The inhibitor effect of probencid and structural analogues on organic anions and chloride permeabilities in ox erythrocytes.

Probenecid inhibits anion movements (organic anions and chloride) in ox erythrocytes. The I50 is 4. 10(-5) M. Structural analogues such as carinamide, p-carboxybenzene sulfonamide and p-carboxy N,N-diethyl benzene sulfonamide, which are drugs of the sulfonamide class, were also found to inhibit anion transport. These results reinforce the previously discussed view based on structural considerations, that sulfonamides act on the red cell membrane as competitors of anion transport. It is possible that probenecid and carinamide act in a similar way in the kidney.

Inhibition of anion transport in the red blood cell by anionic amphiphilic compounds. II. Chemical properties of the flufenamate-binding site on the band 3 protein.

Flufenamate is a powerful inhibitor of anion exchange in red blood cells. It binds to the band 3 protein involved in the transport as discussed in the preceding paper (Cousin, J.-L. and Motais, R. (1982) Biochim. Biophys. Acta 687, 147-155). The present study is concerned with the chemical properties of the inhibitory binding site. Structure-activity studies were performed with two sets of compounds derivated from anthranilate (considered as the basic structure of flufenamate). The molar concentrations required to produce 50% inhibition (I50) varied over more than a 10(4) range. The inhibitory activity was quantitatively correlated with the hydrophobic character of the molecules and the electron-withdrawing capacity of the substituents. Comparison between the inhibitory potency of flufenamate analogs made a definition of the contribution of each part of the molecule in the binding to the receptor possible. The results suggest that anionic inhibitors bind to a site which presents a positively charged groups at the water-protein interface whereas the hydrophobic part of the molecule is inserted into an hydrophobic and electron-donor region of the protein. The specificity of amphiphilic compounds towards anion transport is discussed.

Inhibition of anion transport in the red blood cell by anionic amphiphilic compounds. I. Determination of the flufenamate-binding site by proteolytic dissection of the band 3 protein.

Flufenamate, non-steroidal anti-inflammatory drug, is a powerful inhibitor of anion transport in the human erythrocyte (I50 = 6 . 10(-7) M). The concentration dependence of the binding to ghosts reveals two saturable components. [14C]Flufenamate binds with high affinity (Kd1 = 1.2 . 10(-7) M) to 8.5 . 10(5) sites per cell (the same value as the number of band 3 protein per cell); it also binds, with lower affinity (Kd2 = 10(-4) M) to a second set of sites (4.6 . 10(7) per cell). Pretreatment of cells with 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), a specific inhibitor of anion transport, prevents [14C]flufenamate binding only to high affinity sites. These results suggest that high affinity sites are located on the band 3 protein involved in anion transport. Extracellular chymotrypsin and pronase at low concentration cleave the 95 kDa band 3 into 60 kDa and 35 kDa fragments without affecting either anion transport of [14C]flufenamate binding. Splitting by trypsin at the inner membrane surface of the 60 kDa chymotryptic fragment into 17 kDa transmembrane fragment and 40 kDa water-soluble fragment does not affect [14C]flufenamate binding. In contrast degradation at the outer membrane surface of the 35 kDa fragment by high concentration of pronase or papain decreases both anion transport capacity and number of high affinity binding sites for [14C]flufenamate. Thus it appears that 35 kDa peptide is necessary is necessary for both anion transport and binding of the inhibitors and that the binding site is located in the membrane-associated domain of the band 3 protein.

Inhibition of anion and glucose permeabilities by anesthetics in erythrocytes. The mechanisms of action of positively and negatively charged drugs.

(1) The mode of action of anesthetics as inhibitors of Cl- and glucose transports in human red cells was studied. The term anesthetic is taken in its broad meaning as defined by Seeman (Seeman, P. (1972) Pharmacol. Rev. 24, 583-655) and covers anionic and cationic liposoluble compounds which reversibly block the rising phase of the action potential, without effect on the resting membrane potential. (2) Phenothiazine derivatives were chosen as prototypes of anesthetics because they represent a set of compounds having the same basic chemical structure, the phenothiazine ring, but with either a positive or a negative charge. (3) The Cl- self-exchange is inhibited by both cationic and anionic derivatives. However, to obtain the same level of inhibition, it is necessary to use a concentration 10-100 times higher with cationic than with anionic drugs. (4) At a concentration which inhibits Cl- permeability, cationic derivatives induce a very strong morphological change (cup-shaped cells: stomatocytes or spherostomatocytes) and protect erythrocytes against osmotic hemolysis, signifying that the membrane is fully expanded. Conversely, with anionic derivatives, inhibition occurs at a concentration which does not induce any apparent shape change or protect against osmotic hemolysis: there is no significant membrane expansion. (5) Glucose permeability, measured by glucose exit, is inhibited by cationic and anionic phenothiazine, but always at a concentration which fully expands the membrane as indicated by morphological changes and anti-hemolytic effects. It is interesting to point out that whilst glucose exit shows inhibition by cationic derivatives, glucose exchange flux is scarcely altered. (6) It is concluded that cationic and anionic anesthetics are general inhibitors of transmembrane solute movements involving a facilitated-diffusion process. However, the mechanism of inhibition is not identical for all: inhibition of glucose permeability by anionic and cationic anesthetics, as well as inhibition of Cl- permeability by cationic anesthetics may be of a non-specific nature and result from their interaction with the bilayer (this indirect effect is discussed); on the other hand, inhibition of Cl- permeability by anionic anesthetics may result from a specific perturbation of the transport mechanism according to recent evidence in some cases (Cousin, J.L. and Motais, R. (1979) J. Membrane Biol. 46, 125-153; Zaki, L., Ruffing, W. Gärtner, E.M., Fasold, H., Motais, R. and Passow, H. (1977) 11th FEBS Meeting, Copenhagen, A4 17-671.

Red cell volume regulation: the pivotal role of ionic strength in controlling swelling-dependent transport systems.

A volume increase of trout erythrocytes can be induced either by beta-adrenergic stimulation of a Na+/H+ antiport in an isotonic medium (isotonic swelling) or by suspending red cells in an hypotonic medium (hypotonic swelling). In both cases cells regulate their volume by a loss of osmolytes via specific pathways. After hypotonic swelling several volume-dependent pathways were activated allowing K+, Na+, taurine and choline to diffuse. All these pathways were fully inhibited by furosemide and inhibitors of the anion exchanger (DIDS, niflumic acid), and the K+ loss was mediated essentially via a 'Cl(-)-independent' pathway. After isotonic swelling, the taurine, choline and Na+ pathways were practically not activated and the K+ loss was strictly 'Cl(-)-dependent'. Thus cellular swelling is a prerequisite for activation of these pathways but, for a given volume increase, the degree of activation and the degree of anion-dependence of the K+ pathway depend on the nature of the stimulus, whether hormonal or by reduction of osmolality. It appears that the pattern of the response induced by hormonal stimulation is not triggered by either cellular cAMP (since it can be reproduced in the absence of hormone by isotonic swelling in an ammonium-containing saline) or by the tonicity of the medium in which swelling occurs since after swelling in an isotonic medium containing urea, the cells adopt the regulatory pattern normally observed after hypotonic swelling. We demonstrated that the stimulus is the change in cellular ionic strength induced by swelling: when ionic strength drops, the cells adopt the hypotonic swelling pattern; when ionic strength increases, the isotonic swelling pattern is activated. To explain this modulating effect of ionic strength a speculative model is proposed, which also allows the integration of two further sets of experimental results: (i) all the volume-activated transport systems are blocked by inhibitors of the anion exchanger and (ii) a Cl(-)-dependent, DIDS-sensitive K+ pathway can be activated in static volume trout red cells (i.e., in the absence of volume increase) by the conformational change of hemoglobin induced by the binding of O2 or CO to the heme.

Regulation of Cl-dependent K transport by oxy-deoxyhemoglobin transitions in trout red cells.

The oxygenation of trout red cells opens a Cl-dependent K pathway inhibited by furosemide, and by inhibitors of the erythrocyte anion exchanger such as DIDS and niflumic acid. The trigger is the deoxy-oxy conformational change of hemoglobin. The binding of carbon monoxide to heme, which induces a similar conformational change, mimics the effect of oxygen. The possible mechanisms enabling molecular oxygen to control the transport protein are discussed. This oxygenation-activated K transport appears to play a regulatory role in the control of the extracellular K concentration.

The chloride transport induced by triaklyl-tin compound across erythrocyte membrane.

The effect of tripropyl-tin chloride on anion permeability was studied using red cells previously treated with a covalent binding inhibitor 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) to inhibit completely and irreversibly the natural anion transport system. It was demonstrated that the tin compound can mediate chloride-hydroxide and chloride-chloride exchanges across the "impermeabilised" erythrocyte membrane. In the non hemolytic range, the rate of exchange increased with the concentration of the tin compound in a non linear fashion, and no saturation effect was seen. The temperature profile of the chloride self exchange induced by tripropyl-tin was studied and the apparent activation energy found was 29 Kcal/mol. The tripropyl-tin chloride cannot mediate a chloride-bicarbonate exchange. Because of this discriminatory effect between hydroxide and bicarbonate, the tin compound can be useful in certain experimental conditions as seen for the study of the anion "carrier" of the red cell membrane ("cousin, J.L., Motais, R. and Sola, F. (1975) J. Physiol. Lond. 253, 385-399).

Uncouplers of oxidative phosphorylation. A structure-activity study of their inhibitor effect on passive chloride permeability.

Uncoupling agents inhibit chloride transport in red blood cells, which is a metabolism-independent process. An analysis of the molecular requirements shows that this inhibitory activity is closely correlated with the electronic and the hydrophobic bonding properties of phenols: the more lipophilic and the more electron-attracting the substituent groups are, the greater the activity they confer on the parent molecule. A recent structure-activity study concerning various classes of reversible inhibitors of chloride transport led to the same conclusion (Motais, R. and Cousin, J.L. (1977) in International Conference on Biological Membranes: Drugs, Hormones and Membranes (Bolis, L., Hoffman, J.F. and Straub, R.W., eds.), Raven Press, New York, in the press). The effects of substituents on the activity of phenols as uncouplers have been recently examined (Stockdale, M. and Selwyn, M.J. (1971) Eur. J. Biochem. 21, 565). The comparison of these results with our data shows that uncoupling depends more on electronic properties of phenols than does choloride inhibition.

Desensitization by external Na of the cyclic AMP-dependent Na+/H+ antiporter in trout red blood cells.

The erythrocytes of the trout, Salmo gairdneri, react to beta-adrenergic stimulation by activating a cyclic AMP-dependent and amiloride-sensitive Na+/H+ antiporter (see Borgese, F., F. Garcia-Romeu, and R. Motais, Journal of General Physiology, 1986, 87:551-566). The present study traces the kinetic behavior of the unidirectional Na fluxes after stimulation by isoproterenol. A very considerable increase (100-fold) of the unidirectional Na influx (JNa(in)) follows the addition of isoproterenol to the erythrocyte suspension. After 1.5 min, JNa(in) falls suddenly, and asymptotically diminishes toward the nonstimulated flux level. The unidirectional Na efflux (JNa(out)) proceeds according to similar kinetics. The decrease of JNa(in) and JNa(out)is not linked to either a change in the driving forces of the transported ions or a decrease of the cyclic AMP concentration but to a desensitization of the Na+/H+ antiporter. This desensitization is dependent on the external Na concentration and is not controlled by internal Na, cell swelling, or external Ca.

Exchange diffusion effect and euryhalinity in teleosts.

The sea water (SW)-adapted euryhaline Platichthys flesus, and the marine Serranus exchange about 50% of their internal sodium with the external sodium per hour. This rate of exchange decreases with decreasing salinity of the adaptation medium. When the flounder is transferred from SW to FW an instantaneous 90% reduction of the Na and Cl outflux is observed. About 30 min later a second, progressive, reduction occurs. The outflux reductions appear to result from two types of regulatory mechanisms reducing gill permeability and preventing excessive salt loss. The first reduction corresponds to independent "Na- and Cl-free effects" as shown by transfers to artificial media containing either Na or Cl with an impermeant co-ion. The pattern of simultaneous rapid variations of Na influx and outflux for a range of salinity changes in flounder adapted to SW, 1/2 SW, or 1/4 SW has been studied. The data are compatible with the hypothesis of an exchange diffusion mechanism characterized by a coupling of both unidirectional fluxes. The affinity of the exchange diffusion carrier for sodium has been measured (Km approximately equal to 400 mM). The delayed reduction would result from a progressive diminution of the quantity of carrier available but without modification of its affinity for sodium. When the stenohaline marine perch is transferred from SW to FW, a 40% reduction of the outflux is observed. But it is not the result of an exchange diffusion effect as it is related to the external osmolarity change and not to the NaCl concentration change. Furthermore no delayed reduction is observed after transfer into FW. This transfer is accompanied by a heavy loss of electrolytes resulting in a rapid decline of the plasma electrolyte level and death. A comparative survey of the relative importance of these regulatory mechanisms has been made.

The control of Na+/H+ exchange by molecular oxygen in trout erythrocytes. A possible role of hemoglobin as a transducer.

It has previously been shown that addition of catecholamines to a suspension of trout erythrocytes induces an enlargement of the cells owing to an uptake of NaCl mediated by a cAMP-dependent, amiloride-sensitive Na+/H+ exchange. In this article, we show that the change in cell volume induced by catecholamines is much greater when the erythrocytes are incubated in N2 than when they are in O2. This difference is explained by an inhibition of the cAMP-dependent Na+/H+ exchange by O2. The inhibition is not reversed in cells incubated in O2 but poisoned with cyanide. It cannot be explained by a difference in the content of cAMP in O2 and in N2. In a CO atmosphere, in which the cells are anoxic, swelling and Na permeability are not increased as they are in N2: in CO, the cells behave as they do in O2. Moreover, cells previously exposed to CO and then put in an N2 atmosphere do not show the expected increase in Na+/H+ exchange. This strongly indicates that the binding of CO to hemoglobin, which persists during the subsequent exposure to N2, is the primary event responsible for the inhibition. As CO substitutes for O2 in binding to hemoglobin, the effect of O2 in the control of Na+/H+ exchange is probably explained by this interaction with heme. (Allen and McManus [1968. Biophysical Journal. 8:125a] previously described a similar effect of CO on passive Na permeability in duck red cells.) It is proposed that the hemoglobin, by interacting differently, according to its degree of oxygenation, with the cytoplasmic segment of band 3 protein, may influence some transport function, such as Na+/H+ exchange. The physiological significance of a control of Na+/H+ exchange by molecular O2 is discussed.

Catecholamine-induced transport systems in trout erythrocyte. Na+/H+ countertransport or NaCl cotransport?

It has previously been shown (Baroin, A., F. Garcia-Romeu, T. Lamarre, and R. Motais. 1984a, b. Journal of Physiology. 350:137, 356:21; Mahé, Y., F. Garcia-Romeu, and R. Motais. 1985. European Journal of Pharmacology. 116:199) that the addition of catecholamines to an isotonic suspension of nucleated red blood cells of the rainbow trout first stimulates a cAMP-dependent, amiloride-sensitive Na+/H+ exchange. This stimulation seems to be transient. It is followed by a more permanent activation of a coupled entry of Na+ and Cl-, which is inhibited by amiloride but also by inhibitors of band 3 protein (DIDS, furosemide, niflumic acid). The coupled entry of Na+ and Cl- could therefore result from the parallel and simultaneous exchange of Na+out for H+in (via the cAMP-dependent Na+/H+ antiporter) and Cl- out for HCO3- in (via the anion exchange system located in band 3 protein). However, in view of the following arguments, it had been proposed that NaCl uptake does not proceed by the double-exchanger system but via an NaCl cotransport: (a) Na+ entry requires Cl- as anion (in NO3- medium, the Na uptake is strongly inhibited, whereas NO3- is an extremely effective substitute for Cl- in the anion exchange system); (b) Na uptake is not significantly affected by the presence of HCO3- in the suspension medium despite the fact that in red cells, Cl-/HCO3- exchange occurs more readily than the exchanges of Cl- for basic equivalents in a theoretically CO2-free medium (the so-called Cl-/OH- exchanges). The purpose of the present paper was a reassessment of the two models by using monensin, an ionophore allowing Na+/H+ exchange. From this study, it appears that NaCl entry results from the simultaneous functioning of the Na+/H+ antiporter and the anion exchange system. The apparent Cl dependence is explained by the fact that, in these erythrocytes, NO3- clearly inhibits the turnover rate of the Na+/H+ antiporter. As Na+/H+ exchange is the driving component in the salt uptake process, this inhibition explains the Cl requirement for Na entry. The lack of stimulation of cell swelling by bicarbonate is explained by the fact that the rate of anion exchange in a CO2-free medium (Cl-/OH- exchange) is roughly equivalent to that of Na+/H+ exchange and thus in practice is not limiting to the net influx of NaCl through the two exchangers.(ABSTRACT TRUNCATED AT 400 WORDS)

Glutaraldehyde fixation of the cAMP-dependent Na+/H+ exchanger in trout red cells.

It has been shown that the addition of a beta-adrenergic catecholamine to a trout red blood cell suspension induces a 60-100-fold increase of sodium permeability resulting from the activation of a cAMP-dependent Na+/H+ antiport. Subsequent addition of propranolol almost instantaneously reduces the intracellular cAMP concentration, and thus the Na permeability, to their basal values (Mahé et al., 1985). If glutaraldehyde (0.06-0.1%) is added when the Na+/H+ exchanger is activated after hormonal stimulation, addition of propranolol no longer inhibits Na permeability: once activated and fixed by glutaraldehyde, the cAMP dependence disappears. Glutaraldehyde alone causes a rapid decrease in the cellular cAMP concentration. In its fixed state the antiporter is fully amiloride sensitive. The switching on of the Na+/H+ exchange by cAMP is rapidly (2 min) followed by acute but progressive desensitization of the exchanger (Garcia-Romeu et al., 1988). The desensitization depends on the concentration of external sodium, being maximal at a normal Na concentration (145 mM) and nonexistent at a low Na concentration (20 mM). If glutaraldehyde is added after activation in nondesensitizing conditions (20 mM Na), transfer to a Na-rich medium induces only a very slight desensitization: thus the fixative can "freeze" the exchanger in the nondesensitizing conformation. NO3- inhibits the activity of the cAMP-dependent Na+/H+ antiporter of the trout red blood cell (Borgese et al., 1986). If glutaraldehyde is added when the cells are activated by cAMP in a chloride-containing medium, the activity of the exchanger is no longer inhibited when Cl- is replaced by NO3-. Conversely, after fixation in NO3- medium replacement of NO3- by Cl- has very little stimulatory effect. This indicates that the anion dependence is not a specific requirement for the exchange process but that the anion environment is critical for the switching on of the Na+/H+ exchanger and for the maintenance of its activated configuration.

Arrestin from nucleated red blood cells binds to bovine rhodopsin in a light-dependent manner.

Using a panel of monoclonal antibodies, it has previously been demonstrated that the cytosol of nucleated red cells (trout and turkey) contains a protein similar to arrestin, a soluble protein found so far only in the photosensitive cells and which, by binding to photoexcited rhodopsin, inhibits the phototransduction process. The role of this arrestin-like protein in non-photosensitive cells is questionable. In this report we present evidence that partially purified red blood cell arrestin (RBC arrestin) behaves functionally like bovine retinal arrestin: it binds to phosphorylated bovine rhodopsin only when this receptor has been photoactivated. Thus RBC arrestin and bovine retinal arrestin are closely related both structurally and functionally. By analogy with the function of retinal arrestin, it is proposed that RBC arrestin is involved in desensitization of membrane transport proteins and/or adrenergic receptors.

Immunological detection of arrestin, a phototransduction regulatory protein, in the cytosol of nucleated erythrocytes.

Cytosolic extracts of trout and turkey erythrocytes were tested for their immunoreactivity with polyclonal and monoclonal antibodies to retinal arrestin (S-antigen), a cytosolic protein of photoreceptor cells involved in the desensitization of rhodopsin. After adsorption or immunoaffinity chromatography of the extracts, these antibodies specifically recognized a protein having a molecular weight similar to that of retinal arrestin. Because the G-protein-mediated transduction systems, such as visual and beta-adrenergic systems, display a high degree of structural and functional homology, the presence of arrestin-like proteins in non-photosensitive cells suggests that these proteins are involved in the transduction of chemical signals, with a possible role in receptor desensitization.

The erythrocyte Na+/H+ exchangers of eel (Anguilla anguilla) and rainbow trout (Oncorhynchus mykiss): a comparative study

Trout and eel red blood cell Na+/H+ exchangers show widely different regulatory properties. Catecholamines, cyclic AMP and phorbol esters, which activate the trout red cell antiporter, do not affect the eel exchanger. Unlike the trout red cell exchanger, the eel red cell exchanger is strongly activated by cell shrinkage, allowing a remarkable cell volume recovery. These different regulatory properties probably indicate the existence of different isoforms of the exchangers in nucleated erythrocytes, since sensitivity to catecholamines is known to be dependent upon the presence of protein kinase A consensus sites on the cytoplasmic domain of the antiporter. After shrinkage of eel erythrocytes, the Na+/H+ exchange rate gradually increases to reach a maximum value after about 10 min. The magnitude of activation is a graded function of cell shrinkage. Deactivation, like activation, is induced by a volume change and occurs after some delay (lag time). The response of the trout antiporter (betaNHE) to cell shrinkage is much reduced compared with that of the eel antiporter. In addition, the antiporter is deactivated prior to restoration of the normal control volume, leaving cell volume regulation notably defective. The trout red cell antiporter, which is desensitized and enters a refractory state following hormonal activation, is only deactivated (it can be reversibly reactivated) after shrinkage-induced activation. This dual control may occur by both phosphorylation-dependent and phosphorylation-independent mechanisms. In view of the similarities in the regulatory properties of eel and salamander (Amphiuma sp.) Na+/H+ exchangers, the expression of a putative K+/H+ exchange mediated by the N+/H+ exchanger was sought in eel erythrocytes. However, neither osmotic swelling nor calyculin-A-dependent phosphorylation revealed such a K+/H+ exchange.

Inhibition by amiloride of both adenylate cyclase activity and the Na+/H+ antiporter in fish erythrocytes.

In fish erythrocytes isoproterenol stimulates cellular accumulation of cyclic adenosine 3':5'-monophosphate (cyclic AMP) and produces a large increase in sodium permeability which corresponds to the activation of Na+/H+ exchanges and chloride-dependent sodium uptake. The stimulation of sodium transport by isoproterenol was reproduced by adding cyclic AMP or forskolin to the medium and was blocked by propranolol. This increase in sodium permeability was completely inhibited by amiloride at the relatively high levels (0.1-1 mM) of the diuretic required to inhibit the activity of the Na+/H+ exchanger under physiological conditions in various biological systems. It was shown that amiloride inhibited cyclic AMP accumulation. This effect, which was reversible and dose-dependent (ED50 6 X 10(-6) M-maximal effect 0.5 mM), resulted from the inhibition of the catalytic unit of adenylate cyclase. Amiloride also directly inhibited the sodium entry system but the Na transporter was less sensitive than adenylate cyclase to amiloride (ED50 6 X 10(-5) M). It appears from the data presented in this report that the inhibition of sodium permeability observed in fish erythrocytes in the presence of amiloride can result either from the effect of the diuretic on the adenylate cyclase system or from the effect on the sodium transport system, depending on the conditions in which amiloride is used. Thus, caution is required when interpreting amiloride action in terms of inhibition of specific transport processes.

Sodium movements in high-sodium beef red cells: properties of a ouabain-insensitive exchange diffusion.

1. The relative importance of the Na efflux components in beef red cells has been evaluated. The component which is insensitive to ouabain, which does not require external K but depends on the presence of external Na, accounts for about 90% of the total Na efflux.2. The experiments reported in this paper are consistent with the presence of an ouabain-insensitive Na(+)-Na(+) exchange process accounting for this ouabain-insensitive external Na dependent efflux.3. A strictly parallel behaviour of influx and efflux is observed when the pH is altered. The exchange diffusion process is inhibited over 90% by a decrease in pH from range pH 8.0-5.5.4. Both Na efflux and influx are markedly increased by raising the temperature from 27 to 37 degrees C.5. Energy depleted cells and fresh cells behave similarly in respect to Na movements. In depleted resealed ghosts, a large Na-dependent efflux occurs. No chemical energy and no special nucleotide is required for the Na(+)-Na(+) exchanges.6. When the external or internal Na concentrations are changed, a parallel behaviour of influx and efflux is observed.7. The relation between the magnitude of the exchange diffusion flux and the external or internal Na concentration fits quite well the Michaelis-Menten equation suggesting that only one Na(+) reacts with the transport mechanism. The affinity for Na is lower however at the outer surface than at the inner border of the membrane.8. The relation between this exchange process, the ouabain-insensitive Na-Na exchanges found in human red cell, and Ussing's model of exchange diffusion is discussed.

Characteristics of a sulphydryl group essential for sodium exchange diffusion in beef erythrocytes.

1. In the beef red blood cell, the component of the Na efflux which is insensitive to ouabain but depends on the presence of external Na, is not affected by furosemide but is reduced by several agents: ethacrynic acid, dinitrofluorobenzene, p-chloromercuribenzene (PCMB), N-ethylmaleimide and p-chloromercuribenzene sulphonate (PCMBS). Some of these agents increased a parallel passive permeability which could mask the reduction of efflux.2. N-ethylmaleimide and PCMBS, which in our experimental conditions (initial concentration 5 x 10(-4)M and 5 x 10(-6)M respectively, haematocrit 7.7%) do not increase the leak, inhibit Na influx and efflux markedly and equally. This provides further evidence of the existence of a typical ouabain-insensitive Na exchange diffusion in beef red blood cell.3. Inhibition of the exchange diffusion mechanism by N-ethylmaleimide or PCMBS is not total and their inhibitory effects are slightly additive. Various arguments suggest that their effects on exchange diffusion can be attributed to a reaction with sulphydryl groups.4. These sulphydryl groups are rapidly titrable by a poorly penetrating agent such as PCMBS, and the inhibitory effect is rapidly reversible. Thus, it is assumed that the sulphydryl groups containing proteins are superficially located on the outer border of the membrane.5. After inhibition, there is no change in half saturation constant for the complexing reaction for transfer, suggesting that the inhibited sites are no longer functioning but that the uninhibited sites are in every way normal.6. N-ethylmaleimide and PCMBS act similarly in sheep red blood cells.7. PCMBS does not affect sodium movement in human erythrocytes, but N-ethylmaleimide inhibits markedly the ouabain-insensitive Na efflux.

Effect of catecholamines on deformability of red cells from trout: relative roles of cyclic AMP and cell volume.

1. In the presence of catecholamine the nucleated red blood cells of trout show a large increase in cell volume as a result of an accumulation of sodium and chloride due to activation of an amiloride-sensitive, cyclic AMP-dependent Na+-H+ exchanger allowing Na+ to enter in exchange for internal H+. 2. The activation of this cyclic AMP-dependent Na+-H+ exchange is considered to be involved in an adaptive response to hypoxia by increasing the oxygen-carrying capacity of erythrocytes. But cell swelling could increase resistance to blood flow and thus impair the expected physiological advantages for oxygen transport. The effect of catecholamine on the deformability properties of the red blood cells has been studied by measuring the rate at which blood flows through a Nucleopore filter (5 microM). 3. The results show that stimulation by catecholamine in fact increases the erythrocyte deformability, a response which must favour the supply of oxygen at the tissue level. 4. Hormonal stimulation increases the cellular cyclic AMP content (and cyclic AMP-dependent phosphorylation of cytoskeleton proteins could influence cell deformability) and the cell volume. It has been shown that when cellular cyclic AMP content is increased under conditions where the cell cannot swell, the erythrocyte becomes more rigid and not more deformable. Conversely the results show a systematic coincidence between cell swelling and deformability increase. The precise way in which volume change and deformability are interrelated needs more study.