SUMMARY1. The addition of isoprenaline to an isotonic suspension of red blood cells of rainbow trout induces an amiloride-sensitive Na+ transport which is independent of Cl-and insensitive to 4,4'-diisothiocyano-2,2'-stilbene disulphonic acid (DIDS) and furosemide.2. Na+ uptake is accompanied by amiloride-sensitive H+ release. The H+ efflux is dependent upon the external Na+ concentration, the Ko.5 value for Na+ being 16 mm.3. In the presence of DIDS, when the coupled NaCl entry (NaCl co-transport) induced by catecholamine is blocked, the results provide evidence for a linked movement of Na+ and H+, with a stoicheiometry of 1 :1.4. Exchange of H+ for Na+ induces osmotic swelling of the cells which is due to the replacement of a bound proton by an osmotically active Na+ cation.5. In the absence of DIDS when the bulk of the Na+ uptake is the result of a coupled entry of Na+ and Cl-, H+ extrusion still occurs and the magnitude of acid excretion is identical to that found in DIDS-treated cells. This suggests that Na+-H+ exchange remains active.6. Addition of isoprenaline first stimulates the Na+-H+ exchange but only transiently. This is followed by a more permanent stimulation ofthe NaCl co-transport.

In order to fix spin-labeled acids at the boundary layer of membrane-bound proteins, spin-labeled long-chain derivatives (m,n)MSL (general formula, CH,(CH2),R-(CH2),COO(CH2)2-M, where R is an oxazolidine ring containing a nitroxide and M is a maleimide residue) were synthesized. The spin-labeled molecules bind covalently to at least two different classes of sulfhydryl groups on rhodopsin in disc membrane fragments from bovine retina. One class of sites is hydrophilic and corresponds to the two SH groups labeled readily by N-ethylmaleimide; the second class of sites is only reached by hydrophobic probes. (10,3)MSL binds equally well to the two classes of sites on rhodopsin, whereas (1 , I 4)MSL, more hydrophobic, binds preferentially to the hydrophobic sites. Apparently a third class of SH groups can be labeled if a very large excess of (m,n)MSL is employed, but proteins may be denatured in this latter case. Labels not covalently bound are removed from the membranes by incubation with fatty acid free bovine serum albumin. However, it is found that the probes do not bind only to rhodopsin in the disc membranes. (m,n)MSL also binds covalently to phosphatidylethanolamine in the rod outer segments or in liposomes. This covalent binding to phospholipids is demonstrated by lipid extraction and thin-layer chromatographic analysis. In order to obtain the pure EPR spectra of the spin-labeled fatty acids bound to the protein, the spectra corresponding to phospholipid-bound spin labels have been I t is often admitted that intrinsic membrane proteins are surrounded by a boundary layer or "annulus" of rigidly bound lipid. The immobilization of this shell of lipid has been deduced essentially from EPR experiments involving spin-labeled fatty acids incorporated into reconstituted systems containing variable lipid to protein ratios. Jost et al. (1973) were the first to propose from spin-label experiments the model of a boundary layer of lipid surrounding an intrinsic membrane protein, namely cytochrome oxidase. Later, Hesketh et al. (1976) reported similar experiments with Ca2+-ATPase, while Chapman et al. (1977) showed that gramcidin A can lead to the same EPR results, if this polypeptide is dissolved in a small amount of lipid.Rhodopsin was also tested with spin-labeled fatty acids. Pontus and Delmelle (1975) found evidence of a rigid boundary layer around this hydrophobic protein. However, previously, Hong & Hubbell (1972) had reached a different conclusion from spin-label experiments with rhodopsin reincorporated into phospholipid; they suggested that the acerage viscosity of the membranes was dependent on the lipid to protein ratio. This is in good agreement with recent results put foward by Cherry et al. (1977) from very different experiments involving bacteriorhodopsin. Finally, using proton manuscript receiced A'ocember 21, 1978. This investigation was supported by research grants from the Centre National de la Recherche Scientifique (ERA 690) and the DelEgation G&n&rale a la Recherche Scientifique et Technique (Comm...

On the addition of isoprenaline to an isotonic suspension of red blood cells of rainbow trout (Salmo gairdneri), the cell volume increases. This increase in volume is the result of net uptake of Na+ and osmotically obligated water. Two different pathways are involved in the salt uptake. The minor component of Na+ entry (about 20%) corresponds to a Na+ uptake independent of Cl‐ and is inhibited by amiloride, yet is insensitive to DIDS, furosemide and niflumic acid. It could result from Na+/H+ countertransport. The major component of salt uptake is due to Na+ entry which requires Cl‐ as anion, and is electroneutral, independent of extracellular K+, sensitive to amiloride, DIDS, niflumic acid and furosemide, but insensitive to other loop diuretics such as piretanide or bumetanide. These characteristics, as well as the response of valinomycin‐treated cells to isoprenaline and some other properties (ionic selectivity, drug sensitivity) of the anion exchange system of volume‐static trout red cells, permit the definition of the nature of this Cl‐‐dependent pathway. The findings are inconsistent with the electrically silent double antiporter model (proposed in amphibian red cells by Cala, 1980) and with the co‐migration of Cl‐ with Na+ through parallel conductive pathways, but strongly suggest a symport mechanism. Striking differences, mainly pharmacological, exist between this NaCl co‐transport and the duck red blood cell Na+/K+/2Cl‐ co‐transport (Kregenow, 1977, 1978; McManus & Schmidt, 1978).

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