Studies on avian erythrocyte metabolism. Effect of organic phosphates on oxygen affinity of embryonic and adult-type hemoglobins of the chick embryo.

The effects of 2,3 diphosphoglyceric acid (2,3-DPG), adenosine triphosphate (ATP), and inositol hexaphosphate (IHP) on the oxygen affinity of whole “stripped” hemoglobin (WSH), hemoglobin H (Hb-H), hemoglobin A (Hb-A) and hemoglobin D (Hb-D) isolated from 18-day chick embryo blood have been determined. The effect of the three organic phosphates upon the oxygen dissociation curves is similar and the following order of decreasing oxygen affinity of the organic phosphates was observed for each hemoglobin: 2,3-DPG < ATP < IHP. 2,3-DPG appears to have a slightly greater effect upon the P₅₀ of Hb-H than upon that of either of the two adult-type hemoglobins. However, this effect seems insufficient to suggest a preferential interaction of 2,3-DPG with Hb-H which would account for either the large amounts of 2,3-DPG in the erythrocytes of embryos or the higher oxygen affinity of the whole blood. The effects of the organic phosphates upon the Hill constant of the purified hemoglobins are variable. It is concluded that since the distribution of hemoglobins H, A, and D in the erythrocytes during the developmental period from 18-day embryos to 6-day chicks remains fairly constant, the previously described progressive decrease in oxygen affinity of the whole blood during this period results from changes in the total amount and distribution of the intraerythrocytic organic phosphates.²     

Cryopreservation of cyanate-treated sickle erythrocytes

Metabolic features and in vivo recovery of cryopreserved cyanate-treated erythrocytes from patients with sickle cell anemia were studied. Red cells were treated with the anti-sickling agent sodium cyanate, glycerolized, and frozen at ?80 °C. Cyanate increased post-thaw hemolysis of both normal and sickle erythrocytes. The thawed carbamylated sickle erythrocytes maintained high oxygen affinity but lost more than half of their ATP content. Addition of the metabolic nutrients adenine, pyruvate, and inosine (rejuvenation) during cyanate incubation prevented ATP loss. Rejuvenation also increased red cell 2,3-DPG and opposed the cyanate effect by lowering oxygen affinity. Yet cyanate improved by nearly 50% the intravascular recovery of thawed rejuvenated sickle erythrocytes in a rat transfusion model. Cryopreservation of autologous cyanate-treated erythrocytes could lead to their use as an extracorporeal treatment of sickle cell disease.     

Blood respiratory properties of rainbow trout,Salmo gairdneri: Responses to hypoxia acclimation and anoxic incubation of blood in vitro

Summary Blood respiratory properties of rainbow trout were determined following acclimation to normoxia and two levels of hypoxia.The most prominent response appeared to be an increase in blood O₂ affinity graded to the level of hypoxia. TheP ₅₀ values (at pH 7.8 and 20°C) were 24.1 21.7 and 16.8 mm Hg when specimens were acclimated to water O₂ tensions of 150, 80 and 50 mm Hg, respectively. The blood O₂ affinity was closely correlated with the erythrocytic ATP concentration. The stepwise correlation of ATP andP ₅₀, when trout were exposed to graded oxygen lack in the water, indicates that the blood O₂ affinity is precisely regulated.Anoxic incubation of trout blood in vitro induced a rapid reduction in erythrocytic ATP concentration (t _1/2=75 min), which was closely correlated to theP ₅₀ value. The drop inP ₅₀ value during anoxic exposure can be explained partly by the direct allosteric effect of a decreased erythrocytic ATP concentration and partly by the modified Donnan distribution of protons across the red cell membrane. Reoxygenation of the incubated blood, however, only partly re-established the erythrocytic ATP concentration, with a concurrent rise inP ₅₀ value.The results invite discussion about the mechanism, by which fish regulate their blood O₂ affinity. It is concluded, that it is regulated at the organismal rather than at the red cell level.Abbreviation (E) erythrocytes, erythrocytic     

Oxygen equilibria and structural characteristics of the tetrameric and polymeric intracellular hemoglobins from the bivalve molluscBarbatia reeveana

Summary The arcid bivalveBarbatia reeveana contains within its erythrocytes two hemoglobins with remarkably different structures and oxygen equilibrium properties. A tetrameric hemoglobin (M _r about 60,000) with non-identical subunits (₂₂) constitutes about 60% of the erythrocytic heme protein. This hemoglobin has a relatively low oxygen affinity (P ₅₀=19 Torr at 20°C, pH 7.2), shows cooperativityn _H=2.2, shows no Bohr effect between pH 6.8 and 7.6 and a heat of oxygenation (H) of –5.4 kcal/mole between 15 and 35°C. Its oxygen affinity appears to be insensitive to ATP.B. reeveana erythrocytes also contain another hemoglobin withM _r=430,000, the largest intracellular hemoglobin known in any organism. The subunit of this hemoglobin is unusual, having aM _r of 32–34,000 and two heme oxygen binding sites per polypeptide chain. The large hemoglobin has a very low oxygen affinity (P ₅₀=33 Torr at 20°C, pH 7.2), shows slight cooperativity,n _H=1.8, and no Bohr effect (Grinich and Terwilliger 1980). The H at pH 7.2 equals –2.9 kcal/mole, a low value for most hemoglobins, and its O₂ affinity appears to be insensitive to ATP. The two hemoglobins ofB. reeveana, so different in their structure, are also different in their functional properties.     

Vanadate increases oxygen affinity and affects enzyme activities and membrane properties of erythrocytes

Incubation of blood with vanadate markedly increases the affinity of hemoglobin for oxygen, decreases the deformability of erythrocytes, reduces their osmotic fragility and alters their morphology, determining the appearance of equinocytic forms. Since vanadate is easily taken up by the erythrocytes and binds hemoglobin, these effects might result from interactions of vanadate with hemoglobin and with membrane proteins at the glycerate-2, 3-P₂ and/or ATP binding site. In addition, vanadate inhibits phosphoglycerate mutase, phosphoglucomutase and adenylate-kinase activities from hemolysates, suggesting a possible inhibitory effect on erythrocyte metabolism     

Prostaglandin E2 effects on cation flux in sickle erythrocyte ghosts

Prostaglandin E₂ has previously been shown to enhance the shape transformation of sickle prone erythrocytes (8) and to reduce the oxygen resaturation of Hemoglobin SS within intact sickle cell erythrocytes after deoxygenation (15). In view of the recent importance attributed to calcium transport in maintaining erythrocyte shape and viability (10) and the suggestion that prostaglandins may act via a calcium ionophore mechanism (9) on cell membranes, erythrocyte ghosts were prepared following the method of Lepke and Passow (12) from normal and sickle cell anemia erythrocytes. These two classes of ghosts are shown to display differing patterns of sodium and calcium transport, with calcium influx being preferentially stimulated by prostaglandin E₂ in sickle cell ghosts. It is suggested that in hypoxic, stasis conditions , prostaglandins may play a role in accelerating sickling of sickle prone erythrocytes via stimulation of calcium influx.     

Studies on avian erythrocyte metabolism: IX. Relationship of changing organic phosphate composition to whole blood oxygen affinity during development of the ostrich (Struthio camelus camelus)

(1) 2,3-Diphosphoglyceric acid (2,3-DPG) is present in erythrocytes (RBC) of the 37-day ostrich embryo at a concentration of 3.7 μmole/ml RBC, representing 23.5% of the cell phosphate. (2) Inositol tetraphosphate (inositol-P₄) is absent in red cells of the 37-day embryo, appears in the RBC about 63 days after hatch, and thereafter gradually accumulates to a level of 2.8 μmole/ml RBC in the adult bird, representing 30–35% of the cell phosphate. (3) Inositol pentaphosphate (inositol-P₅) is present in the erythrocytes of the 37-day embryo at a concentration of 0.8 μmole/ml RBC, reaches a peak level of 2.9 μmole/ml RBC by Day 63 after hatch, and thereafter declines to a level of 1.1 μmole/ml RBC in the adult bird. (4) The crossover time where the molar concentrations of inositol-P₅ and inositol-P₄ are equal in the erythrocyte appears to be about 150 days after hatch. (5) The p₅₀ of whole blood from the 37-day ostrich embryo, 5-day ostrich chick, and adult ostrich was 15.4, 31.8, and 24.9 Torr. The p₅₀ of whole blood immediately after hatch correlates best with an abrupt rise in ATP concentration but after 54 days posthatch correlates best with the appearance of increasing concentrations of inositol-P₄ and the decrease in concentrations of ATP and inositol-P₅. (6) The appearance of inositol-P₄ in the cells could be an adaptive mechanism for regulating oxygen supply by switching to a modulator which maintains a higher oxygen affinity than would a predominance of inositol-P₅.     

Thermoacclimatory changes in blood oxygen binding properties and gill secondary lamellar structure ofSalmo gairdneri

Summary The blood oxygen binding properties and gill secondary lamellar structure of rainbow trout acclimated to several temperatures were studied. The blood oxygen carrying capacity decreased as acclimation temperature increased from 2 to 15 °C; the decrease was probably caused by an increase in plasma volume. Also the blood oxygen affinity decreased as the acclimation temperature increased from 2 to 15 °C. This change had no effect on the oxygen loading in gills, since the efferent arterial oxygen tension was adequate for approximately 100% erythrocytic O₂ saturation at all acclimation temperatures, but facilitated the oxygen unloading in tissues. At the highest acclimation temperature (18 °C) the oxygen loading in gills was facilitated by the changes in the secondary lamellar structure; the proportion of erythrocytes in the secondary lamellar capillaries was higher than at the other acclimation temperatures (2 and 10 °C).     

Hormone action at the membrane level VI. Binding of (—)-[3H]dihydroalprenolol and (±)-[3H]isoproterenol to turkey erythrocytes and correlation with adenylate cyclase activity

The binding of (±)-[³H]isoproterenol and (—)-[³H]dihydroalprenolol to intact turkey erythrocytes was studied using a rapid centrifugation technique. The binding of both ligands is rapid, dissociable, stereospecific and inhibited by (—)-propranolol. The total number of isoproterenol binding sites is 2800 sites/ cell. This consists of a low and high affinity site both of which show stereospecific binding. The high affinity isoproterenol site has a K_d of 15.5—19.5 nM and has 600 sites/cell. The low affinity isoproterenol site has a K_d of 195 nM and has 2200 sites/cell. The binding of (—)-[³H]dihydroalprenolol shows one type of site with a K_d of 7.8 nM and has 2500 sites/cell. The agonists epinephrine, norepinephrine, soterenol and p-hydroxyphenylisoproterenol which were tested by competition for binding showed a 6—25-fold greater affinity for the high affinity site determined by (±)-[³H]isoproterenol as compared to the (—)-[³H]dihydroalprenolol binding site. However, the antagonists propranolol, practolol and metrapolol showed similar affinities for the binding sites as determined by competition of binding of either labeled isoproterenol or dihydroalprenolol. These studies indicate that isoproterenol binding can recognize two independent stereospecific β-adrenergic receptors or can recognize two different conformational states of a single receptor. Provisional calculations are made on the turnover number of adenylate cyclase under physiological conditions using intact erythrocytes. The turnover number is 4000 molecules of cyclic AMP/10 min per high affinity receptor.     

Respiratory adaptations in carp blood influences of hypoxia, red cell organic phosphates, divalent cations and CO2 on hemoglobin-oxygen affinity

Summary This study concerns the adaptation of oxygen transporting function of carp blood to environment hypoxia, tracing the roles played by erythrocytic cofactors, inorganic cations, carbon dioxide and hemoglobin multiplicity.Carp acclimated to hypoxia ( 30 mmHg) display striking increases in blood oxygen affinity compared to normoxic ( =120–150 mm) specimens (P ₅₀'s are 3.0 and 7.0 mm, respectively, at pH 7.9 and 20°C). This correlates with a marked decrease in erythrocytic concentrations of NTP (nucleoside triphosphates) (Figs. 1, 2, Table 1), permitting investigation of the time-course of the response (Fig. 3). That GTP (guanosine triphosphate) plays a greater role than ATP in the allosteric regulation of blood oxygen affinity, follows from greater decreases in its concentration during hypoxia, and its greater effect on oxygen affinity of the hemoglobin (Figs. 1, 5). It is furthermore shown that divalent cations (which complex with NTP) inhibit the regulatory role of GTP on O₂ affinity to a lesser extent than that of ATP (Fig. 7). However, the divalent cation, Mg²⁺, occurs in similarly high concentrations in the erythrocytes of hypoxic and normoxic fish (Table 1). CO₂ specifically depresses the O₂ affinity of carp hemoglobin, but below pH 8.3, its effect is obliterated by ATP and GTP suggesting that the chains are the main sites for CO ₂ ^– binding. Four carp hemoglobin components are isolated and their oxygen-binding properties compared with those of the cofactor-free hemolysate (Figs. 4, 8, 9). The results are discussed comparatively with special reference to hemoglobin function in fish and mammals.     

Effects of acclimation to altitude on oxygen affinity and organic phosphate concentrations in pigeon blood.

Hematocrit ratio, hemoglobin concentration and blood oxygen affinity, Bohr effect factor and Hill coefficient, adenosine triphosphate and inositol pentaphosphate (IPP) concentrations were studied in blood of adult pigeons exposed first at 140 m, and then for 3 weeks at 4000 m in an altitude chamber. At altitude, the hematocrit ratio and hemoglobin concentration significantly increased, IPP concentration decreased, and P₅₀ did not change. A lower mean red cell age and a higher hemoglobin concentration may account for the unchanged P₅₀. Adaptation to hypoxia of the tissue oxygen supply was shown by a greater blood O₂ capacitance (ΔC_HbO₂/Δ_o2) in the physiological range of oxygen partial pressures.     

Babesia microti: Biochemistry and function of hamster erythrocytes infected from a human source

Babesia microti, a protozoan parasite of mammalian erythrocytes was obtained from the blood of an infected human and maintained in golden hamsters, in which a parasitemia of 70% was obtained regularly. The hamsters' response—a subacute, hemolytic anemia—was studied with regard to oxygen affinity and red cell organic phosphate content. In addition, the reduced glutathione status of infected erythrocytes was observed because of the possible importance of this metabolite to parasite growth and red cell integrity. Infected animals developed a severe anemia with reticulocytosis; there occurred a 4-mm decrease in whole blood oxygen affinity without any change in erythrocytes' 2,3-diphosphoglycerate levels. The glutathione content of the infected animals' erythrocytes increased twofold during the course of the infection. In uninfected animals, in which anemia and reticulocytosis had been produced by bleeding, all changes seen in infected animals were reproduced. It was concluded that the changes in the infected animals were due to the anemia and reticulocytosis alone, and that the parasite played no role in these changes apart from being a cause of anemia and reticulocytosis.     

The fatty acid composition of phospholipids and absorption spectra of lipid extracts from lamprey,frog, and rat erythrocytes

The work studies the content and fatty acid composition of phospholipids as well as the absorption spectra of lipid extracts from red blood cells of poikilothermal and homoiothermal animals at different evolutionary levels. The objects of study include two poikilothermal species, the river lamprey (Lampetra fluviatilis) that uses oxygen dissolved in water, and the common frog (Rana temporaria) that consumes oxygen both from water and from air. A homoithermal animal is the white rat (Rattus rattus) that inhabits the terrestrial-aerial environment. The animals are studied in winter and spring. The phospholipid content in lamprey blood plasma is found to be twice higher than that in its erythrocytes. In the frog and the rat, the ratio is reverse. Determination of the fatty acid lipid composition of red blood cell phospholipids suggests that membranes in the lamprey are denser than in the frog. As for the fatty acids in the erythrocyte fraction of rat blood, they appear to be less diverse, with a double prevalence of saturated acids over unsaturated ones and devoid of long chain (C22) ω3 fatty acids. All of this results in a lower degree of unsaturation and a denser packing of fatty acids in the membrane structures of rat erythrocytes. The mechanism of reversible binding of O₂ molecules to hemoglobin in erythrocytes is discussed. Presumably, the mechanism of interaction between molecules of O₂ and molecules of water prevents the exchange interaction of electrons of the hemoglobin iron atoms with an oxygen molecule. This is confirmed by our obtained absorption spectra, which show that in the lipid extract almost totally devoid of water the heme isolated from erythrocytes is converted to hemin.     

Induction of Oxidant Stress by Iron Available in Advanced Forms of Plasmodium Falciparum

Oxidative stress has been incriminated as a deleterious factor in the development of malaria parasites. Various chemical reductones which can undergo cyclic oxidation and reduction, such as ascorbate have been shown to cause oxidative stress to red blood cells. This, naturally-occurring and redox-active compound, can induce the formation of active oxygen derived species, such as superoxide radicals (.O^?₂), hydrogen peroxide (H₂O₂) and hydroxyl radical (OH.), The formation of the hydroxyl radical, the ultimate deleterious species, is mediated by the redox-active and available transition metals iron and copper in the Haber-Weiss reaction.

During the development of the parasite, hemoglobin is progressively digested and a concurrent release of high levels of iron-containing breakdown products takes place within the red blood cell. Indications for the progressive increase in redox-active iron during the growth of P. falciparum have been recently found in our lab: a) adventitious ascorbatc proved highly detrimental to the parasite when added to the mature forms. In contrast, if the parasitized erythrocytes were in the early phase following invasion, and only low levels of iron-containing structures had been liberated. then the observed effect was a small promotion of parasite development. b) erythrocytes containing mature parasites were more potent than erythrocytes containing ring forms as a source for redox-active iron in the acerbate-driven metal-mediated degradation of DNA. The addition of extracts from parasitized erythrocytes and ascorbate to DNA causcd a dose and time dependent DNA degradation. Non-infected erythrocytes had no effect. These findings could also propose that the parasite-dependent accumulation of redox-active forms of iron within the erythrocytes serve as a biological clock triggering the rupture of the red blood cell membrane at the right moment, when the parasite reaches its maturity.     

Ethanol-induced alterations in human erythrocyte shape and surface properties: Modulatory role of prostaglandin E1

Summary Exposure of human erythrocytes to ethanol (1 to 20% by vol) in Ca²⁺ and Mg²⁺-free phosphate-buffered saline, pH 7.4, transformed biconcave discs into spiculated echinocytes within 3 min at 25°C. The effects of ethanol were concentration- and time-dependent, but reversible by washing in the incubation buffer system within 60 min of initial exposure to ethanol. After prolonged ethanol exposure (180 min), washing of cells resulted in the formation of stomatocytes (cup-forms). Ethanol-induced echinocytosis was also accompanied by a 30% enhancement in the agglutinability of erythrocytes by ligands with high affinity for negative surface charge (poly-l-lysine and wheat germ agglutinin, 20 l/ml) without any alterations in surface charge topography. Concomitant exposure of erythrocytes to prostaglandin E₁ (100nm) selectively prevented the enhancement of ligand-mediated agglutinability, but did not modify cell shape. These data indicate that certain erythrocyte surface properties may not be directly influenced by cell shape and suggest a unique modulatory action of prostaglandin E₁ on shape-transformed cells.     

Effects of nitrite-induced methaemoglobinaemia on oxygen affinity of carp blood

Synopsis The oxygen transport characteristics and the acid-base status of carp blood was studied in vitro by equilibration of blood samples with and without addition of 5mmol l^–1 of nitrite for 30 min at various Po₂ values in combination with Pco₂ of 1.5 and 5.7mmHg (0.2 and 0.76kPa). After equilibration pH, Po₂, Pco₂, and Co₂ as well as methaemoglobin and HCO₃ ^– concentration were determined and oxygen dissociation curves established. At Pco₂ of 1.5mmHg (0.2kPa) oxygen affinity, expressed by a normal P₅₀ of 3.3mmHg (0.44kPa) was unaffected by nitrite exposure, whereas at Pco₂ 5.7 (0.76kPa), nitrite exposure shifted P₅₀ from 7.59mmHg (1.01kPa) to 21.9mmHg (2.92kPa). Methaemoglobin formation was greater at the higher Pco₂ and increased with falling Po₂. Erythrocyte shrinkage and rising plasma [HC0₃ ^–] during nitrite exposure indicated that the erythrocyte osmoregulation was significantly affected. The present results indicate significantly reduced oxygen affinity upon exposure of carp blood to nitrite. This result contrasts with findings in mammalian blood, where oxygen affinity is greatly enhanced.     

The P2X7 receptor mediates the uptake of organic cations in canine erythrocytes and mononuclear leukocytes: comparison to equivalent human cell types

We previously demonstrated that canine erythrocytes express the P2X₇ receptor, and that the function and expression of this receptor is greatly increased compared with human erythrocytes. Using ⁸⁶Rb⁺ (K⁺) and organic cation flux measurements, we further compared P2X₇ in erythrocytes and mononuclear leukocytes from these species. Concentration response curves of BzATP- and ATP-induced ⁸⁶Rb⁺ efflux demonstrated that canine P2X₇ was less sensitive to inhibition by extracellular Na⁺ ions compared to human P2X₇. In contrast, canine and human P2X₇ showed a similar sensitivity to the P2X₇ antagonists KN-62 and Mg²⁺. KN-62 and Mg²⁺ also inhibited ATP-induced choline⁺ uptake into canine and human erythrocytes. BzATP and ATP but not ADP or NAD induced ethidium⁺ uptake into canine monocytes, T- and B-cells. ATP-induced ethidium⁺ uptake was twofold greater in canine T-cells compared to canine B-cells and monocytes. KN-62 inhibited the ATP-induced ethidium⁺ uptake in each cell type. P2X₇-mediated uptake of organic cations was 40- and fivefold greater in canine erythrocytes and lymphocytes (T- and B-cells), respectively, compared to equivalent human cell types. In contrast, P2X₇ function was threefold lower in canine monocytes compared to human monocytes. Thus, P2X₇ activation can induce the uptake of organic cations into canine erythrocytes and mononuclear leukocytes, but the relative levels of P2X₇ function differ to that of equivalent human cell types.     

The effect of prostaglandin E2-induced echinocytic transformation on the optassium loss,viscosity and osmotic fragility of normal and sickle cell erythrocytes

Prostaglandin E₂ (PGE₂)-induced discocyte → echinocytic transformation has no effect om the viscosity or osmotic fragility of normal or stickle cell erythrocytes. Membrane permeability, reflected, reflected as potassium efflux, is significantly affected in normal erythrocytes when >90% of the cells are morphologically transformed to the enchinocytic III stage (PGE₂ concentration of 1–2×10⁶ ng/ml blood). This potassium loos is significant in sickle erythrocytes when 50–70% of the cell population has been transformed (PGE₂ concentration, 5×10⁵ ng/ml blood). This change in membrane permeability reprensents one-half to one-third the flux that occurs with sickling (i.e., >80% of the erythrocytes sickled).