms.

An underlying mechanism for improved liver preservation with a combined histidine-lactobionate-raffinose flush solution.

In previous experimental liver transplant studies, it was possible to extend cold ischaemic time (CIT) by using a flush/storage solution combining histidine, lactobionate and raffinose (HLR). In this study, energy metabolism, glycolytic substrate (glucose) and anaerobic end-product (lactate) were examined in rat liver over 24 h of cold storage to determine the mechanism of action of the HLR solution. In livers subjected to simple flush and storage with the HLR solution, levels of ATP and ADP were considerably higher than livers stored with modified UW throughout 24 h of storage; at 4 h of storage, ATP and ADP levels were 1.1 and 3.1 mumol/g for HLR solution versus 0.18 and 0.81 mumol/g for UW solution. Total adenylate contents (TA = ATP + ADP + AMP) also remained 1-2 mumol/g higher in HLR-treated livers than those preserved in UW; TA values ranged from 3.8 to 5.7 mumol/g. Glucose increased to 20-35 mumol/g by 10-24 h of storage (similar to the UW group). Lactate rose to almost twice that in livers stored in UW; total lactate accumulation was approximately 10.0 mumol/g. This study demonstrated that the combined HLR solution is able to prolong the maximum 'safe' CIT by increasing anaerobic metabolism and consequently preserving liver energetics. The second part of the experiment examined the effect of continuous perfusion (with/without O2) over the 1st h of cold ischaemia. Under current methods of liver flushing and excision, the 1st h of cold storage may be the critical time of metabolic 'adjustment' since most of the pH and ATP changes occur during this period.(ABSTRACT TRUNCATED AT 250 WORDS)

Response of potato tubers to hypoxia followed by re-aeration

Potato tubers kept under hypoxia (1%) showed improved viability in comparison to anoxia, which was associated with the maintenance of intermediate adenylate energy charge values (A.E.C.=0.6) and stable adenylate pools at 50% of the initial levels. Re-admission of oxygen to the tuber resulted in an almost full recovery of adenylate energy charge and total adenylates after up to 3 days of hypoxic pretreatment. Tubers exhibited a mixed fermentation. The high lactate, ethanol and acetaldehyde levels proved to be non toxic. Ethanol was degraded to acetaldehyde during re-aeration. Posthypoxic lipid peroxidation was indicated by malondialdehyde and ethane formation. Both products occurred with a temporary delay and in lower amounts compared to post-anoxia. Ethylene release was also considerably smaller. Severe hypoxia and posthypoxia postponed tissue death compared to anoxia. Survival was correlated with an improved energy supply which stabilized membranes.

An underlying mechanism for improved liver preservation with a combined histidine-lactobionate-raffinose flush solution

Abstract In previous experimental liver transplant studies, it was possible to extend cold ischaemic time (CIT) by using a flush/storage solution combining histidine, lactobionate and raffinose (HLR). In this study, energy metabolism, glycolytic substrate (glucose) and anaerobic end-product (lactate) were examined in rat liver over 24 h of cold storage to determine the mechanism of action of the HLR solution. In livers subjected to simple flush and storage with the HLR solution, levels of ATP and ADP were considerably higher than livers stored with modified UW throughout 24 h of storage; at 4 h of storage, ATP and ADP levels were 1.1 and 3.1 μmol/g for HLR solution versus 0.18 and 0.81 μmol/g for UW solution. Total adenylate contents (TA = ATP + ADP + AMP) also remained 1–2 μmol/g higher in HLR-treated livers than those preserved in UW; TA values ranged from 3.8 to 5.7 μmol/g. Glucose increased to 20–35 μmol/g by 10–24 h of storage (similar to the UW group). Lactate rose to almost twice that in livers stored in UW; total lactate accumulation was approximately 10.0 μmol/g. This study demonstrated that the combined HLR solution is able to prolong the maximum ‘safe’ CIT by increasing anaerobic metabolism and consequently preserving liver energetics. The second part of the experiment examined the effect of continuous perfusion (with/without O2) over the 1st h of cold ischaemia. Under current methods of liver flushing and excision, the 1st h of cold storage may be the critical time of metabolic ‘adjustment’ since most of the pH and ATP changes occur during this period. Therefore, we tested the hypothesis that the combination of a simple flush with an additional brief 1-h perfusion period prior to storage would enhance the maintenance of hepatic energetics. There was no beneficial effect of 1 h of perfusion without O2 compared to simple HLR flush and storage. However, perfusion with O2 resulted in prolonged maintenance of high energy adenylates and total adenylates; at 10 h of storage ATP was 1.0, ADP 3.3, and TA 5.7 μmol/g. However, any improvement in ultimate viability following long-term storage of the livers in these two groups needs to be tested in an animal transplant model.

Disturbed energy balance in skeletal muscle of patients with untreated primary hypertension.

It has been shown that the distribution of Na+ and Ca2+ in various cells is abnormal in patients with untreated primary hypertension, indicating an altered membrane permeability in these cells. This would activate certain ion pumps and thereby enhance ATP turnover. We investigated possible alterations in energy economy of skeletal muscle tissue. Skeletal muscle energetics were studied in vitro and in vivo in patients with untreated primary hypertension. Phosphocreatine (PCr), energy charge (EC) and total adenylate values were assessed. The study was performed at the outpatient clinic of a general hospital and at a university clinical chemistry department and at a specialized bioenergetic laboratory. Altogether, 17 patients with untreated primary hypertension were examined together with matched, healthy and normotensive controls with normal body-mass index. Skeletal muscle biopsies were obtained from 10 patients and 10 controls for analysis of high energy phosphate compounds. Another seven patients were enrolled for in vivo NMR spectroscopy. We found a decrease of 30% (P < 0.01) of PCr content in the patients, whilst EC and total adenylates were unchanged. Nuclear magnetic resonance spectroscopy showed an abnormal decrease of PCr during exercise followed by a markedly slower regeneration of PCr during post-exercise recovery parallelled by a slower recovery of pH. This phenomenon was mirrored by a more pronounced decrease of ATP/Pi in patients during exercise and a slower recovery of ATP/Pi. The data are compatible with an increased ATP turnover in skeletal muscle cells of patients with untreated primary hypertension although ATP was favoured and kept at a normal resting level at the expense of the PCr store.

Stimicrolation of Glycolysis by Histidine Buffers in Mammalian Liver during Cold Hypoxia

This study was designed to address the reasons why glycolysis in mammalian liver is unable to function more efficiently during periods of cold hypoxia. Our hypothesis was that control of intracellular pH, by use of amino acid buffers with high pKa values, would allow prolonged flux through glycolysis and better maintenance of liver high-energy adenine nucleotide pool. The effects of two concentrations of histidine (90 and 180 mM) and one of carnosine (90 mM), a histidyl dipeptide, on energy metabolism and levels of glycolytic substrate (glucose) and anaerobic endproduct (lactate) were investigated during cold hypoxia using rat livers to model the mammalian system. The transition to anaerobic metabolism was apparent by an immediate rise in lactate levels upon entry into cold hypoxia. By 10-14 h hypoxia, contents of the endproduct had increased by 10, 13.5, and 14.5 μmol/g in buffers containing 90 and 180 mM. histidine and 90 mM carnosine, respectively. As well, ATP, total adenylate contents, and "energy charge" ratios exhibited a rapid decline from initial values of 2.3-3.3 μmol/g, 4.3-5.5 μmol/g, and 0.64-0.75, respectively, over the first 2-4 h of cold hypoxia. With respect to efficacy, the 180 mM histidine buffer exhibited the most positive maintenance of adenylate levels, followed closely by 90 mM carnosine, and finally 90 mM histidine as the least effective of the three buffers. Nevertheless, all three buffers examined in this study showed positive effects compared to similarly treated livers stored in a solution of minor buffering capacity (a citrate-based solution) over the same time period. The data support the hypothesis that glycolytic flux and cellular energetics can be maintained by the inclusion of efficient buffering agents during periods of cold hypoxia.

Fermentation and Adenylate Metabolism of Hedychium coronarium J. G. Koenig (Zingiberaceae) and Acorus calamus L. (Araceae) under Hypoxia and Anoxia

Rhizomes of wetland plants are subjected to periods of hypoxia and/or anoxia by the seasonal or permanent waterlogging of their growing sites. Hedychium coronarium, the White Ginger, and Acorus calamus, the Sweet Flag, have their origin in India and were introduced into Latin America and Europe, respectively, more than three centuries ago. The White Ginger grows in humus-rich, shaded or semi-shaded areas subjected to waterlogging but it is never totally submersed, while the Sweet Flag grows at lake margins and is totally submersed during winter. Winter rhizomes of both species were cultivated in water culture in a greenhouse. The end products of fermentation (ethanol, lactic acid, malic acid), overall rhizome pH, the adenylate pool of nucleotides, the energy charge and their capacity to resume growth, were measured after periods of 1, 2, 4, 8 and 16 days of anoxia and hypoxia. In all cases metabolic responses were also determined in rhizomes allowed to recover for 24 h in air. Ethanol was the main fermentation end product in both species, reaching higher concentrations in the anoxia-treated rhizomes. In H. coronarium, there was also a significant increase in the levels of lactic acid, with a considerable drop in overall rhizome pH. Anoxia and hypoxia induced, in both species, a significant drop in the energy charge values. Control plant rhizomes and rhizomes allowed to recover in air for 24 h had energy charge values of around 0.8. In rhizomes subjected to stress these values were lower, around 0.50 in A. calamus and as low as 0.3 in H. coronarium. Although in both species there is also a decrease in the amount of total nucleotides, it was much more drastic in the case of anoxia treated rhizomes of H. coronarium. The pH drop was most probably the underlying cause of the metabolic disarray that lead to a depletion of the adenylate pool and, finally, failure to regenerate after 16 days of anoxia. The results also show that energy charge values without measurements of the total adenylate pool may give a misleading impression of fitness. Thus, the anaerobic metabolism of H. coronarium is less efficient and more harmful than that of A. calamus and, although considerably tolerant to hypoxia, it does not tolerate strict anoxia as the latter species does.

1015-71 Lower Adenosine Levels During Early Ischemia: Cause of Increased Injury in the Aging Heart?

Myocardial injury following ischemia and reperfusion is increased in the aging heart, including in the aging 24 mo Fischer 344 rat (mean survival 29 mo) compared to the Fischer 344 adult (6 mo). Adenosine (ADO) has been increasingly appreciated as a cardioprotective agent in myocardial ischemia. We hypothesized that a potential mechanism of the increased injury in the aging heart is decreased production of ADO in response to ischemia. Isolated buffer perfused (glucose 5 mM — insulin 5 μ /1) hearts from aging and adult Fischer 344 rats were subjected to stop-flow ischemia for either 2, 5, 10, 15 or 25 min after a 15 min equilibration phase. ADO and hypoxanthine (HX) were measured by HPLC. Tissue total adenylates, ATP, glycogen and lactate were measured. Ischemia Pre-ischemia 2 min 5 min 10 min 15 min 25 min (n = 5) (n = 5) (n = 5) (n = 5) (n = 5) (n = 5) ADO 6 mo 0.2 ± 0.1 0.7 ± 0.1 0.7 ± 0.1 2.2 ± 0.2 37 ± 0.6 4.5 ± 0.3 24 mo 0.2 ± 0.1 05 ± 0.1 0.3 ± 0.1 * 1.1 ± 0.3 * 2.1 ± 0.3 * 3.5 ± 0.4 HX 6 mo 2.0 ± 0.2 1.6 ± 0.2 2.3 ± 0.3 2.2 ± 0.1 25 ± 0.2 2.8 ± 0.2 24 mo 1.9 ± 0.2 1.4 ± 0.1 1.4 ± 0.2 * 2.1 ± 0.2 2.2 ± 0.8 3.5 ± 0.3 (Mean ± SE, nmol/mg protein) * p &lt; 0.05 vs 6 mo ADO levels were 50% lower in the aging heart at 5 and 10 min, remained depressed at 15 min and had not fully equalized to adult levels by 25 min of ischemia. HX was decreased at 5 min, consistent with decreased ADO production at that time. The change in total adenylate pool was similar in both groups. The decrease in tissue glycogen and increase in lactate were similar during ischemia in both groups, suggesting comparable glycolytic activity. ATP levels were decreased in aging hearts at 5 min (11.1 ± 2.3 vs 18.7 ± 1.9 nmol/mg protein), but reached levels similar to adult hearts as ischemia progressed. Thus, decreased ADO levels during early ischemia may reflect reduced production of ADO in the aging heart, and increase the susceptibility of the aging heart to damage during ischemia and reperfusion.

Obstruction of proximal tubules initiates cytoresistance against hypoxic damage

Following acute tubular necrosis (ATN), cytoresistance to further renal injury results. However, the initiating events and the subcellular determinants of this phenomenon have not been defined. Since tubular obstruction is a consequence of ATN, this study evaluated whether it alters tubular susceptibility to hypoxic damage. Extrarenal obstruction (ureteral ligation in rats) was used for this purpose to dissociate obstructive effects from those of ATN. Twenty-four hours following ureteral ligation or sham surgery, cortical proximal tubular segments (PTS) were isolated and subjected to hypoxic (15 or 30 min)/reoxygenation injury. Since oxidant stress, cell Ca2+ overload, and PLA2 attack are purported mediators of hypoxic/reoxygenation injury, degrees of FeS04, Ca2+ ionophore, and phospholipase A2-induced PTS damage also were assessed. The cell injury (% LDH release) which resulted from each of the above was consistently less in PTS obtained from obstructed kidneys. This cytoresistance: (a) did not require prior uremia to develop (seen with unilateral obstruction); (b) it did not appear to correlate with a tubular proliferative response (assessed by proliferating cell nuclear antigen expression); (c) it was uninfluenced by early tubular repair (unchanged by 24 hrs of obstruction release); and (d) it occurred without increased heat shock protein (HSP-70) or antioxidant enzyme (superoxide dismutase, catalase) expression. Total adenylate pools were higher in obstructed versus control PTS during injury; however, this appeared to be a correlate of the protection, rather than a mediator of it. In contrast, obstructed tubules manifested a primary increase in plasma membrane resistance to PLA2 attack (approximately 3-fold less lysophosphatidylcholine and free fatty acid generation in obstructed vs. control PTS during incubation with exogenous PLA2). In sum, these results indicate that: (1) tubular obstruction protects PTS from injury, suggesting that its development during ATN may initiate cytoresistance; and (2) this cytoresistance appears to be mediated, at least in part, by a direct increase in plasma membrane resistance to PLA2 and potentially other forms (such as, oxidant stress, cytosolic Ca2+ loading) of attack.

IMP metabolism in human skeletal muscle after exhaustive exercise

This study addressed whether AMP deaminase (AMPD)myosin binding occurs with deamination during intense exercise in humans and the extent of purine loss from muscle during the initial minutes of recovery. Male subjects performed cycle exercise (265 +/- 2 W for 4.39 +/- 0.04 min) to stimulate muscle inosine 5'-monophosphate (IMP) formation. After exercise, blood flow to one leg was occluded. Muscle biopsies (vastus lateralis) were taken before and 3.6 +/- 0.2 min after exercise from the occluded leg and 0.7 +/- 0.0, 1.1 +/- 0.0, and 2.9 +/- 0.1 min postexercise in the nonoccluded leg. Exercise activated AMPD; at exhaustion IMP was 3.5 +/- 0.4 mmol/kg dry muscle. Before exercise, 16.0 +/- 1.6% of AMPD cosedimented with the myosin fraction; the extent of AMPD:myosin binding was unchanged by exercise. Inosine content increased about threefold during exercise and twofold more during recovery; by 2.9 min postexercise it was 0.43 +/- 0.02 mmol/kg dry muscle. IMP decreased 2.1 +/- 0.3 mmol/kg dry muscle with no change in total adenylates. Total purines declined significantly (P < 0.05) during the recovery period in the nonoccluded leg, consistent with a loss of purines to the circulation, whereas total purines were unchanged in the occluded leg. Regulation of muscle purine content is a dynamic process that must accommodate rapid changes due to degradation and efflux.

Biogenic Sediment Compounds in Relation to Marine Meiofaunal Abundances

AbstractCorrelations between a series of biogenic sediment compounds, commonly used in ecological studies, and a major component of the benthic infauna, the meiofauna, were studied on the continental margin off Southwest Africa (Angola) and in a central oceanic region of the Atlantic Ocean (Mid Oceanic Ridge). Biogenic sediment compounds chosen for this investigation (electron‐transport‐system activity, total adenylates and energy charge, particulate proteins, chloroplastic pigments) are obviously not suitable for a quick and rough estimation of meiofaunal abundances. Nevertheless, biogenic sediment compounds might reflect quite well the activity and biomass of the total benthic infauna, including all size classes (from bacteria to macrofaunal organisms) and/or the total particulate organic matter within the sediments. Furthermore, analyzing biogenic sediment compounds leads to a better understanding of environmental conditions and biological activities of benthic organisms. Consequently, despite their limitations, biochemical sediment parameters may be very useful in benthic ecological studies to obtain rapid information on the eco‐status of the benthic system.

Glycolysis and energetics in organs of hibernating mice (Zapus hudsonius)

Hibernation-induced changes in the concentrations of glycolytic intermediates, creatine phosphate, and adenylates were monitored in brain and skeletal muscle of the meadow jumping mouse, Zapus hudsonius, after both short (24 h) and long (5 – 7 d) periods of hibernation. Levels of hexose phosphates were greatly reduced in both organs after 24 h of hibernation, suggesting strong suppression of carbohydrate catabolism early in hibernation and indicating enzymatic regulation at the level of carbohydrate input into glycolysis. Both organs showed large changes in energy status during hibernation. Creatine phosphate content declined progressively over time to levels in 5 – 7 d hibernating animals that were 50 and 60% of control values in brain and muscle, respectively. Total adenylates and ATP levels also fell sharply during hibernation, but the net effect of changing adenylate levels on energy charge was minimal; energy charge was 0.94 in both organs of control animals and fell to 0.88 –0.90 in hibernating animals. The overall reduction in the pool sizes of phosphagen and adenylates in organs of hibernating animals may be one factor involved in metabolic suppression during hibernation.

Respiratory Characteristics inPyricularia oryzaeExposed to a Novel Alkoxyiminoacetamide Fungicide

(E)-2-Methoxyimino-N-methyl-2(2-phenoxyphenyl) acetamide (SSF-126) inhibited respiration of Pyricularia oryzae mycelia by blockade of the electron flux through the cytochrome bc 1 segment of the mitochondrial respiratory chain. However, the mycelia again began to respire at 20 min after treatment. The recovered respiration was sensitive to salicylhydroxamic acid, a potent inhibitor of cyanide-resistant respiration, but not to potassium cyanide, indicating that SSF-126 had the ability to induce cyanideresistant respiration in P. oryzae. The recovered respiration was partially linked to energy transduction because SSF-126 could not completely suppress the mycelial growth. SSF-126 caused a rapid depletion of ATP content, but the ATP levels showed recovery, resulting in improvement of energy charge values. However, the total adenylate pool gradually decreased, resulting in only 60% remaining 48 h after treatment. SSF-126 markedly suppressed the mycelial growth, the duration for mass doubling in cultures incubated with SSF-126 being 2.4 times that in the control ones. Thus, SSF-126 works deleteriously on the supply of metabolic energy in P. oryzae

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.