Lower ATP Content Research Articles

Neurologic deficit, blood flow and biochemical sequelae of reversible focal cerebral ischemia in cats

Temporary focal cerebral ischemia was induced in 23 cats by occluding the left middle cerebral artery (MCA) for 2 h. Animals then were divided into groups for unforced reperfusion of varying duration ranging from 2 to 48 h. Regional blood flow (rCBF) at the borders of the ischemic area was measured repeatedly using the hydrogen clearance technique, and neurological ratings were obtained, both during ischemia and reperfusion. At the scheduled end of reperfusion brains were frozen in situ with liquid nitrogen, and regional distributions of biochemical substrate contents as well as tissue pH were visualized using bioluminescence and fluorescence techniques. During focal ischemia collateral flow in the border zone dropped to 55 ± 20.3% of control level, and all animals developed a neurologic deficit with a median of 6 points on a disability scale from 0 to 10, rCBF and functional impairment being closely correlated ( τ = −0.47, P 1 < 0.005). After reopening of the MCA there was an immediate and rather uniform increase in border zone flow to 105 ± 25.7% of control level, while neurologic recovery was quite variable. In all but one animal reversible ischemia led to persistent disturbances in the energy-producing metabolism as demonstrated by the low regional ATP content, which in part was accompanied by a diminished NADH fluorescence and an alkaline pH shift at high tissue glucose levels. These findings suggest that disturbances in cerebral energy metabolism induced by temporary ischemia may be caused by inhibition of the glycolytic pathway that is hardly reversed by unforced reperfusion and, therefore, results in permanent damage.

The gelation kinetics of platelet extracts.

The kinetics of the gelation process that occurs upon warming cold platelet extracts were studied using a sensitive rheometer. At micromolar or less free Ca2+ concentrations and in the presence of 1 mM ATP, the gel rigidity curves showed several peaks, indicating that platelet extract proteins went through network assembling/disassembling cycles during gelation. The gelation kinetics were accelerated by increasing the free Ca2+ concentration up to about 2 microM. At 4-15 microM free Ca2+, the gelation cycles were completely abolished except for the first peak. The gelation process became one of monotonically increasing elastic modulus at millimolar free Ca2+ concentrations. Trifluoperazine (50 microM), a calmodulin inhibitor, did not affect gelation at micromolar free Ca2+ concentrations. Except for the first gelation step, which was completed within 5 min after warming, the rest of the gelation process was found to be affected by K+, ATP, cytochalasin E and colchicine. K+ at concentrations higher than 10 mM retarded the gelation kinetics. Extracts prepared with low (0.1 mM) ATP content showed impaired gelation, and this was partially reversed by adding 1 mM ATP, but not 1 mM adenylylimidodiphosphate (p[NH]ppA). Both cytochalasin E (1 microM) and colchicine (1 mM) interfered with the gelation process.

赤血球膜蛋白への血清脂質とリポ蛋白の関与

The impairment of blood fluidity facilitates the formation of ischemic lesion with respect to decrease in blood supply to tissues under the participation of changes in arterial wall and local blood pressure gradient. Functional compensation due to augmentation of blood fluidity for decrease in blood supply to tissues would protect even elderly adults with systemic arteriosclerotic changes from the occurrence of thrombotic diseases. Blood fluidity is influenced by corpuscular and plasma components in the blood. Erythrocytes participate to low blood fluidity by increasing viscosity and decreasing the deformability induced by low stroma ATP content and quality changes in membrane constituents affected by plasma lipids and lipoproteins. This study was intended to clarify fundamental association of serum lipids with erythrocyte membrane proteins as a part of lipid and hemorheological research in thrombotic cerebrovascular disorders.Seventy-nine healthy adults with normal physical findings, blood pressures, urinalysis, blood sedimentation rate, chest films, ECG, liver function tests and serum lipids were selected for this study in the annual screening for circulatory disease. Blood specimens were drawn in the fasting state early in the morning. Erythrocyte membrane was separated by the method of Dodge et al., the membrane proteins were extracted and analyzed qualitatively using SDS-polyacrylamide gel disc electrophoresis by the method of Weber et al., Total cholesterol (TCh), triglyceride and phospholipid were determined by conventional methods, HDL-cholesterol (HDL-Ch) by MgCl2-dextran sulfate method, LDL and VLDL by the precipitating method, and apo A-I, apo A-II and apo B by single radial immunodiffusion method.Subjects aged above 60 years revealed significantly higher levels of TCh, LDL and apo B, and significantly lower levels of HDL-Ch and HDL-Ch/TCh ratio compared with those aged 20 to 39 years. Erythrocyte membrane band 2.1 connecting with spectrin affecting immediately erythrocyte deformobility was lower in subjects aged above 60 years than in those aged 20 to 39 years. In subjects aged 20 to 39 years, serum TCh, esterified Ch and LDL were directly proportional to spectrin 1, spectrin 2 and inversely to glucose 3 phosphate dehydrogenase, and HDL-Ch directly to band 4.1, 4.2, 8 and actin (Table 3). LDL was directly proportional to spectrin (band 1+2) (Fig. 2). In subjects aged 40 to 59 years, apo A-I was inversely proportional to actin, apo A-II directly to band 4.1 (Table 4). In subjects aged above 60 years, A-I was directly proportional to band 2.1 (Fig. 3). Neither of age groups showed significant relationships between serum unesterified Ch and membrane proteins.Erythrocyte lipids are almost located in the membrane. The mature erythrocyte is incapable of the denovo synthesis of cholesterol. A notable feature of blood cholesterol is its ability to exchange between erythrocyte and plasma. Morphological observations have shown the appearance of various kinds of abnormal erythrocyte, which impaires hemorheological behavior following the changes of membrane lipids with regard to the membrane protein and stroma ATP. Although membrane lipids analysis is lacking in our study, intimate associations of serum lipids and lipoproteins with the membrane protein were shown. Therefore serum lipids should be regarded as one of important factors in the development of hemorheological disorders as well as the participation in atherosclerosis in thrombotic disorders.

Energy metabolism and ATP turnover time during the cell cycle in roentgen irradiated Ehrlich ascites tumour cells.

The cell-cycle related energy metabolism after roentgen irradiation of in vivo growing Ehrlich ascites tumour cells was investigated in cell fractions obtained by elutriator centrifuging. The oxygen consumption and the lactate and pyruvate production, measured in vitro after 4.5, 5 and 9 Gy up to 24 h, were undisturbed, while the decrease in the in vivo ATP content was dose-independent in all parts of the cell cycle. On the basis of these data the ATP turnover time was found to be decreased. The decrease in the ATP content is considered to be less likely to be due to membrane leakage or increased ATP consumption than to reduced ATP production. Since in vitro incubation normalizes the ATP content, it is suggested that environmental factors in the ascites liquid after irradiation cause a decrease in the ATP production. Low ATP contents of the cells do not appear to influence the irradiation-induced changes in the cell flow through the cell cycle.

Red blood cell filterability and occlusive arterial disease.

In 1976 Ehrly and co-workers as well as Reid and co-workers investigated the filterability of blood from patients with chronic occlusive vascular diseases. Using micropore filters there was a markedly reduced filterability in spite of differences in the filterability techniques as filter type, hydrostatic pressure and others.The results were explained by an increased ridgidity of red cells which increase the resistance to flow through the filter pores. These findings where discussed as one of the pathophysiological mechanisms of chronic occlusive arterial diseases, but brought up a number of questions such as the mechanism of the reduced filterability and the significance of an impaired deformability of red cells in vivo. Another question is if the impaired filterability of blood is the cause or the consequence of the underlying disease.If we consider the red cell deformability to be an important factor for the pathophysiology of vascular diseases, it seems clear that not only the mechanical property of the less deformable red cell in the capillary will be most important, but also rigid red cell as a cause for an inhomogenous blood flow in the microcirculation (microcirculatory blood maldistribution).From the therapeutic point of view it seems important to know the mechanism which leads to an impaired flexibility of red cells, so, for example, a low ATP-content in the cells, hypermolarity or acidosis of blood.In vitro tests, like filtration of patients blood through micropore filters, may indicate the value of drugs said to improve the deformability of red cells. More sophisticated methods like the measurement of the oxygen tension of the muscle tissue can give us an answer as to whether an improvement of the deformability of red cells also leads to an improvement of the tissue supply.

A possible mechanism of glycolytic impairment after adenosine triphosphate deplection in the perfused rat heart.

1. When the heart is deprived of substrate and O2 for a long period, the ATP content falls to very low levels, with associated changes in ADP and AMP content, and a fall in intracellular pH. 2. These changes appear sufficient to block the hexokinase reaction, outweighing the normal mechanisms controlling this enzyme. Anaerobic glycolysis then remains blocked, even when glucose supply is restored. The block in anaerobic glycolysis can be overcome, however, by a brief period of oxidative metabolism, apparently because the improvement in adenine nucleotide levels serves to 're-prime' the system. 3. When the cell is severely depleted of ATP, the resulting impairment of glycolysis tends to reinforce the low ATP content, establishing a vicious cycle. Metabolic effects of this kind may cause irreversible loss of function, and may contribute to the mechanism of cell death.

Mode of action of alpha-chlorohydrin as a male anti-fertility agent. Inhibition of the metabolism of ram spermatozoa by alpha-chlorohydrin and location of block in glycolysis.

1. The effect of alpha-chlorohydrin on the metabolism of glycolytic and tricarboxylate-cycle substrates by ram spermatozoa was investigated. The utilization and oxidation of fructose and triose phosphate were much more sensitive to inhibition by alpha-chlorohydrin (0.1-1.0mm) than lactate or pyruvate. Inhibition of glycolysis by alpha-chlorohydrin is concluded to be between triose phosphate and pyruvate formation. Oxidation of glycerol was not as severely inhibited as that of the triose phosphate. This unexpected finding can be explained in terms of competition between glycerol and alpha-chlorohydrin. A second, much less sensitive site, of alpha-chlorohydrin inhibition appears to be associated with production of acetyl-CoA from exogenous and endogenous fatty acids. 2. Measurement of the glycolytic intermediates after incubation of spermatozoal suspensions with 15mm-fructose in the presence of 3mm-alpha-chlorohydrin showed a ;block' in the conversion of glyceraldehyde 3-phosphate into 3-phosphoglycerate. alpha-Chlorohydrin also caused conversion of most of the ATP in spermatozoa into AMP. After incubation with 3mm-alpha-chlorohydrin, glyceraldehyde 3-phosphate dehydrogenase and triose phosphate isomerase activities were decreased by approx. 90% and 80% respectively, and in some experiments aldolase was also inhibited. Other glycolytic enzymes were not affected by a low concentration (0.3mm) of alpha-chlorohydrin. Loss of motility of spermatozoa paralleled the decrease in glyceraldehyde 3-phosphate dehydrogenase activity. alpha-Chlorohydrin, however, did not inhibit glyceraldehyde 3-phosphate dehydrogenase or triose phosphate isomerase in sonicated enzyme preparations when added to the assay cuvette. 3. Measurement of intermediates and glycolytic enzymes in ejaculated spermatozoa before, during and after injection of rams with alpha-chlorohydrin (25mg/kg body wt.) confirmed a severe block in glycolysis in vivo at the site of triose phosphate conversion into 3-phosphoglycerate within 24h of the first injection. Glyceraldehyde 3-phosphate dehydrogenase activity was no longer detectable and both aldolase and triose phosphate isomerase were severely inhibited. Spermatozoal ATP decreased by 92% at this time, being quantitatively converted into AMP. At 1 month after injection of alpha-chlorohydrin glycolytic intermediate concentrations returned to normal in the spermatozoa but ATP was still only 38% of the pre-injection concentration. Motility of spermatozoa was, however, as good as during the pre-injection period. The activity of the inhibited enzymes also returned to normal during the recovery period and 26 days after injection were close to pre-injection values. 4. An unknown metabolic product of alpha-chlorohydrin is suggested to inhibit glyceraldehyde 3-phosphate dehydrogenase and triose phosphate isomerase of spermatozoa. This results in a lower ATP content, motility and fertility of the spermatozoa. Glycidol was shown not to be an active intermediate of alpha-chlorohydrin in vitro.

Utilization of glutamate for phospho enolpyruvate and aspartate synthesis in kidney cortex mitochondria of rabbit

1. 1. Utilization of glutamate for both phospho enolpyruvate and aspartate synthesis in kidney cortex mitochondria of the rabbit was studied under three metabolic conditions (state 3, uncoupled state and in the presence of uncoupler and oligomycin) in order to induce a high respiratory activity with an oxidized state of the nicotinamide dinucleotides but with varied intramitochondrial ATP levels. 2. 2. The highest rates of glutamate deamination and thus of phospho enolpyruvate formation were observed in uncoupled mitochondria incubated without oligomycin, i.e. under conditions of low intramitochondrial ATP content. Aspartate synthesis was high in both State 3 and the uncoupled state. 3. 3. In contrast to liver, addition of oligomycin to uncoupled kidney cortex mitochondria in order to increase the mitochondrial ATP/ADP ratio resulted in an inhibition of glutamate deamination, phospho enolpyruvate formation and aspartate synthesis. Atractyloside augmented an inhibitory effect of oligomycin. 4. 4. Addition of either ATP trapping system or ADP to mitochondria incubated in uncoupled-plus-oligomycin state resulted in the abolishing of the inhibition of both phospho enolpyruvate and aspartate synthesis, due to a depletion of intramitochondrial ATP content. 5. 5. In contrast to liver, leucine, which activates glutamate dehydrogenase, caused an acceleration of glutamate deamination in kidney cortex mitochondria, accompanied by an enhancement of phospho enolpyruvate formation. On the other hand, aspartate synthesis was not affected by leucine. 6. 6. In the uncoupled-plus-oligomycin state, leucine abolished an inhibitory effect of ATP and/or GTP on both glutamate deamination and phospho enolpyruvate synthesis. However, it did not relieve the inhibition of aspartate formation. 7. 7. In the presence of malate in addition to glutamate, oligomycin inhibited neither aspartate formation nor phospho enolpyruvate synthesis, since under these conditions an accumulation of energy did not occur in mitochondria due to an efficient phospho enolpyruvate formation. 8. 8. The data indicate that in contrast to liver, in kidney cortex mitochondria, utilizing glutamate as a substrate, the rates of both phospho enolpyruvate and aspartate synthesis depend greatly upon the mitochondrial energy level and the availability of both oxaloacetate and leucine. d

Restriction of receptor mobility and the agglutination of cells by concanavalin a

Summary Transformed hamster fibroblasts with a high ATP content which are not agglutinated by concanavalin A (ConA) can be induced to agglutinate by decreasing their ATP content, mixing of glutaraldehyde prefixed ConA-coated cells with unfixed uncoated cells, or mixing of unfixed ConA-saturated cells with uncoated cells. The decrease in ATP content required for a gain of ConA agglutination between unfixed cells, was associated with a decrease in the short range rapid lateral mobility (RLM) of ConA surface receptors and with an increased exposure of the surface membrane phospholipids. The transformed fibroblasts with a high ATP content were agglutinated by the lectin from wheat germ (WGA) and after neuraminidase treatment also by the lectin from soybean (SBA). Agglutination by these two lectins was not effected by changes in the cellular ATP content. The half maximum rate ( V m ) of ConA-induced agglutination of cells with a low ATP content required 75% saturation of the cell receptors for ConA and was temperature sensitive. The half V m of agglutination between prefixed ConA-coated and unfixed uncoated cells, like the WGA- and SBA-induced agglutination of unfixed cells, required only about 15% saturation and was less temperature sensitive. The results indicate, that in cells with a high RLM of ConA receptors, agglutination induced by ConA required an appropriate degree of restriction of receptor mobility to allow the alignment of surface receptors that is required for agglutination. This restriction can be obtained by lowering the ATP content, mixing of prefixed lectin coated cells with unfixed uncoated cells, or saturating unfixed cells with ConA before mixing with unfixed uncoated cells. The results also suggest that the receptors for ConA are differently embedded in the lipid phase of the surface membrane than the receptors for WGA and SBA.

Effect of Reserpine on &lt;sup&gt;14&lt;/sup&gt;C Serotonin Uptake by Thrombasthenic Platelets

The incorporation of &lt;sup&gt;14&lt;/sup&gt;C-5HT by platelets was found to be abnormal in two groups of thrombasthenia. In the first group of patients, which have a low platelet fibrinogen content, incorporation was initially faster than in normal platelets. With the exception of one patient who was subsequently discovered to possess an antibody which inhibited ADP-induced aggregation of control platelets, the rate of incorporation of &lt;sup&gt;14&lt;/sup&gt;C-5HT in group I thrombasthenic platelets reached a maximum and then decreased. In the control platelets incorporation of &lt;sup&gt;14&lt;/sup&gt;C-5HT was continuous. In a patient of the second group of thrombasthenia, with a low ATP content, incorporation of &lt;sup&gt;14&lt;/sup&gt;C-5HT was also continuous but was at a much lower level than normal. Results obtained with reserpine suggest that this compound affects the active incorporation of &lt;sup&gt;14&lt;/sup&gt;C-5HT into platelet granules and, possibly, is associated with a prevention of 5-HT release.

Membrane Changes and Adenosine Triphosphate Content in Normal and Malignant Transformed Cells

Transformed fibroblasts had a low content of ATP when grown at a high cell density and a high content of ATP when grown at a low cell density. Concanavalin A agglutinated transformed cells with a low, but not those with a high, ATP content. Transformed cells with a high ATP content gained agglutinability after ATP depletion by inhibitors of the energy-generating systems, and those with a low ATP content lost their agglutinability after restoration of a high ATP content by glucose. Fixation of the surface membrane by formaldehyde, glutaraldehyde, or LaCl(3), inhibited agglutination of cells with an ATP content that allows agglutination. Normal fibroblasts grown at a high or a low cell density were not agglutinated by concanavalin A. Depletion of the cellular ATP content of normal cells induced agglutination only in cells grown at a high, but not at a low, cell density. A similar number of concanavalin A molecules was bound to the surface membrane of agglutinating and nonagglutinating fibroblasts. It is suggested that a high content of ATP inhibits the movement of concanavalin A binding sites, and that a low content of ATP allows, in transformed cells, a new distribution of binding sites to form the clusters required for cell agglutination. Agglutinability of transformed cells is determined by ATP content, and in normal cells changes in the content of ATP are by themselves not sufficient to induce agglutination. Transformed cells, therefore, do not have a control, presumably for membrane stability, that exists in normal cells.

Lysolecithin metabolism in erythrocyte membranes. Lysolecithin acylation and lysophospholipase in aging erythrocytes

Human erythrocytes have a life span of about 120 days. One can easily separate young and aged cells by density centrifugation. For these reasons they are especially suited for studying ageing processes. There is general agreement that the activity of several glycolytic enzymes decreases during ageing. However, nothing is known about typical membrane enzymes in connection with ageing, and since it was felt that membrane alterations might be of primary importance, a comparative study of phospholipid-metabolizing enzymes was undertaken. The enzymes studied in this investigation were: a) the structure-bound acid: CoA ligase (AMP) and acyl-CoA: Iysolecithin-O-acyltransferase which together incorporate lysolecithin and fatty acid into membrane bound lecithin. b) lysophospholipase (lysolecithin acyl-hydrolase) which degrades lysolecithin into glycerophosphorylcholine plus fatty acid. Human erythrocytes from healthy donors were separated by simple ultracentrifugation and by albumin density centrifugation according to age. The mean volume of the young erythrocytes was 109 μ3, while that of the aged cells was 74 μ3. The enzyme activities of freeze-thawed cells were determined and corrected for cell numbers. Furthermore, the enzyme activities-in hemoglobin-free stroma were measured. [14C]-labelled substrates were used to determine the enzyme activities: [1-14C]oleic acid for the acylation of lysolecithin and [1-acyl-14C]lysolecithin for the lysophospholipase reaction. The results showed that the activities of both enzymic reactions as measured with old cells were 40 to 50% lower than the activities found in young cells. In hemolysates from young and old cells the following enzyme activities were determined: the acylating activity decreased from 117 to 67 MolX10−20/cell×min, and the lysophospholipase activity from 185 to 123 Mol×10−20/cellXmin. The ATP content of old cells was only slightly lower than that of young cells. Comparative analyses of erythrocytes stored in acid citric dextrose for two months at 4° gave different results: these cells which had been “aged in vitro” had an extremely low ATP content, while the activities of the lysolecithin metabolizing enzymes remained unchanged. These findings suggest that the lysolecithin metabolism has a significance in cell ageing processes. It remains to be shown, however, that lysolecithin produced by macrophages lining the RES sinuses is the decisive factor responsible for the death and removal of aged cells.