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The erythrocytes of the echidna (Tachyglossus aculeatus) and platypus (Ornithorhynchus anatinus), which are practically devoid of intracellular ATP content (1), were examined for active Rb86influx and for the presence of Na+K+Mg ATPase. We found that intact erythrocytes of both species possess the ability to actively transport cations. Ouabain sensitive Rb86influx in the echidna was approximately 0.17 μmoles/ml cells × hr, whereas the platypus exhibited a higher value of 0.43 μmoles/ml cells × hr. Surprisingly, ouabain sensitive Na+K+Mg ATPase activity of isolated membranes was high amounting to some 15 to 25 fold higher than the human erythrocyte counterpart determined under identical conditions. These findings suggest that a trace amount of ATP is sufficient to maintain active cation transport across the monotreme cell membranes.

The erythrocytes of the monotrenes, having a trace amount of ATP, can metabolize glucose to lactate at a rate comparable to human and other mammalian erythrocytes. The echidna energy metabolism is unique in that adenosine can stimulate glycolytic carbon flow, resulting in a nearly 20-fold net synthesis of ATP.

A four-fold increase in red blood cell 2,3-diphosphoglycerate (DPG) levels occurs within 1–2 weeks after birth resulting in a decrease in oxygen affinity in the newborn pig. In this study, we found that although newborn pig red blood cells seem to be equally capable of net DPG synthesis as adult pig cells when challenged by a mixture of inosine, pyruvate and phosphate, the cells from newborns degrade DPG much faster than do cells from adults. As a result, DPG levels in the newborn’s cells do not seem to increase in comparison to the adult’s cells. Using a density centrifugation technique, it was found that the postnatal cells initially entering the circulation have a relatively low rather than high DPG content. However, DPG content quickly increases as they age. These findings suggest that the rapid rise of DPG seen in the growing piglet is brought about in part (a) by an elimination of the fetal cells with low DPG, and (b) by an emergence of postnatal cells having the capacity to augment their DPG content while in circulation.

Perchloric acid extracts of radiation-induced fibrosarcoma (RIF-1) tumors grown in mice have been analyzed by multinuclear NMR spectroscopy and by various chromatographic methods. This analysis has permitted the unambiguous assignment of the 31P resonances observed in vivo to specific phosphorus-containing metabolites. The region of the in vivo spectra generally assigned to sugar phosphates has been found in RIF-1 tumors to contain primarily phosphorylethanolamine and phosphorylcholine rather than glycolytic intermediates. Phosphocreatine was observed in extracts of these tumor cells grown in culture as well as in the in vivo spectra, indicating that at least some of the phosphocreatine observed in vivo arises from the tumor itself and not from normal tissues. In the 31P-NMR spectra of the perchloric acid extract, resonances originating from purine and pyrimidine nucleoside di- and triphosphate were resolved. HPLC analyses of the nucleotide pool indicate that adenine derivatives were the most abundant components, but other nucleotides were present in significant amounts. The 1H and 13C resonance assignments of the majority of metabolites present in RIF-1 extracts have also been made. Of particular importance is the ability to observe lactate, the levels of which may provide a noninvasive measure of glycolysis in these cells in both the in vivo and in vitro states. In addition, the aminosulfonic acid, taurine, was found in high levels in the tumor extracts.

We examined the activation of genes induced by erythropoietin (Epo) in erythroid progenitor cells that were isolated from the spleens of mice infected with anemia-inducing strain of the Friend virus. These erythroid progenitor cells, termed FVA cells, undergo in vitro differentiation to erythrocytes under the influence of Epo within 2 to 3 days. We used a differential hybridization procedure to screen a cDNA library constructed from FVA cells that were treated with Epo 2U/mL in the absence of serum for 2 hours. Of 20,000 recombinant phages, 47 plaques hybridized preferentially to cDNA probe prepared from Epo-stimulated cells. We found at least three different Epo-responsive genes (ERGs) and one of them corresponds to the mouse virus-like (VL30) element, similar to already reported BVL-1. The induction of VL30, which was evident within 30 minutes after Epo exposure, reached a maximum by 1 hour and remained stable for up to 4 hours. The treatment of FVA cells with cycloheximide (CHX) 10 µg/mL, which in itself activates the expression of VL30 caused a superinduction of the Epo signal. Changes in intracellular Ca>2+concentrations, either raised by ionomycin or depleted by EGTA, had no effect on the Epo-induced VL30 expression. In addition, protein kinase C (PKC) inhibitors such as staurosporine (3 µmol/L) or H7 (20 µmol/L) and a tyrosine kinase inhibitor, genistein (200 µmol/L), did not inhibit the Epo-induced expression of VL30. TPA (100 ng/mL), a PKC agonist, did not induce VL30 expression. Although the physiologic role of VL30 in the differentiation of erythroid progenitor cells is not known, our findings demonstrate that VL30 is an early ERG, and may be a useful indicator of the initial molecular actions of Epo.

Metabolic development was examined in erythroid precursor cells, which were isolated from the spleens of mice infected with the anemia-inducing strain of Friend virus (FVA cells). FVA cells undergo differentiation in vitro from the proerythroblast stage through the reticulocyte stage over a 48-hour period in the presence of erythropoietin. Concomitant with marked decreases in cellular size and energy demand, metabolic capacities of both glycolysis and oxygen consumption diminish after 48 hours in culture by 7- and 18-fold, respectively. Because the oxidative capacity decreases more than glycolytic ability does, the metabolic machinery increasingly shifts toward anaerobic metabolism. During the 48-hour period of differentiation, the 2,3-diphosphoglyceric acid (DPG) content per cell and 2,3-DPG mutase activity per cell increased eightfold and threefold, respectively. Freshly har- vested FVA cells have adenosine triphosphate (ATP) levels of 7.23 ± 2.52 µmol/1010 cells or 3.76 ± 1.31 µmol/mL cell water which are 12- or 2.3-fold higher, respectively, than the ATP levels of mature red blood cells. In the course of FVA cell differentiation, ATP content per cell decreases by fourfold, but ATP concentration in cell water remains unchanged because of a corresponding decrease in cellular size and water content during differentiation. These studies show that in the face of dramatic decreases in cell size and cellular energy demand, terminally differentiating erythroid cells maintain a constant ATP level by undergoing an involution of their glycolytic machinery as well as by losing their aerobic metabolic capacity.