1. Replacement of chloride by foreign anions in the suspending medium of trout erythrocytes can affect in a complex manner both the activation by catecholamines of the latent Na(+)‐H+ exchanger and its subsequent desensitization. These changes are discussed in relation to other cellular modifications (distribution of permeant anions and accumulation of cyclic AMP) induced by foreign anions. 2. The transfer of trout erythrocytes from a chloride‐containing medium to media containing lyophilic permeable anions, NO3‐ or SCN‐, immediately induces a decrease of distribution ratios of permeable anions across the red cell membrane (i.e. Donnan ratios). It is probable that the binding of lyophilic anions to haemoglobin, by altering the amount of negative fixed charges, results in changes of distribution of permeant anions across the membrane. 3. The effectiveness of anions in decreasing both the activation of the Na(+)‐H+ exchanger and the Donnan ratio follows the same sequence in both cases, i.e., SCN‐ greater than NO3‐ greater than Cl‐ = propionate. It was demonstrated that a change in Donnan ratio affects antiport activity possibly through a shift in intracellular pH; such a mechanism however cannot account for all the effects of foreign anions on antiport activity. 4. The present results show that lyophilic anions do not modify the affinity of the antiporter for sodium ions but greatly decrease the transport capacity of the exchange system. This is interpreted as indicating that the binding of lyophilic anions to some component of the transport system prevents antiporters from establishing their activated configuration once stimulated. Since the inhibitory effect of anions on Na(+)‐H+ exchange has been demonstrated in all erythrocytes studied but in no other cell, the crucial substance involved in this inhibition could well be haemoglobin, which appears to control antiport activity in erythrocytes. 5. Some anions affect desensitization of the exchanger. This effect is not related to the lyophilic character of the anion and is not mediated by a change in intracellular cyclic AMP. 6. Propionate and acetate drastically reduce the intracellular level of cyclic AMP and seem to facilitate the activated configuration of the exchanger.

The anion transport protein of the human erythrocyte membrane, band 3, was solubilized and purified in solutions of the non-ionic detergent nonaethylene glycol lauryl ether and then reconstituted in spherical egg phosphatidylcholine bilayers as described earlier (U. Scheuring, K. Kollewe, W. Haase, and D. Schubert, J. Membrane Biol. 90, 123-135 (1986)). The resulting paucilamellar proteoliposomes of average diameter 70 nm were transformed into smaller vesicles by French press treatment and fractionated according to size by gel filtration. The smallest protein-containing liposomes obtained had diameters around 32 nm; still smaller vesicles were free of protein. All proteoliposome samples studied showed a rapid sulfate efflux which was sensitive to specific inhibitors of band 3-mediated anion exchange. In addition, the orientation of the transport protein in the vesicle membranes was found to be "right-side-out" in all samples. This suggests that the orientation of the protein in the vesicle membranes is dictated by the shape of the protein's intramembrane domain and that this domain has the form of a truncated cone or pyramid.

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.

Biochemical studies of the plasma membrane and the cytoskeleton of nucleated erythrocytes are strongly limited by the difficulties encountered in enucleating large quantities of cells. We describe an easily built hydrodynamic system which allows rapid preparation of large amounts of avian and fish erythrocyte plasma membranes. The contents of two 25-ml syringes containing hemolyzed nucleated erythrocytes are forced through four capillaries to a specially designed mixing chamber which fills a collecting syringe. The 50-ml erythrocyte suspension can be processed in 2 s. The high speed flow is achieved with a hand-activated piston. The turbulences in the mixing chamber are carried to an optimal efficiency by the vis-à-vis disposition of the four mixing jets. The enucleated membranes are separated from the nuclei and residual nucleated cells by differential centrifugations. They do not show contamination with nuclear material. Erythrocytes from chicken and trout have been used. They present striking differences in their stability toward hydrodynamic disruption, erythrocytes from chicken being far more stable. Ninety-five percent of trout erythrocytes are enucleated after only one run through the mixing chamber. Two runs performed at the maximal flow rate are necessary to enucleate chicken erythrocytes with a yield of 80%. In the former case most of the purified enucleated plasma membranes are fragmented in small vesicles while they retain a large size in the case of chicken erythrocytes. The proteins of the membranes thus prepared are characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis: we found that erythrocyte membranes from trout are remarkable for their small spectrin content compared to those from chicken.

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 β‐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.

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.

The anion transport system of the human erythrocyte membrane was reconstituted in unilamellar phosphatidylcholine vesicles, and a vesicle subpopulation of a narrow size distribution was isolated from the sample by gel filtration. In this subpopulation, the turnover number of the transport protein (the band 3 protein) for sulfate transport was determined. It was found that, in the reconstituted system, the protein transports sulfate 5–10‐times faster than in the human erythrocyte membrane.

The anion transport protein of the human erythrocyte membrane, band 3, was incorporated into unilamellar sphingomyelin vesicles. The vesicles showed a rapid sulfate efflux which could be inhibited by specific inhibitors of the erythrocyte anion transport system. All band 3 molecules contributing to the inhibitor-sensitive flux component were arranged 'right-side-out'. The turnover number of the transport protein for sulfate transport was virtually identical to that in phosphatidylcholine bilayers and around 6 times larger than in human erythrocyte membranes. Thus, in contrast to other claims, sphingomyelin does not inhibit the erythrocyte anion transport system.

The anion transport system of the human erythrocyte membrane was reconstituted in phosphatidylcholine vesicles by a new procedure. The reconstituted system shows all major properties of the sulfate transport system found in the erythrocyte membrane (Scheuring, U., K. Kollewe, W. Haase, and D. Schubert: J. Membrane Biol. 90, 123-135 (1986)). Modifications of the reconstitution method have now led to increased values of the internal volumes of the protein-lipid vesicles and of the average turnover number of the transport protein (which is now larger than in the erythrocyte membrane). Gel filtration on Sephacryl S-1000 has allowed the isolation of vesicles of a narrow size distribution.