Trout Red Cells Research Articles

Leaky Cl − –HCO 3 − exchangers: cation fluxes via modified AE1

The abundant membrane protein AE1 normally functions as an obligate anion exchanger, with classical carrier properties, in human red blood cells. Recently, four single point mutations of hAE1 have been identified that have lost the anion exchange function, and act as non-selective monovalent cation channels, as shown in both red cell flux and oocyte expression studies. The red cell transport function shows a paradoxical temperature dependence, and is associated with spherocytic and stomatocytic red cell defects, and haemolytic anaemias. Other forms of AE1, including the native AE1 in trout red cells, and the human mutation R760Q show both channel-like and anion exchange properties. The present results point to membrane domains 9 and 10 being important in the functional modification of AE1 activity.

Phylogeny of anion exchangers: Could trout AE1 conductive properties be shared by other members of the gene family?

A phylogenetic tree of anion exchangers (AE) was performed in order to better understand relationships between the different known AE and how they arose. Indeed, the different known AE1 from mammals or fish do not exhibit the same transport features: all studied anion exchangers 1 (AE1) catalyse an electroneutral Cl −/HCO 3 − exchange through the plasma membrane; however, trout AE1 (tAE1) is able to spontaneously form an anion conductive pathway permeable to some inorganic cations (Na + and K +) as well as to organic osmolytes such as taurine. Therefore, it has been proposed that this major erythrocyte membrane protein could play a key role for the cell volume regulation of trout red cells. By analogy, it was envisioned that other fish anion exchangers could play a similar role in osmolyte loss induced by erythrocyte swelling. We have cloned AE1 from Raja erinacea and Danio rerio and studied their properties after expression in Xenopus laevis oocytes. In this study, we show that none of them is able to induce any conductive pathway or taurine permeability in Xenopus oocytes. Our phylogenetic analyses show that, first, all present AE1 genes have a common ancestor distinct from that of AE2 and AE3 and second, tAE1 is a true AE1 ortholog. The question of whether tAE1 conductive properties are a derived character in the trout lineage within Euteleostei or whether other AE1 members can share these properties is then discussed.

Oxygen sensing and K+‐Cl− cotransport

Oxygen modulates membrane transport in erythrocytes of a number of vertebrate species. In red cells of horse, sheep, human and trout, raising oxygen tension enhances K+-Cl− cotransport (KCC). Swelling, among other treatments, activates this transporter. Swelling-activated KCC promotes KCl efflux, water follows by obligatory osmotic coupling, and cell volume is decreased. In this manner regulation of cell volume is promoted. Direct activation of KCC is probably by a serine/threonine phosphatase. Regulation is afforded by a volume-sensitive kinase: cell swelling inhibits the kinase, allowing the phosphatase to activate KCC (see Jennings, 1999, for references). Since O2 stimulates KCC, there must be a sensor of O2 that signals KCC. The most obvious sensor for oxygen is haemoglobin (Hb). See Gibson et al. (2000) for a recent review. In this volume of The Journal of Physiology, Berenbrink et al. (2000) present studies on O2 modulation of K+ transport in trout red cells; most of the measured K+ transport was KCC. Changes in KCC were measured as functions of O2 partial pressure (PO2) and pH, varied independently. At physiological pH, raising PO2 to atmospheric pressure increased K+ transport several-fold. At lower pH, stimulation of K+ flux by O2 was reduced. At pH 7.9, the PO2 for half-maximal stimulation of K+ transport, P50, was ∼10-fold higher than the P50 for equilibrium binding of O2 to Hb. Stimulation of K+ flux by O2 was not cooperative, whereas the curve for Hb-O2 binding was sigmoid, with a Hill coefficient of ∼3. These results are strong evidence that bulk Hb is not the O2 sensor in trout red cells. Whatever the O2 sensor, it has a low O2 affinity and a high pH sensitivity (Berenbrink et al. 2000). Oxygen affects a diverse array of processes in all cells. There are sensing mechanisms based on haem proteins shared by most if not all cells (Bunn & Boyton, 1996). In cells such as erythropoietin-producing hepatocytes and type I cells of the carotid body, O2 sensing is their central function. Type I cells are a pertinent example because changes in PO2 provoke changes in membrane transport. The current view is that low PO2 is sensed by a haem protein which causes closing of a class of K+ channel. The resultant depolarization opens Ca2+ channels; the increased Ca2+ activity causes secretion of neurotransmitter. The post-synaptic neurons transmit signals to the brain stem where respiration is regulated (Gonzalez et al. 1994). The identity of the haem sensor is uncertain; the non-mitochondrial cytochrome b558 was proposed (Cross et al. 1990). There are several possible mechanisms of signalling from the sensor, including direct coupling of the sensor to the channel and action of a reactive O2 species, e.g. superoxide, produced from O2 interacting with the sensor. The signals may alter the function of a protein kinase and/or phosphatase. Serine/threonine phosphatases types 1 and 2A are metalloproteins (Barford, 1996) and could be targets of superoxide. A protein kinase regulated by haem has been reported (Chen & London, 1995). In these terms, a signalling pathway in carotid body type 1 cells can be envisioned. O2 modulation of KCC in trout as well as mammalian red cells may operate by similar signalling mechanisms. Trout red cells are nucleated and have mitochondria, and probably have the full complement of haem proteins as possible O2 sensors. In mammalian red cells, as in trout red cells, there is evidence that Hb is not the O2 sensor. Activation of KCC in horse red cells requires 70 % saturation of Hb with O2 (Honess et al. 1996), similar to the situation in trout red cells (Berenbrink et al. 2000): low affinity of the sensor for O2. There are no known haem proteins in mammalian red cells other than Hb that are likely candidates for an O2 sensor. It would make sense if the sensor were bound to the membrane to afford access to the cotransporter. It turns out that Hb binds to band 3, the membrane anion exchanger, from which it could interact with KCC. Deoxy-Hb binds preferentially (Chetrite & Cassoly, 1985). With an elevated fraction of deoxy-Hb over oxy-Hb at most PO2 levels, bound Hb would have a lower O2 affinity than bulk Hb. This low affinity is a characteristic of the O2 sensor regulating KCC (Honess et al. 1996; Berenbrink et al. 2000). The lack of cooperativity of O2 activation of KCC in trout red cells still makes it unlikely that Hb is the sensor in these cells. However, the O2 activation curve of KCC in horse red cells is strongly sigmoid (Speake et al. 1997), so Hb, membrane-bound, remains a candidate O2 sensor in mammalian red cells.

Cell volume regulation: the role of taurine loss in maintaining membrane potential and cell pH.

1. In response to a hypo-osmotic stress cells undergo a regulatory volume decrease (RVD) by losing osmotically active solutes and obliged water. During RVD, trout red cells lost taurine, K+ and Cl- but gained Na+ and Cl-. Over the full time course of RVD the chloride concentration in the cell water remained remarkably constant. Thus membrane potential and cell pH, which depends on the ratio of internal to external chloride concentration ([Cl-]i:[Cl-]o), remained fixed. 2. When cell volume decreases it is only possible to keep the chloride concentration in the cell water constant if an equal percentage of the cell chloride pool and of the cell water pool are lost simultaneously. Quantitative analysis of our data showed that this requirement was fulfilled because, over the full time course of RVD, cells lost osmotically active solutes with a constant stoichiometry: 1 Cl-:1 positive charge:2.35 taurine. Any change in taurine permeability, by modifying the stoichiometric relationship, would affect the amount of water lost and consequently cell chloride concentration. 3. Experiments carried out with different cations as substitutes for external Na+ suggest that the constancy of the chloride concentration is not finely tuned by some mechanism able to modulate the channel transport capacity, but results in part from the fact that the swelling-dependent channel constitutively possesses an adequately fixed relative permeability for cations and taurine. However, as a significant fraction of K+ and Cl- loss occurs via a KCl cotransporter, the contribution of the cotransport to the stoichiometric relationship remains to be defined. 4. The large amount of taurine released during RVD (50 % of all solutes) was shown to be transported as an electroneutral zwitterion and not as an anion. How the channel can accommodate the zwitterionic form of taurine, which possesses a high electrical dipole, is considered.

Regulatory volume response of erythrocytes exposed to a gradual and slow decrease in medium osmolality.

A sudden decrease in external medium osmolality (90 mosmol/kg) causes an immediate swelling of trout erythrocytes, followed by a regulatory volume decrease (RVD) due to activation of both a KCl cotransporter and a taurine transport pathway. Here, we determined how trout red cells respond when they are exposed to a gradual and slow decrease in medium osmolality (80 mosmol/kg at a rate of 0.7 mosmol/kg per min). Erythrocytes were unable to regulate their volume efficiently when swollen gradually and it increased continuously throughout the experimental period (120 min). As long as volume was increased slowly by 15-25%, regulatory pathways remained essentially inactivated, erythrocytes losing no significant amount of intracellular osmotically active solutes. Above this swelling threshold, a response was triggered but the quantity of solutes lost via the regulatory pathways was still not sufficient to counterbalance the continuous entry of water due to the slow and gradual decrease in medium tonicity.

Transport of uncharged organic solutes in Xenopus oocytes expressing red cell anion exchangers (AE1s).

When expressed in Xenopus oocytes, the trout red cell anion exchanger tAE1, but not the mouse exchanger mAE1, elicited a transport of electroneutral solutes (sorbitol, urea) in addition to the expected anion exchange activity. Chimeras constructed from mAE1 and tAE1 allowed us to identify the tAE1 domains involved in the induction of these transports. Expression of tAE1 (but not mAE1) is known to generate an anion conductance associated with a taurine transport. The present data provide evidence that (i) the capacity of tAE1 and tAE1 chimeras to generate urea and sorbitol permeability also was associated with an anion conductance; (ii) the same inhibitors affected both the permeability of solutes and anion conductance; and (iii) no measurable water transport was associated with the tAE1-dependent conductance. These results support the view that fish red blood cells, to achieve cell volume regulation in response to hypotonic swelling, activate a tAE1-associated anion channel that can mediate the passive transport of taurine and electroneutral solutes.

Regulation of Na+/H+ antiporter in trout red blood cells.

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.

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.

Catecholamine-induced changes in oxygen affinity of carp and trout blood

Carp and trout blood maintained at low constant oxygen and carbon dioxide tensions was β-stimulated. This activated the Na + H +- exchanger of the red cell membrane, leading to increases in red cell pH (pHi) and cell water content, the latter resulting in dilution of hemoglobin and organic phosphates. The increase in pHi was rapid and maintained throughout the experimental period, the trout red cells showing the largest increase. Likewise swelling of the red cells was larger in trout than in carp blood. As a consequence of β-stimulation the oxygen affinity of the blood increased. In trout the intracellular Bohr factor of unstimulated blood combined with the pHi increase upon stimulation could account for 85% of the increase in oxygen affinity, whereas it only covered 65% of the increase in carp blood. We therefore conclude that blood oxygen affinity is dependent on the red cell hemoglobin concentration in both species, the effect being more marked in carp.

Glycine uptake by trout (Salmo trutta) red blood cells

The present study demonstrates the presence of different amino acid carriers in the membrane of trout red cells. Most glycine is taken up through the Na(+)-dependent system ASC, although the nearly specific Gly system is also active. Besides these carriers, glycine is taken up by means of Na(+)-independent transporters, system L being the most important. A system asc of high affinity and low capacity has been found, and band 3 is unable to transport glycine under physiological conditions. These results suggest that although all these carriers are already present in primitive vertebrates, several differences exist in their properties with respect to those found in mammalian cells.

Role of protein phosphorylation and dephosphorylation in activation and desensitization of the cAMP-dependent Na+/H+ antiport.

The Na+/H+ antiporter of trout erythrocytes is activated by agents raising intracellular cAMP, whereas other Na+/H+ exchangers are insensitive to or inhibited by cAMP. Cloning of the beta agonist-activated exchanger (beta NHE) reveals the presence of two consensus sites for phosphorylation by the cAMP-dependent protein kinase A (cAMP-PKA) on the cytoplasmic loop. Transfected to fibroblasts, beta NHE can no longer be activated by cAMP when these consensus sites are removed, indicating regulation through cAMP-PKA. Moreover, it has been shown that activation of the exchanger is rapidly followed by its desensitization. To further investigate the role of phosphorylation in these processes, we examined the effects of protein kinase and phosphatase inhibitors on the antiporter activation and desensitization in trout red cells. Na+/H+ exchange was not induced by strong acidification, indicating that beta NHE is normally in a nonfunctional state, whereas cAMP did activate the system by forcing beta NHE into a functional conformation; preincubation of cells with the kinase inhibitor H89 blocked cAMP-activation, confirming the role of cAMP-PKA in the activation process. The protein phosphatase inhibitor okadaic acid (OA) neither activated the exchange when added on unstimulated cells nor prevented deactivation of beta agonist-activated beta NHE by propranolol. Hence, the cAMP-dependent phosphorylation involved in the activating process is controlled by an OA-insensitive phosphatase. beta NHE activated by beta agonist or cAMP shifts rapidly into a refractory state, accounting for the previously described desensitization. Desensitization was blocked and reversed by OA, indicating a control by an OA-sensitive phosphatase of the phosphorylation level of a site critical for the desensitizing process. Phosphorylation of this (site 2) and of the activating site (site 1) is mediated by cAMP-PKA, as demonstrated by the effects of both intracellular cAMP concentration and kinase inhibitor H89 on the Na+/H+ exchange activity. Based on these data, we proposed that beta NHE can exist in three different states (inactive I, activated A, and desensitized D). Conversion of I to A needs the simultaneous phosphorylation by cAMP-PKA of sites 1 and 2. These two sites might constitute the two neighboring cAMP-PKA sites located on the cytoplasmic loop as deduced from the oligonucleotide sequence. Dephosphorylation of site 2 and subsequent binding of an arrestin-like protein are assumed to account for desensitization of the antiport.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence for a K(+)-H+ exchange in trout red blood cells.

1. Exposure of trout red blood cells to beta-adrenergic agonist isoprenaline activates a cAMP-dependent Na(+)-H+ antiport, the movements of protons being compensated by a Cl(-)-OH- (or HCO3-) exchange mediated by band 3 protein. The absorption of water osmotically linked to sodium and chloride induces cell swelling. 2. In the presence of acetazolamide, anionic exchange is inhibited and activation of cationic exchange resulted in the first 2 min in a strong external acidification and a large internal alkalinization leading to a reversal of the transmembrane pH gradient. Then, for at least 1 h and despite the inhibition of Cl- entry, a net Na+ uptake occurred which was balanced by an equivalent K+ loss, with the result that cell volume and pH gradient remained unchanged. 3. In such conditions, the inactivation of the Na(+)-H+ exchanger by a beta-antagonist, propranolol, blocked Na+ entry while K+ continued to be lost. This volume-independent K+ efflux, which is thus independent of the Na(+)-H+ exchanger, was not accompanied by a Cl- efflux but was associated with large internal and external pH changes consistent with K(+)-H+ exchange. 4. The K+ loss and the related pH changes are inhibited by compounds which are known to inhibit the K(+)-anion co-transporter in trout red cells, i.e. 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (Dids) and niflumic acid.

Inhibition of adrenergic proton extrusion in rainbow trout red cells by nitrite-induced methaemoglobinaemia.

The effects of nitrite-induced methaemoglobinaemia on adrenergic proton extrusion from rainbow trout red blood cells were studied using the pH-stat method. In control conditions adrenergic proton extrusion was completely inhibited by amiloride and was greater in deoxygenated than in oxygenated erythrocytes. Nitrite-induced methaemoglobinaemia was associated with a pronounced reduction in the catecholamine-stimulated proton efflux from both deoxygenated and oxygenated erythrocytes. In deoxygenated erythrocytes the initial proton efflux upon catecholamine stimulation decreased by 60-70%, while the percentage of methaemoglobin in the red cells increased from the control level of 1-3% to 20%. In oxygenated erythrocytes the decrease was 30% at the same methaemoglobin percentage range. It is suggested that the pronounced influence of nitrite-induced methaemoglobinaemia on adrenergic proton efflux results from an inhibition of the red cell sodium/proton exchanger by the R-like haemoglobin conformations.

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 β-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 O 2 or CO to the heme.

Effect of noradrenaline on the methaemoglobin concentration of rainbow trout red cells

We studied the effect of noradrenaline on the methaemoglobin (metHb) concentration in rainbow trout red cells. The erythrocytes were incubated in physiological medium with or without noradrenaline and the percentage of metHb of total Hb content was measured. Noradrenaline lowered the metHb content significantly as compared to controls. To study if the effect of noradrenaline was caused by adrenergic intracellular alkalinization, cells were treated with noradrenaline + carbonic anhydrase or noradrenaline + acetazolamide. Carbonic anhydrase inhibits the adrenergic increase in intracellular pH, but did not reduce the effect of noradrenaline on the metHb concentration. Acetazolamide accentuates the increase in intracellular pH. However, there was no difference in the methaemoglobin content of noradrenaline-incubated and noradrenaline + acetazolamide-incubated cells. These results show that the effect of noradrenaline on the methaemoglobin content is independent from the adrenergic increase in intracellular pH. However, amiloride treatment inhibited the effect of noradrenaline on the methaemoglobin content, suggesting that the protein mediating sodium/proton exchange may also be involved in controlling cellular methaemoglobin levels.

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.

Glycolytic and Associated Enzymes of Rainbow Trout (Oncorhynchus Mykiss) Red Cells: In Vitro And In Vivo Studies

ABSTRACT Studies were undertaken in vitro and in vivo to assess the maximal activities of 26 glycolytic and associated enzymes of rainbow trout (Oncorhynchus mykiss) red cells. The red cells possess a complete sequence of glycolytic enzymes capable of anaerobic oxidation of glucose to lactate. Red cell pyruvate kinase (PK) was inhibited by ATP (I50 ≈ 5 mmol I 1−1), but was not sensitive to alanine inhibition or fructose-1,6-bisphosphate activation. The properties of red cell PK were similar to those of the muscle-type enzyme. Lactate dehydrogenase (LDH) from the trout erythrocyte resembled LDH from trout heart in terms of pyruvate inhibition in vitro. Enzymes associated with phosphagen and amino acid metabolism as well as the pentose phosphate shunt were also present. However, enzyme indicators of glycogenolytic and gluconeogenic potential were either absent or present at very low levels. The capacity for aerobic respiration via the tricarboxylic acid (TCA) cycle was suggested by the presence of citrate synthase activity. The association of glycolytic enzymes with the particulate fraction of the red cell was assessed for six enzymes. No binding was detected for hexokinase, PK and LDH; low levels of binding by phosphofructokinase (5%), aldolase (1%) and glyceraldehyde-3-phosphate dehydrogenase (3%) were not altered by strenuous exercise. Glycolytic enzyme binding, therefore, does not appear to be an important regulator of energy metabolism in these cells. Intracellular glucose levels, in contrast, appeared to be regulated following exercise stress. An increase in intra-erythrocytic lactate levels at this time may reflect the importance of exogenously produced lactate as a substrate for ATP production. The description of trout red cell metabolism presented here provides a basis for further study of the relationships between organismic gas exchange and molecular-level adaptation of nucleated red cell function.

Adrenergic Control of Red Cell pH in Salmonid Fish: Roles of the Sodium/Proton Exchange, Jacobs-Stewart Cycle and Membrane Potential

ABSTRACT We investigated the mechanisms by which adrenergic activation of sodium/proton exchange reduces the pH gradient across the membrane of rainbow trout red cells. In untreated cells, adrenergic stimulation caused a significant increase in the proton distribution ratio ([H+]e/[H+]i) across the red cell membrane. The increase in the proton distribution ratio caused by adrenergic stimulation was inhibited by the protonophore 2,4-dinitrophenol (2,4-DNP). Thus, sodium/proton exchange displaces protons from electrochemical equilibrium. Active regulation of intracellular pH by sodium/proton exchange is possible, because the extracellular dehydration of carbonic acid to carbon dioxide is uncatalyzed. The increase in proton distribution ratio caused by adrenergic stimulation was inhibited in red cell suspensions to which extracellular carbonic anhydrase had been added before stimulation. In contrast, inhibition of intracellular carbonic anhydrase markedly increased the pH changes induced by adrenergic stimulation, suggesting that the net direction of the intracellular hydration/dehydration reaction may markedly affect the intracellular pH changes. Membrane potential changes are not a necessary component of the adrenergic response. The increases in red cell volume and sodium and chloride concentrations induced by adrenergic stimulation were unaffected in cells ‘voltage-clamped’ by valinomycin.

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.

The response of trout red cells to adrenaline during seasonal acclimation and changes in temperature

Adult rainbow trout were acclimated to three different temperature and photoperiod regimes: 17°C, 14 h light: 10 h dark (summer); 7° C, 14 h light: 10 h dark; and 5° C, 8 h light: 16 h dark (winter). Blood was collected from these fish after 40 days acclimation, and the response of red blood cells to in vitro adrenergic stimulation was assessed. To examine potential seasonal variations in endogenous levels of circulating catecholamines, plasma levels of adrenaline (Ad) and noradrenaline (NAd) were measured at rest and after exercise. At rest, there were no differences between groups in plasma levels of either Ad or NAd, but, after exercise, the pattern of catecholamine elevation differed. In fish acclimated to 17 and 7° C in summer, Ad and NAd increased by about the same amount (10–15 times). In fish acclimated to 5° C in winter, NAd increased about three‐fold, compared to the near 50‐fold increase in Ad levels. Whether this difference between groups can be attributed to seasonal influences is unclear. At both low (0·5%) and high (2%) PCO2, adrenergic stimulation (2 × 10‐7 M Ad) of trout red cells in vitro led to a significant reduction in MCHC (mean cell [haemoglobin]), compared to non‐stimulated cells. However, only at the high PCO2 were pHe and red cell pHi significantly different from those in the non‐stimulated cells: the latter was higher and the former lower in the stimulated cells. There were no differences in the response of red cells to adrenergic stimulation between groups of fish. Under the conditions of the present study no influence of season and/or temperature on the in vitro response of trout red cells to adrenergic stimulation was shown.