Simulated biological effects of microgravity on phospholipid and energy metabolism of chicken embryonic brain cells studied by 31P-NMR spectroscopy

Levels of phosphomonoester (PME), phosphodiester (PDE), ATP and pH in brain cells of chicken embryos rotated for 24 h in a clinostat during the period of hatching the 13th day (E13) and 15th day (E15) embryos were investigated by using 31P-NMR spectroscopy. Significant increases in the values of PME, ATP and pH were identified after E13 rotating for 24 h. With the same treatment, differences were obtained in the phospholipid and energy metabolism of E15, but no significant levels have been reached . The calorimetric assay (malachite green method) was used for measuring the activity of total ATPase. A dramatic decrease was evident in the activity of ATPase in brain cells of rotated E13 and E15. The former is more sensitive than the latter. All the levels mentioned above could restore in 24 h after the rotation stopped, except that the level of ATP was still higher than the control.     

The effects of hyperthermia and hyperthermia plus microwaves on rat brain energy metabolism

The effects of hyperthermia, alone and in conjunction with microwave exposure, on brain energetics were studied in anesthetized male Sprague-Dawley rats. The effect of temperature on adenosine triphosphate concentration [ATP] and creatine phosphate concentration [CP] was determined in the brains of rats that were maintained at 35.6, 37.0, 39.0, and 41.0 degrees C. At 37, 39, and 41 degrees C brain [ATP] and [CP] were down 6.0, 10.8, and 29.2%, and 19.6, 28.7, and 44%, respectively, from the 35.6 degrees C control concentrations. Exposure of the brain to 591-MHz radiation at 13.8 mW/cm2 for 0.5, 1.0, 3.0, and 5.0 min caused further decreases (below those observed for 30 degrees C hyperthermia only) of 16.0, 29.8, 22.5, and 12.3% in brain [ATP], and of 15.6, 25.1, 21.4, and 25.9% in brain [CP] after 0.5, 1.0, 3.0, and 5.0 min, respectively. Recording of brain reduced nicotinamide adenine dinucleotide (NADH) fluorescence before, during, and after microwave exposure showed an increase in NADH fluorescence during microwave exposure that returned to preexposure levels within 1 min postexposure. Continuous recording of brain temperatures during microwave exposures showed that brain temperature varied between -0.1 and +0.05 degrees C. Since the microwave exposures did not induce tissue hyperthermia, it is concluded that direct microwave interaction at the subcellular level is responsible for the observed decrease in [ATP] and [CP].     

Microwave effects on energy metabolism of rat brain

Rat brain was exposed to 591-MHz, continuous-wave (CW) microwaves at 13.8 or 5.0 mW/cm² to determine the effect on nicotinamide adenine dinucleotide, reduced (NADH), adenosine triphosphate (ATP) and creatine phosphate (CP) levels. On initiation of the in vivo microwave exposures, fluorimetrically determined NADH rapidly increased to a maximum of 4.0%–12.5% above pre-exposure control levels at one-half minute, then decreased slowly to 2% above control at three minutes, finally increasing slowly to 5% above control level at five minutes. ATP and CP assays were performed on sham- and microwave-exposed brain at each exposure time. At 13.8 mW/cm², brain CP level was decreased an average of 39.4%, 41.1%, 18.2%, 13.1%, and 36.4% of control at exposure points one-half, one, two three, and five minutes, respectively, and brain ATP concentration was decreased an average of 25.2%, 15.2%, 17.8%, 7.4%, and 11.2% of control at the corresponding exposure periods. ATP and CP levels of rat brain exposed to 591-MHz cw microwaves at 5 mW/cm² for one-half and one minute were decreased significantly below control levels at these exposure times, but were not significantly different from the 13.8 mW/cm² exposures. For all exposures, rectal temperature remained constant. Heat loss through the skull aperture caused brain temperature to decrease during the five-minute exposures. This decrease was the same in magnitude for experimental and control subjects. Changes in NADH, ATP, and CP levels during microwave exposure cannot be attributed to general tissue hyperthermia. The data support the hypothesis that microwave exposure inhibits mitochondrial electron transport chain function, which results in decreased ATP and CP levels in brain.     

The influence of Methylprednisolone on the energy metabolism of Ehrlich ascites tumour cells

Using Ehrlich ascites tumour cells, the short-term effects of the therapeutic glucocorticoid Methylprednisolone (MP) on the cellular energy metabolism were studied. ATP-consuming processes involved in the rapid MP effects were identified indirectly from the effects of MP on cellular oxygen consumption related to the inhibition of respiration by selective inhibitors of Ca²⁺-ATPase and protein synthesis. The effects of MP on plasma membrane permeability for Ca²⁺ ions and phospholipid turnover were studied directly by using confocal laser scanning microscopy and tracerkinetic measurements, respectively. MP inhibited cellular oxygen consumption, suppressed the inhibitory effect of lanthanum but not that of cycloheximide on oxygen consumption, blocked the [Ca²⁺]_i rise in response to calcium ionophore A 23187, and decreased phospholipid turnover. MP acted instantly in a dose-dependent manner.The observed effects of MP are discussed in relation to the hypothesis that the drug has direct membrane effect affecting plasma membrane permeability and function.     

The differential effects of 200, 591, and 2,450 MHz radiation on rat brain energy metabolism

Three key compounds in brain energy metabolism have been measured during and after exposure to continuous wave radiofrequency radiation at 200, 591, and 2,450 MHz. Frequency-dependent changes have been found for all three compounds. Changes in NADH fluorescence have been measured on the surface of a surgically uncovered rat brain during exposure. At 200 and 591 MHz, NADH fluorescence increased in a dose-dependent manner between approximately 1 and 10 mW/cm2, then became constant at higher exposures. There was no effect at 2,450 MHz. Levels of ATP and CP were measured in whole brain after exposure. The ATP levels were decreased at 200 and 591 MHz but not at 2,450 MHz. The CP levels decreased only at 591 MHz. The effect of duration of exposure (up to 5 min) was investigated for all compounds at 200 MHz and 2,450 MHz, and exposures to 20 minutes were examined at 591 MHz. Temperature in the rat brain was essentially constant for all exposures. A general mechanism for inhibition of the mitochondrial electron transport chain and the CP-kinase reaction pathway by radiofrequency radiation has been proposed.     

The effects of insulin on myocardial metabolism and acidosis in normoxia and ischaemia: A 31P-NMR study

1. The effect of insulin on the perfused rat heart during normoxia and total ischaemia was studied by ³¹P-NMR. 2. During normoxic perfusion, insulin increased the phosphocreatine to ATP ratio at the expense of P_i, when glucose was the substrate. No change was observed when acetate was used as the sole substrate. The intracellular pH (as measured from the position of the 2-deoxyglucose 6-phosphate resonance peak) was unaffected by insulin treatment. 3. Infusion of insulin prior to ischaemia caused an increase in the rate and extent of acidosis during the period of no flow while the rate of ATP depletion was decreased. 4. Freezeclamped studies showed an increase in glycogen levels upon insulin treatment of the glucose perfused rat heart. During ischaemia, a decrease in glycogen content concomitant with an increase in lactate was observed. The accessibility of glycogen to phosphorylase during ischaemia is increased as a result of insulin treatment. The control of glycolysis during ischaemia is discussed with respect to the content and structure of glycogen in heart tissue.     

Effects of continuous-wave, pulsed, and sinusoidal-amplitude-modulated microwaves on brain energy metabolism

A comparison of the effects of continuous-wave, sinusoidal-amplitude-modulated, and pulsed square-wave-modulated 591-MHz microwave exposures on brain energy metabolism was made in male Sprague-Dawley rats (175-225 g). Brain NADH fluorescence, adenosine triphosphate (ATP) concentration, and creatine phosphate (CP) concentration were determined as a function of modulation frequency. Brain temperatures of animals were maintained between -0.1 and -0.4 degrees C from the preexposure temperature when subjected to as much as 20 mW/cm2 (average power) CW, pulsed, or sinusoidal-amplitude modulated 591-MHz radiation for 5 min. Sinusoidal-amplitude-modulated exposures at 16-24 Hz showed a trend toward preferential modulation frequency response in inducing an increase in brain NADH fluorescence. The pulse-modulated and sinusoidal-amplitude-modulated (16 Hz) microwaves were not significantly different from CW exposures in inducing increased brain NADH fluorescence and decreased ATP and CP concentrations. When the pulse-modulation frequency was decreased from 500 to 250 pulses per second the average incident power density threshold for inducing an increase in brain NADH fluorescence increased by a factor of 4--ie, from about 0.45 to about 1.85 mW/cm2. Since brain temperature did not increase, the microwave-induced increase in brain NADH and decrease in ATP and CP concentrations was not due to hyperthermia. This suggests a direct interaction mechanism and is consistent with the hypothesis of microwave inhibition of mitochondrial electron transport chain function of ATP production.     

Transport and metabolism of glucose by dissociated brain cells: Effects of trypsin

A study was carried out to determine the effect of trypsin on glucose transport into brain cells. Two suspensions of dissociated cells were prepared from the two brain hemispheres of adult rats—one using only mechanical means to dissociate the cells and one using trypsin. The use of trypsin for preparation of dissociated brain cells caused a marked reduction in the rate of transport of [1,2-³H]-2-deoxy-d-glucose compared to uptakes of this glucose analog by cells prepared without trypsin. Responses of the two cell preparations to inhibitors of glucose transport (cytochalasin B and phloretin) were similar. Rates of oxidation of [6-¹⁴C]glucose to¹⁴CO₂ by trypsin-treated cells were nearly double those in cells prepared without trypsin. Electron microscopic examination of the two preparations revealed much less preservation of structural integrity if trypsin was used to prepare the cells. The findings suggest that trypsin alters cell structure and affects receptor-regulated events in brain cells.     

Mg~(2+)和SeO_3~(2-)对鸡胚软骨细胞受模拟微重力不良影响的拮抗

在回转模拟微重力条件下 ,研究了鸡胚负重软骨细胞骨架的微管系统和碱性磷酸酶活性两项指标的变化 ,以及 1mg/L亚硒酸钠和 5mmol/LMg2 + 对这些指标的影响 .流式细胞仪对微管含量的测定显示回转后微管蛋白含量的减少 ,说明微管系统受到不良影响 .碱性磷酸酶活性比对照组明显降低 ,表明模拟微重力能降低软骨细胞的钙化能力 .如果在回转前加入SeO2 -3 和Mg2 + ,发现SeO2 -3 可以在一定程度上拮抗模拟微重力引起的微管蛋白及碱性磷酸酶活性改变 ,而Mg2 + 基本上可以完全拮抗模拟微重力对这两项指标的不良影响     

Hymenolepis diminuta: effects of amoscanate on energy metabolism and ultrastructure

Amoscanate possesses chemotherapeutic activity against schistosomes, and in higher doses against many other helminths including filariids and Hymenolepis diminuta. The primary mode of action of this compound is unknown. Effects of the drug on the carbohydrate metabolism as well as on the tegumental and nephridial epithelia of H. diminuta were examined. At various time intervals after administration of the drug to rats infected with H. diminuta, the parasites were recovered and incubated in glucose-salts medium for 90 min. Chemotherapy resulted in decreases in succinate, lactate, and acetate recoveries, while ATP levels dropped. In addition, glycogen levels were depressed in drug-treated worms which were homogenized immediately upon isolation. Glycogen synthase I activity was inhibited 16-61% in cestodes obtained from Amoscanate-treated animals and homogenized immediately, but returned to normal levels after incubation for 90 min in glucose-salts medium prior to homogenization and assay. Phosphorylase a activity was found to be 25-30% higher in preparations of worms from drug-treated rats, which correlates with the rapid depletion of glycogen in parasites exposed to the drug. However, in contrast with glycogen synthase activity, the elevation of phosphorylase a activity in H. diminuta exposed to the drug was not readily reversible. Attempts to demonstrate activity of the drug in vitro by incubating intact cestodes directly with Amoscanate were unsuccessful. Thin sections of parasites obtained from Amoscanate-treated rats and examined by transmission electron microscopy revealed surface alterations of the tegument and nephridial canals. Alterations included bleb formation and erosion of microtriches from the tegument, as well as disappearance of microvilli from nephridial canals. However, these effects became manifest only after 4 or more hr exposure of the rat to the drug. Biochemical effects, on the other hand, were significant after 3 hr exposure.     

Inducible expression of phospholipid transfer protein (PLTP) in transgenic mice: acute effects of PLTP on lipoprotein metabolism

One main determinant in high-density lipoprotein (HDL) metabolism is phospholipid transfer protein (PLTP), a plasma protein that is associated with HDL. In transgenic mice overexpressing human PLTP we found that elevated plasma PLTP levels dose-dependently increased the susceptibility to diet-induced atherosclerosis. This could be mainly due to the fact that most functions of PLTP are potentially atherogenic, such as decreasing plasma HDL levels. To further elucidate the role of PLTP in lipoprotein metabolism and atherosclerosis we generated a novel transgenic mouse model that allows conditional expression of human PLTP. In this mouse model a human PLTP encoding sequence is controlled by a Tet-On system. Upon induction of PLTP expression, our mouse model showed a strongly increased PLTP activity (from 3.0 ± 0.6 to 11.4 ± 2.8 AU, p < 0.001). The increase in PLTP activity resulted in an acute decrease in plasma cholesterol of 33% and a comparable decrease in phospholipids. The decrease in total plasma cholesterol and phospholipids was caused by a 35% decrease in HDL-cholesterol level and a 41% decrease in HDL-phospholipid level. These results demonstrate the feasibility of our mouse model to induce an acute elevation of PLTP activity, which is easily reversible. As a direct consequence of an increase in PLTP activity, HDL-cholesterol and HDL-phospholipid levels strongly decrease. Using this mouse model, it will be possible to study the effects of acute elevation of PLTP activity on lipoprotein metabolism and pre-existing atherosclerosis.     

Differentiation-promoting effects of mammary-derived growth inhibitor (MDGI) on pluripotent mouse embryonic stem cells

Pluripotent embryonic stem cells (line BLC6), when cultivated in vitro as embryoid bodies and injected subcutaneously into syngeneic mice, form teratocarcinomas consisting of embryonal carcinoma cells and differentiated tissues of all three primary germ layers. In order to study the possible effects of the mammary-derived growth inhibitor (MDGI) on the differentiation pattern of the tumors developing in the mice, BLC6 cell-derived embryoid bodies were treated in vitro for 4 days with either MDGI or a synthetic peptide composed of the C-terminal 11 amino acids of MDGI. In those tumors, significantly more differentiated neural tissue and lesser proportions of undifferentiated embryonic carcinoma cells (ECC) were found in the MDGI-and peptide-treated groups, compared with controls. The results are discussed with respect to a possible differentiation-promoting capacity of MDGI.     

Ontogenesis of catabolic and energy metabolism capacities during the embryonic development of spotted wolffish (Anarhichas minor)

The catabolic and energy metabolism capacities during spotted wolffish (Anarhichas minor) embryogenesis were investigated. We assessed the embryo's ability to catabolize proteins (trypsin-like proteases) and lipids (triglyceride lipase) and examined the development of metabolic capacities using enzymatic assays: ability to use carbohydrates (pyruvate kinase), amino acids (aspartate aminotransferase) and fatty acids (hydroxyacyl-CoA dehydrogenase) for energy production, and aerobic (citrate synthase) and anaerobic (lactate dehydrogenase) energy production. Functional enzymatic systems were detected from the eyed stage (350 degree-days), except for fatty acids, which was detected from 540 degree-days. To compare the development of 1) aerobic and anaerobic pathways and 2) the capacity to mobilize the different energy substrates, enzymatic ratios were calculated. Anaerobic capacity appeared to increase at a significantly higher rate than the aerobic capacity. Ratios revealing the relative capacity to use specific energy substrates showed a significantly slower increase during development in the capacity to use carbohydrates than amino acids and fatty acids. The end of embryogenesis was characterized by a significant decrease in the use of carbohydrates for aerobic energy production but an increasing capacity to use amino acids. Egg survival as affected by the variability in metabolic parameters is discussed.     

Expression of the 49 human ATP binding cassette (ABC) genes in pluripotent embryonic stem cells and in early- and late-stage multipotent mesenchymal stem cells

The 49-member human ATP binding cassette (ABC) gene family encodes 44 membrane transporters for lipids, ions, peptides or xenobiotics, four translation factors without transport activity, as they lack transmembrane domains, and one pseudogene. To understand the roles of ABC genes in pluripotency and multipotency, we performed a sensitive qRT-PCR analysis of their expression in embryonic stem cells (hESCs), bone marrow-derived mesenchymal stem cells (hMSCs) and hESC-derived hMSCs (hES-MSCs). We confirm that hES-MSCs represent an intermediate developmental stage between hESCs and hMSCs. We observed that 44 ABCs were significantly expressed in hESCs, 37 in hES-MSCs and 35 in hMSCs. These variations are mainly due to plasma membrane transporters with low but significant gene expression: 18 are expressed in hESCs compared with 16 in hES-MSCs and 8 in hMSCs, suggesting important roles in pluripotency. Several of these ABCs shared similar substrates but differ regarding gene regulation. ABCA13 and ABCB4, similarly to ABCB1, could be new markers to select primitive hMSCs with specific plasma membrane transporter^low phenotypes. ABC proteins performing basal intracellular functions, including translation factors and mitochondrial heme transporters, showed the highest constant gene expression among the three populations. Peptide transporters in the endoplasmic reticulum, Golgi and lysosome were well expressed in hESCs and slightly upregulated in hMSCs, which play important roles during the development of stem cell niches in bone marrow or meningeal tissue. These results will be useful to study specific cell cycle regulation of pluripotent stem cells or ABC dysregulation in complex pathologies, such as cancers or neurological disorders.     

Effects of CGP 28014 on the in vivo release and metabolism of dopamine in the rat striatum assessed by brain microdialysis

The effects on rat striatal dopamine (DA) metabolism of systemic and local administration of CGP 28014, an inhibitor of catechol-O-methyl-transferase (COMT), were studied by in vivo microdialysis. CGP 28014 (30 mg/kg i.p.) significantly reduced the levels of homovanillic acid (HVA), but did not modify DA and 3,4-dihydroxyphenylacetic acid (DOPAC). The intrastriatal administration (via the microdialysis probe) of 5, 7.5, 10, and 20 mM of CGP 28014 elicited a concentration-dependent, several-fold increase in extracellular DA but did not alter the levels of HVA and DOPAC. Thus, the effects of CGP 28014 observed after i.p. injection (decrease in HVA levels) are different from those measured after intrastriatal administration (increase in DA release). Therefore, the inhibition of COMT is likely to be due to the action of a metabolite of CGP 28014 formed in the periphery and not in the brain.     

In vivo effects of adenosine A(2) receptor agonist and antagonist on neuronal and astrocytic intermediary metabolism studied with ex vivo (13)C MR spectroscopy.

The effect of adenosine A(2) receptor agonist 2-[p-(2-carboxyethyl)phenylethylamino]-5'-ethylcarboxamidoadenosine (CGS 21680) and antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) on [1-(13)C]glucose and [1,2-(13)C]acetate metabolism was studied in rats by (13)C magnetic resonance (MR) spectroscopy and HPLC. In the cortex a significant reduction was observed in the amounts of [2-(13)C]GABA and [3-(13)C]aspartate from [1-(13)C]glucose in CGS 21680. In the subcortex the concentration of labelled [4-(13)C]glutamate was increased in both treatment groups. The amounts of [2 + 3-(13)C]succinate and [3-(13)C]lactate were increased in the CGS 21680 group compared to control, and the DMPX group showed an increase in the total amount of [6-(13)C]N-acetyl aspartate compared to control in the subcortex. Astrocyte metabolism was only affected in the cortex as shown by a decrease in the pyruvate carboxylase/pyruvate dehydrogenase ratio in glutamate and glutamine in the treatment groups. Labelling from [1,2-(13)C]acetate was not much affected by CGS 21680 or DMPX. However, the amount of [1,2-(13)C]acetate in cortex and subcortex was reduced in the DMPX group. In the cortex a reduction in the labelling of [3-(13)C]GABA in the DMPX group compared to control and an increase in the total amount of taurine in both treatment groups was detected. The present study shows that A(2) receptor agonist and antagonist have similar effects; however, in cortex GABAergic neurones and astrocytes were affected in contrast to subcortex, where glutamatergic neurones showed the greatest changes.     

Effect of chronic hypernatremic dehydration and rapid rehydration on brain carbohydrate, energy, and amino acid metabolism in weanling mice

Abstract: This is a study of the effects of chronic hypernatremic dehydration and rehydration on carbohydrate, energy, and amino acid metabolism in the brains of weanling mice. Chronic hypernatremic dehydration induced by 4 days of water deprivation and salt loading was associated with severe weight loss (no other observed clinical effects), increased brain Na⁺ levels, and a decreased brain water content. Changes in the concentrations of brain glucose, glycolytic and citric acid cycle metabolic intermediates, and phosphocreatine were compatible with reduced cerebral metabolic rate. In adaptation to chronic hypernatremia, there was a significant increase in the content of the measured brain amino acids. Rapid rehydration over a 4-h period with 2.5% dextrose in water returned plasma Na⁺ levels and brain Na⁺ and water contents to normal. After rehydration, metabolites were altered in a manner consistent with increased fluxes through the glycolytic pathway and citric acid cycle; the brain glycogen content almost tripled. Brain taurine and glutamine levels were not lowered by rehydration, and the total content of the measured amino acids in brain was still significantly higher than in controls. We speculate that these metabolic perturbations may relate to the development of cerebral edema and seizures or coma following rapid rehydration of humans with chronic hypernatremic dehydration.     

Hydrocortisone regulates arylsulfatase A (cerebroside-3-sulfate-3-sulfohydrolase) by decreasing the quantity of the enzyme in cultures of cells dissociated from embryonic mouse cerebra

Previous work from our laboratory (Biochem. J. 219:689–697 (1984) had shown that hydrocortisone stimulated the net accumulation of the myelin-specific sulfolipid in cultures of cells dissociated from embryonic mouse cerebra. This accumulation caused by hydrocortisone was shown to be due to a decrease of sulfolipid degradation by arylsulfatase A (ASA) and not due to a stimulation of its synthesis by a sulfotransferase. Both ASA activity and the turnover of sulfolipid were decreased by hydrocortisone to 60–62% of untreated cells. In current work the same decrease in enzyme activity was obtained and enzyme linked immunosorbent assays demonstrate that hydrocortisone decreased the number of ASA protein molecules to 61% of untreated cells [(-)hydrocorcortisone 0.31±0.06 ng ASA/g protein; (+)hydrocortisone: 0.18±0.04 ng ASA/g protein]. This decrease in the number of ASA molecules correlates well with the decrease in both the enzyme activity and the sulfolipid turnover, which suggests that the major mode of inhibition of ASA activity by hydrocortisone involves a decrease in the concentration of ASA in the cells rather than some other mechanism of inhibition.The material in this paper has been included in a dissertation submitted by A.J.M. in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Temple University.     

Culture of adult rat lung cells: Benzo(a)pyrene metabolism and mutagenesis

Summary A method is described for obtaining and culturing large numbers of lung cells from normal adult male rats. The lungs were perfused in situ to remove blood cells and then perfused via the trachea with a trypsin-collagenase solution to initiate tissue digestion. The tissue was further digested in the enzyme solution and approximately 2×10⁸ viable lung cells were obtained per animal. Primary cultures contained a mixed cell population. Through eight subcultures about 70% of the cell population possessed an epithelial-like morphology, whereas the remaining 30% was fibroblast-like. Three clones of epithelial-like cells were isolated at the fourth subculture. The mass culture lung cells and the epithelial-like clone that was studied retained a normal karyotype and did not grow in soft agar. Both the mass culture cells and the epithelial clone metabolized the lung carcinogen benzo(a)pyrene (BP) to water-soluble products. Furthermore, the mass culture lung cells metabolized BP to intermediate(s) which mutated Chinese hamster V79 cells from ouabain sensitivity to ouabain resistance. These lung cell cultures have potential use in cell transformation, mutation and carcinogen metabolism studies. Visiting scientist from Hungary. This research was supported by Grant 5 R01 CA20022 and Public Health Service Contract N01 CP33278 from the Division of Cancer Cause and Prevention, National Cancer Institute, National Institutes of Health.