Some physiological aspects of genetic variation in the blood of sheep.

The principal genetic variants in sheep red cells and plasma are listed. Current hypotheses as to how the L blood group antigen affects active potassium transport across the red cell membrane are summarized. Recent work on an inherited defect in amino acid transport which results in a red cell GSH deficiency is also described.     

Complete dissociation of DOCA-salt hypertension and red cell ion transport alterations.

Our previous study revealed major ion transport alterations that resulted in a pronounced elevation of red cell Na+ content in DOCA-salt treated homozygous vasopressin-deficient (DI) Brattleboro rats in which only a moderate increase of systolic blood pressure occurred. In contrast, no changes of red cell Na+ content were observed in heterozygous vasopressin-secreting (non-DI) Brattleboro rats with a severe DOCA-salt hypertension. Using a chronic supplementation of DI rats with an antidiuretic agonist dDAVP (1-desamino-8-D-arginine vasopressin) we did not demonstrate any significant changes of red cell ion transport in dDAVP-treated DI rats with a fully developed DOCA-salt hypertension. The absence of ion transport alterations seems to be mainly due to dDAVP-induced correction of altered K+ metabolism seen in DOCA-salt treated DI animals. It can be concluded that DOCA-salt hypertension can develop even without red cell ion transport alterations which are usually caused by cell K+ depletion.     

Quench-flow analysis reveals multiple phases of GluT1-mediated sugar transport

Standard models for carrier-mediated nonelectrolyte transport across cell membranes do not explain sugar uptake by human red blood cells. This means that either (1) the models for sugar transport are incorrect or (2) measurements of sugar transport are flawed. Most measurements of red cell sugar transport have been made over intervals of 10 s or greater, a range which may be too long to measure transport accurately. In the present study, we examine the time course of sugar uptake over intervals as short as 5 ms to periods as long as 8 h. Using conditions where transport by a uniform population of cells is expected to be monophasic (use of subsaturating concentrations of a nonmetabolizable but transported sugar, 3-O-methylglucose), our studies demonstrate that red cell sugar uptake is comprised of three sequential, protein-mediated events (rapid, fast, and slow). The rapid phase is more strongly temperature-dependent than the fast and slow phases. All three phases are inhibited by extracellular (maltose or phloretin) or intracellular (cytochalasin B) sugar-transport inhibitors. The rate constant for the rapid phase of uptake is independent of the 3-O-methylglucose concentration. The magnitude (moles of sugar associated with cells) of the rapid phase increases in a saturable manner with [3-O-methylglucose] and is similar to (1) the amount of sugar that is retained by red cell membrane proteins upon addition of cytochalasin B and phloretin and (2) the d-glucose inhibitable cytochalasin B binding capacity of red cell membranes. These results are consistent with the hypothesis that previous studies have both under- and overestimated the rate of erythrocyte sugar transport. These data support a transport mechanism in which newly bound sugars are transiently sequestered within the translocation pathway where they become inaccessible to extra- and intracellular water.     

Influence of increased membrane cholesterol on membrane fluidity and cell function in human red blood cells

Cholesterol and phospholipid are the two major lipids of the red cell membrane. Cholesterol is insoluble in water but is solubilized by phospholipids both in membranes and in plasma lipoproteins. Morever, cholesterol exchanges between membranes and lipoproteins. An equilibrium partition is established based on the amount of cholesterol relative to phospholipid (C/PL) in these two compartments. Increases in the C/PL of red cell membranes have been studied under three conditions: First, spontaneous increases in vivo have been observed in the spur red cells of patients with severe liver disease; second, similar red cell changes in vivo have been induced by the administration of cholesterol-enriched diets to rodents and dogs; third, increases in membrane cholesterol have been induced in vitro by enriching the C/PL of the lipoprotein environment with cholesterol-phospholipid dispersions (liposomes) having a C/PL of >1.0. In each case, there is a close relationship between the C/PL of the plasma environment and the C/PL of the red cell membrane. In vivo, the C/PL mole ratio of red cell membranes ranges from a normal value of 0.9–1.0 to values which approach but do not reach 2.0. In vitro, this ratio approaches 3.0. Cholesterol enrichment of red cell membranes directly influences membrane lipid fluidity, as assessed by the rotational diffusion of hydrophobic fluorescent probes such as diphenyl hexatriene (DPH). A close correlation exists between increases in red cell membrane C/PL and decreases in membrane fluidity over the range of membrane C/PL from 1.0 to 2.0; however, little further change in fluidity occurs when membrane C/PL is increased to 2.0–3.0. Cholesterol enrichment of red cell membranes is associated with the transformation of cell contour to one which is redundant and folded, and this is associated with a decrease in red cell filterability in vitro. Circulation in vivo in the presence of the slpeen further modifies cell shape to a spiny, irregular (spur) form, and the survival of cholesterol-rich red cells is decreased in the presence of the spleen. Although active Na-K transport is not influenced by cholesterol enrichment of human red cells, several carrier-mediated transport pathways are inhibited. We have demonstrated this effect for the cotransport of Na + K and similar results have been obtained by others in studies of organic acid transport and the transport of small neutral molecules such as erythritol and glycerol. Thus, red cell membrane C/PL is sensitive to the C/PL of the plasma environment. Increasing membrane C/PL causes a decrease in membrane fluidity, and these changes are associated with a reduction in membrane permeability, a distortion of cell contour and filterability and a shortening of the survival of redcells in vivo.     

Heterogeneity in dog red blood cells: sodium and potassium transport

After incubation in isotonic KCl, dog red blood cells can be separated by centrifugation into subgroups which assume different cell volumes and possess different transport characteristics. Those red cells which swell in isotonic KCl exhibit a higher permeability to K and possess a greater volume dependence for transport of K than those red cells which shrink. A high Na permeability characterizes cells which shrink in isotonic KCl and these cells exhibit a larger volume-dependent Na flux than those red cells which swell. These two subgroups of red cells do not seem to represent two cell populations of different age. The results indicate that the population of normal cells is evidently heterogeneous in that the volume-dependent changes in Na and K permeability are distributed between differnt cell types rather than representing a single cell type which reciprocally changes its selectivity to Na and K.     

Target analysis studies of red cell water and urea transport

Radiation inactivation was used to determine the nature and molecular weight of water and urea transporters in the human red cell. Red cells were frozen to -50 degrees C in a cryoprotectant solution, irradiated with 1.5 MeV electrons, thawed, washed and assayed for osmotic water and urea permeability by stopped-flow light scattering. The freezing and thawing process did not affect the rates of water or urea transport or the inhibitory potency of p-chloromercuribenzenesulfonate (pCMBS) on water transport and of phloretin on urea transport. Red cell urea transport inactivated with radiation (0-4 Mrad) with a single target size of 469 +/- 36 kDa. 40 microM phloretin inhibited urea flux by approx. 50% at each radiation dose, indicating that urea transporters surviving radiation were inhibitable. Water transport did not inactivate with radiation; however, the inhibitory potency of 2.5 mM pCMBS decreased from 86 +/- 1% to 4 +/- 9% over a 0-2 Mrad dose range. These studies suggest that red cell water transport either required one or more low-molecular-weight proteins, or is lipid-mediated, and that the pCMBS-binding site which regulates water flow inactivates with radiation. These results also suggest that red cell urea transport is mediated by a specific, high-molecular-weight protein. These results do not support the hypothesis that a band 3 dimer (190 kDa) mediates red cell osmotic water and urea transport.     

Iron Metabolism in the Anaemia of Chronic Renal Failure. Effects of Dialysis and of Pareuteral Iron

Serial studies of iron transport in patients on maintenance dialysis showed normal or raised values in almost all subjects and a transient increase soon after the start of dialysis in three. These patients, who were seldom or never transfused, had low serum iron levels and normal iron-binding capacity with low saturation. Iron transport was substantially increased by parenteral iron-dextran treatment. Tracer studies showed good iron utilization, with transport to the marrow rather than to the liver. In these circumstances iron therapy is safe and beneficial, and a useful rise in red cell mass was shown to result from it. The packed cell volume was found to be a valid index of red cell mass in these patients. Red cell loss in the dialysers was insufficient to account for the observed reduction in red cell survival.     

Electron microscopic examination of common red cell ghosts: cytoplasmic contamination.

Red blood cell ghost preparations are often cited as providing unequivocal or convincing evidence for the active transport of solutes from a solution of low concentration across a membrane to a solution of higher concentration. Electron microscopic examination of the more widely used ghost preparations show that a considerable quantity of cytoplasmic macromolecules (including hemoglobin) remain within the treated red blood cells. That is, many of the ghost preparations are not hollow membrane perparations. It is concluded that the problem of active solute transport in red blood cell ghost preparations should be reexamined. Furthermore, experiments with ghost preparations purporting to demonstrate active transport should include electron photomicrographs of the preparation utilized.     

Adaptation of the Blood Antioxidant Defence Mechanisms of Sheep with a Genetic Lesion Resulting in Low Red Cell Glutathione Concentrations

Finnish Landrace sheep with a genetic lesion which results in restricted cysteine transport across the red cell membrane have total glutathione concentrations in their red blood cells that are approximately 40% of those in normal sheep of the same breed. However, dimethyldisulphide-challenged red blood cells from both phenotypes produce an ESR-spin adduct at similar rates. The resistance of the low glutathione phenotype red cells to oxidant challenge is reflected by increases in the activities of antioxidant enzymes. Sheep with a genotypic disorder in cysteine transport may be a suitable model for studying the genetic expression of antioxidant enzymes in response to oxidant loads.     

Comparative aspects of sulfate transport in the erythrocytes of different mammalian species

The pH dependence of sulfate transport through the red cell membranes of ten mammalian (nucleated and non-nucleated) species was studied by measuring tracer exchange. The characteristic maximum of the sulfate transfer system, observed previously in the red cells of man and a number of other mammalian red cell species was found to be present in all species investigated. Although the volumes of the red cells of the various species differ by no more than a factor of 2 and the ratios of volume/surface even less, the rate constants at the respective maxima vary from 0.33 x 10(-2) min-1 (beef) to 15.50 x 10(-2) min-1 (eagle), i.e. about 45 fold. This suggests that either the number of anion transport molecules per cell and/or the turnover numbers of the individual transport molecules show considerable variations.     

Phosphorylation of the red blood cell membrane during the active transport of C++

The phosphorylation of red blood cell membrane fragments (RBCMF) during Ca++ transport was investigated. When red cell membrane fragments are incubated with [gamma-32P]ATP under the experimental condition which minimizes the phosphorylation of Na+-K+-ATPase, RBCMF are labeled in the presence of Mg++ without Ca++. When Ca++ is added, the labeling decreases due to dephosphorylation of RBCMF. The initial reaction of phosphorylation is reversed in the presence of excess ADP. The treatment of RBCMF with n-ethylmaleimide (NEM) does not interfere with the initial phosphorylation reaction, but blocks the dephosphorylation in the presence of Ca++. These data suggest that the enzymatic sequence of the Ca++ transport mechanism may be very similar to that of the Na+ transport mechanism.     

Binding of DTNB to band 3 in the human red cell membrane

Inhibition of red cell water transport by the sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) has been reported by Naccache and Sha'afi ((1974) J. Cell Physiol. 84, 449-456) but other investigators have not been able to confirm this observation. Brown et al. ((1975) Nature 254, 523-525) have shown that, under appropriate conditions, DTNB binds only to band 3 in the red cell membrane. We have made a detailed investigation of DTNB binding to red cell membranes that had been treated with the sulfhydryl reagent N-ethylmaleimide (NEM), and our results confirm the observation of Brown et al. Since this covalent binding site does not react with either N-ethylmaleimide or the sulfhydryl reagent pCMBS (p-chloromercuribenzenesulfonate), its presence has not previously been reported. This covalent site does not inhibit water transport nor does it affect any transport process we have studied. There is an additional low-affinity (non-covalent) DTNB site that Reithmeier ((1983) Biochim. Biophys. Acta 732, 122-125) has shown to inhibit anion transport. In N-ethylmaleimide-treated red cells, we have found that this binding site inhibits water transport and that the inhibition can be partially reversed by the specific stilbene anion exchange transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS), thus linking water transport to anion exchange. DTNB binding to this low-affinity site also inhibits ethylene glycol and methyl urea transport with the same KI as that for water inhibition, thus linking these transport systems to that for water and anions. These results support the view that band 3 is a principal constituent of the red cell aqueous channel, through which urea and ethylene glycol also enter the cell.     

Effects of p-nitrophenylphosphate on Ca2+ transport in inside-out vesicles from human red-cell membranes

Ca2+-ATPase activity and Ca2+ uptake in inside-out vesicles from human red cell membranes are changed in parallel by p-nitrophenylphosphate. This indicates that, unlike the Ca2+ pump of sarcoplasmic reticulum, the Ca2+ pump of the red cell membrane does not utilize p-nitrophenylphosphate hydrolysis to drive Ca2+ transport.     

Effects of low-amplitude pulsed magnetic fields on cellular ion transport

Pulsed magnetic fields (PMFs) are widely used to treat difficult fractures of bone and other disorders of connective tissue. It is not clear how they interact with tissue metabolism, although it has been proposed that induced currents or electric fields impinging on cell membranes may modify their ion transport function. This hypothesis was tested by treating in vitro models for ion transport processes with short-term exposure to PMFs. No change occurred in active transport of potassium or calcium in human red cells or in calcium transport through an epithelial membrane. We considered less direct action on red cell membranes, that their permeability might be modified after PMF treatment, and also that PMFs might alter the extracellular ionic activity within connective tissue by interacting with its Donnan potential. Each of these studies proved negative, and we conclude that the PMF waveforms used here do not exert a general short-term effect on cellular ion transport.     

Changes in alanine and glutamine transport during rat red blood cell maturation

Alanine and glutamine transport have been studied during red blood cell maturation in the rat. Kinetic parameters of Na⁺-dependent L-alanine transport were:K _m 0.43 and 1.88 mM andV _max 158 and 45 nmoles/ml ICW/min for reticulocytes and erythrocytes, respectively. During red cell maturation in the rat there is a loss of capacity and affinity of the system ASC for L-alanine transport. The values for Na⁺-dependent L-glutamine transport in reticulocytes wereK _m 0.51 mM andV _max 157 nmoles/ml ICW/min. On the other hand, a total loss of L-glutamine transport mediated by both N and ASC systems is demonstrated in mature red cells. This seems to indicate that during rat red cell maturation the system N disappears. Furthermore, the system ASC specificity in mature cells changes, and glutamine enters the red cell by non-mediated diffusion processes.     

Elevated free tyrosine in rhinoceros erythrocytes

Red blood cells of African black rhinoceroses (Diceros bicornis) are highly sensitive to oxidant-induced hemolysis and they possess a number of enzymatic and biochemical features that differ radically from other mammals. Here we show concentrations of free tyrosine in rhinoceros red blood cells which can approach levels as high as 1 mM, 50-fold higher than in human red blood cells. Elevated levels of tyrosine are also observed in red blood cells of other members of the order Perissodactyla such as the horse and zebra. Captive black rhinoceroses have significantly lower levels of red blood cell tyrosine than black rhinoceroses in the wild. Tyrosine transport studies indicate that black rhinoceros red blood cells have lost the ability to transport tyrosine as efficiently as human red blood cells.     

Autocrine production of insulin-like growth factor-I (IGF-I) affects paracellular transport across epithelial cells in vitro

Autocrine production of growth factors can have significant effects on cell activity. We report for the first time that autocrine production of insulin-like growth factor-I (IGF-I) alters paracellular transport across bovine mammary epithelial cells in vitro. Paracellular transport was assessed by measuring phenol red transport across mammary alveolar cells-large T antigen (MAC-T cells) derived from parental mammary epithelial cells, cultured on porous membranes and compared with two different transfected MAC-T cell lines that constitutively secrete IGF-I. Phenol red transport was essentially blocked in parental cell culture after six days, while IGF-I secreting cells provided essentially no barrier. Surprisingly, neither co-culture studies between parental and IGF-I-secreting cells nor addition of exogenous IGF-I or IGF-binding protein-3 reversed the phenol red transport properties. IGF-I-secreting cells did however express lower levels of the junction components occludin and E-cadherin than parental cells, suggesting that localized autocrine IGF-I activity might lead to increased permeability via changes in both the tight and adherens junction protein levels.     

Some factors affecting pulmonary oxygen transport

Equations governing oxygen transport from alveolar gas to red blood cells flowing through pulmonary capillaries are written down. Some analytical predictions are made on factors affecting the rate at which this process takes place. Numerical simulations are carried out to investigate the effect of red blood cell shape, capillary dimensions, haematocrit and choice of oxygen dissociation curve on pulmonary oxygen transport. These factors all have an effect on pulmonary transport, with the effect being much more marked for simulations with low oxygen levels, typical of those seen in some subjects with respiratory disease.     

Relation between red cell anion exchange and water transport

A new distilbene compound, 4',4'-dichloromercuric-2,2,2',2'-bistilbene tetrasulfonic acid (DCMBT), has been synthesized for use in studies of anion and water transport in the human red cell. DCMBT combines features of both the specific stilbene anion transport inhibitor, DIDS, and the mercurial water transport inhibitor, pCMBS. This new compound inhibits anion transport almost completely with a Ki of 15 microM. DCMBT also inhibits water transport by about 15-20% with a Ki of about 8 microM. Treatment of red cells with DIDS inhibits the effect of DCMBT on water transport, suggesting that anion transport and water transport are mediated by the same protein.     

Autocrine Production of Insulin-like Growth Factor-I (IGF-I) Affects Paracellular Transport Across Epithelial Cells In Vitro

Autocrine production of growth factors can have significant effects on cell activity. We report for the first time that autocrine production of insulin-like growth factor-I (IGF-I) alters paracellular transport across bovine mammary epithelial cells in vitro. Paracellular transport was assessed by measuring phenol red transport across mammary alveolar cells-large T antigen (MAC-T cells) derived from parental mammary epithelial cells, cultured on porous membranes and compared with two different transfected MAC-T cell lines that constitutively secrete IGF-I. Phenol red transport was essentially blocked in parental cell culture after six days, while IGF-I secreting cells provided essentially no barrier. Surprisingly, neither co-culture studies between parental and IGF-I-secreting cells nor addition of exogenous IGF-I or IGF-binding protein-3 reversed the phenol red transport properties. IGF-I-secreting cells did however express lower levels of the junction components occludin and E-cadherin than parental cells, suggesting that localized autocrine IGF-I activity might lead to increased permeability via changes in both the tight and adherens junction protein levels.