The trout red blood cell Na+/H+ antiporter (beta NHE) plays two interesting properties: it is the only NHE own to be activated by cyclic AMP, and the activation process is followed by a desensitisation of the transport system itself. Cloning and expression of beta NHE have provided inificant information about Na+/H+ activation, in particular that activation by cyclic AMP is directly dependent upon the presence of two protein kinase A consensus sites in the cytoplasmic tail of the antiporter. Expression of beta NHE in fibroblasts demonstrates that the protein kinase A (PKA) and protein kinase C (PKC) activation pathways are independent and do not converge a common kinase. Moreover, the hydrophilic C-terminal fragment is essential to the mediation of the various hormonal responses. NHE1 (the human ubiquitous isoform) is not activated by cyclic AMP, but a "NHE1 transmembrane domain/beta NHE cytoplasmic domain' chimera is fully activated by cyclic AMP. In red cells, activation of beta NHE is the result of phosphorylation by PKA of at least two independent sites. Desensitisation, inhibited by the phosphatase inhibitor okadaic acid, may consist of the dephosphorylation of one of these two sites. Furthermore, Calyculin A (CIA), another specific protein phosphatase inhibitor, induces in unstimulated cells a Na+/H+ exchange activity whose exchange properties are very different from those of the adrenergically stimulated antiporter. It is suggested that CIA may be able to revive "sequestered' antiporters. We propose that the molecular events underlying beta NHE desensitisation could be similar to those involved in rhodopsin desensitisation. Antibodies were generated against trout red cell arrestin in order to analyse the binding of arrestin to the activated exchanger. Recombinant trout arrestin was produced in a protease-deficient strain of Escherichia coli and its functionality tested in a reconstituted rhodopsin assay.

In response to swelling, cells recover their initial volume by releasing intracellular solutes via volume-sensitive pathways. There is increasing evidence that structurally dissimilar organic osmolytes (amino acids, polyols, methyl amines), which are lost from cells in response to swelling, share a single pathway having the characteristics of an anion channel. However, the molecular identity of this pathway remains to be established. It has been suggested that the erythrocyte anion exchanger (AE1) or some AE1-related proteins could be involved. A direct evaluation of this possibility has been made by comparing the functional properties of two AE1s when expressed in Xenopus laevis oocytes: tAE1 is from a fish erythrocyte which releases taurine when swollen, and mAE1 is from a mammalian erythrocyte which does not regulate its volume when swollen. While mAE1 performs exclusively Cl-/Cl- exchange, tAE1 behaves as a bifunctional protein with both anion exchange and Cl-/taurine channel functions. Construction of diverse tAE1/mAE1 chimaeras allows the identification of protein domains associated with this channel activity. Thus, some AE1 isoforms could act as a swelling-activated osmolyte channel, a result having a potentially important implication in malaria. This review also discusses the possibility that several different proteins might function as swelling-activated osmolyte channels.

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.

1. In all marine fish, both stenohaline and euryhaline, the net sodium excretion by the gill, compensating for intestinal absorption, is the result of a Na int -K ext exchange. 2. In a euryhaline fish, such as the eel, in which a sodium exchange diffusion (linkage of sodium influx and outflux) also occurs, the two kinds of exchange (Na-Na and Na-K) can be regarded as alternative modes of behaviour of the same transport system. In normal sea water both exchanges take place at the same time; in K-free sea water the system exchanges Na for Na exclusively and in Na-free solution containing K the system exchanges Na for K exclusively. 3. The transport system is located on the external membrane since it is inhibited by ouabain added in the external medium. 4. The Na-K process is ouabain-sensitive but K can antagonize the inhibitory action of the glycoside. The Na-Na process on the other hand is not ouabain-sensitive.

1. The temperature dependence of diffusional permeability to water was studied in freshwater-adapted and seawater-adapted eels. The pattern of temperature acclimation is discussed. 2. The ratio of water permeability to sodium permeability is approximately 3 at 25°C, but falls to 1 at 5°C. This drop is mainly due to a diminution of the water permeability, the temperature coefficients being much higher for water than for sodium. 3. The relatively independent variations of water and sodium permeabilities in the seawater-adapted fish probably indicate a certain dissociation between water movements and salt movements. 4. In the freshwater-adapted eel the osmotic permeability is considerably higher than the diffusional permeability, which supports the previously advanced hypothesis concerning the presence of water-filled channels in the branchial epithelium. 5. In the seawater-adapted eel the osmotic permeability is lower than the diffusional permeability, this difference being greater the lower the temperature. This surprising result must signify either that the osmotic pressure difference between blood and sea water does not represent the true osmotic gradient across the membrane, or that a reabsorption of water linked with a movement of solutes occurs in a specialized region of the gill.

Adaptive changes of the water permeability of the teleostean gill epithelium in relation to external salinity. For SW Anguilla 8 9.3 ± 2.4 Read SW Anguilla 8 29.3 ± 2.4

1. Cannulation of afferent and efferent branchial vessels in the eel permitted studies of tritiated water clearance. It was observed that most of the diffusional water flow occurs through the gills. 2. Diffusional and osmotic water flows have been measured in a fresh-water ( Carassius ), a marine ( Serranus ) stenohaline fish and in two euryhaline species ( Platichthys and Anguilla ) adapted to either fresh water or sea water, and are found to be lower than in any comparable epithelia so far studied. 3. The diffusional water flow deduced from THO turnover is significantly smaller in the sea-water fish. 4. The osmotic water flow, determined indirectly by measuring drinking rate and urine flow, is smaller in the sea-water fishes despite a greater osmotic gradient across the gills. 5. Attempts to compare diffusional and osmotic permeabilities for the gill are hindered by our ignorance of the extent of solute (salt)-solvent interaction in the epitheium. It is suggested that the gill of the fresh-water-adapted fishes is semi permeable, while that of the sea-water teleosts may not be, because of the very high ionic exchange across the gill. 6. The surprisingly low diffusional and osmotic permeabilities of the gill epithelium in sea-water fish may be possibly related to the absence of water-filled pores.