Measurement of the Elastic Modulus for Red Cell Membrane Using a Fluid Mechanical Technique

Red cells which adhere to a surface in a parallel plate flow channel are stretched when acted on by a fluid shear stress. Three types of stretching are studied: whole cell stretching, the stretching of a red cell evagination, and tether (long, thin membrane process) stretching. In addition, the stretching of a large scale model cell attached to a surface is studied in a Couette flow channel. The results indicate that the uniaxial stretching of red cell membrane can be described by a linear stress-strain relationship. Simple theories developed from free body diagrams permit the calculation of a value for the modulus of elasticity of cell membrane in each of the three experiments. In all cases the value for the modulus is on the order of 10⁴ dyn/cm² for an assumed membrane thickness of 0.01 μm. It was also observed that red cell tethers steadily increase in length when the fluid shear stress is greater than approximately 1.5 dyn/cm² and tether lengths in excess of 200 μm have been achieved. Tethers appear to possess both fluid and elastic properties.     

Analysis by partitioning in aqueous two-phase systems of the loss of transferrin-binding capacity during maturation of rat reticulocytes

A decrease in the number of binding sites for¹²⁵I-transferrin, without an apparent modification of the association constant, has been observed during the maturation of reticulocytes into erythrocytes. As an experimental model, different red cell populations from phenylhydrazinic anaemic rates (95% to 12% reticulocyte-rich) have been used. The fractionation by multiple partition in two-phase systems of these red cell populations has been applied here to show the relationship between number of transferrin receptors and rate of reticulocyte maturation.     

On the problem of dehydration and intracellular crystallization during freezing of cell suspensions.

The kinetic equation of the process of cell dehydration during freezing has been obtained. It is used to assess the degree of protoplasmic supercooling as a function of the cooling rate and cell parameters.The suggested model of dehydration cannot be applied to cells with permeability coefficients for water molecules more than 10^?5 cm/sec · bar, in particular to erythrocytes.The peculiarities of intracellular crystallization in red cells have been studied. The results show that red cells are likely to start freezing at cooling rates slower than those supposed from calculations of Mazur (9).     

Peculiarities of osmoregulation of circulating red blood cells in steno-and euryhaline marine fish species under hypoosmotic conditions

The peculiarities of osmoregulation of circulating red blood cells of the stenohaline giant gobyGobius cobitis and the euryhaline toad gobyGobius batrachocephalus have been studied under experimental conditions. In the giant goby, volume of the red blood cells increased steadily by 10.6–18.1% (p < 0.05) after reduction of the medium salinity from 15–17 to 6.0–6.8‰ and this volume increase remained during the entire experimental period (40–45 days). Lysis of red blood cells was noticed in some cases, which was indicated by a decrease of the number of red blood cells and an increase of concentration of free hemoglobin in the blood plasma. No similar reactions were observed in the euryhaline toad goby; the mean cell volume did not change statistically significantly. The volume regulation resulted in K⁺ efflux from red blood cells. The blood red cells of the toad goby had a high resistance to osmotic stress. The Na⁺,K⁺-ATPase activity in the red blood cell membranes of the toad goby was higher by 18.8% (p < 0.001) than in the giant goby.     

Peculiarities of osmoregulation of circulating red blood cells in steno- and euryhaline marine fish species under hypoosmotic conditions

The peculiarities of osmoregulation of circulating red blood cells of the stenohaline giant gobyGobius cobitis and the euryhaline toad gobyGobius batrachocephalus have been studied under experimental conditions. In the giant goby, volume of the red blood cells increased steadily by 10.6–18.1% (p (WENA) 0.05) after reduction of the medium salinity from 15–17 to 6.0–6.8‰ and this volume increase remained during the entire experimental period (40–45 days). Lysis of red blood cells was noticed in some cases, which was indicated by a decrease of the number of red blood cells and an increase of concentration of free hemoglobin in the blood plasma. No similar reactions were observed in the euryhaline toad goby; the mean cell volume did not change statistically significantly. The volume regulation resulted in K⁺ efflux from red blood cells. The blood red cells of the toad goby had a high resistance to osmotic stress. The Na⁺,K⁺-ATPase activity in the red blood cell membranes of the toad goby was higher by 18.8% (p (WENA) 0.001) than in the giant goby.     

Density distribution and cation composition of red blood cells in newborn puppies

The density distribution and cation composition of red blood cells from newborn puppies have been studied. The density distribution of red cells from a newborn puppy in a bovine serum albumin density gradient resembles a normal distribution with a peak density at a region less than that found for adult dog red cells. In two weeks the whole distribution shifts toward a more dense region, and a second cell peak appears so that the distribution becomes bimodal. This second cell peak is smaller than the original peak, and it appears at a region of lower density. In nine weeks the distribution becomes a normal one again, but the peak density corresponds to the peak density of the second cell peak which first appeared at two weeks. Evidence has been obtained to show that fetal red cells are located in the more dense cell peak and neonatal cells are in the less dense second peak. These results were obtained by labeling fetal cells with Cr⁵¹ and neonatal cells with Fe⁵⁹. The analysis of the cation content of these cells shows that fetal cells contain more K and Na and have a higher K/Na ratio than adult red cells. Furthermore, neonatal cells contain considerably less cation and hemoglobin than do fetal cells. From a study of the cation and hemoglobin content of red cells appearing in various density fractions it is concluded that fetal cells lose K and Na during the first two weeks after birth. Thus, the change in the density disribution of the erythrocytes is thought to be due to two factors: (1) An increase in the density of fetal cells due to the loss of K and Na and, hence, water during the first two weeks after birth, and (2) the entry of less dense neonatal cells into the circulation.     

The novel role of peroxiredoxin-2 in red cell membrane protein homeostasis and senescence

Peroxiredoxin-2 (Prx2), a typical two-cysteine peroxiredoxin, is the third most abundant protein in red cells. Although progress has been made in the functional characterization of Prx2, its role in red cell membrane protein homeostasis is still under investigation. Here, we studied Prx2^−/− mouse red cells. The absence of Prx2 promotes (i) activation of the oxidative-induced Syk pathway; (ii) increased band 3 Tyr phosphorylation, with clustered band 3; and (iii) increased heat shock protein (HSP27 and HSP70) membrane translocation. This was associated with enhanced in vitro erythrophagocytosis of Prx2^−/− red cells and reduced Prx2^−/− red cell survival, indicating the possible role of Prx2 membrane recruitment in red cell aging and in the clearance of oxidized hemoglobin and damaged proteins through microparticles. Indeed, we observed an increased release of microparticles from Prx2^−/− mouse red cells. The mass spectrometric analysis of erythroid microparticles found hemoglobin chains, membrane proteins, and HSPs. To test these findings, we treated Prx2^−/− mice with antioxidants in vivo. We observed that N-acetylcysteine reduced (i) Syk activation, (ii) band 3 clusterization, (iii) HSP27 membrane association, and (iv) erythroid microparticle release, resulting in increased Prx2^−/− mouse red cell survival. Thus, we propose that Prx2 may play a cytoprotective role in red cell membrane protein homeostasis and senescence.     

Different red cell populations in newborn, genetically low potassium sheep: relation to hematopoietic, immunologic and physiologic differentiation

Three red cell populations have been distinguished in genotypically low potassium (LK) newborn sheep by an improved electrical sizing method and were best approximated by a logarithmic normal distribution. Labeling studies with ⁵¹Cr and ⁵⁹Fe exclude transformation of the three red cell populations into each other. Population I, consisting of large red cells (mean volume 36 μm³), with a comparatively slow electrophoretic mobility is present at birth and disappears within three to four weeks from circulation. These cells possess a high potassium (HK) steady state concentration, a K⁺ pump influx activity at least 5-fold greater than observed in adult LK red cells, very low amounts of the L antigens generally associated with the LK property, and do not respond to the stimulatory action of the L antibody. The first population is gradually replaced by population II comprising small red cells (mean volume 28 μm³) of intermediate electrophoretic mobility and with a peak production around day 20 after birth. The potassium concentration, [K⁺]_c, in these cells appears to be lower than in the cells of population I but the L antigen content is increased. Formation of population III (mean volume 30 μm³ and comparatively fast electrophoretic mobility) follows closely that of population II and is preceded by a sharp increase in reticulocytosis. The red cells of population III exhibit parameters characteristic for adult LK cells: low [K⁺]_c and K⁺ pump activity, fully developed L antigen content, and an almost maximal response to the K⁺ pump stimulating effect of anti-L. In L and M antigen positive LK red cells of newborn sheep, the development of the M antigen parallels that of the L antigen. The data are consistent with the hypothesis that cellular replacement and not maturation is the major factor in controlling the HK-LK transition in newborn sheep.     

C-peptide,Na+,K+-ATPase,and Diabetes

Na⁺,K⁺-ATPase is an ubiquitous membrane enzyme that allows the extrusion of three sodium ions from the cell and two potassium ions from the extracellular fluid. Its activity is decreased in many tissues of streptozotocin-induced diabetic animals. This impairment could be at least partly responsible for the development of diabetic complications. Na⁺,K⁺-ATPase activity is decreased in the red blood cell membranes of type 1 diabetic individuals, irrespective of the degree of diabetic control. It is less impaired or even normal in those of type 2 diabetic patients. The authors have shown that in the red blood cells of type 2 diabetic patients, Na⁺,K⁺-ATPase activity was strongly related to blood C-peptide levels in non–insulin-treated patients (in whom C-peptide concentration reflects that of insulin) as well as in insulin-treated patients. Furthermore, a gene-environment relationship has been observed. The alpha-1 isoform of the enzyme predominant in red blood cells and nerve tissue is encoded by the ATP1A1 gene.Apolymorphism in the intron 1 of this gene is associated with lower enzyme activity in patients with C-peptide deficiency either with type 1 or type 2 diabetes, but not in normal individuals. There are several lines of evidence for a low C-peptide level being responsible for low Na⁺,K⁺-ATPase activity in the red blood cells. Short-term C-peptide infusion to type 1 diabetic patients restores normal Na⁺,K⁺-ATPase activity. Islet transplantation, which restores endogenous C-peptide secretion, enhances Na⁺,K⁺-ATPase activity proportionally to the rise in C-peptide. This C-peptide effect is not indirect. In fact, incubation of diabetic red blood cells with C-peptide at physiological concentration leads to an increase of Na⁺,K⁺-ATPase activity. In isolated proximal tubules of rats or in the medullary thick ascending limb of the kidney, C-peptide stimulates in a dose-dependent manner Na⁺,K⁺-ATPase activity. This impairment in Na⁺,K⁺-ATPase activity, mainly secondary to the lack of C-peptide, plays probably a role in the development of diabetic complications. Arguments have been developed showing that the diabetesinduced decrease in Na⁺,K⁺-ATPase activity compromises microvascular blood flow by two mechanisms: by affecting microvascular regulation and by decreasing red blood cell deformability, which leads to an increase in blood viscosity. C-peptide infusion restores red blood cell deformability and microvascular blood flow concomitantly with Na⁺,K⁺-ATPase activity. The defect in ATPase is strongly related to diabetic neuropathy. Patients with neuropathy have lower ATPase activity than those without. The diabetes-induced impairment in Na⁺,K⁺-ATPase activity is identical in red blood cells and neural tissue. Red blood cell ATPase activity is related to nerve conduction velocity in the peroneal and the tibial nerve of diabetic patients. C-peptide infusion to diabetic rats increases endoneural ATPase activity in rat. Because the defect in Na⁺,K⁺-ATPase activity is also probably involved in the development of diabetic nephropathy and cardiomyopathy, physiological C-peptide infusion could be beneficial for the prevention of diabetic complications.     

AN EARLY RESPONSE OF THE MORPHOLOGICALLY RECOGNIZABLE ERYTHROID PRECURSORS TO BLEEDING

⁵⁵Fe autoradiography of the peripheral red blood cells has been used to study the proliferation of the recognizable erythroid precursors in bled animals. The transit time of the recognizable erythroid precursors present in the bone marrow and labelled with ⁵⁵Fe 6 hr before bleeding, remains unchanged, but the number of red cells produced by these precursors is significantly greater than normal. It is deduced that the increased red cell production is brought about by an increase in the number of divisions that the cells undergo during maturation and that a shortening in the red cell cycle time is implied. The possibility that the transit time of the progeny of cells differentiating into pro-erythroblasts after bleeding may be shorter than the transit time of the precursors already differentiated before bleeding, is briefly discussed.     

Effect of Ferriprotoporphyrin IX and Non-heme Iron on the Ca2+ Pump of Intact Human Red Cells

Previous studies have shown that ferriprotoporphyrin IX (FP) and non-heme iron have a marked inhibitory effect on the Ca²⁺-Mg²⁺-ATPase activity of isolated red cell membranes, the biochemical counterpart of the plasma membrane Ca²⁺ pump (PMCA). High levels of membrane-bound FP and non-heme iron have been found in abnormal red cells such as sickle cells and malaria-infected red cells, associated with a reduced life span. It was important to establish whether sublytic concentrations of FP and non-heme iron would also inhibit the PMCA in normal red cells, to assess the possible role of these agents in the altered Ca²⁺ homeostasis of abnormal cells. Active Ca²⁺ extrusion by the plasma membrane Ca²⁺ pump was measured in intact red cells that had been briefly preloaded with Ca²⁺ by means of the ionophore A23187. The FP and nonheme iron concentrations used in this study were within the range of those applied to the isolated red cell membrane preparations. The results showed that FP caused a marginal inhibition (∼20%) of pump-mediated Ca²⁺ extrusion and that non-heme iron induced a slight stimulation of the Ca²⁺ efflux (11–20%), in contrast to the marked inhibitory effects on the Ca²⁺-Mg²⁺-ATPase of isolated membranes. Thus, FP and non-heme iron are unlikely to play a significant role in the altered Ca²⁺ homeostasis of abnormal red cells. Received: 22 November 1999/Revised: 29 February 2000     

Local Membrane Deformations Activate Ca2+-Dependent K+ and Anionic Currents in Intact Human Red Blood Cells

Background

The mechanical, rheological and shape properties of red blood cells are determined by their cortical cytoskeleton, evolutionarily optimized to provide the dynamic deformability required for flow through capillaries much narrower than the cell''s diameter. The shear stress induced by such flow, as well as the local membrane deformations generated in certain pathological conditions, such as sickle cell anemia, have been shown to increase membrane permeability, based largely on experimentation with red cell suspensions. We attempted here the first measurements of membrane currents activated by a local and controlled membrane deformation in single red blood cells under on-cell patch clamp to define the nature of the stretch-activated currents.

Methodology/Principal Findings

The cell-attached configuration of the patch-clamp technique was used to allow recordings of single channel activity in intact red blood cells. Gigaohm seal formation was obtained with and without membrane deformation. Deformation was induced by the application of a negative pressure pulse of 10 mmHg for less than 5 s. Currents were only detected when the membrane was seen domed under negative pressure within the patch-pipette. K⁺ and Cl⁻ currents were strictly dependent on the presence of Ca²⁺. The Ca²⁺-dependent currents were transient, with typical decay half-times of about 5–10 min, suggesting the spontaneous inactivation of a stretch-activated Ca²⁺ permeability (PCa). These results indicate that local membrane deformations can transiently activate a Ca²⁺ permeability pathway leading to increased [Ca²⁺]_i, secondary activation of Ca²⁺-sensitive K⁺ channels (Gardos channel, IK1, KCa3.1), and hyperpolarization-induced anion currents.

Conclusions/Significance

The stretch-activated transient PCa observed here under local membrane deformation is a likely contributor to the Ca²⁺-mediated effects observed during the normal aging process of red blood cells, and to the increased Ca²⁺ content of red cells in certain hereditary anemias such as thalassemia and sickle cell anemia.     

Cation Transport in Dog Red Cells

Studies have been made on the cation transport system of the dog red cell, a system of particular interest because it has been shown that there is a marked dependence of cation fluxes on the cell volume. We have found that a 10% decrease in cell volume causes a large increase in 1 hr uptake of ²⁴Na as well as a considerable inhibition of ⁴²K uptake. This effect cannot be produced by a difference in medium osmolality but rather requires the cell volume to change. Dog red cell uptake of ²⁴Na is not inhibited by iodoacetate. Phloretin inhibits ²⁴Na uptake and lactate production, and virtually abolishes the volume effect on Na uptake. These several observations may be accounted for in terms of a working hypothesis which presupposes a cation carrier complex which pumps K into and Na out of cells of normal volume. When the cells are shrunken the carrier specificity shifts to an external Na-specific mode and there is a large increase in ²⁴Na uptake, driven by the inwardly directed Na electrochemical potential gradient.     

Antibodies to pig kidney (Na++K+)-ATPase inhibit the Na+ pump in human red cells provided they have access to the inner surface of the cell membrane

Antisera from rabbits that had been immunized with a highly active membrane preparation of (Na⁺ + K⁺)-ATPase from the outer medulla of pig kidney strongly inhibited (Na⁺ + K⁺)-ATPase activity in various tissues. When the antiserum was incorporated into released human red cell ghosts, the ouabain-sensitive efflux of Na⁺ into both 15 mM K⁺ and K⁺-free high Na⁺ media was completely abolished. This effect was not observed when non-immune serum was used, or when the immune serum was allowed access only to the outer surface of the red cell membranes.     

The emergence of Cochlodinium along the California Coast (USA)

A sudden and nearly synchronous emergence of the red tide forming dinoflagellate Cochlodinium along more than 800 km of California coastline was initially observed in late summer 2004. Thereafter high cell concentrations have been detected on an annual basis. Here, we present quantitative and semi-quantitative data indicating that Cochlodinium was uncommon in the phytoplankton community in California prior to 2004 and is now persisting as a more regular component and one that seasonally can cause red tides. The quantitative portion of this study was primarily conducted in Monterey Bay, where cell densities reached at least 6 × 10⁴ cells L⁻¹ during the initial outbreak. A semi-quantitative comparison of California coastal counties by the California Department of Health Services (CDHS) was also made: of the 15 counties surveyed (most with multiple sites per county), cells were detected only from Los Angeles County in the south to San Mateo County in the central region (seven counties), but not in the northern part of the state (six counties). Two counties in the central region of the state, San Luis Obispo and Santa Cruz, displayed intense and frequent periods of elevated Cochlodinium cell abundances. Although not observed in the state-wide CDHS survey, we occasionally found cells in San Diego County with densities up to 2.7 × 10⁴ cells L⁻¹. Though these colonial dinoflagellates have been recognized in California for over 80 years, with several “blooms” recorded prior to 2004, the species’ geographic range and abundance in recent years suggest significant shifts in the nearshore phytoplankton community of this region of the eastern Pacific.     

Gene frequencies of adenylatekinase polymorphism in the Roman population

Summary The red cell AK phenotypes of 841 hemolysates from unrelated individuals living in Rome have been determined by the starch gel electrophoretic method of Fildes and Harris (1966). The observed gene frequencies were: AK ¹=96.3 and AK ²=3.7.Supported by a grant of the Consiglio Nazionale delle Ricerche (CNR).     

Plasmodium berghei: uptake of clindamycin and its metabolites by mouse erythrocytes with clindamycin-sensitive and clindamycin-resistant parasites.

Tritiated Clindamycin was used to compare the uptake of Clindamycin in plasma and red cells of mice infected with clindamycin-sensitive or clindamycin-resistant Plasmodium berghei and in uninfected mice. Red cells infected with either sensitive or resistant parasites have a higher concentration of [³H]clindamycin and its active metabolites 1 hr after drug administration than uninfected red blood cells. There was no significant difference in uptake of Clindamycin by red blood cells parasitized by sensitive or resistant parasites. Levels of Clindamycin and its metabolites were consistently higher in red cells than in plasma, both in infected and uninfected mice, but the drug was readily removed by washing red cells with phosphate buffered saline in either case. It is concluded that resistance to Clindamycin is not due to an impaired uptake of the drug by the parasitized red cell as has been shown for chloroquine resistance in P. falciparum and P. berghei.     

Membrane mediated link between ion transport and metabolism in human red cells

When 10^?6 M oubain is added to human red cells that have been incubated without glucose for two hours, there is a significant shift in the ³¹P nuclear magnetic resonances of both phosphate groups of cellular 2,3-diphosphoglycerate, which is not found in control cells incubated with glucose. This means that an effect induced by ouabain on the outside of the red cell membrane is transmitted through the membrane to alter the environment of an intracellular metabolite. Experiments with glycolytic cycle inhibitors have indicated that the intracellular ligand responsible for the resonance shifts is monophosphoglycerate mutase which requires 2,3-diphosphoglycerate as a cofactor for the reaction it catalyzes. To account for this finding a hypothesis is presented that the (Na⁺ + K⁺)-ATPase in human red cells is linked to monophosphoglycerate mutase through the agency of phosphoglycerate kinase. Evidence is presented for the existence of phosphoglycerate kinase/monophosphoglycerate mutase in solution. It is shown that this complex can interact with the cytoplasmic face of (Na⁺ + K⁺)-ATPase at the outside surface of inside out red cell vesicles, and that this interaction is inhibited when 10^?6 M ouabain is contained within the vesicle. Neither monophosphoglycerate mutase nor phosphoglycerate kinase is significantly bound to the inside surface of the intact human red cell, but glyceraldehyde 3-phosphate dehydrogenase is; it is shown that this enzyme also interacts with the cytoplasmic face of the (Na⁺ + K⁺)-ATPase and that the interaction is inhibited by 10^?6 M ouabain.     

Modulation of 2,3-diphosphoglycerate 31P-NMR resonance positions by red cell membrane shape

Na⁺ transport in the red cells of the dog is dependent on cell volume, a 20% change in cell volume leading to a 25-fold increase in apparent Na⁺ flux; the effect is dependent upon metabolic energy. We have found that swelling and shrinking dog red cells causes a shift in the ³¹P-NMR peak of 2,3-diphosphoglycerate, which is present in dog red cells at 5.5 mM. Control experiments indicate that the 2,3-diphosphoglycerate resonance peak shifts may not be attributed to: interaction with hemoglobin, changes in cell pH, ionic strength, diamagnetic susceptibility or small changes in the Mg²⁺/2,3-diphosphoglycerate ratio. Experiments with chlorpromazine and pentanol which alter red cell membrane area by a mechanism different from osmotic swelling suggest that 2,3-diphosphoglycerate interacts with a binding site in the cell that is dependent upon the physical condition of the dog red cell membrane.     

Ion and water shifts produced by ammonium chloride in high K and low K red cells

The effect of ammonium chloride on the cellular Na⁺, K⁺ and water has been examined in human and horse (high K), cow (medium K) and cat (low K) red cells. It was found that high K red cells, especially those of the horse, gained water an Na⁺, whereas the net movement of K⁺ was negligible. There was a correlation between the increase of cellular Na⁺ concentration and of the packed red cell volume. In contrast, the packed cell volume of low K red cells increased slightly or not at all, and Na⁺ ions leaked out from the cells. The high K cells had a lower Cl^? concentration and higher buffer capacity than the low K cells. The results obtained with the medium K (cow) cells usually lay between those of the other two cell types. In all the cases both the plasma and cell pH decreased resulting from the addition of ammonium chloride. The mechanism of movements of water and Na⁺ ions in high K cells remained unsolved, but the response of low K cells to ammonium chloride was near that of a cation exchange resin.