A quantitative description in three dimensions of oxygen uptake by human red blood cells

Oxygen uptake by human erythrocytes has been examined both experimentally and theoretically in terms of the influence of unstirred solvent layers that are adjacent to the cell surface. A one-dimensional plane sheet model has been compared with more complex spherical and cylindrical coordinate schemes. Although simpler and faster, the plane sheet algorithm is an inadequate representation when unstirred solvent layers are considered. The cylindrical disk model most closely represents the physical geometry of human red cells and is required for a quantitative analysis. In our stopped-flow rapid mixing experiments, the thickness of the unstirred solvent layer expands with time as the residual turbulence decays. This phenomenon has been quantified using a formulation based on previously developed hydrodynamic theories. An initial 10(-4) cm unstirred layer is postulated to occur during mixing and expand rapidly with time by a (t)0.5 function when flow stops. This formula, in combination with the three-dimensional cylinder scheme, has been used to describe quantitatively uptake time courses at various oxygen concentrations, two different external solvent viscosities, and two different internal heme concentrations.     

Regulation of 5-phosphoribosyl 1-pyrophosphate and of hypoxanthine uptake and release in human erythrocytes by oxypurine cycling.

Uptake and release of purines by red blood cells has been shown to be markedly sensitive to changes in pH, inorganic phosphate (Pi), and oxygen concentration (Berman, P., Black, D., Human, L., and Harley, E. (1988) J. Clin. Invest. 82, 980-986). The mechanism of this regulation has been further studied. We have shown that incubation of red cells in medium containing xanthine oxidase rapidly and completely depletes intracellular hypoxanthine and causes accumulation of 5-phosphoribosyl 1-pyrophosphate (PRPP) at physiological Pi concentrations. Hypoxanthine release from intracellular IMP is strictly dependent on PRPP depletion, induced by either alkalinizing the cells or by adding excess adenine. Xanthine oxidase abolishes this dependence. Oxygen depletion enhances adenine uptake and prevents hypoxanthine release. The results suggest that hypoxanthine release is governed by PRPP-dependent recycling of hypoxanthine to IMP. We propose that PRPP accumulation in red cells is regulated by a substrate cycle, comprising hypoxanthine, IMP, and inosine. Cycle flux is controlled by Pi inhibition and 2,3-bisphosphoglycerate activation of purine-5'-nucleotidase, which converts IMP to inosine. Oxypurine cycling may account for the sensitive control of purine uptake and release by changes in pH and oxygen tension that occur physiologically.     

Heme inhibits transferrin endocytosis in immature erythroid cells

The inhibitory effect of heme on iron uptake from transferrin by rat and rabbit reticulocytes and erythroid cells from the fetal rat liver was studied in vitro. Addition of hemin was shown to cause a decrease in the rate of transferrin endocytosis, the degree of inhibition being proportional to the reduction in iron uptake. The heme synthesis inhibitors, isoniazid and succinylacetone, stimulated the rate of transferrin endocytosis by 15-30% and caused a proportional increase in the rate of iron uptake, possibly by reducing the intracellular free heme concentration. It is concluded from these results that heme affects iron uptake by influencing the rate of transferrin endocytosis and recycling.     

Intracellular movement of cell surface receptors after endocytosis: resialylation of asialo-transferrin receptor in human erythroleukemia cells

The intracellular movement of cell surface transferrin receptor (TfR) after internalization was studied in K562 cultured human erythroleukemia cells. The sialic acid residues of the TfR glycoprotein were used to monitor transport to the Golgi complex, the site of sialyltransferases. Surface-labeled cells were treated with neuraminidase, and readdition of sialic acid residues, monitored by isoelectric focusing of immunoprecipitated TfR, was used to assess the movement of receptor to sialyltransferase-containing compartments. Asialo-TfR was resialylated by the cells with a half-time of 2-3 h. Resialylation occurred in an intracellular organelle, since it was inhibited by treatments that allow internalization of surface components but block transfer out of the endosomal compartment. Moreover, roughly half of the resialylated molecules were cleaved when cells were retreated with neuraminidase after culturing, indicating that this fraction of the molecules had returned to the cell surface. These results suggest that TfR is transported from the cell surface to the Golgi complex, the intracellular site of sialyltransferases, and then returns to the cell surface. This pathway, which has not been previously described for a cell surface receptor, may be different from the route followed by TfR in iron uptake, since reported rates of transferrin uptake and release are significantly more rapid than the resialylation of asialo-TfR.     

Modelling of hexadecane degradation in continuous-flow cultures

Microorganisms of Wadden Sea sediments are able to degrade hydrocarbons in suspensions. (Berthe-Corti, L., Bruns, A., Hulsch, R., 1997. J. Microb. Methods 29, 129-137) have observed in continuous culture experiments that the growth rate of microorganisms increases roughly proportional to the dilution rate. The growth rate is nearly independent of the oxygen saturation down to about 0.5%. Even at very low oxygen supply, corresponding to an oxygen saturation far below 0.1%, growth takes place at a reduced rate. In this paper, a model is presented which can reproduce the results of these experiments. The model treats the following processes, selection of the active fraction of microorganisms growing on hexadecane, uptake of hexadecane and transformation into palmitate as a first metabolic step, synthesis of biomass, respiration and exudation. The processes are regulated by the substrate concentration, the internal palmitate quota, the exudates' concentration and an inhibiting factor. For the experiments under very low oxygen conditions, the observed growth with reduced O(2)-consumption and CO(2)-production is modelled by assuming an anoxic metabolic pathway.     

Turbulent flow of red cells in dilute suspensions. Effect on kinetics of O2 uptake.

The turbulent flow properties of dilute (0.06% by volume) suspensions of human red blood cells in 4-mm-bore glass tubing were estimated by laser anemometry. The flow properties of the dilute red cell suspension were similar to those of a dilute suspension of polystyrene spheres (0.5 micron diameter) in isotonic NaCl solution. Flow was found to be laminar when the Reynolds number was below 2,000, transitional in the range of Reynolds numbers from 2,000 to 3,000, and fully turbulent above Reynolds number 3,000. These results differ from previous studies of more concentrated red cell suspensions. The length scales of the turbulence were also estimated: at a Reynolds number near 4,000 the macroscale is about 1.25 mm, the Taylor microscale is about 0.85 mm, and the Kolmogoroff scale is near 0.075 mm. The results are discussed in relation to previous measurements of the rate of oxygen uptake by dilute red cell suspensions in the flow-type rapid reaction apparatus. Our results suggest that under the conditions of most of these oxygen uptake measurements, the turbulent flow is characterized by eddies about 1 mm across, mixing with each other on a time scale of about 45 ms. Since most of the reported oxygen uptake measurements involve a similar time scale, it is possible that an effective "unstirred layer" influenced the reported rate of oxygen uptake.     

Relationship between outer membrane protein and lipopolysaccharide profiles and serotypes of enterotoxigenic Escherichia coli isolated in Brazil

Abstract When the yeast Saccharomyces cerevisiae was grown under aerobic continuous culture conditions with a medium containing ethanol as carbon source, an autonomous sustained metabolic oscillation appeared. This oscillation was observed in rates and concentrations of various parameters such as, ethanol, oxygen uptake rate, carbon dioxide evolution rate, NaOH addition rate for pH control, acetate, and intracellular pH. No changes were observed in concentrations of stock carbohydrates. Intracellular pH changes were out of phase with oxygen uptake rate, which was reverse of the results with glucose-based oscillation. These results suggested that changes in glycolytic flux and intracellular pH were not regulating the oscillation. Analysis suggested that one of the oscillatory regulation points was located in the ethanol assimilation pathway.     

Heme transfer between phospholipid membranes and uptake by apohemoglobin

The incorporation of CO-heme into single bilayer, egg lecithin vesicles was examined by following the spectral changes that occur when the porphyrin becomes embedded in the membranes. The rate of CO-heme uptake by liposomes is extremely fast (t1/2 less than or equal to 20 ms at 10 degrees C), and the maximum extent is roughly 1 heme/5 phospholipid molecules. This limiting stoichiometry is due to unfavorable electrostatic interactions between the propionate groups of the bound CO-heme. This effect was treated theoretically by attenuating the intrinsic heme partitioning equilibrium constant with an exponential term reflecting the surface potential of the membranes. The surface potential was assumed to be proportional to the concentration of CO-heme in the membranes, and the final expression is Kp = Kop exp[-AHb/VpCp], where Kp is the observed partition constant; Kop, the intrinsic constant; Hb, the concentration of bound heme in the suspension; Vp, the partial molar volume of egg lecithin; Cp, the concentration of lipid phosphate; and A, an empirical constant representing the capacitance of the membrane for heme. For the analysis of kinetic data, the electrostatic term is assumed to apply only to the membrane dissociation rate constant, k-1, and not the association rate constant, k1. The dissociation rate was measured independently either by following the transfer of CO-heme from one vesicle fraction to another or by monitoring heme efflux from the membranes and incorporation into apohemoglobin at high protein concentrations. The data for all three sets of experiments, heme uptake, transfer, and incorporation into globin at 10 degrees C, were fitted quantitatively to the partitioning mechanism using A = 15 M-1, Kop = 5 X 10(5), k1 = 2 X 10(6) s-1, and k0(-1) = 4 s-1. Thus, heme can spontaneously migrate across lipid-water interfaces and hence diffuse rapidly from the mitochondrial inner membrane where it is synthesized to the rough endoplasmic reticulum where it is incorporated into hemoglobin.     

Physiological factors affecting O2 transport by hemoglobin in an in vitro capillary system

O2 transport was examined by measuring the fractional saturation of concentrated hemoglobin solutions flowing through an artificial capillary that was approximately 27 micron in diameter and embedded in a silicone rubber film approximately 170 micron thick. The effects of pH, hemoglobin concentration, O2 tension, temperature, and organic phosphate were measured and analyzed quantitatively by a rigorous mathematical model that included the geometry of the capillary in the silicone film, parabolic flow velocity distributions inside the lumen, and cooperative O2 binding by hemoglobin. The rates of both oxygenation and deoxygenation were limited by diffusion and governed by the magnitude of the O2 gradient between the intracapillary fluid phase and the external gas space. In uptake experiments, O2 flux is determined primarily by the external O2 tension (16-160 mmHg in our experiments) because the internal O2 pressure is kept small due to chemical combination with hemoglobin. In release experiments, the external O2 tension is maintained at zero, and the transport rate is determined by the intracapillary partial pressure of O2 that is proportional to the O2 half-saturation pressure of hemoglobin value of the hemoglobin sample. As a result, factors that change the affinity of hemoglobin for O2, such as pH, temperature, and organic phosphate concentration, influence strongly the rate of O2 release but have little effect on the rate of O2 uptake. These properties are physiologically advantageous, since a decrease in pH or an increase in temperature during exercise increases both the rate and extent of deoxygenation while not altering the kinetics of oxygenation.     

Free and hemophore-bound heme acquisitions through the outer membrane receptor HasR have different requirements for the TonB-ExbB-ExbD complex

Many gram-negative bacteria have specific outer membrane receptors for free heme, hemoproteins, and hemophores. Heme is a major iron source and is taken up intact, whereas hemoproteins and hemophores are not transported: the iron-containing molecule has to be stripped off at the cell surface, with only the heme moiety being taken up. The Serratia marcescens hemophore-specific outer membrane receptor HasR can transport either heme itself or heme bound to the hemophore HasA. This second mechanism is much more efficient and requires a higher TonB-ExbB-ExbD (TonB complex) concentration than does free or hemoglobin-bound heme uptake. This requirement for more of the TonB complex is associated with a higher energy requirement. Indeed, the sensitivity of heme-hemophore uptake to the protonophore carbonyl cyanide m-chlorophenyl hydrazone is higher than that of heme uptake from hemoglobin. We show that a higher TonB complex concentration is required for hemophore dissociation from the receptor. This dissociation is concomitant with heme uptake. We propose that increasing the TonB complex concentration drives more energy to the outer membrane receptor and speeds up the release of empty hemophores, which, if they remained on receptors, would inhibit heme transport.     

Variations in tumor cell growth rates and metabolism with oxygen concentration, glucose concentration, and extracellular pH.

Tumors and multicellular tumor spheroids can develop gradients in oxygen concentration, glucose concentration, and extracellular pH as they grow. In order to calculate these gradients and assess their impact on tumor growth, it is necessary to quantify the effect of these variables on tumor cell metabolism and growth. In this work, the oxygen consumption rates, glucose consumption rates, and growth rates of EMT6/Ro mouse mammary tumor cells were measured at a variety of oxygen concentrations, glucose concentrations, and extracellular pH levels. At an extracellular pH of 7.25, the oxygen consumption rate of EMT6/Ro cells increased by nearly a factor of 2 as the glucose concentration was decreased from 5.5 mM to 0.4 mM. This effect of glucose concentration on oxygen consumption rate, however, was slight at an extracellular pH of 6.95 and disappeared completely at an extracellular pH of 6.60. The glucose consumption rate of EMT6/Ro cells increased by roughly 40% when the oxygen concentration was reduced from 0.21 mM to 0.023 mM and decreased by roughly 60% when the extracellular pH was decreased from 7.25 to 6.95. The growth rate of EMT6/Ro cells decreased with decreasing oxygen concentration and extracellular pH; however, severe conditions were required to stop cell growth (0.0082 mM oxygen and an extracellular pH of 6.60). Empirical correlations were developed from these data to express EMT6/Ro cell growth rates, oxygen consumption rates, and glucose consumption rates, as functions of oxygen concentration, glucose concentration, and extracellular pH. These empirical correlations make it possible to mathematically model the gradients in oxygen concentration, glucose concentration, and extracellular pH in EMT6/Ro multicellular spheroids by solution of the diffusion/reaction equations. Computations such as these, along with oxygen and pH microelectrode measurements in EMT6/Ro multicellular spheroids, indicated that nutrient concentration and pH levels in the inner regions of spheroids were low enough to cause significant changes in nutrient consumption rates and cell growth rates. However, pH and oxygen concentrations measured or calculated in EMT6/Ro spheroids where quiescent cells have been observed were not low enough to cause the cessation of cell growth, indicating that the observed quiescence must have been due to factors other than acidic pH, oxygen depletion, or glucose depletion.     

Effects of anaerobic exercise accompanying catch-and-release fishing on blood-oxygen affinity of the sandbar shark (Carcharhinus plumbeus, Nardo)

Recovery from anaerobic exercise is thought to be prolonged in elasmobranchs because they lack several mechanisms for maintaining or increasing oxygen delivery that are present in teleosts. For example, teleosts increase hematocrit and maximal blood-oxygen carrying capacity through red cell ejection from the spleen. Teleosts also counteract the reduction in hemoglobin oxygen affinity resulting from metabolic acidosis through an adrenergic-mediated increase in red cell Na⁺-H⁺ exchanger activity. To begin to assess the consequences of anaerobic exercise accompanying catch-and-release fishing occurring within the estuarine nursery habitats of juvenile sandbar sharks (Carcharhinus plumbeus, Nardo), we constructed blood-oxygen equilibrium curves using samples from individuals 1 h after capture by hook and line (exercise-stressed) and samples from fully-recovered animals maintained in a shore-side tank (control sharks). We also compared exercise-stressed and control sharks for hemoglobin concentration, hematocrit, red cell count, intracellular pH, and nucleoside triphosphate concentration ([NTP]). In contrast to results from previous studies on elasmobranchs, we found an elevation in both hematocrit (≈ 21%) and blood hemoglobin concentration (≈ 10%) in exercise-stressed sharks. There was also clear evidence of red cell swelling. Mean red cell volume was ≈ 28% higher and mean cell hemoglobin concentration was ≈ 10% lower in exercise-stressed sharks. Most important, in spite of significant metabolic acidosis (0.3 pH units), blood from exercise-stressed sharks had an oxygen affinity equivalent to that of blood from control sharks. This was a direct consequence of intracellular pH being alkalinized by approximately 0.15 pH units relative to plasma pH in exercise-stressed sharks. Our results using isolated hemoglobin solutions showed that the observed reduction (≈ 15%) in intracellular [NTP] also contributed to the leftward shift in the oxygen equilibrium curves. As expected, we found sandbar shark red cells to be unresponsive to exogenous catecholamines. Regardless, sandbar sharks appear able to prevent the decrease in blood-oxygen affinity resulting from anaerobic exercise (and the concomitant decreases in plasma pH), as has been well-documented in teleosts. Our results suggest, therefore, that oxygen delivery following exhaustive exercise is not necessarily compromised in juvenile sandbar sharks, and that hook and line capture and subsequent release do not increase rates of mortality, although both are yet to be directly confirmed.     

Effect of Growth Conditions on Yield and Heme Content of Vitreoscilla

Vitreoscilla, a gliding bacterium in the Beggiatoaceae, is an obligate aerobe in which cytochrome o functions as the terminal oxidase. Protoheme IX is the only heme type present in this organism. The yield and heme content of Vitreoscilla cells grown in yeast extract, peptone, and acetate were dependent on growth conditions. Cells harvested in early stationary phase contained roughly three times as much heme as cells in early log phase. There was an optimal shaking rate for maximum heme content of cells harvested in stationary phase at fixed initial nutrient concentration. The heme content of cells grown at a fixed shaking rate increased from 5 nmol/g (wet weight) in media which had low nutrient concentration to a maximum of 45 nmol/g (wet weight) in media which had high nutrient concentration, and there was a corresponding sixfold increase in cytochrome o content and an eightfold increase in respiratory rate, evidence that some of the additional heme was incorporated into respiratory pigments. Heme content may be controlled jointly by competition for oxygen and availability of nutrients. Temperature and initial pH affected the growth rate but not the final yield or heme content. Growth rate was optimal at pH 8.0 to 8.5. A defined medium for Vitreoscilla, which is based on glutamate as the carbon source, is described; the other organic components of this medium are acetate, tryptophan, thiamine, biotin, and riboflavin.     

Cytochrome c turnover in rat skeletal muscles.

Exercise induces an increase in cytochrome c concentration in skeletal muscle. This adaptation provides an approach to studying the turnover of cytochrome c that avoids the problem of reutilization encountered with isotopic tracers. The half-life of cytochrome c was estimated from the time course of the increase in its concentration to a new, higher, steady state level in response to exercise training, and from the decrease in cytochrome c after cessation of exercise. The half-time of the increase in cytochrome c concentration was approximately 6 days, while the half-time of the decrease was 7 to 8 days in the fast red and slow red types of muscle. The finding that the half-times of the increase and of the decrease in cytochrome c concentration are similar provides evidence that the exercise-induced increase in cytochrome c is due to an increase in its rate of synthesis. These half-times are much shorter than those obtained with isotopic tracers. It had been thought that the heme precursor delta-aminolevulinate is not reutilized. However, the half-time of the decrease in radioactivity of cytochrome c labeled with delta-aminol[14C]levulinate was 45 days, and increased to 60 days in response to exercise, in fast red muscle. The half-time of the decrease in radioactivity of cytochrome c labeled with [(3H)]leucine in gastrocnemius muscle was shorter than with delta-amino[14C]levulinate (18 days compared to 38 days). These results indicate that when delta-amino(14C)levulinate is used to label heme, reutilization is a serious problem in skeletal muscle.     

Effect of an unstirred layer on the membrane permeability of anandamide

To study the effect of an unstirred layer (UL), we have investigated the exchange efflux kinetics of anandamide at 0 degrees C, pH 7.3, from albumin-free as well as from albumin-filled human red blood cell ghosts to media of various BSA concentrations ([BSA](o)). The rate constant (k(m)) of unidirectional flux from the outer membrane leaflet to BSA in the medium increased with the square root of [BSA](o) in accordance with the existence of a UL, which is a water layer adjacent to the membrane that is not subject to the same gross mixing that takes place in the rest of the medium. From k(m), it is possible to calculate the rate constant of anandamide dissociation from BSA (k(1)) if we know the membrane binding of anandamide, the equilibrium dissociation constant of BSA-anandamide complexes, and the diffusion constant of anandamide. We estimated k(1) to be 3.33 +/- 0.27 s(-1). The net flux of [(3)H]anandamide is balanced by an equal and opposite movement of nonradioactive anandamide in exchange efflux experiments. This means that our results are also valid for uptake. We show that for anandamide with rapid membrane translocation, UL causes a significant resistance to cellular uptake. Depicting the rate of anandamide uptake as a function of equilibrium water phase concentrations results in a parabolic uptake dependence. Such apparent "saturation kinetics" is often interpreted as indicating the involvement of transport proteins. The validity of such an interpretation is discussed.     

Nitric oxide scavenging by red blood cells as a function of hematocrit and oxygenation

The reaction rate between nitric oxide and intraerythrocytic hemoglobin plays a major role in nitric oxide bioavailability and modulates homeostatic vascular function. It has previously been demonstrated that the encapsulation of hemoglobin in red blood cells restricts its ability to scavenge nitric oxide. This effect has been attributed to either factors intrinsic to the red blood cell such as a physical membrane barrier or factors external to the red blood cell such as the formation of an unstirred layer around the cell. We have performed measurements of the uptake rate of nitric oxide by red blood cells under oxygenated and deoxygenated conditions at different hematocrit percentages. Our studies include stopped-flow measurements where both the unstirred layer and physical barrier potentially participate, as well as competition experiments where the potential contribution of the unstirred layer is limited. We find that deoxygenated erythrocytes scavenge nitric oxide faster than oxygenated cells and that the rate of nitric oxide scavenging for oxygenated red blood cells increases as the hematocrit is raised from 15% to 50%. Our results 1) confirm the critical biological phenomenon that hemoglobin compartmentalization within the erythrocyte reduces reaction rates with nitric oxide, 2) show that extra-erythocytic diffusional barriers mediate most of this effect, and 3) provide novel evidence that an oxygen-dependent intrinsic property of the red blood cell contributes to this barrier activity, albeit to a lesser extent. These observations may have important physiological implications within the microvasculature and for pathophysiological disruption of nitric oxide homeostasis in diseases.     

Kinetic analyses of calcium movements in HeLa cell cultures. I. Calcium influx

Calcium influx was studied in monolayers of HeLa cells to determine the number of exchangeable and nonexchangeable pools and the rate constant of the different fluxes. Of the two exchangeable pools, one has a very fast rate of exchange with a half-time of 1.54 min, a compartment size of 1.06 mµmoles/mg cell protein, and an exchange rate of 474 µµmoles/(mg protein\·min). This compartment is likely to be extracellular and could represent calcium exchange between the extracellular fluids and surface binding sites of the cell membrane. The second exchangeable pool has a half-time of exchange of 31 min, a compartment size of 2.69 mµmoles/mg cell protein (0.224 millimole calcium/kg cell water), and a flux rate of 0.0546 µµmole cm^-2 sec^-1. This compartment can be considered to be the intracellular pool of exchangeable calcium. An unexchangeable intracellular pool of calcium of 3.05 mµmoles/mg cell protein was detected implying that only 45% of the intracellular calcium is exchangeable. In addition, a large extracellular pool of calcium has been found to be unexchangeable, probably a part of the cell glycocalix. Finally, dinitrophenol 10^-3 M does not affect the slow component of the calcium uptake curve which brings new evidence that calcium entry into the cell is not a metabolically dependent process.     

The effect of intracellular pH on the rate of hexose uptake in Chlorella.

The rate of hexose uptake by Chlorella is reduced by uncouplers such as carbonyl cyanide p-trifluoromethoxyphenyl hydrazone or dinitrophenol even before concentration equilibrium is reached. The addition of uncouplers changes the membrane potential and the intracellular pH. The membrane potential does not influence the initial velocity of net sugar uptake, whereas manipulation of the cell pH by means of dimethyloxazolidinedione or by butyric acid uncovered a dramatic influence of cell pH on the rate of hexose uptake: at pH values of 7.5--6.8 maximal rate of uptake is observed but at more acid pH a strong inhibition takes place with virtually total blockage of uptake at pH 6.1. The decrease of cell pH to 6.1 in the presence of carbonyl cyanide p-trifluoromethoxyphenyl hydrazone could therefore account for the decrease in hexose transport rate. It was shown that the intracellular pH as such determines the rate of uptake and not the pH difference between inside and outside; the transport rate did not correlate with delta pH.     

A thermodynamic study of electroneutral K-Cl cotransport in pH- and volume-clamped low K sheep erythrocytes with normal and low internal magnesium

Swelling-induced human erythrocyte K-Cl cotransport is membrane potential independent and capable of uphill transport. However, a complete thermodynamic analysis of basal and stimulated K-Cl cotransport, at constant cell volume, is missing. This study was performed in low K sheep red blood cells before and after reducing cellular free Mg into the nanomolar range with the divalent cation ionophore A23187 and a chelator, an intervention known to stimulate K- Cl cotransport. The anion exchange inhibitor 4,4''diisothiocyanato- 2,2''disulfonic stilbene was used to clamp intracellular pH and Cl or NO3 concentrations. Cell volume was maintained constant as external and internal pH differed by more than two units. K-Cl cotransport was calculated from the K effluxes and Rb (as K congener) influxes measured in Cl and NO3, at constant internal K and external anions, and variable concentrations of extracellular Rb and internal anions, respectively. The external Rb concentration at which net K-Cl cotransport is zero was defined as flux reversal point which changed with internal pH and hence Cl. Plots of the ratio of external Rb concentrations corresponding to the flux reversal points and the internal K concentration versus the ratio of the internal and external Cl concentrations (i.e., the Donnan ratio of the transported ions) yielded slopes near unity for both control and low internal Mg cells. Thus, basal as well as low internal Mg-stimulated net K-Cl cotransport depends on the electrochemical potential gradient of KCl.     

Red cells of newborn rats have low bisphosphoglyceromutase and high pyruvate kinase activities in association with low 2,3-bisphosphoglycerate

2,3-Bisphosphoglycerate is a physiologically important regulator of red cell oxygen affinity during mammalian development. The rat has no fetal hemoglobin, but the newborn red cell has low 2,3-bisphosphoglycerate and high ATP concentrations, and high oxygen affinity. This report shows that red cell bisphosphoglyceromutase activity increases from near zero in the newborn rat to very high levels by four weeks of age. This increase roughly parallels the increase in red cell 2,3-bisphosphoglycerate concentration. Red cell pyruvate kinase activity declines ten-fold from birth to four weeks of age. This decrease is associated with a changeover in red cell populations from larger to smaller cells. The glycolytic rate is at least 50% higher in newborn than adult rat red cells. The data suggest that high pyruvate kinase activity and glycolytic rate contribute to the high ATP concentration in newborn rat red cells, but that their low 2,3-bisphosphoglycerate concentration is due primarily to low bisphosphoglyceromutase activity.