Stopped-flow kinetics of oxygen uptake and release by embryonic red blood cells.

The processes of O2 uptake and release by the three embryonic haemoglobins contained within early mouse embryonic red blood cells have been studied using dual-wavelength stopped-flow kinetic spectroscopy. The rate of O2 uptake in the pseudo-spherical, nucleated, embryonic red blood cells exhibits a greater than first-order dependence on O2 concentration. The time courses for the release from the red blood cells into dithionite-containing solutions tends towards a limiting rate at high dithionite concentrations. The rates of both the uptake and release processes observed in the embryonic cells are compared with those previously seen for adult mouse red blood cells. A new mathematical model is described which accurately simulates both uptake and release experimental data for the nucleated embryonic red blood cells.     

Effect of oxygen on ascorbic acid uptake and concentration in embryonic chick brain

The effects of oxygen on ascorbic acid concentration and transport were studied in chick embryo (Gallus gallus domesticus). During normoxic incubations, plasma ascorbic acid concentration peaked on fetal day 12 and then fell, before increasing again on day 20 when pulmonary respiration began. In contrast, cerebral ascorbic acid concentration rose after day 6, was maintained at a relatively high level during days 8–18, and then fell significantly by day 20. Exposure of day 16 embryos for 48 h to 42% ambient O₂ concentration decreased ascorbic acid concentration by four-fifths in plasma and by one-half in brain, compared to values in normoxic (21% O₂) or hypoxic (15% O₂) controls. Hyperoxic preincubation of embryos also inhibited ascorbic acid transport, as evidenced by decreased initial rates of saturable and Na⁺-dependent [¹⁴C]ascorbic acid uptake into isolated brain cells. It may be concluded that changes in ascorbic acid concentration occur in response to oxidative stress, consistent with a role for the vitamin in the detoxification of oxygen radicals in fetal tissues. However, changing O₂ levels have less effect on ascorbic acid concentration in brain than in plasma, indicating regulation of the vitamin by brain cells. Furthermore, the effect of hyperoxia on cerebral vitamin C may result, in part, from inhibition of cellular ascorbic acid transport.     

Characteristics of hematin uptake in malignant, embryonic and normal cells

The characteristics of hematin uptake were examined in three malignant cell lines [L1210 leukemia, 745 murine erythroleukemia (MEL) and Walker carcinoma (W256)], a cell line derived from normal rat liver (BRL-3A) and a normal embryonic cell, chick embryo fibroblasts (CEF). Uptake in the normal liver cell line was slight and occurred at a slow rate in contrast to the rapid uptake, which was more rapid and of greater magnitude in the three tumor cell lines, Saturation of the heme uptake mechanism was observed in MEL cells at an extra-cellular hematin concentration of 160 micro M and in L1210 cells at 300 micro M. At saturation L1210 cells achieved a cellular heme concentration nine times as high as MEL cells. Hematin uptake in MEL cells was markedly augmented by pretreatment with DMSO, procaine, detergent or proteolytic enzymes or by increases in the pH of the medium from 8 to 9.5. In contrast to MEL cells where SA inhibits growth by lowering cellular heme, the inhibition of growth of L1210 cells by SA appears to operate by a mechanism independent of heme. In gradual increase in hematin uptake capacity in MEL cells over a period of days. Afer exposure of MEL cells to a high concentration of hematin in the medium, the egress of heme was followed under various conditions. Of the various agents studied, only cyanide produced a loss of heme from MEL cells.     

Real‐time monitoring of specific oxygen uptake rates of embryonic stem cells in a microfluidic cell culture device

Oxygen plays a key role in stem cell biology as a signaling molecule and as an indicator of cell energy metabolism. Quantification of cellular oxygen kinetics, i.e. the determination of specific oxygen uptake rates (sOURs), is routinely used to understand metabolic shifts. However current methods to determine sOUR in adherent cell cultures rely on cell sampling, which impacts on cellular phenotype. We present real‐time monitoring of cell growth from phase contrast microscopy images, and of respiration using optical sensors for dissolved oxygen. Time‐course data for bulk and peri‐cellular oxygen concentrations obtained for Chinese hamster ovary (CHO) and mouse embryonic stem cell (mESCs) cultures successfully demonstrated this non‐invasive and label‐free approach. Additionally, we confirmed non‐invasive detection of cellular responses to rapidly changing culture conditions by exposing the cells to mitochondrial inhibiting and uncoupling agents. For the CHO and mESCs, sOUR values between 8 and 60 amol cell^?1 s^?1, and 5 and 35 amol cell^?1 s^?1 were obtained, respectively. These values compare favorably with literature data. The capability to monitor oxygen tensions, cell growth, and sOUR, of adherent stem cell cultures, non‐invasively and in real time, will be of significant benefit for future studies in stem cell biology and stem cell‐based therapies.     

Is resting muscle oxygen uptake controlled by oxygen availability to cells?

To estimate oxidative capacity of noncontracting rat skeletal muscle, the isolated gracilis muscle was perfused at various high flow rates with high-PO2 (88 kPa) saline-albumin solution and simultaneously perifused at either low (6.3 kPa) or high PO2 in a calorimeter at 28 degrees C. Under low-PO2 perifusion, specific O2 consumption and heat production rates (MO2 and E, respectively) were flow-rate dependent. E values were all larger than those obtained on blood-perfused preparations at 28 degrees C. MO2 reached 0.47 mumol.min-1.g muscle-1 and E reached 4 mW/g. Normalized to 36 degrees C by means of activation energies determined from 30 and 36 degrees C measurements on nonperfused gracilis strips, these maxima correspond to three times the largest MO2 measured by other authors in blood-autoperfused gracilis. Increasing perifusion PO2 from 6.3 to 88 kPa sharply decreased MO2. These results confirm that MO2 of blood-perfused skeletal muscles in vitro (and a fortiori in vivo) is kept much below its maximum for a noncontracting organ; they also suggest that this maximum MO2 is not necessarily an effect of unphysiologically high PO2 in the tissue cells.     

GABA uptake in embryonic palate mesenchymal cells of two mouse strains

To obtain further evidence that the inhibitory neurotransmitter GABA functions in palate development, the presence of an active GABA uptake mechanism was sought using primary cultures of embryonic palate mesenchymal cells. Uptake was compared from cells of two inbred mouse strains in which the SWV strain shows greater sensitivity than the AJ strain to effects of GABA on palate morphogenesis and of diazepam in producing cleft palate (1). Palate cells were capable of accumulating [³H]GABA by saturable uptake mechanisms characteristic of a high and a low affinity active transport as indicated by temperature, Na⁺ ion and carrier dependence as well asK _m andV _max values that were comparable to other biological systems. TheV _max of the high-affinity uptake system from cells of the SWV strain was 1.8 fold higher than that of the AJ. GABA uptake was also observed in fibroblasts from various sources including embryonic mouse limb cells, human skin fibroblasts and 3T3 cells When active GABA uptake was measured in skin fibroblasts from the mouse SWV and AJ strains, the rate of uptake from SWV cells under high affinity conditions was also 1.8 fold greater than in AJ cells. Thus active GABA uptake appears to be genetically regulated in non-neural cells which may contribute to differential resonses to GABA.     

ANG II increases 2-deoxyglucose uptake in mouse embryonic stem cells

It is now suggested that all components of the renin-angiotensin system are present in many tissues, including the embryo and may play a major role in embryo development and differentiation. However, little is known regarding whether ANG II regulates glucose transport in mouse embryonic stem (ES) cells. Thus, the effects of ANG II on [3H]-2-deoxyglucose (2-DG) uptake and its related signal pathways were examined in mouse ES cells. ANG II significantly increased cell proliferation and 2-DG uptake in concentration- and time-dependent manner (>18 h, >10(-8) M) and increased mRNA and protein level of GLUT1 by 31+/-7% and 22+/-5% compared to control, respectively. Actinomycin D and cycloheximide completely blocked the effect of ANG II on 2-DG uptake. ANG II-induced increase of 2-DG uptake was blocked by losartan, an ANG II type 1 (AT1) receptor blocker, but not by PD 123319, an ANG II type 2 (AT2) receptor blocker. In addition, ANG II-induced stimulation of 2-DG uptake was attenuated by phospholipase C (PLC) inhibitors, neomycin and U 73122 and ANG II increased inositol phosphates (IPs) formation by 37+/-8% of control. Protein kinase C (PKC) inhibitors, staurosporine, bisindolylmaleimide I, and H-7 also blocked ANG II-induced stimulation of 2-DG uptake. Indeed, ANG II activated a PKC translocation from the cytosolic to membrane fraction, suggesting a role of PKC. A 23187 (Ca2+ ionophore) increased 2-DG uptake and nifedifine (L-type Ca2+ channel blocker) blocked it. In conclusion, ANG II increased 2-DG uptake by PKC activation via AT1 receptor in mouse ES cells.     

Rates of oxygen uptake by embryonic anterior horn tissue isolated at various developmental stages.

Neuroepithelial cells of the presumptive spinal cord (stage 11) consume oxygen, albeit at a low rate. As neurons differentiate in the presumptive motor horns the rate of oxygen consumption increases to approximately 70 mumoles/g wet wt/hr by stage 26. It is suggested that the rate of oxygen consumption per unit volume of neuron then remains constant as subsequent development ensues but since the neurons become more widely spaced the oxygen consumption per unit volume of anterior horn tissue decreases.     

RYBP represses endogenous retroviruses and preimplantation- and germ line-specific genes in mouse embryonic stem cells

Polycomb repressive complexes (PRCs) are important chromatin regulators of embryonic stem (ES) cell function. RYBP binds Polycomb H2A monoubiquitin ligases Ring1A and Ring1B and has been suggested to assist PRC localization to their targets. Moreover, constitutive inactivation of RYBP precludes ES cell formation. Using ES cells conditionally deficient in RYBP, we found that RYBP is not required for maintenance of the ES cell state, although mutant cells differentiate abnormally. Genome-wide chromatin association studies showed RYBP binding to promoters of Polycomb targets, although its presence is dispensable for gene repression. We discovered, using Eed-knockout (KO) ES cells, that RYBP binding to promoters was independent of H3K27me3. However, recruiting of PRC1 subunits Ring1B and Mel18 to their targets was not altered in the absence of RYBP. In contrast, we have found that RYBP efficiently represses endogenous retroviruses (murine endogenous retrovirus [MuERV] class) and preimplantation (including zygotic genome activation stage)- and germ line-specific genes. These observations support a selective repressor activity for RYBP that is dispensable for Polycomb function in the ES cell state. Also, they suggest a role for RYBP in epigenetic resetting during preimplantation development through repression of germ line genes and PcG targets before formation of pluripotent epiblast cells.     

Blood rheology and oxygen uptake

Continuously measured oxygen uptake during constant work exercise (15' 50W) reveals increasing oxygen consumption in individuals with elevated blood viscosity parameters, indicating persistent contribution of anaerobic glycolysis during steady state exercise far below expected "anaerobic threshold". Improvement of viscosity parameters by prostaglandin E1--infusion (Prostavasin) 40 micrograms i.v., naftidrofurylhydrogenoxalat (Dusodril pi) 400 mg i.v. or hemodilution with 500 ml 6% hydroxyethylamylum MW 40000 (Onkohaes) in 5 patients results in significant reduction of this oxygen gradient in subsequent exercise test. Integrated VO2 during exercise above the mean value at rest or the quotient of VO2 during 15 min by VO2 during 30 min (including recovery time) are not differing significantly due to high variations inter- and intraindividually. Oxygen gradient during submaximal constant exercise permits direct clinical determination of microcirculatory performance in involved muscle tissue as a function of blood viscosity.     

Effects of lactate and ammonium on the oxygen uptake rate of human cells

Effects of lactate and ammonium on the respiration rate and specific growth rate of HL-60 and RPMI 8226 human cells were studied. These compounds inhibited cell propagation, but did not affect the oxygen uptake of the cells even after two days of cultivation in their presence.     

Morphological and physiological factors affecting oxygen uptake and release by red blood cells

The kinetics of oxygen uptake and release by human, salamander (Amphiuma means), and artificially constructed red cells were measured under a variety of physiological conditions using stopped-flow, rapid mixing techniques. The results were analyzed quantitatively using the generalized, three-dimensional disc model that was developed in two previous publications (Vandegriff, K. D., and Olson, J. S. (1984) Biophys. J. 45, 825-835 and Vandegriff, K. D., and Olson, J. S. (1984) J. Biol. Chem. 259, 12609-12618). The apparent rate of gas exchange is governed primarily by the oxygen flux at the red cell surface. In the case of uptake, this flux is roughly independent of intracellular chemical reaction parameters and inversely proportional to the thickness of the unstirred solvent layer which is adjacent to the red cell surface. For release experiments in the presence of high concentrations of sodium dithionite, the flux at the cell surface is inversely proportional to the oxygen affinity of the intracellular hemoglobin and roughly independent of the thickness of the external unstirred solvent layer. As a result, the effects of cell size, internal heme concentration, and pH are expressed differently in the two types of kinetic experiments. The rate of oxygen uptake depends on roughly the second power of the surface area to volume ratio of the erythrocyte, whereas the rate of release is much less dependent on the size and shape of the red cell. The half-time of oxygen uptake is directly proportional to intracellular heme concentration for cells of equivalent geometries; the half-time of oxygen release is linearly dependent on internal heme concentration but, at low heme concentrations, is determined primarily by the rate of oxygen dissociation from hemoglobin. The rate of cellular oxygenation is roughly independent of pH and internal 2,3-diphosphoglycerate concentration; in contrast, the rate of deoxygenation depends markedly on these conditions. As the pH is lowered or the internal diphosphoglycerate concentration is raised, the overall oxygen affinity of the cell suspension decreases severalfold, and the rate of oxygen release increases by roughly the same extent.