Nucleoside transport and metabolism in erythrocytes from the Yucatan miniature pig. Evidence that inosine functions as an in vivo energy substrate

Erythrocytes from the Yucatan miniature pig, like those from the normal domestic pig, lack functional glucose transporters and were unable to utilize plasma glucose as an energy source. In contrast, inosine and adenosine entered the cells rapidly. The nucleoside transporter responsible for this uptake was identified as a band 4.5 polypeptide (5000 copies per cell; apparent Mr 45 000-66 000). Inosine concentrations in the physiological plasma range (1.6-2.5 microM) were found to maintain normal erythrocyte ATP levels and ATP/ADP ratios during prolonged in vitro incubation of cells at 37 degrees C, an effect that was blocked by the specific nucleoside transport inhibitor, nitrobenzylthioguanosine. In the absence of extracellular nucleoside, cells 'protected' themselves against some of the consequences of deprivation of energy substrate by glycolyzing the ribose moiety of inosine produced during ATP catabolism. Although erythrocytes from the miniature pig were capable of utilizing extracellular adenosine as an energy substrate, plasma samples from these animals contained less than 0.4 microM adenosine. It is concluded that inosine is a major physiological energy source of pig erythrocytes.     

A symbiotic relationship of energy metabolism between a ‘non-glycolytic’ mammalian red cell and the liver

The red cell of newborn pig loses the ability to carry out glycolysis within a month after birth. The metabolic energy source for this ‘non-glycolytic’ mammalian red cell is unknown. Hepatectomy of an adult pig results in the loss of red cell ATP with a characteristic half-time of 7–8 h which is identical to the rate with which ATP disappears in the pig cells under in vitro substrate-free incubation. Exposure of pig red cells with either normal or depleted levels of ATP to isolated hepatocytes causes a net synthesis of red cell ATP during a 12 h incubation. These findings suggest that a symbiotic relationship of energy metabolism may exist between the red cell and the liver of the pig.     

Inosine from liver as a possible energy source for pig red blood cells

Adult pig red blood cells are unable to metabolize glucose due to a membrane permeability barrier to glucose developed shortly after birth. $\text{In}$$\text{vitro}$, pig red cells incubated in their own plasma are unable to maintain normal ATP levels, thus the question has been raised as to the nature of the metabolic energy source. We have suggested that organs, such as the liver, might supply low levels of substrate to the red cells as they transit through the organ. In this paper, evidence is presented to show that perfused pig livers supply a metabolic substrate used by pig red cells. This substrate has been tentatively identified as inosine.     

Preservation of red blood cells with purines and nucleosides. III. Synthesis of adenine, guanine, and hypoxanthine nucleotides

Purine nucleotides of fresh human red cells and of red cells during storage at 4 degrees and 25 degrees C with additions of adenine, guanine, guanosine and inosine were estimated by HPLC. Six nucleotides were found in red cells: ATP, ADP, AMP, GTP, GDP, and IMP. The adenine nucleotides represented 92 per cent of the total purine nucleotides, guanine nucleotides 7 per cent and IMP less than 1 per cent. In red cells stored with adenine the total concentration of purine nucleotides increased to 125 per cent of the normal value. An adenine-free but guanine and guanine + inosine containing medium caused a decrease of the concentration of purine nucleotides by 10 to 20 per cent. When red cells were stored without adding guanine or guanosine the content of the guanine nucleotides decreased from 0.32 to 0.17 mumol/g Hb due to the decrease in the GTP content, but the GDP concentration increased slightly. In CPD-AG blood, however, the concentration of guanine nucleotides increased considerably up to 0.6 mumol/g Hb. IMP was estimated in all investigated stored red cells. In CPD-A and in CPD-AG blood 0.4 mumol/g Hb were produced during 3 weeks of storage, but twice of that in CPD-AI blood. The principles of the synthesis and the degradation of purine nucleotides in stored red cells are discussed in detail.     

Inosine metabolism in the rat

1. Uptake and subsequent metabolism of purine and ribose moieties was monitored after intravenous administration of doubly labelled inosine. 2. More than 95% was cleared from the plasma within 5 min, and 99% within 20 min. 3. Approx. 50% of the 160 mumol total was rapidly incorporated into liver and kidney. Kidney removed the greatest amount (21 mumol/g wet wt.), about 10-fold more than heart, lung or liver. Lung and heart accounted for only 3%. These tissues then lost radioactivity during the remainder of the experiment. Radioactivity in the skeletal muscle, in contrast, increased from 8% of the injected dose at 5 min to 40% at 60 min. 4. In liver, kidney, heart and lung there was a significant difference in the fate of inosine. After initial incorporation of inosine, kidney predominantly lost inosine; heart preferentially lost purines; lung preferentially lost ribose radioactivity; and in liver the ribose radioactivity was rapidly lost, whereas purine was retained. Some of the ribose moiety was metabolized to glucose, presumably in the liver, and then released into the blood. Ribose radioactivity (probably as glucose) and radioactive hypoxanthine accumulated in skeletal muscle throughout the experiment. 5. Inosine caused a rapid and prolonged increase in the blood glucose content, from 6 to 15 mM in 60 min. This was accompanied by a small increase in plasma insulin. 6. It is concluded that the purine and ribose radioactivity lost from the kidney, liver and other tissues becomes incorporated into skeletal muscle.     

Studies on the energy metabolism of opossum (Didelphis Virginiana) erythrocytes. I. Utilization of carbohydrates and purine nucleosides

Opossum erythrocytes filtered through cellulose columns were used to estimate their permeability to D-glucose and optimum inorganic phosphate requirement for D-glucose utilization at pH 7.4 and 8.1. D-Glucose readily penetrated opossum red cells; there was no measurable difference whether plasma or electrolyte solution served as the suspending medium. Optimum extracellular inorganic phosphate concentration for glucose utilization as indicated by red cell lactate production was pH-dependent, with a sharp optimum of 30 mmol/liter at pH 8.1. Whereas glucose, fructose, mannose, dihydroxyacetone, adenosine, and inosine were readily utilized at pH 7.4 and Pi 30 mmol/liter as shown by net lactate and ATP production by the red cells, galactose and ribose as substrates were not metabolized. In electrolyte, Pi 30 mmol/liter, and pH 7.4 glucose utilization by opossum red cells averaged 3.5 mumol, at pH 8.1, 9.5 mumol/ml cells/hr were utilized. Red cells suspended in leukocyte-free plasma utilized D-glucose at a rate of 3.0 mumol/ml/hr at pH 7.5. Seven percent of D-glucose flowed through the pentose phosphate pathway; this rate increased 11-fold by methylene blue stimulation. The amount of D-glucose recycled through the pentose phosphate pathway increased 300-fold in the presence of the redox dye.     

NMR studies of intracellular free calcium, free magnesium and sodium in the guinea pig reticulocyte and mature red cell

During the maturation process reticulocytes lose their intracellular organelles and undergo changes in membrane lipid composition and ion transport properties. While several reports indicate differences in the levels of magnesium, sodium and calcium in reticulocytes and erythrocytes, controversy remains concerning the actual magnitude and direction of ionic alterations during reticulocyte maturation. One problem with all of these studies is that the techniques used are invasive and are limited to measuring only the total cell ion content. We have used 31P, 23Na and 19F nuclear magnetic resonance (NMR) spectroscopy to compare the intracellular free ion and phosphometabolite levels in guinea pig reticulocytes and mature red blood cells. In contrast to a sharply decreased concentration of ATP in erythrocytes in comparison to reticulocytes, the intracellular free magnesium, measured using 31P-NMR, was increased by about 65% upon maturation (150 mumol/l cell water in reticulocytes in comparison to 250 mumol/l cell water in erythrocytes). Sizeable but opposite changes in intracellular sodium (5.5 mumol/ml cells in reticulocytes vs. 8.5 mumol/ml cells in erythrocytes) and intracellular free calcium (99 nM vs. 31 nM in reticulocytes and mature red cells, respectively) were also observed, suggesting that alterations in the kinetics of membrane ion transport systems, accompanying changes in phospholipid and cholesterol content, occur during the process of red cell maturation. However, in contrast to dog red blood cells, there was no evidence for the presence of a Na+/Ca2+ exchanger in guinea pig reticulocytes or erythrocytes.     

Decreased thyroid hormone-stimulated oxygen consumption and glucose uptake in mononuclear blood cells from patients with autosomal dominant osteopetrosis type I

Nine patients, from four different families, with autosomal dominant osteopetrosis were investigated. They all had roentgenological type I disease, characterized by universal, symmetrical osteosclerosis and enlarged thickness of the cranial vault. All patients appeared clinically euthyroid. Thyroxine (T4) and tri-iodothyronine (T3) induced oxygen consumption and glucose uptake were studied in vitro in mononuclear blood cells from patients and control persons. Unstimulated oxygen consumption from patients and controls did not differ, and no difference in unstimulated glucose uptake was observed. The increase in T4 and T3 stimulated oxygen consumption was significantly lower in cells from patients with osteopetrosis (T4: 0.007 +/- 0.004 mumol/mg DNA per h, T3: 0.011 +/- 0.004 mumol/mg DNA per h) compared with controls (T4: 0.017 +/- 0.003 mumol/mg DNA per h, T3: 0.023 +/- -0.013 mumol/mg DNA per h; p less than 0.05, p less than 0.05). Cellular glucose uptake after T4 and T3 stimulation was significantly lower in patients (T4: 0.032 +/- 0.017 mmol/l per mg DNA per h, T3: 0.02 +/- 0.017 mmol/l per mg DNA per h) compared with controls (T4: 0.09 +/- 0.017 mmol/l per mg DNA per h, T3: 0.08 +/- 0.01 mmol/l per mg DNA per h; p less than 0.05, p less than 0.01). The reduced oxygen consumption and glucose uptake indicate thyroid hormone resistance which may be of pathogenetic importance for the development of autosomal dominant osteopetrosis type I.     

Is inosine the physiological energy source of pig erythrocytes?

Pig erythrocytes are unable to metabolize glucose and their physiological energy source is unknown. These cells have a high-capacity nucleoside transport system with similar properties to that responsible for nucleoside transport in other species. Nucleoside transport is sufficiently rapid to allow the possibility that inosine and/or adenosine may represent major energy substrates for pig erythrocytes in vivo. Normal and adenosine deaminase-deficient pig erythrocytes have similar ATP levels, suggesting that adenosine is not important in this respect. However, it was calculated that an extracellular inosine concentration of only 40 nM could support the cells' entire energy requirement, a value 40-fold lower than plasma levels of this nucleoside.     

The Protective Effects of Inosine Against Chemical Hypoxia on Cultured Rat Oligodendrocytes

Inosine is a purine nucleoside and is considered protective to neural cells including neurons and astrocytes against hypoxic injury. However, whether oligodendrocytes (OLs) could also be protected from hypoxia by inosine is not known. Here we investigated the effects of inosine on primarily cultured rat OLs injured by rotenone-mediated chemical hypoxia, and the mechanisms of the effects using ATP assay, MTT assay, PI-Hoechst staining, TUNEL, and immunocytochemistry. Results showed that rotenone exposure for 24 h caused cell death and impaired viability in both immature and mature OLs, while pretreatment of 10 mM inosine 30 min before rotenone administration significantly reduced cell death and improved the viability of OLs. The same concentration of inosine given 120 min after rotenone exposure also improved viability of injured mature OLs. Immunocytochemistry for nitrotyrosine and cellular ATP content examination indicated that inosine may protect OLs by providing ATP and scavenging peroxynitrite for cells. In addition, immature OLs were more susceptible to hypoxia than mature OLs; and at the similar degree of injury, inosine protected immature and mature OLs differently. Quantitative real-time PCR revealed that expression of adenosine receptors was different between these two stages of OLs. These data suggest that inosine protect OLs from hypoxic injury as an antioxidant and ATP provider, and the protective effects of inosine on OLs vary with cell differentiation, possibly due to the adenosine receptors expression profile. As OLs form myelin in the central nervous system, inosine could be used as a promising drug to treat demyelination-involved disorders.     

Purine accumulation in human fat cell suspensions. Evidence that human adipocytes release inosine and hypoxanthine rather than adenosine

Human adipocytes are of limited viability (7 +/- 2% release of lactate dehydrogenase/h) and contain active ectophosphatases which are capable of sequentially degrading ATP to adenosine. At densities of 30,000-40,000 cells/ml, human fat cell suspensions accumulated adenosine, inosine, and hypoxanthine, and their concentrations were 38 +/- 8, 120 +/- 10, and 31 +/- 7 nmol/liter after 3 h of incubation. Dipyridamole (10 mumol/liter), an inhibitor of nucleoside transport, caused a 5-7-fold increase in adenosine accumulation which was reduced by 85% on inhibition of ectophosphatases by beta-glycerophosphate and antibodies against ecto-5'-nucleotidase or alpha, beta-methylene 5'-adenosine diphosphate (10 mumol/liter), respectively, indicating that most of the adenosine is produced in the extracellular compartment. Accordingly, the spontaneous accumulation of adenosine was reduced beyond 5 nmol/liter on inhibition of ectophosphatase activities or removal of extracellular AMP by AMP deaminase (4 units/ml). Added adenosine (30 nmol/liter) disappeared until its concentration approached 5 nmol/liter. Isoproterenol (1 mumol/liter) had no effect on adenosine accumulation regardless whether purine production from extracellular sources was minimized or not. In contrast to adenosine, the concentrations of inosine and hypoxanthine displayed only a modest decrease (30-50%) on inhibition of ectophosphatase activities. In addition, isoproterenol caused a 2-3-fold increase in inosine and hypoxanthine production which was concentration-dependent and could be inhibited by propranolol. It is concluded that the adenosine that accumulates in human adipocyte suspensions is almost exclusively derived from adenine nucleotides which are released by leaking cells. By contrast, inosine and hypoxanthine are produced inside the cells, and the release of these latter purines appears to be linked to ATP turnover via adenylate cyclase.     

Mobilization of intracellular calcium by extracellular ATP and by calcium ionophores in the Ehrlich ascites-tumour cell

We have studied the changes of the intracellular free calcium concentration ([Ca2+]i) effected by external ATP, which induces formation of inositol trisphosphate, and by the divalent cation ionophores ionomycin and A23187. Both, ATP (40 microM) and ionophores (1-80 mumol/l cells ionomycin; 20-400 mumol/l cells A23187), produced a transient rise of [Ca2+]i which reached its maximum within 15-30 s and declined near resting values (about 200 nM) within 1-3 min. When the [Ca2+]i peak surpassed 500 nM a transient cell shrinkage due to simultaneous activation of Ca2+-dependent K+ and Cl- channels was also observed. The cell response was similar in medium containing 1 mM Ca2+ and in Ca2+-free medium, suggesting that the Ca mobilized to the cytosol comes preferently from the intracellular stores. Treatment with low doses of ionophore (1 mumol/l cells for ionomycin; 20 mumol/l cells for A23187) depressed the response to a subsequent treatment, either with ionophore or with ATP. Treatment with ATP did also inhibit the subsequent response to ionophore, but in this case the inhibition was dependent on time, the stronger the shorter the interval between both treatments. This result suggests that the permeabilization of Ca stores by ATP is transient and that Ca can be taken up again by the intracellular stores. Refill was most efficient when Ca2+ was present in the incubation medium. Addition of either ATP or ionomycin (1-25 mumol/l cells) to cells incubated in medium containing 1 mM Ca2+ decreased drastically the total cell Ca content during the following 3 min of incubation. In the case of ATP the total cell levels of Ca returned to the initial values after 7-15 min, whereas in the case of the ionophore they remained decreased during the whole incubation period. These results indicate that Ca released from the intracellular stores by either ATP or ionophores is quickly extruded by active mechanisms located at the plasma membrane. They also suggest that, under the conditions studied here, with 1 mM Ca2+ outside, the Ca-mobilizing effect of ionophores is stronger in endomembranes than in the plasma membrane.     

Is adenosine a second metabolic substrate for human red blood cells?

Adenosine is present in the micromolar range in human plasma. In this study, metabolism of adenosine, which was maintained between 0.62 +/- 0.03 and 2.92 +/- 0.43 microM by means of a continuous infusion using a Harvard infusion pump, was investigated in human red blood cells. It was found that lactate production increases linearly as the adenosine concentration was raised. Cells infused with an average adenosine concentration of 2 microM produced lactate comparable to that produced by 5 mM glucose. The extent to which ATP concentration is maintained by adenosine also depends on its concentration. After a 4 h infusion with an average adenosine concentration of 0.7 microM, ATP content amounts to 75% of the glucose control. Raising the adenosine infusion concentration to 1.5 microM results in a full maintenance of ATP levels and at concentrations higher than 1.5 microM, adenosine produces a net synthesis of ATP. A net synthesis of ATP also occurs with adenosine concentration below 1.5 microM, if supplemented with glucose. In contrast, inosine infusion provides only a partial support of ATP and fails to produce a net synthesis of ATP in the presence of glucose. In addition, the presence of purine nucleoside and glucose together influence the metabolism of each other, depending on inorganic phosphate content (Pi). At a Pi concentration of 1 mM, the glucose consumption rate is reduced by approx. 25% by purine nucleoside infusion and vice versa. In sharp contrast, glucose consumption at 16 mM Pi is potentiated by adenosine. These findings suggest that plasma adenosine contributes significantly to human red cell energetics, even though it is present at a concentration several orders of magnitude lower than glucose.     

Preservation of red blood cells with purines and nucleosides. II. Uptake and utilization of purines and nucleosides by stored red blood cells

The uptake of adenine, guanine, guanosine and inosine by stored red cells was investigated in whole blood and red cell resuspensions at initial concentrations of 0.25, 0.5 and 0.75 mM for adenine and 0.5 mM for the other additives using a rapid ion-exchange chromatographic microanalysis of purines and nucleosides in plasma and whole blood. Increasing adenine concentrations from 0.25 to 0.75 mM in blood elevated the adenine uptake from 0.3 up to 0.8 mmol/l red cells during 2 hours after collecting blood. The intra-/extracellular distribution ratio changed from 1 : 1.3 to 1: 1.7. Some 2 hours after withdrawing blood into CPD--solution with purines and nucleosides the uptake of adenine and guanine resulted in 40 per cent and 70 per cent respectively and of guanosine and inosine in 80 and 90 per cent respectively. The replacement of plasma by a resuspending solution gave the same uptake rates for purines and nucleosides. The nucleosides were rapidly split to purines and R-1-P and disappeared from blood during one week. Adenine and guanine were utilized to 80 to 90 per cent only after 3 weeks. During the same period the utilization of guanine was smaller by 40 per cent than that of adenine due to the different activity of the purine nucleoside phosphorylase for these substrates. The plasma of all analyzed blood samples contained hypoxanthine and inosine, but guanine and guanosine were detected only in those samples to which one of them was added. After 3 weeks of storage the highest concentration of hypoxanthine was found in CPD-AI blood with 600 microM in plasma and the highest concentration of synthesized inosine in CPD-AG blood with a concentration of 100 microM in plasma. Three ways of utilization of purines by stored red cells were discussed : the synthesis of nucleotide monophosphates, the formation of nucleosides, and the deamination. The portions of these ways change during storage. The most effective concentrations of adenine and guanosine in stored blood seems to be 0.25 and 0.5 mM respectively. The full utilization of the nucleoside requires the addition of inorganic phosphate.     

肌苷产生菌B.Subtilis H841在2升罐中的发酵

枯草芽孢杆菌(Bacillus Subtilis)H841肌苷产生菌是腺嘌呤、组氨酸、硫胺素三重缺陷型菌株,并对8—氮杂乌嘌呤、6—巯基嘌呤有抗性。在摇瓶中产肌胺18.1克/升,在2L自控发酵罐中最高可产肌苷19.6克/升,在流加葡萄糖情况下可产肌苷26.2克/升。控制pH较不控制pH发酵肌苷产量有较大的增加,控制pH发酵并补加营养时,肌苷产量可稳定地增长,但对葡萄糖的转化率是相同的。     

Substrate-induced alterations of high energy phosphate metabolism and contractile function in the perfused heart

The bioenergetic basis by which the Krebs cycle substrate pyruvate increased cardiac contractile function over that observed with the Embden-Meyerhof substrate glucose was investigated in the isovolumic guinea pig heart. Alterations in the content of the high energy phosphate metabolites and the rate of high energy phosphate turnover were measured by 31P NMR. These were correlated to the changes in contractile function and rates of myocardial oxygen consumption. Maximum left ventricular developed pressure (LVDP) and high energy phosphates were observed with 16 mM glucose or 10 mM pyruvate. In hearts perfused with 16 mM glucose, the intracellular phosphocreatine (PCr) concentration was 15.2 +/- 0.6 mM with a PCr/Pi ratio of 10.3 +/- 0.9. The O2 consumption was 5.35 mumol/g wet weight/min, and these hearts exhibited a LVDP of 97 +/- 3.7 mm Hg at a constant paced rate of 200 beats/min. In contrast, when hearts were switched to 10 mM pyruvate, the PCr concentration was 18.3 +/- 0.4 mM, the PCr/Pi ratio was 30.4 +/- 2.2, the O2 consumption was 6.67 mumol/g wet weight/min, and the LDVP increased to 125 +/- 3.3 mm Hg. From NMR saturation transfer experiments, the steady-state flux of ATP synthesis from PCr was 4.9 mumol/s/g of cell water during glucose perfusion and 6.67 mumol/s/g of cell water during pyruvate perfusion. The flux of ATP synthesis from ADP was measured to be 0.99 mumol/s/g of cell water with glucose and calculated to be 1.33 mumol/s/g of cell water with pyruvate. These results suggest that pyruvate quite favorably alters myocardial metabolism in concert with the increased contractile performance. Thus, as a mechanism to augment myocardial performance, pyruvate appears to be unique.     

An Na+-stimulated Mg2+-transport system in human red blood cells

The initial rate of net Mg2+ efflux was measured in human red blood cells by atomic absorption. In fresh erythrocytes incubated in Na+,K+-Ringer's medium this rate was 7.3 +/- 2.8 mumol/l cells per h (mean +/- S.D. of 14 subjects) with an energy of activation of 13 200 cal/mol. Cells with total Mg2+ contents ([ Mg]i) ranging from 1.8 to 24 mmol/l cells were prepared by using a modified p-chloromercuribenzenesulphonate method. Mg2+ efflux was strongly stimulated by increases in [Mg]i and in external Na+ concentrations ([ Na]o). A kinetic analysis of Mg2+ efflux as a function of [Mg]i and [Na]o revealed the existence of two components: an Na+-stimulated Mg2+ efflux, which exhibited a Michaelian-like dependence of free internal Mg2+ content (apparent dissociation constant = 2.6 +/- 1.4 mmol/l cells; mean +/- S.D. of six subjects) and on external Na+ concentration (apparent dissociation constant = 20.5 +/- 1.9 mM; mean +/- S.D. of four subjects) and a variable maximal rate ranging from 35 to 370 mumol/l cells per h, and an Na+-independent Mg2+ efflux, which showed a linear dependence on internal Mg2+ content with a rate constant of (6.6 +/- 0.7) X 10(-3) h-1. Fluxes catalyzed by the Na+-stimulated Mg2+ carrier were partially dependent on the ATP content of the cells and completely inhibited by quinidine (IC50 = 50 microM) and by Mn2+ (IC50 = 0.5-1.0 mM).     

Pig red blood cell hexokinase: Regulatory characteristics and possible physiological role

The regulatory properties of pig erythrocyte hexokinase III have been studied. Among mammalian erythrocyte hexokinases, the pig enzyme shows the highest affinity for glucose and a positive cooperative effect with n_H = 1.5 at all the MgATP concentrations studied (for 0.5 to 5 mm). Glucose at high concentrations is also an inhibitor of hexokinase III. Similarly, the apparent affinity constant for MgATP is independent of glucose concentration. Uncomplexed ATP and Mg are both competitive inhibitors with respect to MgATP. Glucose 6-phosphate, known as a stronger inhibitor of all mammalian erythrocyte hexokinases, is a poor inhibitor for the pig enzyme (K_i = 120 μm). Furthermore, this inhibition is not relieved by orthophosphate as with other mammalian red blood cell hexokinases. A variety of red blood cell-phosphorylated compounds were tested and found to be inhibitors of pig hexokinase III. Of these, glucose 1,6-diphosphate and 2,3-diphosphoglycerate displayed inhibition constants in the range of their intracellular concentrations. In an attempt to investigate the role of hexokinase type III in pig erythrocytes some metabolic properties of this cell have been studied. The adult pig erythrocyte is able to utilize 0.27 μmol of glucose/h/ml red blood cells (RBC) compared with values of 0.56–2.85 μmol/h/ml RBC for the other mammalian species. This reduced capacity to metabolize glucose results from a relatively poor ability of the cell membrane to transport glucose. In fact, all the glycolytic enzymes were present and a low intracellular glucose concentration was measured (0.5 mm against a plasma level of 5 mm). Furthermore, transport and utilization were concentration-dependent processes. Inosine, proposed as the major energy substrate of the pig erythrocyte, at physiological concentrations is not as efficient as glucose in maintaining reduced glutathione levels under oxidative stress. Furthermore, newborn pig erythrocytes (fully permeable to glucose) possess hexokinase type II as the predominant glucose-phosphorylating activity. This fact and the information derived from the study of the regulatory characteristics of hexokinase III and from metabolic studies on intact pig erythrocytes permit the hypothesis that the presence of this peculiar hexokinase isozyme (type III) enables the adult pig erythrocyte to metabolize low but appreciable amounts of glucose.     

Studies on avian erythrocyte metabolism. XVI. Accumulation of 2,3-bisphosphoglycerate with shifts in oxygen affinity of chicken erythrocytes

The ability of the chicken erythrocyte to accumulate 2,3-bisphosphoglycerate (2,3-P2-glycerate) and its effect upon the oxygen affinity (P50) of the cell suspensions have been determined. Erythrocytes from chick embryos, which contain 4-6 mM 2,3-P2-glycerate, and from chickens at various ages, which contain 3-4 mM inositol pentakisphosphate but no 2,3-P2-glycerate, were incubated with inosine, pyruvate, and inorganic phosphate. Red blood cells from 20-day chick embryos incubated in Krebs-Ringer, pH 7.45, containing 20 mM inosine and 20 mM pyruvate had an increase in 2,3-P2-glycerate from 4.3 to 11.9 mM after 4 h. Importantly, as 2,3-P2-glycerate concentration increased there was a corresponding increase in P50 of the cell suspension. Further, erythrocytes from 9- and 11-week, and 7-, 14-, 24-, and 28-month-old chickens when incubated similarly with inosine and pyruvate accumulated 2,3-P2-glycerate with corresponding increases in P50 of the cell suspensions. The ability of the red cell to accumulate this compound under the incubation conditions used apparently decreases with age of the bird (e.g., 11.9 mM in the 20-day embryo to 1.1 mM in the 28-month-old chicken after 4 h incubation). Despite the presence of significant amounts of inositol-P5, the accumulation of 2,3-P2-glycerate markedly decreases oxygen affinity of the cell suspensions. The delta P50/mumol increase in 2,3-P2-glycerate in the red cells of the 20-day chick embryo after 4 h incubation is 1.5 Torr; conversely, the delta P50/mumol decrease in 2,3-P2-glycerate in the red cells of the 17-day embryo after 6 h incubation in the presence of sodium bisulfite is 2.8 Torr. The demonstrated ability of the chicken erythrocyte to accumulate 2,3-P2-glycerate in response to certain substrates suggests that regulation of concentration of this compound could contribute significantly to regulation of blood oxygen affinity in birds.