Red-cell amino acid transport. Evidence for the presence of system ASC in mature human red blood cells.

The properties of Na+-dependent L-alanine transport in human erythrocytes were investigated using K+ as the Na+ substitute. Initial rates of Na+-dependent L-alanine uptake (0.2 mM extracellular amino acid) for erythrocytes from 22 donors ranged from 40 to 180 mumol/litre of cells per h at 37 degrees C. Amino acid uptake over the concentration range 0.1-8 mM was consistent with a single saturable component of Na+-dependent L-alanine transport. Apparent Km and Vmax. values at 37 and 5 degrees C measured in erythrocytes from the same donor were 0.27 and 0.085 mM respectively, and 270 and 8.5 mumol/litre of cells per h respectively. The transporter responsible for this uptake was identified as system ASC on the basis of cross-inhibition studies with a series of 42 amino acids and amino acid analogues. Apparent Ki values for glycine, L-alpha-amino-n-butyrate, L-serine and L-leucine as inhibitors of Na+-dependent L-alanine uptake at 37 degrees C were 4.2, 0.12, 0.16 and 0.70 mM respectively. Reticulocytes from a patient with inherited pyruvate kinase deficiency were found to have a 10-fold elevated activity of Na+-dependent L-alanine uptake compared with erythrocytes from normal donors. Separation of erythrocytes according to cell density (cell age) established that even the oldest mature erythrocytes retained significant Na+-dependent L-alanine transport activity. Amino acid transport was, however, a more sensitive indicator of cell age than acetylcholinesterase activity. Erythrocytes were found to accumulate L-alanine against its concentration gradient (distribution ratio approx. 1.5 after 4 h incubation), an effect that was abolished in Na+-free media. Na+-dependent L-alanine uptake was shown to be associated with L-alanine-dependent Na+ influx, the measured coupling ratio being 1:1.     

Dibasic amino acid interactions with Na+-independent transport system asc in horse erythrocytes. Kinetic evidence of functional and structural homology with Na+-dependent system ASC

Amino acid transport in horse erythrocytes is regulated by three co-dominant allelomorphic genes coding for high-affinity transport activity (system asc1), low-affinity transport activity (system asc2) and transport-deficiency, respectively. The asc systems are selective for neutral amino acids of intermediate size, but unlike conventional system ASC, do not require Na+ for activity. In the present series of experiments we have used a combined kinetic and genetic approach to establish that dibasic amino acids are also asc substrates, systems asc1 and asc2 representing the only mediated routes of cationic amino acid transport in horse erythrocytes. Both transporters were found to exhibit a strong preference for dibasic amino acids compared with neutral amino acids of similar size. Apparent Km values (mM) for influx via system asc1 were L-lysine (9), L-ornithine (27), L-arginine (27), L-alanine (0.35). Corresponding Vmax estimates (mmol/l cells per h, 37 degrees C) were L-lysine (1.65), L-ornithine (2.15), L-arginine (0.54), L-alanine (1.69). Apparent Km values for L-lysine and L-ornithine influx via system asc2 were approximately 90 and greater than 100 mM, respectively, with Vmax values greater than 2 and greater than 1 mmol/l cells per h, respectively. Apparent Km and Vmax values for L-alanine uptake by system asc2 were 14 mM and 6.90 mmol/l cells per h. In contrast, L-arginine was transported by system asc2 with the same apparent Km as L-alanine (14 mM), but with a 77-fold lower Vmax. This dibasic amino acid was shown to cause cis- and trans-inhibition of system asc2 in a manner analogous to its interaction with system ASC, where the side-chain guanidinium group is considered to occupy the Na+-binding site on the transporter. Concentrations of extracellular L-arginine causing 50% inhibition of zero-trans L-alanine influx and half-maximum inhibition of L-alanine zero-trans efflux were 14 mM (extracellular L-alanine concentration 15 mM) and 3 mM (intracellular L-alanine concentration 15.5 mM), respectively. We interpret these observations as evidence of structural homology between the horse erythrocyte asc transporters and system ASC. Physiologically, intracellular L-arginine may function as an endogenous inhibitor of system asc2 activity.     

Basolateral amino acid transport systems in the perfused exocrine pancreas: sodium-dependency and kinetic interactions between influx and efflux mechanisms

Basolateral amino acid transport systems have been characterized in the perfused exocrine pancreas using a high-resolution paired-tracer dilution technique. Significant epithelial uptakes were measured for L-alanine, L-serine, alpha-methylaminoisobutyric acid, glycine, methionine, leucine, phenylalanine, tyrosine and L-arginine, whereas L-tryptophan and L-aspartate had low uptakes. alpha-Methylaminoisobutyric acid transport was highly sodium dependent (81 +/- 3%), while uptake of L-serine, L-leucine and L-phenylalanine was relatively insensitive to perfusion with a sodium-free solution. Cross-inhibition experiments of L-alanine and L-phenylalanine transport by twelve unlabelled amino acids indicated overlapping specificities. Unidirectional L-phenylalanine transport was saturable (Kt = 16 +/- 1 mM, Vmax = 12.3 +/- 0.4 mumol/min per g), and weighted non-linear regression analysis indicated that influx was best described by a single Michaelis-Menten equation. The Vmax/Kt ratio (0.75) for L-phenylalanine remained unchanged in the presence of 10 mM L-serine. Although extremely difficult to fit, L-serine transport appeared to be mediated by two saturable carriers (Kt1 = 5.2 mM, Vmax1 = 7.56 mumol/min per g; Kt2 = 32.8 mM, Vmax2 = 22.9 mumol/min per g). In the presence of 10 mM L-phenylalanine the Vmax/Kt ratio for the two L-serine carriers was reduced, respectively, by 79% and 50%. Efflux of transported L-[3H]phenylalanine or L-[3H]serine was accelerated by increasing perfusate concentrations of, respectively, L-phenylalanine and L-serine, and trans-stimulated by other amino acids. In the pancreas neutral amino acid transport appears to be mediated by Na+-dependent Systems A and ASC, the classical Na+-independent System L and another Na+-independent System asc recently identified in erythrocytes. The interactions in amino acid influx and efflux may provide one of the mechanisms by which the supply of extracellular amino acids for pancreatic protein synthesis is regulated.     

Characterization of a novel variant of amino acid transport system asc in erythrocytes from Przewalski's horse (Equus przewalskii).

In thoroughbred horses, red blood cell amino acid transport activity is Na(+)-independent and controlled by three codominant genetic alleles (h, l, s), coding for high-affinity system asc1 (L-alanine apparent Km for influx at 37 degrees C congruent to 0.35 mM), low-affinity system asc2 (L-alanine Km congruent to 14 mM), and transport deficiency, respectively. The present study investigated amino acid transport mechanisms in red cells from four wild species: Przewalski's horse (Equus przewalskii), Hartmann's zebra (Zebra hartmannae), Grevy's zebra (Zebra grevyi), and onager (Equus hemonius). Red blood cell samples from different Przewalski's horses exhibited uniformly high rates of L-alanine uptake, mediated by a high-affinity asc1-type transport system. Mean apparent Km and Vmax values (+/- SE) for L-alanine influx at 37 degrees C in red cells from 10 individual animals were 0.373 +/- 0.068 mM and 2.27 +/- 0.11 mmol (L cells.h), respectively. As in thoroughbreds, the Przewalski's horse transporter interacted with dibasic as well as neutral amino acids. However, the Przewalski asc1 isoform transported L-lysine with a substantially (6.4-fold) higher apparent affinity than its thoroughbred counterpart (Km for influx 1.4 mM at 37 degrees C) and was also less prone to trans-stimulation effects. The novel high apparent affinity of the Przewalski's horse transporter for L-lysine provides additional key evidence of functional and possible structural similarities between asc and the classical Na(+)-dependent system ASC and between these systems and the Na(+)-independent dibasic amino acid transport system y+. Unlike Przewalski's horse, zebra red cells were polymorphic with respect to L-alanine transport activity, showing high-affinity or low-affinity saturable mechanisms of L-alanine uptake. Onager red cells transported this amino acid with intermediate affinity (apparent Km for influx 3.0 mM at 37 degrees C). Radiation inactivation analysis was used to estimate the target size of system asc in red cells from Przewalski's horse. The transporter's in situ apparent molecular weight was 158,000 +/- 2500 (SE).     

Ouabain-insensitive net sodium influx in erythrocytes of normotensive and essential hypertensive humans

The Na+ content of erythrocytes is elevated in people with essential hypertension. There is conflicting evidence about its cause. The present study was designed to investigate whether the increase in content is due to a defect in a ouabain-resistant Na+ flux. Net Na+ influx was determined from the increase in Na+ content of erythrocytes during incubation in the presence of ouabain. Na+ content of erythrocytes from 24 normotensive Caucasian subjects with no known family history of hypertension was 6.9 +/- 1.3 mmol per litre of cells. It was 7.9 +/- 2.0 mmol per litre of cells in 18 subjects with essential hypertension. The difference was less and not significant when the two non-Caucasian subjects of the hypertensive group were excluded. Net Na+ influx was 1.83 mmol/h per litre of cells in the normotensive group. In eight subjects it was measured on a second occasion after an interval of several months. The coefficient of a variation of the duplicate tests was 2.4%. Net Na+ influx was significantly higher in the hypertensive group, the value was 2.18 +/- 0.15 mmol/h per litre of cells. In 11 of these subjects, Na+ influx was measured on a second occasion. The coefficient of variation was 6.2%, significantly greater than in the control group. In some of these subjects Na+ influx was within the normal range on one of the two occasions. When the groups were compared with use of the mean values from the duplicate tests, net Na+ influx was elevated in 17 of the 18 hypertensive subjects. The findings are discussed with reference to previous work and in relation to the established facilitatory effects of an increased intracellular Na+ concentration on excitable cells that influence blood pressure.     

The binding of calcium ions by erythrocytes and `ghost''-cell membranes

1. Washed human erythrocytes, suspended in iso-osmotic sucrose containing 2.5mm-calcium chloride, bind about 400mug-atoms of calcium/litre of packed cells. Sucrose may be replaced by other sugars. 2. Partial replacement of sucrose by iso-osmotic potassium chloride diminishes the uptake of calcium, 50% inhibition occurring at about 50mm-potassium chloride. 3. Other univalent cations behave like potassium, whereas bivalent cations are much more inhibitory. The tervalent cations, yttrium and lanthanum, however, are the most effective inhibitors of calcium uptake. 4. An approximate correlation exists between the calcium uptake and the sialic acid content of erythrocytes of various species and of human erythrocytes that have been partially depleted of sialic acid by treatment with neuraminidase. However, even after complete removal of sialic acid, human erythrocytes still bind about 140mug-atoms of calcium/litre of packed cells. 5. A Scatchard (1949) plot of calcium uptake at various Ca(2+) concentrations in the suspending media shows the presence of three different binding sites on the external surface of the human erythrocyte membrane. 6. Erythrocyte ;ghost' cells, the membranes of which appear to be permeable to Ca(2+) ions, can bind about 1000mug-atoms of calcium per ;ghost'-cell equivalent of 1 litre of packed erythrocytes. This indicates that there are also binding sites for calcium on the internal surface of the erythrocyte membrane.     

Effect of temperature on the velocity of erythrocyte aggregation

The velocity of the aggregation of human erythrocytes was examined in the range of 5-43 degrees C with a rheoscope combined with a video camera, an image analyzer and a computer. (1) With increasing temperature, the velocity of erythrocyte aggregation induced by fibrinogen, immunoglobulin G and artificial macromolecules (dextran of 70 kDa and poly(glutamic acid) of 50 kDa) increased. However, the relationship between the velocity of erythrocyte aggregation and the temperature was different among these macromolecules. (2) In 70% autologous plasma, the velocity of erythrocyte aggregation was minimum at 15-18 degrees C, and increased at both higher and lower temperatures. (3) The shape of erythrocyte aggregates in 12 mumol/l fibrinogen (containing 770 mumol/l albumin) and in 70% autologous plasma was dependent on temperature: three-dimensional below 15-18 degrees C and one-dimensional (mainly rouleaux) above 15-18 degrees C. However, the shape of aggregates in 27 mumol/l immunoglobulin G (containing 770 mumol/l albumin) was three-dimensional in all temperature ranges. (4) The temperature dependency of erythrocyte aggregation was discussed in terms of the changes of medium viscosity, of erythrocyte properties and of bridging macromolecules.     

Cation and harmaline interactions with Na(+)-independent dibasic amino acid transport system y+ in human erythrocytes and in erythrocytes from a primitive vertebrate the pacific hagfish (Eptatretus stouti).

Transport systems y+, asc and ASC exhibit dual interactions with dibasic and neutral amino acids. For conventional Na(+)-dependent neutral amino acid system ASC, side chain amino and guanido groups bind to the Na+ site on the transporter. The topographically equivalent recognition site on related system asc binds harmaline (a Na(+)-site inhibitor) with the same affinity as asc (apparent Ki range 1-4 mM), but exhibits no detectable affinity for Ha. Although also classified as Na(+)-independent, dibasic amino acid transport system y+ accepts neutral amino acids when Na+ or another acceptable cation is also present. This latter observation implies that the y+ translocation site binds Na+ and suggests possible functional and structural similarities with ASC/asc. In the present series of experiments with human erythrocytes, system y(+)-mediated lysine uptake (5 microM, 20 degrees C) was found to be 3-fold higher in isotonic sucrose medium than in normal 150 mM NaCl medium. This difference was not a secondary consequence of changes in membrane potential, but resulted from Na+ functioning as a competitive inhibitor of transport. Apparent Km and Vmax values for lysine transport at 20 degrees C were 15.2 microM and 183 mumol/l cells per h, respectively, in sucrose medium and 59.4 microM and 228 mumol/l cells per h in Na+ medium. Similar results were obtained with y+ in erythrocytes of a primitive vertebrate, the Pacific hagfish (Eptatretus stouti), indicating that Na(+)-inhibition is a general property of this class of amino acid transporter. At a permeant concentration of 5 microM, the IC50 value for Na(+)-inhibition of lysine uptake by human erythrocytes was 27 mM. Other inorganic and organic cations, including K+ and guanidinium+, also inhibited transport. In parallel with its actions on ASC/asc harmaline competitively inhibited lysine uptake by human cells in sucrose medium. As predicted from mutually competitive binding to the y+ translocation site, the presence of 150 mM Na+ increased the harmaline inhibition constant (Ki) from 0.23 mM in sucrose medium to 0.75 mM in NaCl medium. We interpret these observations as further evidence that y+, asc and ASC represent a family of closely related transporters with a common evolutionary origin.     

Purification and properties of NADH-specific dihydropteridine reductase from human erythrocytes

NADH-specific dihydropteridine reductase (EC 1.6.99.7) has been purified from human erythrocytes in essentially homogeneous form. The molecular weight of the enzyme was estimated to be 46,000 by Sephadex G-100 gel filtration. The enzyme reacted with antiserum against NADH-specific dihydropteridine reductase from bovine liver and formed a single immunoprecipitin line in the Ouchterlony double-diffusion system. This precipitin line completely fused with that formed between the human liver enzyme and the antiserum. With use of 5,6,7,8-tetrahydro-6-methylpterin, Km values of the erythrocyte enzyme for NADH and NADPH were determined to be 0.94 and 47 mumol/l, respectively. Vmax values were 58.7 mumol/min/mg with NADH and 6.41 mumol/min/mg with NADPH. The average activity of NADH-specific dihydropteridine reductase of 9 human blood samples from healthy males (20-25 years old) was calculated to be approximately 600 mU/g of hemoglobin, 1.8 mU per 20 microliters of blood, or 1.9 mU per 10(8) erythrocytes.     

Human erythrocyte myosin: identification and purification

Human erythrocytes contain an Mr 200,000 polypeptide that cross-reacts specifically with affinity-purified antibodies to the Mr 200,000 heavy chain of human platelet myosin. Immunofluorescence staining of formaldehyde-fixed erythrocytes demonstrated that the immunoreactive myosin polypeptide is present in all cells and is localized in a punctate pattern throughout the cell. Between 20-40% of the immunoreactive myosin polypeptide remained associated with the membranes after hemolysis and preparation of ghosts, suggesting that it may be bound to the membrane cytoskeleton as well as being present in the cytosol. The immunoreactive myosin polypeptide was purified from the hemolysate to approximately 85% purity by DEAE-cellulose chromatography followed by gel filtration on Sephacryl S-400. The purified protein is an authentic vertebrate myosin with two globular heads at the end of a rod-like tail approximately 150-nm long, as visualized by rotary shadowing of individual molecules, and with two light chains (Mr 25,000 and 19,500) in association with the Mr 200,000 heavy chain. Peptide maps of the Mr 200,000 heavy chains of erythrocyte and platelet myosin were seen to be nearly identical, but the proteins are distinct since the platelet myosin light chains migrate differently on SDS gels (Mr 20,000 and 17,000). The erythrocyte myosin formed bipolar filaments 0.3-0.4-micron long at physiological salt concentrations and exhibited a characteristic pattern of myosin ATPase activities with EDTA, Ca++, and Mg++-ATPase activities in 0.5 M KCl of 0.38, 0.48, and less than 0.01 mumol/min per mg. The Mg++-ATPase activity of erythrocyte myosin in 0.06 M KCl (less than 0.01 mumol/min per mg) was not stimulated by the addition of rabbit muscle F-actin. The erythrocyte myosin was present in about 6,000 copies per cell, in a ratio of 80 actin monomers for every myosin molecule, which is an amount comparable to actin/myosin ratios in other nonmuscle cells. The erythrocyte myosin could function together with tropomyosin on the erythrocyte membrane (Fowler, V.M., and V. Bennett, 1984, J. Biol. Chem., 259:5978-5989) in an actomyosin contractile apparatus responsible for ATP-dependent changes in erythrocyte shape.     

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.     

Choline kinase activity in Plasmodium-infected erythrocytes: characterization and utilization as a parasite-specific marker in malarial fractionation studies

Choline kinase (EC 2.7.1.32) was investigated in plasmodium falciparum-infected erythrocytes. Disrupted infected erythrocytes had a choline kinase activity of 1.9 +/- 0.2 nmol phosphorylcholine/10(7) infected cells per h, whereas the activity in normal uninfected erythrocytes was less than 6 pmol/10(7) cells per h. A broad alkaline optimal pH (7.9-9.2) was observed. The Km values for choline and ATP were 79 +/- 20 microM, and 1.3 +/- 0.3 mM, respectively. ATP concentrations higher than 12 mM inhibited choline kinase. Maximal activity was registered with a Mg2+ concentration of 10 mM, whereas its replacement by Mn2+, or other divalent cations, involved a decrease in choline kinase activity of at least 75%. Inhibition by products of the reaction, such as phosphorylcholine and ADP was investigated. In plasmodium knowlesi-infected erythrocytes, choline kinase had similar properties, but with a much higher specific activity of 16.4 +/- 2.1 nmol/10(7) infected cells per h. Subcellular fractionation of P. knowlesi-infected erythrocyte suspensions revealed that choline kinase was located exclusively in the cytosol of the parasite. We show that this enzyme is a useful index of parasite cytosolic content leakage, when infected erythrocytes are fractionated by saponin lysis or nitrogen decompression.     

Increased glucose metabolism by enzyme-loaded erythrocytes in vitro and in vivo normalization of hyperglycemia in diabetic mice.

The main metabolic properties of human red blood cells (RBC) overloaded with glucose catabolizing enzymes such as hexokinase and glucose oxidase were evaluated. Human erythrocytes loaded with human hexokinase metabolized 3.1 +/- 0.2 mumol/h/ml RBC of glucose, an amount double that consumed by normal and unloaded cells (1.46 +/- 0.16 mumol/h/ml RBC), while glucose oxidase-loaded erythrocytes consumed up to 5.5 +/- 0.5 mumol/h/ml RBC of glucose but with a time-dependent increase in methemoglobin formation due to the H2O2 produced in the glucose oxidase reaction. This methemoglobin production was greatly reduced while glucose consumption was increased (8.1 +/- 0.4 mumol/h/ml RBC) by coentrapment of hexokinase and glucose oxidase. Similar results were obtained in mouse red blood cells, although the role of hexokinase was less pronounced due to a higher basal level of this enzyme. When administered to diabetic mice the hexokinase/glucose oxidase-overloaded erythrocytes had a circulating half-life of 5 days and were able to regulate blood glucose at near physiological levels. A single intraperitoneal administration of 500 microliters of enzyme-loaded cells maintained a near-normal blood glucose concentration for 7 +/- 1 days, while repeated administrations at 10-day intervals were effective in the regulation of blood glucose levels for several weeks. These results suggest that enzyme-loaded erythrocytes can behave as circulating bioreactors and can provide a new way to reduce abnormally elevated blood glucose.     

Glutathione replenishment capacity is lower in isolated perivenous than in periportal hepatocytes.

The zonal distribution of GSH metabolism was investigated by comparing hepatocytes obtained from the periportal (zone 1) or perivenous (zone 3) region by digitonin/collagenase perfusion. Freshly isolated periportal and perivenous cells had similar viability (dye exclusion, lactate dehydrogenase leakage and ATP content) and GSH content (2.4 and 2.7 mumol/g respectively). During incubation, periportal cells slowly accumulated GSH (0.35 mumol/h per g), whereas in perivenous cells a decrease occurred (-0.14 mumol/h per g). Also, in the presence of either L-methionine or L-cysteine (0.5 mM) periportal hepatocytes accumulated GSH much faster (3.5 mumol/h per g) than did perivenous cells (1.9 mumol/h per g). These periportal-perivenous differences were also found in cells from fasted rats. Efflux of GSH was faster from perivenous cells than from periportal cells, but this difference only explained 10-20% of the periportal-perivenous difference in accumulation. Furthermore, periportal cells accumulated GSH to a plateau 26-40% higher than in perivenous cells. There was no significant difference in gamma-glutamylcysteine synthetase or glutathione synthetase activity between the periportal and perivenous cell preparations. The periportal-perivenous difference in GSH accumulation was unaffected by inhibition of gamma-glutamyl transpeptidase or by 5 mM-glutamate or -glutamine, but was slightly diminished by 2 mM-L-methionine. This suggests differences between periportal and perivenous cells in their metabolism and/or transport of (sulphur) amino acids. Our results suggest that a lower GSH replenishment capacity of the hepatocytes from the perivenous region may contribute to the greater vulnerability of this region to xenobiotic damage.     

Analysis of double knockout mice lacking aquaporin-1 and urea transporter UT-B. Evidence for UT-B-facilitated water transport in erythrocytes

We reported increased water permeability and a low urea reflection coefficient in Xenopus oocytes expressing urea transporter UT-B (former name UT3), suggesting that water and urea share a common aqueous pathway (Yang, B., and Verkman, A. S. (1998) J. Biol. Chem. 273, 9369-9372). Although increased water permeability was confirmed in the Xenopus oocyte expression system, it has been argued (Sidoux-Walter, F., Lucien, N., Olives, B., Gobin, R., Rousselet, G., Kamsteeg, E. J., Ripoche, P., Deen, P. M., Cartron, J. P., and Bailly, P. (1999) J. Biol. Chem. 274, 30228-30235) that UT-B does not transport water when expressed at normal levels in mammalian cells such as erythrocytes. To quantify UT-B-mediated water transport, we generated double knockout mice lacking UT-B and the major erythrocyte water channel, aquaporin-1 (AQP1). The mice had reduced survival, retarded growth, and defective urinary concentrating ability. However, erythrocyte size and morphology were not affected. Stopped-flow light scattering measurements indicated erythrocyte osmotic water permeabilities (in cm/s x 0.01, 10 degrees C): 2.1 +/- 0.2 (wild-type mice), 2.1 +/- 0.05 (UT-B null), 0.19 +/- 0.02 (AQP1 null), and 0.045 +/- 0.009 (AQP1/UT-B null). The low water permeability found in AQP1/UT-B null erythrocytes was also seen after HgCl(2) treatment of UT-B null erythrocytes or phloretin treatment of AQP1 null erythrocytes. The apparent activation energy for UT-B-mediated water transport was low, <2 kcal/mol. Estimating 14,000 UT-B molecules per mouse erythrocyte, the UT-B-dependent P(f) of 0.15 x 10(-4) cm/s indicated a substantial single channel water permeability of UT-B of 7.5 x 10(-14) cm(3)/s, similar to that of AQP1. These results provide direct functional evidence for UT-B-facilitated water transport in erythrocytes and suggest that urea traverses an aqueous pore in the UT-B protein.     

Decline in ribonucleic acid and protein synthesis with loss of viability during the early hours of imbibition of rye (Secale cereale L.) embryos.

The specificity of amino acid transport in normal (high-glutathione) sheep erythrocytes was investigated by studying the interaction of various neutral and dibasic amino acids in both competition and exchange experiments. Apparent Ki values were obtained for amino acids as inhibitors of L-alanine influx. Amino acids previously found to be transported by high-glutathione cells at fast rates (L-cysteine, L-alpha-amino-n-butyrate) were the most effective inhibitors. D-Alanine and D-alpha-amino-n-butyrate were without effect. Of the remaining amino acids studied, only L-norvaline, L-valine, L-norleucine, L-serine and L-2,4-diamino-n-butyrate significantly inhibited L-alanine uptake. L-Alanine efflux from pre-loaded cells was markedly stimulated by extracellular L-alanine. Those amino acids that inhibited L-alanine influx also stimulated L-alanine efflux. In addition, D-alanine, D-alpha-amino-n-biutyrate, L-threonine, L-asparagine, L-alpha, beta-diaminoproprionate, L-ornithine, L-lysine and S-2-aminoethyl-L-cysteine also significantly stimulated L-alanine efflux. L-Lysine uptake was inhibited by L-alanine but not by D-alanine, and the inhibitory potency of L-alanine was not influenced by the replacement of Na+ in the incubation medium with choline. L-Lysine efflux from pre-loaded cells was stimulated by L-alanine but not by D-alanine. It is concluded that these cells possess a highly selective stero-specific amino acid-transport system. Although the optimum substrates are small neutral amino acids, this system also has a significant affinity for dibasic amino acids.     

A one-to-one Mg2+:Mn2+ exchange in rat erythrocytes

Mg2+ efflux in rat erythrocytes was stimulated by increases in external Na+ concentration following a Michaelian-like function with an apparent dissociation constant (KNa) of 11 +/- 3 mM (mean +/- S.D. of three experiments) and a variable maximal rate ranging from 150 to 1200 mumol (liter (1) cells X h)-1. Na+-stimulated Mg2+ efflux was inhibited by quinidine and by ATP depletion. In the absence of external Na+, Mg2+ efflux was stimulated by increases in external Mn2+ concentration following a Michaelian-like function with an apparent dissociation constant (KMn) of 35 +/- 15 microM (mean +/- S.D. of four experiments) and a variable maximal rate ranging from 350 to 1400 mumol (1 cells X h)-1. Mn2+-stimulated Mg2+ efflux was inhibited by quinidine, by ATP depletion, and by increasing the external Na+ concentration. Quinidine-sensitive (or ATP-dependent) Mg2+ efflux exhibited very similar values when compared with quinidine-sensitive (or ATP-dependent) Mn2+ influx. Mn2+ efflux in rat erythrocytes (loaded with total internal Mn2+ contents of 230-450 mumol/l cells) was stimulated by increases in external Na+ concentration and inhibited by quinidine. In the absence of external Na+, Mn2+ efflux was stimulated by increases in external Mg2+ concentration following a Michaelian-like function with an apparent dissociation constant (KMg) of about 35 +/- 5 microM (mean +/- range of two experiments) and a maximal rate of about 60-100 mumol (1 cells X h)-1. In conclusion, the Na+-stimulated Mg2+ carrier of rat erythrocytes may catalyze a one-to-one and reversible Mn2+:Mg2+ exchange in the absence of external Na+.     

Patterns of purine nucleotides in fish erythrocytes.

1. The purine nucleotides were determined in the whole blood of 9 fresh water teleosts and 2 marine selachians. 2. GTP and ATP accounted for 88-99% of the total erythrocytes purines. 3. The ATP/ADP ratio ranged from 11 to 60 in the erythrocytes of the fish examined. 4. GTP is widely distributed in fish erythrocytes but its level ranged from 1 to 33 nmol/mg Hb (0.4 to 9 mumol/ml erythrocyte). 5. Lepomis and Esox exhibited a GTP/ATP ratio as elevated as in Anguilla; moreover the concentration of GTP per mol of Hb (physiologically most indicative) is higher in Lepomis, Esox, Ictalurus and Silurus than in Anguilla.     

Semiautomated enzymic microassay for plasma L-alanine.

Plasma L-alanine is deaminated by bacterial alanine-dehydrogenase; the resulting ammonia is dialyzed out and measured by use of a continuous flow phenol-hypochlorite colorimetric microassay. Concentrations in the range of 10-1,000 mumol alanine/l can be determined in a 50-microliter sample. The optimal conditions for the assay are specified. Study of the analytical qualities of the technique shows high specificity, good reproducibility , and a detection limit of 6 mumol/l. Usual values in human plasma from arterial or venous blood are respectively 296 +/- 166 and 376 +/- 214 mumol alanine/l (x +/- 2 SD). The usual values in rats are close to those found in man.