Band-3 protein function in human erythrocytes: effect of oxygenation-deoxygenation

Sulfate transport by band-3 protein in adult human erythrocytes was shown to be modulated by oxygen pressure. In particular, a higher transport activity was measured under high oxygen pressure than at low one (0.0242±0.0073 vs. 0.0074±0.0010 min⁻¹). Other factors, such as magnesium ions and orthovanadate, which can indirectly affect the binding properties of the cytoplasmic domain of band 3 (cdb3), influence significantly the anion exchanger activity. No effect of oxygen pressure on sulfate transport was found in chicken erythrocytes, which may be related to their lacking the cdb3 binding site. These findings are fully consistent with a molecular mechanism where the oxygen-linked transition of hemoglobin (T→R) could play a key role in the regulation of anion exchanger activity.     

A transport metabolon. Functional interaction of carbonic anhydrase II and chloride/bicarbonate exchangers

The cytoplasmic carboxyl-terminal domain of AE1, the plasma membrane chloride/bicarbonate exchanger of erythrocytes, contains a binding site for carbonic anhydrase II (CAII). To examine the physiological role of the AE1/CAII interaction, anion exchange activity of transfected HEK293 cells was monitored by following the changes in intracellular pH associated with AE1-mediated bicarbonate transport. AE1-mediated chloride/bicarbonate exchange was reduced 50-60% by inhibition of endogenous carbonic anhydrase with acetazolamide, which indicates that CAII activity is required for full anion transport activity. AE1 mutants, unable to bind CAII, had significantly lower transport activity than wild-type AE1 (10% of wild-type activity), suggesting that a direct interaction was required. To determine the effect of displacement of endogenous wild-type CAII from its binding site on AE1, AE1-transfected HEK293 cells were co-transfected with cDNA for a functionally inactive CAII mutant, V143Y. AE1 activity was maximally inhibited 61 +/- 4% in the presence of V143Y CAII. A similar effect of V143Y CAII was found for AE2 and AE3cardiac anion exchanger isoforms. We conclude that the binding of CAII to the AE1 carboxyl-terminus potentiates anion transport activity and allows for maximal transport. The interaction of CAII with AE1 forms a transport metabolon, a membrane protein complex involved in regulation of bicarbonate metabolism and transport.     

Reversible DIDS binding to band 3 protein in human erythrocyte membranes

Reversible binding of DIDS [4,4'-diisothiocyanato-2,2'-stilbenedisulphonate] to Band 3 protein, the anion exchanger located in erythrocyte plasma membrane, was studied in human erythrocytes. For this purpose, the tritiated form of DIDS ([3H]DIDS) has been synthesized and the filtering technique has been used to follow the kinetics of DIDS binding to the sites on Band 3 protein. The obtained results showed monophasic kinetics both for dissociation and association of the 'DIDS--Band 3' complex at 0 degree C in the presence of 165 mM KCl outside the cell (pH 7.3). A pseudo-first order association rate constant k+1 was determined to be (3.72 +/- 0.42) x 10(5) M-1 s-1, while the dissociation rate constant K-1 was determined to be (9.40 +/- 0.68) x 10(-3) s-1. The dissociation constant KD, calculated from the measured values of k-1 and k+1, was found to be 2.53 x 10(-8) M. The standard thermodynamics parameters characterizing reversible DIDS binding to Band 3 protein at 0 degree C were calculated. The mean values of the activation energies for the association and dissociation steps in the DIDS binding mechanism were determined to be (34 +/- 9) kJ mole-1 and (152 +/- 21) kJ mole-1, respectively. The results provide, for the first time, evidence for the reversibility of DIDS binding to Band 3 protein at 0 degree C. The existence of a stimulatory site is suggested, nearby the transport site on the Band 3 protein. The binding of an anion to this site can facilitate (through electrostatic repulsion interaction between two anions) the transmembrane movement of another anion from the transport site.     

Kinetic properties of sodium transport pathways in the river lamprey Lampetra fluviatilis erythrocytes

To activate Na+/H+ exchange, intracellular pH (pHi) of erythrocytes of the river lamprey Lampetra fluviatilis were changed from 6 to 8 using nigericin. The Na+/H+ exchanger activity was estimated from the values of amiloride-sensitive components of Na+ (22Na) inflow or of H+ outflow from erythrocytes. Kinetic parameters of the carrier functioning were determined by using Hill equation. Dependence of Na+ and H+ transport on pHi value is described by hyperbolic function with the Hill coefficient value (n) close to 1. Maximal rate of ion transport was within the limits of 9-10 mmol/l cells/min, and the H+ concentration producing the exchanger 50% activation amounted to 0.6-1.0 microM. Stimulation of H+ outcome from acidified erythrocytes (pHi 5.9) with increase of H+ concentration in the incubation medium is described by Hill equation with n value of 1.6. Concentration of Na+: for the semimaximal stimulation of H+ outcome amounted to 19 mM. The obtained results indicate the presence in lamprey erythrocytes of only one binding site for H+ from the cytoplasm side and the presence of positive cooperativity in Na+ binding from the extracellular side of the Na+/H+ exchanger. Its efflux from cells in the Na+ -free medium did not change at a 10-fold increase of H+ concentration in the incubation medium. The presented data indicate differences of kinetic properties of the lamprey erythrocyte Na+/H+ exchanger and of this carrier isoforms in mammalian cells. In intact erythrocytes the dependence of the amiloride-sensitive Na+ inflow on its concentration in the medium is described by Hill equation with n 1.5. The Na+ concentration producing the 50% transport activation amounted to 39 mM and was essentially higher as compared with that in acidified erythrocytes. These data confirm the concept of the presence of two amiloride-sensitive pathways of Na+ transport in lamprey erythrocytes.     

Structure and stability of hereditary spherocytosis mutants of the cytosolic domain of the erythrocyte anion exchanger 1 protein

Anion exchanger 1 (AE1, Band 3) is the predominant membrane protein of erythrocytes. Its 52 kDa C-terminal domain functions as a chloride-bicarbonate exchanger, while its 43 kDa N-terminal cytosolic domain (cdb3) anchors the cytoskeleton to the membrane. Several proteins bind to cdb3, including protein 4.2, a cytoskeletal protein. Three mutations in cdb3 are associated with hereditary spherocytosis (HS) and decreased levels of protein 4.2 in erythrocytes. In this study, these cdb3 mutants (E40K, G130R, and P327R) were expressed in and purified from Escherichia coli. Sedimentation experiments showed that the wild-type and mutant proteins are dimers. No difference in secondary structure between mutant and wild-type proteins was detected using circular dichroism (CD) analysis. The wild-type and mutant proteins underwent similar pH-dependent conformational changes when monitored by intrinsic tryptophan fluorescence. Urea denaturation of proteins monitored by intrinsic fluorescence showed no significant differences in the sensitivity of the proteins to this chemical denaturant. Thermal denaturation monitored by CD and by calorimetry revealed that only the P327R mutant had a significantly lower midpoint of transition (approximately 5 degrees C) than the wild-type protein, suggesting a modest decrease in stability. The results show that the HS mutant cdb3 proteins do not differ to any great extent in structure from the wild-type protein, suggesting that the HS mutations may directly affect protein 4.2 binding.     

Anion exchange in human erythrocytes has a large activation volume

Sulfate equilibrium exchange in human red cells has an activation volume of +150 +/- 20 cm3/mol over the pressure range 0.1 to 83 MPa (15 to 12000 lb/in2) at 30 degrees C. This value greatly exceeds the expected contribution from sulfate binding to the anion exchanger. We suggest that the activation volume reflects conformational changes during the transport cycle.     

Expression of band 3 anion exchanger induces chloride current and taurine transport: structure-function analysis.

Most, but not all, cell types release intracellular organic solutes (e.g. taurine) in response to cell swelling to achieve cell volume regulation. Although this efflux is blocked by classical inhibitors of the electroneutral anion exchanger band 3 (AE1), it is thought to involve an anion channel. The role of band 3 in volume-dependent taurine transport was determined by expressing, in Xenopus oocytes, band 3 from erythrocytes which do (trout) or do not (mouse) release taurine when swollen. AE1 of both species elicited anion exchange activity, but only trout band 3 showed chloride channel activity and taurine transport. Chimeras constructed from trout and mouse band 3 allowed the identification of some protein domains critically associated with channel activity and taurine transport. The data provide evidence that swelling-induced taurine movements occur via an anion channel which is dependent on, or controlled by, band 3. They suggest the involvement of proteins of the band 3 (AE) family in cell volume regulation.     

Determination of structural models of the complex between the cytoplasmic domain of erythrocyte band 3 and ankyrin-R repeats 13-24

The adaptor protein ankyrin-R interacts via its membrane binding domain with the cytoplasmic domain of the anion exchange protein (AE1) and via its spectrin binding domain with the spectrin-based membrane skeleton in human erythrocytes. This set of interactions provides a bridge between the lipid bilayer and the membrane skeleton, thereby stabilizing the membrane. Crystal structures for the dimeric cytoplasmic domain of AE1 (cdb3) and for a 12-ankyrin repeat segment (repeats 13-24) from the membrane binding domain of ankyrin-R (AnkD34) have been reported. However, structural data on how these proteins assemble to form a stable complex have not been reported. In the current studies, site-directed spin labeling, in combination with electron paramagnetic resonance (EPR) and double electron-electron resonance, has been utilized to map the binding interfaces of the two proteins in the complex and to obtain inter-protein distance constraints. These data have been utilized to construct a family of structural models that are consistent with the full range of experimental data. These models indicate that an extensive area on the peripheral domain of cdb3 binds to ankyrin repeats 18-20 on the top loop surface of AnkD34 primarily through hydrophobic interactions. This is a previously uncharacterized surface for binding of cdb3 to AnkD34. Because a second dimer of cdb3 is known to bind to ankyrin repeats 7-12 of the membrane binding domain of ankyrin-R, the current models have significant implications regarding the structural nature of a tetrameric form of AE1 that is hypothesized to be involved in binding to full-length ankyrin-R in the erythrocyte membrane.     

Band 3 protein function in teleost fish erythrocytes: effect of oxygenation-deoxygenation

During vertebrate evolution, structural changes in red blood cells (RBC) and hemoglobin (Hb), have probably resulted in the importance of blood carbon dioxide transport. The chloride/bicarbonate exchange across the RBC membrane, which is an integral part of the blood CO(2) transport process in vertebrates, has been examined on two different species of teleost fish, Euthynnus alletteratus and Thunnus thynnus, at several oxygenation states of erythrocyte HOS (high-oxygenation state, about 90 % of saturation) and LOS (low-oxygenation state, about 15 % of saturation). The results were compared with those observed in human RBC under the same experimental conditions and with the chicken (Gallus gallus) erythrocytes, which have particular modifications at the N-terminus of the band 3 protein (B3). In fish the kinetic measurements have shown a different anion transport in several oxygenation states of erythrocytes, indicating that also at lower levels of vertebrate evolution there exists a modulation of the anionic flow affected by oxygen. The functional correlation of anion transport to changes of parts of the hemoglobin sequence responsible for alterations in the interactions with the cytoplasmic domain of band 3 protein (cdb3) allowed us to suggest a hypothesis about fish physiology. The highest values of kinetic measurements observed in fish have been attributed to the metabolic need of the RBC in response to the removal of CO(2) that in teleosts is also of endogenous origin.     

An intramolecular transport metabolon: fusion of carbonic anhydrase II to the COOH terminus of the Cl(-)/HCO(3)(-)exchanger, AE1

Anion exchanger 1 (AE1) is the plasma membrane Cl(-)/HCO(3)(-) exchanger of erythrocytes. Carbonic anhydrases (CA) provide substrate for AE1 by catalyzing the reaction, H(2)O + CO(2) ? HCO(3)(-) + H(+). The physical complex of CAII with AE1 has been proposed to maximize anion exchange activity. To examine the effect of CAII catalysis on AE1 transport rate, we fused either CAII-wild type or catalytically inactive CAII-V143Y to the cytoplasmic COOH terminus of AE1 to form AE1.CAII and AE1.CAII-V143Y, respectively. When expressed in transfected human embryonic kidney 293 cells, AE1.CAII had a similar Cl(-)/HCO(3)(-) exchange activity to AE1 alone, as assessed by the flux of H(+) equivalents (87 ± 4% vs. AE1) or rate of change of intracellular Cl(-) concentration (93 ± 4% vs. AE1), suggesting that CAII does not activate AE1. In contrast, AE1.CAII-V143Y displayed transport rates for H(+) equivalents and Cl(-) of 55 ± 2% and of 40 ± 2%, versus AE1. Fusion of CAII to AE1 therefore reduces anion transport activity, but this reduction is compensated for during Cl(-)/HCO(3)(-) exchange by the presence of catalytically active CAII. Overexpression of free CAII-V143Y acts in a dominant negative manner to reduce AE1-mediated HCO(3)(-) transport by displacement of endogenous CAII-wild type from its binding site on AE1. To examine whether AE1.CAII bound endogenous CAII, we coexpressed CAII-V143Y along with AE1 or AE1.CAII. The bicarbonate transport activity of AE1 was inhibited by CAII-V143Y, whereas the activity of AE1.CAII was unaffected by CAII-V143Y, suggesting impaired transport activity upon displacement of functional CAII from AE1 but not AE1.CAII. Taken together, these data suggest that association of functional CAII with AE1 increases Cl(-)/HCO(3)(-) exchange activity, consistent with the HCO(3)(-) transport metabolon model.     

Binding properties of the stilbene disulfonate sites on human erythrocyte AE1: kinetic, thermodynamic, and solid state deuterium NMR analyses.

A novel stilbene disulfonate, 4-trimethylammonium-4'-isothiocyanostilbene-2,2'-disulfonic acid (TIDS), has been chemically synthesized, and the interaction of this probe with human erythrocyte anion exchanger (AE1) was characterized. Covalent labeling of intact erythrocytes by [N(+)((14)CH(3))(3)]TIDS revealed that specific modification of AE1 was achieved only after removal of other ligand binding sites by external trypsinization. Following proteolysis, (1.2 +/- 0.4) x 10(6) TIDS binding sites per erythrocyte could be blocked by prior treatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a highly specific inhibitor of AE1. Inhibition of sulfate equilibrium exchange by TIDS in whole cells was described by a Hill coefficient of 1.10 +/- 0.06, which reduced to 0.51 +/- 0.01 following external trypsinization. The negative cooperativity of TIDS binding following external trypsinization suggests that trypsin-sensitive proteins modulate allosteric coupling between AE1 monomers. Thermodynamic analysis revealed that TIDS binding induces smaller conformational changes in AE1 than is observed following DIDS binding. The similar inhibitory potencies of both TIDS (IC(50) = 0.71 +/- 0.48 microM) and DIDS (IC(50) = 0.2 microM) imply that there is no correlation between the ability of stilbene disulfonates to arrest anion exchange function and the magnitude of ligand-induced conformational changes in AE1. Solid state (2)H NMR analysis of a [N(+)(CD(3))(3)]TIDS-AE1 complex in both unoriented and macroscopically oriented membranes revealed that large amplitude "wobbling" motions describe ligand dynamics. The data are consistent with a model where TIDS bound to AE1 is located exofacially in contact with the bulk aqueous phase.     

Intracellular pH regulation in rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) hepatocytes: the activity of sodium/proton exchange is oxygen-dependent

We studied pH regulation in freshly isolated rainbow trout hepatocytes using microspectrofluorometry with the fluorescent dye BCECF. In accordance with earlier data on rainbow trout hepatocytes, ion substitution (N-methyl D-glucamine for sodium and gluconate for chloride) and transport inhibitor [10 microM M methyl isobutyl amiloride (MIA) to inhibit sodium/proton exchange and 100 microM DIDS to inhibit bicarbonate transport] studies in either Hepes-buffered or bicarbonate/carbon dioxide-buffered media (extracellular pH 7.6) indicated a role for sodium/proton exchange, sodium-dependent bicarbonate transport, and sodium-independent anion exchange in the regulation of hepatocyte pH. In Hepes-buffered medium, the activity of the sodium/proton exchanger (i.e. proton extrusion inhibited by MIA) was greater at 1% than at 21% oxygen. The oxygen dependency of the sodium/proton exchange is not caused by hydroxyl radicals, which appear to mediate the oxygen sensitivity of potassium-chloride cotransport in erythrocytes.     

Adenosine cyclic 3',5'-monophosphate uptake and regulation of membrane protein kinase in intact human erythrocytes

The uptake of adenosine cyclic 3',5'-monophosphate (cAMP) and stimulation of membrane-associated protein kinase in mature human erythrocytes were investigated. cAMP transport across the membrane was temperature dependent, and cAMP binding to the isolated membrane had less temperature dependence. More than 99% of the [3H]-cAMP taken up by erythrocytes was nonmembrane bound. Maximal stimulation of membrane protein kinase and maximal occupancy of membrane cAMP binding sites by extracellular cAMP cccurred at 30 degrees C within 30 min after initiation of the incubation of erythrocytes with cAMP. The concentration of extracellular cAMP that gave half-maximal stimulation of membrane protein kinase was 5.4 X 10-4 M, a value consistent with the concentrations of cAMP (5.2 X 10-4 M) found to occupy half-maximally the membrane cAMP binding sites in erythrocytes. Extracellular cAMP and to a lesser extent guanosine cyclic 3',5'-monophosphate and inosine cyclic 3',5'-monophosphate stimulated membrane protein kinase in erythrocytes. The cAMP uptake by human erythrocytes as well as cAMP binding to membranes in the erythrocyte was blocked by an inhibitor [4,4'-bis(isothiocyano)stilbene-2,2-disulfonate] of the anion channel. These studies indicate that cAMP can be transported across membranes into human erythrocytes and can bind to membranes to activate membrane protein kinase. It appears that there is a shared transport channel for cAMP and anion transport.     

Kinetics of uptake and deacetylation of N-acetylcysteine by human erythrocytes

Overproduction of reactive oxygen species associated with several diseases including sickle cell anaemia reduces the concentration of glutathione, a principal cellular antioxidant. Glutathione depletion in sickle erythrocytes increases their conversion to irreversible sickle cells that promote vaso-occlusion. Therapeutically, N-acetylcysteine partially restores glutathione concentrations but its mode of action is controversial. Following glutathione depletion, glutathione synthesis is limited by the supply of cysteine and it has been assumed that deacetylation of N-acetylcysteine within erythrocytes provides cysteine to accelerate glutathione production. To determine whether this is the case we studied the kinetics of transport and deacetylation of N-acetylcysteine. Uptake of N-acetylcysteine had a first order rate constant of 2.40+/-0.070min(-1) and only saturated above 10mM. Inhibition experiments showed that 56% of N-acetylcysteine transport was via the anion exchange protein. Deacetylation, measured using (1)H NMR, had a K(m) of 1.49+/-0.16mM and V(max) of 2.61+/-0.08micromolL(-1)min(-1). Oral doses of N-acetylcysteine increase glutathione concentrations in sickle erythrocytes at plasma N-acetylcysteine concentrations of approximately 10microM. At this concentration, calculated rates of N-acetylcysteine uptake and deacetylation were approximately 5% of the rate required to maintain normal glutathione production. We concluded that on oral administration, intracellular deacetylation of N-acetylcysteine supplies little of the cysteine required for accelerated glutathione production. Instead, N-acetylcysteine acts by freeing bound cysteine in the plasma that then enters the erythrocytes. To be effective, intracellular cysteine precursors must be designed to enter erythrocytes rapidly and employ enzymes with high activity within erythrocytes to liberate the cysteine.     

Estimation of the number and turnover rate of Na+/H+ exchangers in lymphocytes. Effect of phorbol ester and osmotic shrinking

Rat thymic lymphocytes possess an amiloride-sensitive Na+/H+ exchanger in their plasma membrane. Kinetic studies revealed that 5-(N-methyl-N-isobutyl)amiloride (MIA) was a more potent inhibitor of the antiport than amiloride (cf. apparent Ki of 174 nM and 6 microM, respectively). Inhibition by MIA was rapid (less than 5 s) and readily reversible. [3H]MIA binding to whole cells was assayed by rapid centrifugation following short (5 s) incubations to minimize nonspecific binding. A saturable binding component (Kd approximately equal to 170 nM) which was displaced by amiloride was detected. In contrast, there was no significant amiloride-displaceable binding to human erythrocytes, which have comparatively little Na+/H+ exchange activity. In lymphocytes, the ability of amiloride and several of its analogs to displace [3H]MIA correlated with their potency as inhibitors of the antiport. Both kinetic and binding studies revealed that extracellular H+, but not Na+, inhibited the interaction of MIA with its receptor(s). Taken together, these data suggest that [3H]MIA binds to the Na+/H+ exchanger. Scatchard analysis revealed that [3H]MIA bound to a maximum of 8000 high affinity sites/cell. Activation of Na+/H+ exchange by osmotic shrinking or by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate was not accompanied by a significant change in [3H]MIA binding. Given an upper limit of 8000 functional sites/thymocyte, we estimate that the turnover number of each maximally activated exchanger is at least 2000 cycles/s.     

Ankyrin regulation: an alternatively spliced segment of the regulatory domain functions as an intramolecular modulator.

This study of two forms of ankyrin (protein 2.1 and 2.2) from human erythrocytes has revealed a role for alternate exon usage at the level of regulation of protein interactions. The smaller form of ankyrin (protein 2.2), which lacks a portion of the regulatory domain due to alternative splicing of pre-mRNA, exhibits increased affinity for the cytoplasmic domain of the anion exchanger, spectrin, and tubulin. Direct evidence that at least one of these associations is modulated by the alternatively spliced segment of the regulatory domain is provided by experiments utilizing a polypeptide that is comprised of residues 1513-1674 corresponding to the portion of the regulatory domain missing from protein 2.2. Addition of this regulatory domain polypeptide to binding assays reversed the increase in affinity of protein 2.2 for the anion exchanger. The inhibitory activity of the regulatory domain polypeptide in these assays is accompanied by a direct interaction with a site that is available on the smaller form of ankyrin and is distinct from the binding site for the anion exchanger. These results support the idea that the alternatively spliced segment within the regulatory domain of erythrocyte ankyrin performs a repressor function and acts through an allosteric mechanism involving interaction(s) at a site separate from the binding site for the anion exchanger.     

Chinese hamster ovary cell mutants deficient in an anion exchanger functionally similar to the erythroid band 3

Studies in Chinese hamster ovary cells demonstrate the presence of an anion exchanger, which has some of the properties of the band 3 transporter in erythrocytes. 1) Extracellular chloride is a competitive inhibitor of sulfate influx and stimulates sulfate efflux, suggesting that the mechanism of uptake is SO2-(4)/Cl- exchange. 2) The anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibits sulfate uptake in a dose-dependent manner. Half-maximal inhibition is achieved at 0.06 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. 3) Low extracellular pH markedly stimulates sulfate uptake. A 6-fold decrease in the apparent Km is observed at pHout 5.5 as compared to pHout 7.5. However, studies carried out over a broad range of extracellular SO2-(4) concentrations indicate the presence of three components of this transport activity in Chinese hamster ovary cells: two high affinity low capacity systems, one in the range 0.5 microM less than [SO2-(4)]out less than 50 microM and one in the range 50 microM less than [SO2-(4)]out less than 150 microM, and a low affinity, high capacity system (at [SO2-(4)]out greater than 150 microM). These properties have not been previously reported for the erythroid band 3 transporter. The availability of mutants deficient in these activities has enabled us to carry out studies which suggest that the high affinity systems are functionally independent of the low affinity system, but that all systems are dependent on the same anion exchange protein. Studies in a mutant which lacks all components of the transport activity indicates that the anion exchanger may be instrumental in the regulation of the intracellular pH in Chinese hamster ovary cells.     

Oxidative damage by phenylhydrazine diminishes erythrocyte anion transport

Human erythrocytes were exposed to oxidative stress by treatment with the slowly hemolytic drug phenylhydrazine. Phenylhydrazine has been previously shown to trigger the production of toxic oxygen metabolites including O-2 and H2O2 and the formation of Heinz bodies. The concentration-dependent formation of Heinz bodies was confirmed using optical microscopy. Heinz body formation was accompanied by surface protuberances as shown by scanning electron microscopy. The formation of Heinz bodies was accompanied by inhibition of anion translocation. Anion translocation was measured using the anionic fluorescent substrate analog N-(2-aminoethylsulfonate)-7-nitrobenz-2-oxa-1,3-diazole (NBD-taurine). The efflux of NBD-taurine was measured by continuous monitoring of transport by fluorescence (CMTF). The mean value of the kinetic rate constant for transport, k, was found to be -0.090 +/- 0.017 min-1. Phenylhydrazine was found to decrease k to less than one-half of control values in a dose-dependent fashion. The disruption of anion translocation may be related to the oxidative effects of phenylhydrazine and to the generation of Heinz bodies, which bind to the N-terminal domain of band 3.     

Action of neltenexine on anion secretion in human airway epithelia

Neltenexine has been applied to human lung diseases such as chronic obstructive pulmonary disease (COPD) as a mucolytic agent. However, we have no information on the neltenexine action in bronchial epithelial cells. We studied the neltenexine action on the ion transport in human submucosal serous Calu-3 cells. Under a hyper-secreting condition caused by terbutaline (a beta2-adrenergic agonist), neltenexine diminished anion secretion by inhibiting the Cl- and HCO3- uptake via Na+/K+/2Cl- cotransporter and Na+/HCO3- cotransporter without blockade of the cystic fibrosis transmembrane conductance regulator (CFTR) channel, and also diminished anion secretion via stimulation of Cl-/HCO3- exchanger, which facilitates the extrusion of more CFTR-permeant anion, Cl-, with the uptake of less CFTR-permeant anion, HCO3-. Thus, neltenexine reduced the hyper-secretion to keep an appropriate fluid level in the airway, providing a possibility that neltenexine can be an effective drug in airway obstructive diseases by decreasing the airway resistance under a hyper-secreting condition.     

Control and consequences of adrenergic activation of red blood cell Na+/H+ exchange on blood oxygen and carbon dioxide transport in fish.

The catecholamines, adrenaline and noradrenaline, are released into the circulation of fish during a variety of physical and environmental disturbances that share the common feature of a requirement for enhanced blood oxygen transport. Indeed, the dominant factor controlling the mobilization of catecholamines from chromaffin tissue is a depression of blood oxygen content usually coinciding with a reduction of hemoglobin-O2 (Hb-O2) binding to 50-60% saturation. The elevation of plasma catecholamine levels, under such conditions, activates a beta-adrenergic cyclic AMP-dependent Na+/H+ exchanger on the red blood cell (rbc) membrane. The adrenergic responsiveness AMP-dependent Na+/H+ exchanger on the red blood cell (rbc) membrane. The adrenergic responsiveness of the rbc Na+/H+ exchanger to catecholamines varies both within and between species. Such inter- and intra-specific differences may reflect, in part, the availability of cell surface beta-adrenoceptors that are functionally coupled to adenylate cyclase. The activation of rbc Na+/H+ exchange and the accompanying profound adjustments of intracellular and extracellular acid-base status, nucleoside triphosphate (NTP) levels, and cooperativity of Hb-O2 binding have important consequences on both O2 and CO2 transfer and transport in the blood that vary markedly at the sites of oxygenation (the gill) and deoxygenation (the tissues) thereby enabling simultaneous amelioration of O2 loading and unloading. At the gill, oxygen transfer is enhanced owing to increases in Hb-O2 affinity and capacity while at the tissues, oxygen delivery is facilitated by a reduction of Hb-O2 affinity. This reduction in affinity at the tissues is a consequence of the combined effects of increased cooperativity of Hb-O2 binding and a rise in venous PCO2 (PvCO2) caused by the titration of HCO3- by H+ extruded by the rbc Na+/H+ exchanger. This elevation of PvCO2 may contribute to the rise in arterial PCO2 (PaCO2) observed after adrenergic activation of rbc Na+/H+ exchange that is caused primarily by impairment of rbc CO2 excretion related to modification of the intracellular acid-base status.