Adenylate Pool Research Articles

Increased activities of antioxidant enzymes and decreased ATP concentration in cultured myoblasts with the 3243A→G mutation in mitochondrial DNA

The MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) is most commonly caused by the 3243A→G mutation in mitochondrial DNA, resulting in impaired mitochondrial protein synthesis and decreased activities of the respiratory chain complexes. These defects may cause a reduced capacity for ATP synthesis and an increased rate of production of reactive oxygen species. Myoblasts cultured from controls and patients carrying the 3243A→G mutation were used to measure ATP, ADP, catalase and superoxide dismutase, which was also measured from blood samples. ATP and ADP concentrations were decreased in myoblasts with the 3243A→G mutation, but the ATP/ADP ratio remained constant, suggesting a decrease in the adenylate pool. The superoxide dismutase and catalase activities were higher than in control cells, and superoxide dismutase activity was slightly, but not significantly higher in the blood of patients with the mutation than in controls. We conclude that impairment of mitochondrial ATP production in myoblasts carrying the 3243A→G mutation results in adenylate catabolism, causing a decrease in the total adenylate pool. The increase in superoxide dismutase and catalase activities could be an adaptive response to increased production of reactive oxygen species due to dysfunction of the mitochondrial respiratory chain.

Flight-muscle adenylate pool responses to flight demands and thermal constraints in individual Colias eurytheme (Lepidoptera, pieridae).

We study here the connections among body temperature variation, flight performance and flight 'fuel' metabolism in Colias eurytheme butterflies, to begin re-examining the metabolic reasons for animal thermoregulation. Methods are presented for (a) stable extraction of adenylates (and other metabolites) from the flight muscles of individual Colias eurytheme, (b) automated separation and quantitative analysis of individual adenylate samples by high-pressure liquid chromatography and (c) reliable, low-variance assay of inorganic phosphate levels in the same extracts. Correlations among metabolite concentrations and two indices of muscle cytosol ATP maintenance occur as expected on general metabolic principles. [ATP] and [ATP]/[ADP] decline from resting levels to reach a plateau in the first minute of free, interrupted flight, while [AMP] increases at the same time; these concentrations do not vary further for up to 6 min total flight time. In an initial test of the alternative metabolic bases of the thermoregulation of Colias eurytheme, we find that [ATP]/[ADP] rises between a body temperature, T(b), of 31 and 35 degrees C, at the base of the behavioral thermal optimum for flight, but then decreases again at T(b)=39 degrees C, at the top of the behavioral thermal optimum and well short of damaging temperatures. This is not consistent with the view that metabolic effectiveness increases monotonically up to the lower limits of thermal damage to enzymes, but supports an alternative hypothesis that the narrowness of thermoregulation results from a system-based constraint on the breadth of temperature over which maximal energy processing is possible.

Adenine incorporation in human and rat endothelium

Adenine (ADE) reutilisation is an important pathway of adenylate pool regeneration. Data on the rate of this process in different types of cells, its regulation and the importance of species differences is limited. In this study we evaluated adenine incorporation rate and the effect of metabolic factors on this process in human and rat endothelium and compared it to adenine phosphoribosyltransferase (APRT) activity. Microvascular endothelial cells from human (HE) and rat (RE) hearts and a transformed human microvascular endothelial cell line (HMEC-1) were investigated. The rate of adenine incorporation into the adenine nucleotide pool under control conditions was 3.1±0.3, 82.8±11.1 and 115.1±11.2 pmol/min per mg protein for HE, RE and HMEC-1, respectively. In the presence of 2.5 mM ribose or elevated inorganic phosphate concentration in the medium (4.8 mM), few changes were observed in all types of cells. In the presence of both ribose and high inorganic phosphate, the rate of adenine incorporation for RE and HMEC-1 was not significantly different from control, while in HE the rate of adenine incorporation into adenine nucleotides was increased by 75%. Activities of APRT in RE and HMEC-1 were 237.7±23.2 and 262.0±30.6 pmol/min per mg protein respectively while the activity in HE was markedly lower 48.7±3.0 pmol/min per mg protein. In conclusion, nucleotide synthesis from adenine seems to be a slow process in human cardiac microvascular endothelium but it is fast and efficient in rat heart microvascular endothelial cells. Low APRT activity in normal human endothelial cells seems to be the most likely mechanism for this. However, adenine incorporation rate and APRT activity could be greatly enhanced in human endothelium, as demonstrated in transformed cells.

The bioenergetics of embryonic diapause in an annual killifish, austrofundulus limnaeus

The annual killifish Austrofundulus limnaeus inhabits ephemeral ponds that dry out on a seasonal basis, thereby killing the adult and juvenile forms. Populations persist because diapausing embryos become embedded in the pond sediments. The rate of oxygen consumption of diapause II embryos is depressed by up to 90 % compared with that of developing embryos, and a parallel reduction is observed in heart rate. Developmental arrest was identified by cessation of somite proliferation and blockage of the ontogenetic increase in DNA content. Surprisingly, the arrest of metabolism and development is temporally offset as embryos reach diapause II; metabolic rate begins to decline 12 days prior to arrest of development. Release of embryos from diapause II is facilitated by increasing the light phase of the photoperiod. The rate of oxygen consumption of diapause III embryos is 84 % lower than the value preceding diapause III. The total energy flow of diapause II embryos apparently includes a contribution from anaerobic processes on the basis of calorimetric/respirometric ratios that are above the oxycaloric equivalent. Accumulations of lactate and ethanol at the expense of glycogen reserves are small or undetectable and do not account for the excess heat signal. Diapause II embryos maintain high [ATP]/[ADP] ratios and adenylate energy charge during diapause, consistent with a simultaneous depression of energy use and demand. Levels of AMP increase during early development and diapause II despite the highly charged adenylate pool. High values for [AMP]/[ATP] ratios in diapause II embryos are correlated with decreased rates of oxygen consumption and heat dissipation, which suggests a role for AMP in the depression of metabolism during early development and diapause II.

Effects of indomethacin, with vitamin A or β-carotene, on the rat gastric mucosa: Extra-and intracellular membrane-bound ATP mechanisms

Indomethacin (IND) is a widely used non-steroidal anti-inflammatory agent in the treatment of various inflammatory disorders, which causes gastrointestinal injury in humans and animal experiments. Vitamin A and beta-carotene prevent the IND-induced gastric mucosal injury. These compounds modify the membrane-bound ATP-dependent energy systems. The aims of this investigation were: (1) To study the IND-induced gastric mucosal damage and its prevention by vitamin A and beta-carotene; (2) to measure the biochemical compounds of the gastric mucosa ATP, ADP, ATP/ADP, AMP, ATP+ADP+AMP, 'energy charge' (ATP + 0.5 ADP)/(ATP+ADP+AMP), cAMP, lactate under the circumstances mentioned above; (3) to analyze the extra- and intracellular regulatory mechanisms between the membrane-bound ATP-dependent energy systems. The observations were carried out with CFY (Sprague-Dawstrein rats, weighing 180-210 g). The gastric mucosal damage was produced by IND (20 mg/kg sc. administration) and it was prevented by vitamin A (or beta-carotene), given in doses of 0.01-0.1 to 1.0-10.0 mg/kg ig. Different biochemical compounds (ATP, ADP, AMP, cAMP, lactate) and parameters (ATP/ADP, adenylate pool, 'energy charge') were measured and calculated. (1) Vitamin A and beta-carotene prevented dose-dependently the IND-induced gastric mucosal damage; (2) the extent of ATP-ADP transformation was increased significantly, while the ATP-cAMP decreased in the gastric mucosa after IND-treatment; (3) vitamin A and beta-carotene enhanced the extent of ATP-cAMP transformation, while the ATP-ADP transformation was inhibited (the actions were dose-dependent responses); (4) No change was found in 'energy charge' by IND, while its value decreased significantly with vitamin A and beta-carotene. (1) A very complex extra- and intracellular feedback mechanism system exists in the gastric mucosa during IND, IND + vitamin A, and IND + beta-carotene treatments; (2) The gastric mucosal preventive effect of vitamin A and beta-carotene only partly depend on their scavenger properties.

Adenine Nucleotide Synthesis in Human Erythrocytes Depends on the Mode of Supplementation of Cell Suspension with Adenosine

ABSTRACT: In suspensions of washed human erythrocytes, adenosine added in a single dose to concentrations of 0.1–10.0 mmol/l suspension was deaminated at rates ranging from 10 to 50 mmol/l cells h. The sum of adenosine, inosine, and hypoxanthine concentrations in the suspension, as well as the intracellular concentration of ATP, remained constant. In the presence of 25–50 mmol/l orthophosphate, addition of a single dose of adenosine into erythrocyte suspension increased the ATP concentration by up to 280% of the initial level. If the initial adenosine concentrations were greater than 5 mmol/l suspension, ATP increased independently of adenosine concentration to the level determined only by the concentration of orthophosphate. After orthophosphate was returned to its initial level, ATP in erythrocytes began to decrease. In the presence of coformycin, erythrocytes utilised adenosine at a rate of 0.2–0.3 mmol/l cells h. Their adenylate pool increased at a rate of 0.10–0.16 mmol/l cells h for several hours, but intracellular ATP increased only slightly. The energy charge of cells decreased significantly from 0.86±0.05 (control) to 0.82±0.06. Adenosine continuously pumped into erythrocyte suspensions at rates of 0.02–5.0 mmol/l cells h for several hours caused the adenylate pool of erythrocytes and intracellular ATP to increase synchronously at a rate of 0.02–0.35 mmol/l cells h. The energy charge of these erythrocytes increased significantly up to 0.91±0.03. After pumping of adenosine was stopped, the intracellular ATP and the adenylate pool began to decrease, returning sometimes to the initial level in 2–3 h.

The effect of stress and exercise on post‐mortem biochemistry of Atlantic salmon and rainbow trout

Freshwater Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss responded similarly to increase in water flow (exercise), reduction in holding tank water level (stress), or 30 min chasing with water level reduction (stress and exercise). Stress generally resulted in elevated plasma cortisol, above the control. Fish responded to stress and exercise combined, with elevated lactate and [H+] which was sometimes associated with elevated plasma cortisol. These changes were combined with a depletion of the muscle adenylate pool. Post‐mortem, this resulted in an increase in the rate of onset of rigor, and a higher and sometimes sustained muscle proton load. Both species produced predominantly inosine as opposed to hypoxanthine, for up to 72 h of ice storage. This study shows that the physiological disruption in Atlantic salmon and rainbow trout caused by simulated harvest conditions of stress and exercise, results in mostly transient changes in post‐mortem muscle biochemistry. These changes lead to an earlier onset and resolution of rigor, and lower post‐mortem muscle pH in comparison to the control.

The site of oxygen limitation in soybean nodules

In legume nodules the [O2] in the infected cells limits respiration and nitrogenase activity, becoming more severe if nodules are exposed to subambient O2 levels. To identify the site of O2 limitation, adenylate pools were measured in soybean (Glycine max) nodules that were frozen in liquid N2 before being ground, lyophilized, sonicated, and separated on density gradients of nonaqueous solvents (heptane/tetrachloroethylene) to yield fractions enriched in bacteroid or plant components. In nodules maintained in air, the adenylate energy charge (AEC = [ATP + 0.5 ADP]/[ATP + ADP + AMP]) was lower in the plant compartment (0.65 +/- 0.04) than in the bacteroids (0.76 +/- 0.095), but did not change when the nodulated root system was exposed to 10% O2. In contrast, 10% O2 decreased the bacteroid AEC to 0.56 +/- 0.06, leading to the conclusion that they are the primary site of O2 limitation in nodules. To account for the low but unchanged AEC in the plant compartment and for the evidence that mitochondria are localized in O2-enriched microenvironments adjacent to intercellular spaces, we propose that steep adenylate gradients may exist between the site of ATP synthesis (and ADP use) in the mitochondria and the extra-mitochondrial sites of ATP use (and ADP production) throughout the large, infected cells.

Novel Aspects of Tetramer Assembly and N-terminal Domain Structure and Function Are Revealed by Recombinant Expression of Human AMP Deaminase Isoforms

AMP deaminase isoforms purified from endogenous sources display smaller than predicted subunit molecular masses, whereas baculoviral expression of human AMPD1 (isoform M) and AMPD3 (isoform E) cDNAs produces full-sized recombinant enzymes. However, nearly 100 N-terminal amino acid residues are cleaved from each recombinant polypeptide during storage at 4 degreesC. Expression of N-truncated cDNAs (DeltaL96AMPD1 and DeltaM90AMPD3) produces stable recombinant enzymes exhibiting subunit molecular masses and kinetic properties that are similar to those reported for purified isoforms M and E. Conversely, wild type recombinant isoforms display significantly higher Km(app) values in the absence of ATP. Gel filtration analysis demonstrates native tetrameric structures for all recombinant proteins, except the wild type AMPD1 enzyme, which forms aggregates of tetramers that disperse upon cleavage of N-terminal residues at 4 degreesC. These data: 1) confirm that available literature on AMP deaminase is likely derived from N-truncated enzymes and 2) are inconsistent with a new model proposing native trimeric structure of an N-truncated rabbit skeletal muscle AMP deaminase (Ranieri-Raggi, M., Montali, U., Ronca, F., Sabbatini, A., Brown, P. E., Moir, A. J. G., and Raggi, A. (1997) Biochem. J. 326, 641-648). N-terminal residues also influence actomyosin-binding properties of the enzyme, which are enhanced and suppressed by AMPD1 and AMPD3 sequences, respectively. Finally, co-expression of AMPD1 and AMPD3 recombinant polypeptides produces tetrameric enzymes with either isoform-specific or mixed subunits, and also reveals that tetramer assembly is driven by relative polypeptide abundance with no apparent preference for like subunits.

Adenylates and adenylate-energy charge in submerged and planktonic cultures of Salmonella enteritidis and Salmonella typhimurium

Adenine nucleotide values and adenylate energy charge (AEC) were measured during the growth of Salmonella enteritidis and Salmonella typhimurium as submerged colonies in agarose gel and gelatin gel, and as planktonic cells in broth. Growth in all three systems showed similar trends with a ten-fold decrease in total adenylate pool during exponential growth, before attaining a fairly stable value throughout stationary phase. AEC values were generally low, (≈0.66), but did rise slightly during stationary phase. The large proportion of dead cells during early exponential phase may have contributed to the adenosine diphosphate and adenosine monophosphate pools, through cell lysis or excretion, and it is suggested that this was likely to account for the low values of AEC. In agarose and gelatin gelled cultures the percentage of adenosine triphosphate (ATP) in relation to the total adenylates showed random fluctuations. This was contrary to the broth culture where percentage ATP was highest after 12 h and the data formed a smooth curve. These data demonstrated that considerable physiological heterogeneity exists within a colony of bacteria growing in a gel matrix and by analogy a food material also, and that AEC is a poor indicator of cell viability in such systems.

Enhancement of doxorubicin content by the antitumor drug lonidamine in resistant ehrlich ascites tumor cells through modulation of energy metabolism

The effect of the antitumor drug lonidamine (LND) on respiration, aerobic glycolysis, adenylate pool, doxorubicin (DOX) uptake, and efflux in DOX-resistant and DOX-sensitive Ehrlich tumor cells was investigated. The results may be summarized as follows: 1) In both types of cells, LND inhibited both respiration and glycolysis in a dose-dependent manner and lowered the ATP concentration. The effect was more marked in cells incubated in glucose-free medium; 2) LND raised, to a remarkable extent, the intracellular content of DOX in resistant and sensitive cells respiring on endogenous substrates because of reduced ATP availability, whereas in glucose-supplemented medium, where both respiration and glycolysis contributed to ATP synthesis, the increase was lower; and 3) when LND was added to DOX-loaded cells, it failed to significantly inhibit DOX efflux because of time-dependent phenomena. These findings indicated that LND, a drug currently employed in tumor therapy, might also be useful in reducing or overcoming multidrug resistance (MDR) of those cells with a reduced ability to accumulate and retain antitumor drugs.

Disruption of the phosphate-starvation response of oilseed rape suspension cells by the fungicide phosphonate.

The influence of the anti-fungal agent phosphonate (Phi) on the response of oilseed rape (Brassica napus L. cv. Jet Neuf) cell suspensions to inorganic phosphate (Pi) starvation was examined. Subculture of the cells for 7 d in the absence of Pi increased acid phosphate (APase; EC 3.1.3.2) and pyrophosphate (PPi)-dependent phosphofructokinase (PFP; EC 2.7.1.90) activities by 4.5- and 2.8-fold, respectively, and led to a 19-fold increase in Vmax and a 14-fold decrease in Km (Pi) and Pi uptake. Addition of 2 mM Pi to the nutrient media caused dramatic reductions in the growth and Pi content of the Pi-starved, but not Pi-sufficient cells, and largely abolished the Pi-starvation-dependent induction of PFP, APase, and the high-affinity plasmalemma Pi translocator. Immunoblotting indicated the cells contain three APase isoforms that are synthesized de novo following Pi stress, and that Pi treatment represses this process. Phosphonate treatment of Pi-starved cells significantly altered the relative extent of in-vivo 32P-labelling of polypeptides having M(rs) of 66, 55, 45 and 40 kDa. However, Phi had no effect on the total adenylate pool of Pi-starved cells which was about 32% lower than that of Pi-sufficient cells by day 7. Soluble protein levels, and activities of pyruvate kinase (EC 2.7.1.40) and ATP-dependent phosphofructokinase (EC 2.7.1.11) were unaffected by Pi starvation and/or Phi treatment. The effects of Phi on the growth, and APase and PFP activities of Pi-starved B. napus seedlings were similar to those observed in the suspension cells. The results re consistent with the hypothesis that a primary site of Phi action in higher plants is at the level of the signal transduction chain by which plants perceive and respond to Pi stress at the molecular level.

Dynamic compartmentation of vacuolar amino acids in Penicillium cyclopium. Cytosolic adenylates act as a control signal for efflux into the cytosol.

The regulation of amino acid transport from the vacuolar reservoir into the cytoplasm has been studied in hyphal cells of Penicillium cyclopium. To avoid artifacts caused by the isolation of vacuoles, efflux was examined "in situ," i.e. in cells whose plasma membranes were permeabilized for micromolecules by a treatment with nystatin. The ATP-dependent proton gradient and amino acid transport activities at the vacuolar membrane remained intact under these conditions. Accumulation of amino acids in the vacuole proved to be the result of a dynamic equilibrium of active, ATP-dependent uptake and energy-independent efflux. The latter was strongly accelerated after the vacuolar amino acid content had surpassed a threshold level. Efflux of vacuolar amino acids was specifically controlled by extravacuolar adenylates: ATP, 5'-adenylyl imidodiphosphate (an ATPase-resistant ATP-analogue), ADP, or AMP caused a strong inhibition in the concentration range around 200 micromol/liter, whereas both lower and higher concentrations allowed significant efflux rates. Estimates of the cytosolic adenylates (which consisted mainly of ATP) were close to 2 mmol/liter in glucose-metabolizing cells, which concentration allowed maximum rates of both vacuolar uptake and efflux. During 24 h of carbon and nitrogen starvation, the adenylate level decreased toward the efflux-inhibiting region around 200 micromol/liter, whereas 3-4 d of carbon and nitrogen starvation caused a further decline of the adenylate content, leading again to efflux-permitting concentrations. Thus, the cytosolic adenylate pool appears to effectively control the availability of vacuolar amino acids for the cellular metabolism.

Nitrate reductase activation state in barley roots in relation to the energy and carbohydrate status.

The NADH-dependent nitrate reductase (NR, EC 1.6.6.1) in roots of hydroponically grown barley seedlings was extracted, desalted and the activity measured in buffer containing either Mg2+ (10 mM) or EDTA (5 mM). The former gives the actual NR activity (NRact) equivalent to dephospho-NR, whereas the latter gives the maximum NR capacity of the dephospho-form (NRmax). Both values together permit an estimation of the NR-phosphorylation state. Changes in NRact and NRmax were followed in response to root aeration or to shoot illumination or shoot removal, and were correlated with sugar contents and adenylate levels. Ethanol formation was also measured in roots differing in NR activity in order to obtain information on the relation between anaerobic alcoholic fermentation and nitrate reduction. In aerated roots, NR was highly phosphorylated (about 80%) and largely inactive. It was partly dephosphorylated (activated) by anoxia or by cellular acidification (pH 4.8 plus propionic acid). Anaerobic activation (dephosphorylation) of NR was stronger at acidic external pH (5) than at slightly alkaline pH (8), although ATP levels decreased and AMP levels increased at pH 5 and at pH 8 to the same extent. Thus, rapid changes in the NR-phosphorylation state in response to anaerobiosis were not directly triggered by the adenylate pool, but rather by cytosolic pH. Under prolonged darkness (24 h) or after shoot removal. NRmax decreased slowly without a large change in the phosphorylation state. This decrease of NRmax was correlated with a large decrease in the sugar content, and was prevented by glucose feeding, which had only minor effects on the phosphorylation state. Cycloheximide also prevented the decrease in NRmax without affecting the phosphorylation state. In contrast, anaerobiosis or cellular acidification prevented the decrease of NRmax and at the same time decreased the NR-phosphorylation state. It is suggested that NR turnover in roots is controlled by several factors: NR synthesis appears to depend on sugar availability, which has little effect on the phosphorylation state; in addition, NR degradation appears to be strongly affected by the phosphorylation state in such a way that the inactive phospho-NR is a better substrate for NR degradation than the dephospho-form. The rate of anaerobic ethanol formation was not affected by NR activity, indicating that the purpose of NR activation under hypoxia or anoxia is not to decrease or prevent alcoholic fermentation.

Time-related effects of intoxication with cadmium and mercury in the red wood ant

Biochemical and physiological effects of prolonged feeding of the ant Formica aquilonia, in natural conditions with excess of cadmium or mercury, were studied. In all developmental stages metals caused the time-dependent increase of AMP, parallel with a decrease of ATP and the pool size of adenylates. This was reflected by a low adenylate energy charge (AEC) index and accompanied by an inhibition of ATPases. Despite the fall in the adenylate pool of workers, the levels of adenylate energy charge confirmed their ability to maintain energetic balance. Prolonged access to food highly contaminated with Cd, or Cd with Hg, diminished these adaptive abilities, but pre-adaptive reactions (not correlated with the metal load) were induced. Activity patterns of enzymes involved in energy metabolism showed metal dependent inhibitory effects, but repeated contamination evoked some compensatory mechanisms, both in workers and in the pupal stage from the next generations. Cadmium (indirectly) stimulated the esterases and free radical scavengers. Compensatory mechanisms were insufficient in insects contaminated with both Hg and Cd, even causing family disappearance, through increased mortality and migration to other colonies. Compensatory mechanisms expressed by the adult workers appeared to be of a phenotypic origin

Interactions between respiration and inorganic phosphate uptake in phosphate‐limited cells of Chlamydomonas reinhardtii

Phosphate addition to P‐limited cells of Chlamydomonas reinhardtii resulted in an immediate increase in the rate of respiratory O2 consumption. The respiration rate continued to increase for several minutes after the addition of P1. Similar patterns of P1 stimulation of respiratory O2 consumption were observed in the presence of cyanide (cytochrome oxidase inhibitor) and propyl gallate (alternative oxidase inhibitor). Stimulation of O2 consumption was accompanied by rapid changes in levels of glycolytic intermediates. These changes were consistent with activation of ATP‐dependent phosphofructokinase and pyruvate kinase. The adenylate pool exhibited only minor perturbations, P1, uptake resulted in extracellular acidification, which continued for several minutes after the exhaustion of added P1, whereas exhaustion of extracellular P1 resulted in a rapid decline in the O2 consumption rate. These results are consistent with control of respiration in P‐limited cells occurring largely at the level of glycolysis.

Adenylate storage, metabolism and utilization in coelomic cells of the polychaeteNereis virens (Annelida, polychaeta)

Eleocytes are specialized coelomic cells in nereid annelids which assume a central role during germ cell development. They may contain extremely high concentrations of both adenosine monophosphate (AMP) and adenosine diphosphate (ADP) (each >10 μmol/ml of cell vol.), whereas the adenosine triphosphate (ATP) content is comparatively low (0.8 μmol/ml cell vol.).31P nuclear magnetic, resonance (NMR) studies of living eleocytes suggest the compartmentalization of both AMP and ADP in the large acidic vacuole characteristic for this cell type. Eleocytes are thus capable of storing high concentrations of ADP and AMP without inhibiting energy metabolism, by sequestering these compounds in a separate compartment. The high concentrations of both AMP and ADP in the eleocytes decrease in both males and females during the course of maturation. In eleocytes of male animals, the decline of the high nucleotide concentrations was accompanied by a transient increase of two intracellular nucleosides, inosine and guanosine. This suggests the degradation and further metabolism of nucleotides to the corresponding nucleosides. In culture, eleocytes release both inosine and guanosine into the medium. Both nucleosides are also present in the coelomic fluid, the common compartment for both eleocytes and germ cells. Both male and female germ cells incorporate14C-labelled inosine and guanosine in culture. For oocytes, the further incorporation of [14C]inosine into the RNA fraction could be demonstrated. The large adenylate pools in the eleocytes may be regarded as a store for purine compounds for later use by the growing germ cells to supplement nucleic acid synthesis. The supply of nucleic acid precursors seems to be another specific function of eleocytes related to gametogenesis, in addition to their known synthesis of vitellogenin.

Metabolism of glucose, glycogen, and high-energy phosphates during transient forebrain ischemia in diabetic rats: effect of insulin treatment.

Hyperglycemia associated with diabetes mellitus will exacerbate neurologic injury after global brain ischemia. Studies in a rat model of forebrain ischemia (bilateral carotid occlusion plus hypotension for 10 min) discovered that acute restoration of normoglycemia in diabetics, using an insulin infusion, resulted in a neurologic outcome that was similar to normoglycemic rats without diabetes. The current study evaluated cerebral glucose, glycogen, lactate, and high-energy phosphate concentrations to identify metabolic correlates that might account for an alteration in postischemic outcome. Fifty-four pentobarbital-anesthetized Sprague-Dawley rats were assigned to three groups: chronically hyperglycemic diabetic rats (D; N = 18); insulin-treated, acutely normoglycemic diabetic rats (ID; N = 18); and nondiabetic rats (ND; N = 18). These groups were further divided into groups of six rats each that received either no ischemia, forebrain ischemia of 10 min duration without reperfusion, or ischemia plus 15 min of reperfusion. Brains were excised after in situ freezing, and metabolites were measured using enzymatic fluorometric techniques. Before ischemia, D rats had greater concentrations of brain glucose (12.18 +/- 2.67 micromol/g) than did either ID (5.10 +/- 1.33) or ND (3.20 +/- 0.27) rats (P < 0.05). Preischemic brain glycogen was similar in all groups. At the completion of ischemia, brain lactate concentrations in D were 86% greater than in ID and 61% greater than in ND (P < 0.05), reflecting a higher intraischemic consumption of glucose plus glycogen in D (P < 0.05). High-energy phosphate concentrations, as assessed by the energy charge of the adenylate pool, were better preserved in D (energy charge = 0.60 +/- 0.28) than in either ID (0.29 +/- 0.09) or ND (0.36 +/- 0.07; P < 0.05) rats. After 15 min of reperfusion, the energy charge returned to preischemic values (i.e., 0.91-0.92) in all groups. These studies demonstrated greater intraischemic carbohydrate consumption and lactate production in D than in ID or ND rats. Under these conditions, intraischemic-but not postischemic-energy status was better in D rats. Acute insulin therapy in ID rats resulted in a metabolic profile that was similar to that of ND rats. These results suggest that, in this model, primary energy failure during ischemia is not the origin of greater injury in hyperglycemic diabetics, nor is energy enhancement the origin of improved outcome after acute insulin treatment.

A Possible Role of Adenylate Metabolism in Human Erythrocytes: Simple Mathematical Model

A simplified mathematical model of cell metabolism describing ion pump, glycolysis and adenylate metabolism was developed and investigated in order to clarify the functional role of the adenylate metabolism system in human erythrocytes. The adenylate metabolism system was shown to be able to function as a specific regulatory system stabilizing intracellular ion concentration and, hence, erythrocyte volume under changes in the permeability of cell membrane. This stabilization is provided via an increase in adenylate pool in association with ATPases rate elevation. Proper regulation of adenylate pool size might be achieved even in the case when AMP synthesis rate remains constant and only AMP degradation rate varies. The best stabilization of intracellular ion concentration in the model is attained when the rate of AMP destruction is directly proportional to ATP concentration and is inversely proportional to AMP concentration. An optimal rate of adenylate metabolism in erythrocytes ranges from several tenths of a percent to several percent of the glycolytic flux. An increase in this rate results in deterioration of cell metabolism stability. Decrease in the rate of adenylate metabolism makes the functioning of this metabolic system inefficient, because the time necessary to achieve stabilization of intracellular ion concentration becomes comparable with erythrocyte life span.

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.