Pig red blood cell hexokinase: Regulatory characteristics and possible physiological role

The regulatory properties of pig erythrocyte hexokinase III have been studied. Among mammalian erythrocyte hexokinases, the pig enzyme shows the highest affinity for glucose and a positive cooperative effect with n_H = 1.5 at all the MgATP concentrations studied (for 0.5 to 5 mm). Glucose at high concentrations is also an inhibitor of hexokinase III. Similarly, the apparent affinity constant for MgATP is independent of glucose concentration. Uncomplexed ATP and Mg are both competitive inhibitors with respect to MgATP. Glucose 6-phosphate, known as a stronger inhibitor of all mammalian erythrocyte hexokinases, is a poor inhibitor for the pig enzyme (K_i = 120 μm). Furthermore, this inhibition is not relieved by orthophosphate as with other mammalian red blood cell hexokinases. A variety of red blood cell-phosphorylated compounds were tested and found to be inhibitors of pig hexokinase III. Of these, glucose 1,6-diphosphate and 2,3-diphosphoglycerate displayed inhibition constants in the range of their intracellular concentrations. In an attempt to investigate the role of hexokinase type III in pig erythrocytes some metabolic properties of this cell have been studied. The adult pig erythrocyte is able to utilize 0.27 μmol of glucose/h/ml red blood cells (RBC) compared with values of 0.56–2.85 μmol/h/ml RBC for the other mammalian species. This reduced capacity to metabolize glucose results from a relatively poor ability of the cell membrane to transport glucose. In fact, all the glycolytic enzymes were present and a low intracellular glucose concentration was measured (0.5 mm against a plasma level of 5 mm). Furthermore, transport and utilization were concentration-dependent processes. Inosine, proposed as the major energy substrate of the pig erythrocyte, at physiological concentrations is not as efficient as glucose in maintaining reduced glutathione levels under oxidative stress. Furthermore, newborn pig erythrocytes (fully permeable to glucose) possess hexokinase type II as the predominant glucose-phosphorylating activity. This fact and the information derived from the study of the regulatory characteristics of hexokinase III and from metabolic studies on intact pig erythrocytes permit the hypothesis that the presence of this peculiar hexokinase isozyme (type III) enables the adult pig erythrocyte to metabolize low but appreciable amounts of glucose.     

Relationships between membrane lipids and ion transport in red blood cells of Dahl rats

Distinct changes of membrane lipid content could contribute to the abnormalities of ion transport that take part in the development of salt hypertension in Dahl rats. The relationships between lipid content and particular ion transport systems were studied in red blood cells (RBC) of Dahl rats kept on low- and high-salt diets for 5 weeks since weaning. Dahl salt-sensitive (SS/Jr) rats on high-salt diet had increased blood pressure, levels of plasma triacylglycerols and total plasma cholesterol compared to salt-resistant (SR/Jr) rats. Furthermore, RBC of SS/Jr rats differed from SR/Jr ones by increased content of total membrane phospholipids, but membrane cholesterol was not changed significantly. SS/Jr rats had higher RBC intracellular Na+ (Na(i)+) content and enhanced bumetanide-sensitive Rb+ uptake. RBC membrane content of cholesterol and phospholipids correlated positively with RBC Na(i)+ content, with the activity of Na+-K+ pump and Na+-K+-2Cl- cotransport and also with Rb+ leak. The content of phosphatidylserines plus phosphatidylinositols was positively associated with RBC Na(i)+ content, with the activity of Na+-K+ pump and Na+-K+-2Cl- cotransport and with Rb+ leak. The content of sphingomyelins was positively related to Na+-K+-2Cl- cotransport activity and negatively to ouabain-sensitive Rb+-K+ exchange. We can conclude that observed relationships between ion transport and the membrane content of cholesterol and/or sphingomyelins, which are known to regulate membrane fluidity, might participate in the pathogenesis of salt hypertension in Dahl rats.     

Mechanism of anion transport in red blood cells: role of membrane proteins.

A number of anionic chemical probes that inhibit anion permeability of red blood cells are localized in a membrane protein of about 100,000 daltons, known as band 3. The inhibitory site has been explored using a series of disulfonic stilbene compounds. It apparently contains three positive charges, probably amino groups. Two probes, pyridoxal phosphate and N-(4-azido-2-nitropheyny)-2-amino ethyl sulfonate, are transported by the anion system but can be fixed in an irreversible bond under specified conditions (reduction with NaBH4 or exposure to light, respectively). Data obtained with these compounds indicate that the inhibitory site in band 3 is the transport site itself. Band 3 protein is exposed in part on the outside of the cell but it is also hydrophobically associated with membrane lipid. A model is proposed in which the band 3 protein acts as an anion permeation channel through the lipid bilayer. Near the outer aspect of the channel an anion binding site can undergo a local conformational change allowing a one-for-one anion exchange across a diffusion barrier.     

The physiological role of drug transporters

Transporters comprise the largest family of membrane proteins in human organism, including members of solute carrier transporter and ATP-binding cassette transporter families. They play pivotal roles in the absorption, distribution and excretion of xenobiotic and endogenous molecules. Transporters are widely expressed in various human tissues and are routinely evaluated during the process of drug development and approval. Over the past decade, increasing evidence shows that drug transporters are important in both normal physiology and disease. Currently, transporters are utilized as therapeutic targets to treat numerous diseases such as diabetes, major depression, hypertension and constipation. Despite the steady growth of the field of transporter biology, more than half of the members in transporter superfamily have little information available about their endogenous substrate(s) or physiological functions. This review outlines current research methods in transporter studies, and summarizes the drug-transporter interactions including drug-drug and drug-endogenous substrate interactions. In the end, we also discuss the therapeutic perspective of transporters based on their physiological and pathophysiological roles.     

Sodium-phosphate cotransport in human red blood cells. Kinetics and role in membrane metabolism

Orthophosphate (Pi) uptake was examined in human red blood cells at 37 degrees C in media containing physiological concentrations of Pi (1.0- 1.5 mM). Cells were shown to transport Pi by a 4,4'-dinitro stilbene- 2,2'-disulfonate (DNDS) -sensitive pathway (75%), a newly discovered sodium-phosphate (Na/Pi) cotransport pathway (20%), and a pathway linearly dependent on an extracellular phosphate concentration of up to 2.0 mM (5%). Kinetic evaluation of the Na/Pi cotransport pathway determined the K1/2 for activation by extracellular Pi ([Na]o = 140 mM) and extracellular Na [( Pi]o = 1.0 mM) to be 304 +/- 24 microM and 139 +/- 8 mM, respectively. The phosphate influx via the cotransport pathway exhibited a Vmax of 0.63 +/- 0.05 mmol Pi (kg Hb)-1(h)-1 at 140 mM Nao. Activation of Pi uptake by Nao gave Hill coefficients that came close to a value of 1.0. The Vmax of the Na/Pi cotransport varied threefold over the examined pH range (6.90-7.75); however, the Na/Pi stoichiometry of 1.73 +/- 0.15 was constant. The membrane transport inhibitors ouabain, bumetanide, and arsenate had no effect on the magnitude of the Na/Pi cotransport pathway. No difference was found between the rate of incorporation of extracellular Pi into cytosolic orthophosphate and the rate of incorporation into cytosolic nucleotide phosphates, but the rate of incorporation into other cytosolic organic phosphates was significantly slower. Depletion of intracellular total phosphorus inhibited the incorporation of extracellular Pi into the cytosolic nucleotide compartment; and this inhibition was not reversed by repletion of phosphorus to 75% of control levels. Extracellular 32Pi labeled the membrane-associated compounds that migrate on thin-layer chromatography (TLC) with the Rf values of ATP and ADP, but not those of 2,3-bisphosphoglycerate (2,3-DPG), AMP, or Pi. DNDS had no effect on the level of extracellular phosphate incorporation or on the TLC distribution of Pi in the membrane; however, substitution of extracellular sodium with N-methyl-D-glucamine inhibited phosphorylation of the membranes by 90% and markedly altered the chromatographic pattern of the membrane-associated phosphate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purinergic-induced ion current in monolayers of C7-MDCK cells: role of basolateral and apical ion transporters

This study examines purinergic modulation of short-circuit current (I(SC)) in monolayers of C7- and C11-MDCK cells resembling principal and intercalated cells from collecting ducts. In C7 monolayers, basolateral and apical application of ATP led to similar elevation of I(SC), consisting of a transient phase with maximal I(SC) increment of approximately 10 microA/cm2 terminating in 2-3 min, and a sustained phase with maximal I(SC) less than 2 microA/cm2 and terminating in 10 min. ATP-induced I(SC) was insensitive to the presence of Na+, Cl- and inhibitors of K+ (Ba2+, charibdotoxin (ChTX), clotrimazole (CLT), apamin) and Na + (amiloride) channels in the mucosal solution. Inhibitors of Cl- channels, DIDS and NPPB, added to apical membranes at a concentration of 100 microM, did not affect ATP-induced I(SC), whereas at 500 microM, NPPB inhibited it by 70-80%. Substitution of Cl- with NO3- in serosal medium decreased ATP-induced I(SC) by 2-3-fold and elevation of [K+]o from 6 to 60 mM changed its direction. Basolateral NPPB inhibited I(SC) by 10-fold with ED50 of approximately 30 microM, whereas ChTX (50 nM) and CLT (2 microM) diminished this parameter by 30-50%. Suppression of Na+, K+, Cl- cotransport with bumetanide did not affect the transient phase of ATP-induced I(SC) and slightly diminished its sustained phase. ATP increased ouabainand bumetanide-resistant K+ (86Rb) influx across the basolateral membrane by 7-8-fold, which was partially inhibited with ChTX and CLT. ATP-treated C11 cells exhibited negligible I(SC), and their presence did not affect I(SC) triggered by ATP in C7 cells. Thus, our results show that transcellular ion current in ATP-treated C7 cells is mainly caused by the coupled function of apical and basolateral anion transporters providing transient Cl- secretion. These transporters possess different sensitivities to anion channel blocker NPBB and are under the control of basolateral K+ channels(s) inhibited by ChTX and CLT.     

Spatially-resolved eigenmode decomposition of red blood cells membrane fluctuations questions the role of ATP in flickering

Red blood cells (RBCs) present unique reversible shape deformability, essential for both function and survival, resulting notably in cell membrane fluctuations (CMF). These CMF have been subject of many studies in order to obtain a better understanding of these remarkable biomechanical membrane properties altered in some pathological states including blood diseases. In particular the discussion over the thermal or metabolic origin of the CMF has led in the past to a large number of investigations and modeling. However, the origin of the CMF is still debated. In this article, we present an analysis of the CMF of RBCs by combining digital holographic microscopy (DHM) with an orthogonal subspace decomposition of the imaging data. These subspace components can be reliably identified and quantified as the eigenmode basis of CMF that minimizes the deformation energy of the RBC structure. By fitting the observed fluctuation modes with a theoretical dynamic model, we find that the CMF are mainly governed by the bending elasticity of the membrane and that shear and tension elasticities have only a marginal influence on the membrane fluctations of the discocyte RBC. Further, our experiments show that the role of ATP as a driving force of CMF is questionable. ATP, however, seems to be required to maintain the unique biomechanical properties of the RBC membrane that lead to thermally excited CMF.     

Kinetic characteristics of the sulfate self-exchange in human red blood cells and red blood cell ghosts

Summary The sulfate and the chloride self-exchange fluxes were determined by measuring the rate of the tracer efflux from radioactively labeled human red blood cells and red blood cell ghosts. The concentration dependence and the pH-dependence of the sulfate self-exchange flux were studied. In addition, the effects of some monovalent and divalent anions on the sulfate and the chloride self-exchange fluxes were investigated.The sulfate self-exchange fluxes saturate, exhibiting a concentration maximum at sulfate concentrations between 100 and 300mm (25°C). The position of the concentration maximum depends upon pH. At high sulfate concentrations a self-inhibition of the flux becomes apparent. The apparent half-saturation constant and the apparent self-inhibition constant at pH 7.2 were 30mm and 400mm respectively. Within the pH range of 6.3–8.5, both constants decreased with increasing pH. No saturation of the sulfate self-exchange flux was observed if the sulfate concentration was raised by substituting sulfate for isoosmotic amounts of a second salt (NaCl, NaNO₃, Na-acetate, Na-lactate, Na-succinate or Na₂HPO₄). Red blood cells and red blood cell ghosts display the same pattern of concentration responsiveness.The sulfate self-exchange flux exhibits a pH-maximum at about pH 6.2 (37°C). The location of the pH-maximum is little affected by variations of the sulfate concentration. The logarithmic plots (log vs. pH) revealed that the flux/pH relation can be approximated by two straight lines. The slopes of the alkaline branches of the flux/pH curves range from –0.55 to –0.86, the slopes of the branches of the curves range from 0.08 to 1.14 and were strongly affected by changes of the sulfate concentrations. The apparent pK's obtained from the alkaline and from the acidic branches of the flux/pH curves were about 7.0 and 6.0, respectively. Intact red blood cells and red blood cell ghosts display the same type of pH-dependency of the sulfate self-exchange flux.The sulfate self-exchange flux is competitively inhibited by nitrate, chloride, acetate, oxalate and phosphate. The chloride self-exchange flux is competitively inhibited by thiocyanate, nitrate, sulfate and phosphate. The inhibition constants for the various anion species increase in the given sequence.The results of our studies indicate that the sulfate self-exchange flux is mediated by a two-site transport mechanism consisting either of a mobile carrier or a two-site pore. The experiments reported in this paper do not permit distinguishing between both transport mechanisms. The similarities of the sulfate and the chloride self-exchange flux and the mutual competition between sulfate and chloride point to a common transport system for both anion species.     

Loss of membrane phospholipid asymmetry during activation of blood platelets and sickled red cells; mechanisms and physiological significance

Membrane phospholipid asymmetry is considered to be a general property of biological membranes. Detailed information is presently available on the non-random orientation of phospholipids in red cell- and platelet membranes. The outer leaflet of the lipid bilayer membrane is rich in choline-phospholipids, whereas amino-phospholipids are abundant in the inner leaflet. Studies with blood platelets have shown that these asymmetries are not maintained when the cells are activated in various ways. Undoing the normal asymmetry of membrane phospholipids in activated blood cells is presumably mediated by increased transbilayer movement of phospholipids. This process, which leads to increased exposure of negatively charged phosphatidylserine at the outer surface, plays an important physiological role in local blood clotting reactions. A similar phenomenon occurs in sickled red cells. Phospholipid vesicles breaking off from reversibly sickled cells contribute similarly to intravascular clotting in the crisis phase of sickle cell disease.The loss of membrane phospholipid asymmetry in activated platelets seems to be strictly correlated with degradation of cytoskeletal proteins by endogenous calpain. It is remarkable that membrane phospholipid asymmetry can be (partly) restored when activated platelets are treated with reducing agents. This leads to disappearance of phosphatidylserine from the outer leaflet where it was previously exposed during cell activation. These observations will be discussed in relation to two mechanisms which have been recognized to play a role in the regulation of membrane phospholipid asymmetry; i.e. the interaction of aminophospholipids to cytoskeletal proteins, and the involvement of a phospholipid-translocase catalyzing outward-inward transbilayer movement of amino-phospholipids.     

The recognition of red blood cells by macrophages: role of phosphatidylserine and possible implications of membrane phospholipid asymmetry

The recognition of phosphatidylserine (PS) by macrophages was investigated using inside-out (IO) red blood cell (RBC) ghosts and RBC displaying PS in their surface membranes. This was accomplished by employing unmodified pathologic sickle RBC which contain endogenous PS in their outer membrane leaflet, and RBC modified by the transfer of an exogenous fluorescent PS analog. Proper insertion of exogenous PS was confirmed by monitoring the degree to which cell-associated lipid fluorescence was dequenched following transfer of 1-acyl-2-[(N-4-nitro-benzo-2-oxa-1,3 diazole) aminocaproyl] phosphatidylserine (NBD-PS) from a population of self-quenched donor vesicles. Inside-out RBC ghosts were endocytosed approximately 3 times faster than were right side-out control populations. Similarly, using NBD-PS vesicles at concentrations at which dilution of all the cell-associated analog in the recipient RBC could be unequivocally confirmed, we observed that the uptake of NBD-PS treated RBC by macrophages was significantly increased over that of control RBC populations. Fluorescence and electron microscopic observations revealed the formation of typical RBC-macrophage rosettes that were morphologically distinct from opsonized RBC-macrophage rosettes. Enhanced RBC binding to macrophages was also obtained with deoxygenated reversibly sickled cells (RSC); the enhancement correlated with increased exposure of outer leaflet PS in these cells. These findings suggest that PS is recognized by macrophages and that its exposure in the outer leaflet of RBC may have significant pathophysiologic implications.     

Polymerization of membrane components in aging red blood cells

The addition of malonyldialdehyde to red blood cells in vitro causes the formation of fluorescent chromolipids characteristics of those produced during the peroxidation of endogenous membrane phospholipids. Additionally, gel electrophoresis reveals that this agent also causes a decrease in bands 1 and 2 of spectrin as well as an increase in high molecular weight protein polymers. These same changes are observed in membranes of older cell populations fractionated from freshly drawn, untreated blood. The results obtained suggest that polymerization of membrane components, subsequent to the peroxidation of membrane lipids, may contribute to the altered biochemical and mechanical properties of aging cells and to their eventual sequestration.     

Relationship between the shape and the membrane potential of human red blood cells

Summary Microscopic observations of isotonic suspensions of human red blood cells demonstrate that cell shape is unaltered when the transmembrane electrical potential, orE _m , is set in the range –85 to +10 mV with valinomycin at varied external K⁺, or K _o .E _m was measured with the fluorescent potentiometric indicator, diS-C₃(5), as calibrated by a pH method. Repeating Glaser's experiments in which echinocytosis was attributed to hyperpolarization, we found that at low ionic strength the pH-dependent effects of amphotericin B appear to be unrelated toE _m . The effects of increased intracellular Ca²⁺, or Ca _o , on echinocytosis and onE _m are separable. With Ca ionophore A23187 half-maximal echinocytosis occurs at greater Ca _o than that which induces the half-maximal hyperpolarization associated with Ca-induced K⁺ conductance (Gardos effect). Thus, cells hyperpolarized by increased Ca _o remain discoidal when Ca is below the threshold for echinocytosis. With A23187 and higher Ca _o , extensive echinocytosis occurs in cells which are either hyperpolarized or at their resting potential. The Ca-activation curve for echinocytosis is left-shifted by low K _o , a new observation consistent with increased DIDS-sensitive uptake of⁴⁵Ca by hyperpolarized cells. These results support the following conclusions: (1) the shape and membrane potential of human red blood cells are independent under the conditions studied; (2) in cells treated with A23187, the Gardos effect facilitates echinocytosis by increasing Ca.     

Physiological ageing of red blood cells and changes in membrane carbohydrates

Evidence is presented to indicate a generalized role for the terminal sialic acid residues of circulating erythrocytes. After reinjection into their donors, neuraminidase-treated human, rabbit, rat and dog erythrocytes were promptly removed from the circulation : intect erythrocytes, previously incubated under the same conditions but without neuraminidase, were removed after a significantly longer period. The neuraminidase-treated erythrocytes were cleared by the liver and in a little part by the spleen. Old and young human, rabbit, rat erythrocytes contained different quantities of stromal sialic acid, significantly lowered on the old cells. But the half-life of old intact rabbit erythrocytes is sigificantly shorter than that of neuraminidase-treated young erythrocytes with a similar minidase-treated young erythrocytes with a similar sialic acid content. Indeed sialic acid is not the only carbohydrate component of the membrane that is decreased during erythrocyte ageing, the others membranous sugars are decreased too. Theses changes in the carbohydrate moity could have a role in the clearance of the erythrocytes.     

Adaptation of plants to salt stress: the role of the ion transporters

Adaptation to high salinity is achieved by cellular ion homeostasis which involves regulation of toxic sodium ion (Na⁺) and Chloride ion (Cl⁻) uptake, preventing the transport of these ions to the aerial parts of the plants and vacuolar sequestration of these toxic ions. Ion transporters have long been known to play roles in maintaining ion homeostasis. Na⁺ enters the cell through various voltage dependent selective and non-selective ion channels. High Na⁺ concentration in the plasma membrane is balanced either by uptake of potassium ion (K⁺) by various potassium importing channels, by salt exclusion mechanism or by sequestration of Na⁺ in the vacuoles. Therefore, the role of high-affinity potassium transporter, the salt overly sensitive pathway, the most well-defined Na⁺ exclusion pathway that exports Na⁺ from cell into xylem and tonoplast localized cation transporters that compartmentalizes Na⁺ in vacuoles need to be studied in detail and applied to make the plant adaptable to saline soil. Knowledge on the regulation of expression of these transporters by the hormones, microRNAs and other non-coding RNAs can be utilized to manipulate the ion transport. Here, we reviewed paradigm of the ion transporters in salt stress signalling pathways from the recent and past studies aiding transformation of basic knowledge into biotechnological applications to generate engineered salt stress tolerant crops.

     

Ionic strength-dependent alterations of membrane structure of red blood cells

Electron paramagnetic resonance (EPR) measurements using various fatty acid spin labels were performed on membranes of intact human erythrocytes at physiological, and at low ionic strength. In the case of spin probes bearing the nitroxide near the polar head group, a less restricted motion at low ionic strength was seen than with those labels with a nitroxide deeper within the hydrophobic tail of the membrane. Although these data clearly show an influence of ionic strength on membrane structure, and possibly a modified protein-lipid interaction, they cannot be simply discussed in terms of an altered membrane fluidity.