The conversion of plasma HCO 3 − to CO2 by rainbow trout red blood cells in vitro: adrenergic inhibition and the influence of oxygenation status

This study employed a recently developed radioisotopic assay (Wood and Perry 1991) to examine the inhibition, induced by catecholamines, of the conversion of plasma HCO ₃ ^– to CO₂ in acidotic trout blood, and the influence of oxygenation status on the response. Blood was incubated in vitro at P_{CO 2}= 2 torr, and 10^–6 M noradrenaline was employed as the adrenergic stimulus. In particular we investigated whether the inhibition of plasma HCO ₃ ^– conversion could be explained by a limited supply of H⁺s for the intracellular HCO ₃ ^– dehydration reaction because of competition by the adrenergically activated Na /H⁺ exchanger. Hypoxia (P_{O 2}= 15 torr) was employed as a tool to intensify this competition. Hypoxia raised RBC pHi, pHe, and plasma total CO₂ concentration (C_{CO 2}) by the Haldane effect, and increased the magnitude of Na⁺/H⁺ activation, expressed as the change in the transmembrane pH gradient (pHe-pHi). However hypoxia did not alter the inhibition of the conversion of plasma HCO ₃ ^– to CO₂ caused by noradrenaline. Hypoxia itself stimulated the RBC-mediated conversion of plasma HCO ₃ ^– to CO₂ by about 20% in the presence or absence of noradrenaline. The conversion rate was strongly correlated with pHe, pHe-pHi, and plasma C_{CO 2} in these experiments, but not with pHi. We conclude that adrenergically mediated inhibition in the conversion of plasma HCO ₃ ^– to CO₂ by trout RBCs is not due to competitive limitation on intracellular H⁺s, but rather to changes in the electrochemical gradient for HCO ₃ ^– entry and/or to CO₂ recycling from plasma to RBC. The deoxygenated condition helps to promote CO₂ excretion at the level of the RBC.     

The role of cAMP in regulating the β-adrenergic response of rainbow trout (Oncorhynchus mykiss) red blood cells

The -adrenergic response of teleost red blood cells (RBCs) enables the fish to maintain or even enhance the oxygen affinity of haemoglobin during various stress situations. The role of CAMP in the pronounced -adrenergic response of hypoxic rainbow trout RBCs was studied. Rainbow trout RBCs were incubated with three different -agonists (noradrenaline, adrenaline and isoproterenol, 10^–9 - 10^–4 M) at two oxygen tensions (P_{O 2}, 155 and 8 mmHg), and thereafter cAMP accumulation and cellular water content were measured.The cAMP concentration of non-stimulated trout RBCs was ca. 1200 nmol/kg dw. Of the three -agonists used, isoproterenol was the most effective in formation of cAMP, followed by noradrenaline and adrenaline. Oxygen tension affected the accumulation of cAMP in two ways. At physiological catecholamine levels (1–100 nM) there was either no difference between normoxic and hypoxic cells or a slight increase in the normoxic ones. At high catecholamine concentrations the accumulation of cAMP was greater in the hypoxic than in the normoxic cells. Oxygen tension also affected the magnitude of cell swelling but had no effect on the catecholamine concentrations causing half-maximal swelling (EC₅₀-values). The results indicate that, at physiological catecholamine levels, the -adrenergic response of rainbow trout RBCs is mainly regulated on the level of the Na⁺/H⁺ exchange.     

Relationship between otolith and somatic growth:consequence of starvation on acid-base balance inplasma and endolymph in the rainbow trout Oncorhynchus mykiss

The effect of a short period of starvation (5–6 weeks) on thechemistry of plasma and inner ear endolymph (Na⁺,K⁺, Cl^-, total Ca, protein, totalCO₂ concentrations and pH) was studied in trout (Oncorhynchusmykiss). In plasma, only total Ca significantly decreased, by 0.17 mM, instarved trout. A slight drop (10%) in the plasma protein content wasalso observed. Starvation induced a metabolic plasma alkalosis characterizedby a non-signifiacant increase in pH and a significant drop of 22% oftotal CO₂. In endolymph, the Na⁺,K⁺, Cl^- , total Ca and protein concentrationswere not significantly different in control and starved trout, but in thelatter, the pH of the endolymph was less alkaline (significant decrease of0.3 UpH) and the total CO₂ concentration was significantlyreduced by 50%. Experimental starvation was selected as aphysiological situation characterized by a slowing down of both somatic andotolith growth. We propose that the reduced rate of otolith growth isdirectly related to an overall reduction in the alkalinity of theendolymph.     

Quantification of presumptive Na+/H+ antiporters of the erythrocytes of trout and eel

The presumptive Na⁺/H⁺ exchange sites of trout and eel erythrocytes were quantified using amiloride-displaceable 5-(N-methyl-N-[³H]isobutyl)-amiloride (³H-MIA) equilibrium binding to further evaluate the mechanisms of i) hypoxia-mediated modifications in the trout erythrocyte -adrenergic signal transduction system and ii) the marked differences in the catecholamine responsiveness of this system between the trout and eel. MIA was a more potent inhibitor of both trout apparent erythrocyte proton extrusion (IC₅₀ = 20.1 ± 1.1 mol l^–1, N = 6) activity (as evaluated by measuring plasma pH changes after addition of catecholamine in vitro) and specific ³H-MIA binding (IC₅₀ = 257 ± 8.2 nmol l^–1, N = 3) than amiloride, which possessed a proton extrusion IC₅₀ of 26.1 ± 1.6 mol l^–1 (N = 6) and a binding IC₅₀ of 891 ± 113 nmol l^–1 (N = 3). The specific Na⁺ channel blocker phenamil was without effect on adrenergic proton extrusion activity or specific ³H-MIA binding. Trout erythrocytes suspended in Na⁺-free saline and maintained under normoxic conditions possessed 37,675 ± 6,678 (N = 6) amiloride-displaceable ³H-MIA binding sites per cell (B_max, presumptive Na⁺/H⁺ antiporters) with an apparent dissociation constant (K_D) of 244 ± 29 nmol l^–1 (N = 6). Acute hypoxia (PO₂ = 1.2 kPa; 30 min) did not affect the K_D, yet resulted in a 65% increase in the number of presumptive Na⁺/H⁺ antiporters. Normoxic eel erythrocytes, similarly suspended in Na⁺-free saline, possessed only 17,133 ± 3,716 presumptive Na⁺/H⁺ antiporters (N = 6), 45% of that of trout erythrocytes, with a similar K_D (246 ± 41 nmol l^–1, N = 6). These findings suggest that inter- and intra-specific differences in the responsiveness of the teleost erythrocyte -adrenergic signal transduction system can be explained, in part, by differences in the numbers of Na⁺/H⁺ exchange sites.     

The effects of experimental anaemia on CO2 excretionin vitro in rainbow trout,Oncorhynchus mykiss

The effects of severe experimental anaemia on red blood cell HCO₃ ^– dehydrationin vitro were examined in rainbow trout,Oncorhynchus mykiss. After 5 days of anaemia (haematocrit=4.9±1.1%) induced by intraperitoneal injection of phenylhydrazine hydrochloride, fish displayed elevated arterial CO₂ tensions (anaemic PaCO₂=3.19±0.42 torrvs. control PaCO₂=1.35±0.17 torr) and a significant acidosis (anaemic pHa=7.73±0.04vs. control pHa=7.99±0.04). However, after 15–20 days of anaemia (hct=6.6±0.8%) induced by blood withdrawal, the arterial CO₂ tension was significantly lower than the control value, suggesting that physiological adjustments occurred within this time period to compensate for the lowered haematocrit. Compensation probably did not involve alterations in ventilation, which was unaffected by 5 days of anaemia (anaemic ;w=786±187 ml min^–1 kg^–1 vs. control ;w=945±175 min^–1 kg^–1), based on indirect Fick principle measurements.Potential adaptations to longer term anaemia at the level of the red blood cells were investigated using a radioisotopic HCO₃ ^– dehydration assay. Owing to the difference in haematocrits, the HCO₃ ^– dehydration rate for blood from anaemic fish was significantly lower than that for control fish following equilibration at the same CO₂ tension. This difference was eliminated when HCO₃ ^– dehydration rates were measured on blood samples adjusted to the same haematocrit, a result which implies that the intrinsic rate of CO₂ excretion at the level of the red blood cell was not up-regulated during anaemia. The difference was also eliminated by equilibrating the blood samples with CO₂ tensions appropriate for the group from which the sample was obtained,i.e., PCO₂=1.4 torr for control samples and PCO₂=3.2 torr for anaemic samples; each at the appropriate haematocrit. It is concluded that the elevated PaCO₂ helps to reset CO₂ excretion to the control level, but that some additional physiological adjustment occurs to lower the PaCO₂ after 15–20 days of anaemia.     

Volume regulation in glutathione-treated brook trout (Salvelinus fontinalis) erythrocytes

Brook trout erythrocytes that were washed with and suspended in Ringer's solution with reduced glutathione (1.0 mM) maintained steady state cell volume for up to 24h, while those without the thiol-protective agent steadily shrank. Changes in cell volume (measured as packed cell volume, PCV) were evoked by acidic media (Ringer's at pH 6.8), hypoosmotic solutions (or both) and intracellular K⁺ and Cl⁻ concentrations were monitored over 4h. Acid-swollen cells failed to volume regulate or release K⁺ but had significantly elevated intracellular Cl⁻ Osmotically-swollen cells at pH 7.8 but not at pH 6.8 underwent regulatory volume decrease (RVD) and returned to initial levels in 2h, accompanied by release of K⁺ and Cl⁻ In contrast, osmotically-shrunken cells did not show regulatory volume increase. The regulatory volume decrease and concomitant K⁺ release were dependent on Cl⁻ implying a direct or indirect coupling of K⁺ to Cl⁻ transport in volume regulation. RVD was partially blocked by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS, 0.1 mM), an anion exchange blocker, but was unaffected by amiloride (1.0 mM) which blocks Na⁺/H⁺ exchange. Amiloride and DIDS prevented the swelling response to low pH but had no effect on control cells, suggesting involvement of Na⁺/H⁺ and Cl⁻/HCO₃ ⁻ exchanges in acid-induced cell swelling. Quinine (1.0 mM) a known blocker of K⁺ channels, exacerbated the osmotically-induced swelling but had little effect on the subsequent RVD and release of KCl. The results suggest that low extracellular pH inhibits neutral C⁻-dependent K⁺ release and the resultant regulatory volume decrease in osmotically-swollen cells.     

Intestinal HCO3− secretion in marine teleost fish: evidence for an apical rather than a basolateral Cl−/HCO3− exchanger

Intestinal fluid was collected from 11 marine teleost fish from the Baltic sea and the Pacific ocean. The anterior, mid and posterior segments of the intestine contained 33–110 mM of HCO₃ ^– equivalents (with exception of the Atlantic cod which contained only 5–15 mM). Considering literature values of transepithelial potentials and concentration gradients, these high levels of HCO₃ ^– equivalents are probably the result of active HCO₃ ^– transport. Possible HCO₃ ^– transport mechanisms were studied in the Pacific sanddab (Citharichthys sordidus) in vitro. Measurements of net secretion of HCO₃ ^– equivalents across the intestinal epithelium revealed mucosal DIDS sensitivity (10^–4 M) and Cl^–-dependence of the HCO₃ ^– equivalent net flux, but no serosal DIDS (10^–4 M) sensitivity. Net Na⁺ uptake was abolished in the absence of Cl^–, but some Cl^– uptake persisted in the absence of Na⁺, at a rate similar to that of net HCO₃ ^– secretion. Anterior, mid and posterior segments of the intestine performed similarly. These observations support the presence of an apical rather than a basolateral Cl^–/HCO₃ ^– exchanger and thus contrast the currently accepted model for intestinal HCO₃ ^– secretion. This apical Cl^–/HCO₃ ^– exchanger alone, however, is not sufficient for maintaining the observed HCO₃ ^– equivalents gradient in vivo. We suggest a coupling of cytosolic carbonic anhydrase, a basolateral proton pump and the apical Cl^–/HCO₃ ^– exchanger to explain the intestinal HCO₃ ^– transport.     

Ionoregulatory and respiratory responses of brown trout,Salmo trutta,exposed to lethal and sublethal aluminium in acidic soft waters

Soft water acclimated (Ca²⁺ 0.02 mM; Na⁺ 0.03 mM; K⁺ 0.01 mM; pH 7.0), cannulated brown trout (Salmo trutta) were exposed to various pH and aluminium (Al) regimes (pH 7.0, pH 5.0, pH 5.0 plus Al: 50, 25, and 12.5 g l^–1) for up to 5 days in order to determine (i) the sublethal concentration of Al at pH 5.0 for this species (ii) their ionoregulatory and respiratory status. No mortality or physiological disturbances were evident at pH 7.0 or pH 5.0. All trout died within 48 h at pH 5.0 in the presence of Al at 50 g l^–1 and 67% died over the 5 day period at pH 5.0 in the presence of Al at 25 g l^–1. Fish at these lethal Al concentrations showed significant decreases in arterial blood oxygen content (C_aO₂) but no changes in plasma osmolarity or the concentrations of plasma Na⁺, K⁺ and Cl^–. Physiological disturbance was more marked at the 50 g l^–1 Al concentration. The surviving fish at 25 g l^–1 showed few signs of physiological recovery while continually exposed to this regime. No fish died during the exposure to water of pH 5.0 containing 12.5 g l^–1 Al, but physiological disturbance was still apparent. These sublethally-stressed trout showed a transient decline in the plasma concentrations of Na⁺ and Cl^–1. Although C_aO₂ decreased, recovery was evident. The data suggest that in the brown trout, environmental Al concentration is as important as pH and calcium concentration in determining the physiological status of the fish.     

Osmoregulation in the mudskipper,Boleophthalmus boddaerti II. transepithelial potential and hormonal control

Boleophthalmus boddaerti submerged in 10%, 50% and 80% seawater (sw) for 7 days, had whole body transepithelial potentials (TEP) of 3.3, 18.3 and 22.9 mV, respectively. Hypophysectomy significantly decreased the TEP ofB. boddaerti and reversed the polarity of the TEP of the fish exposed to 10% sw.Hypophysectomy also significantly decreased the branchial Na⁺-K⁺ activated adenosine triphosphatase (Na⁺,K⁺-ATPase) activity but increased the activity of branchial HCO₃ ^–-Cl^– stimulated adenosine triphosphatase (HCO₃ ^–,Cl^–-ATPase) inB. boddaerti exposed to 10% sw. However, survival in 10% sw was not significantly impaired by hypophysectomy and no significant change in plasma osmolality and plasma Na⁺ and Cl^– concentrations was observed.Various doses of ovine-prolactin or salmon-prolactin were unable to restore the TEP of hypophysectomizedB. boddaerti in 10% sw to that of the sham-operated fish. However, cortisol increased TEP to a positive value in hypophysectomizedB. boddaerti, though it was still lower than the sham-operated control. Cortisol treatment also affected the plasma osmolality, plasma Na⁺ and Cl^– contents and branchial Na⁺,K⁺-ATPase and HCO₃ ^–,Cl^–-ATPase activities. Overall, the hormonal control of osmoregulation inB. boddaerti appeared to differ from that of other teleosts.     

Growth and physiological changes in scaled carp and blue tilapia under behavioral stress in mono- and polyculture rearing using a recirculated water system

Growth performance, carcass composition, liver and blood parameters ofscaled carp (C), Cyprinus carpio, and blue tilapia (T),Oreochromis aureus, reared for eight weeks in twomonoculture (100%C and 100%T) and two polyculture (60%C–40%T and40%C-60%T) conditions were investigated. In polyculture 40%C–60%T bothspecies achieved the highest levels of specific growth rate and the lowestlevels of food conversion ratio and carcass lipid content. In addition, theyexhibited the highest values of plasma pO₂ and pH and the lowestvalues of plasma pCO₂, cholesterol and albumin, although thedifferences among treatments were not significant in the case of tilapia.Tilapia showed significantly lower plasma Cl^– levels than underthe other conditions. Carp in monoculture and tilapia in polyculture60%C–40%T had the lowest levels of specific growth rate and significantlyhigher levels of liver lipids and plasma triglycerides than in the other groups.In addition, carp in monoculture exhibited a significantly higher haematocritthan in polyculture. No significant variations among treatments were observedconcerning plasma cortisol, glucose, osmolality, Na⁺, K⁺,HCO₃ ^– andHCO₃ ^–/H₂CO₃ in either species.The combination of scaled carp and blue tilapia, in which blue tilapia were themain species, proved to be the best for both species. It was suggested thatgrowth and physiological changes under mono- and polyculture rearing, in anintensive system, seem to be as a result of a different state of stress relatedto fish behavior.     

Reabsorption of urea by the kidney of the freshwater rainbow trout

In light of recent evidence that carrier-mediated transport of urea occurs in the mammalian kidney, this study examined the renal handling of urea in freshwater rainbow trout (Oncorhynchus mykiss). Fish were fitted with indwelling arterial and urinary bladder catheters for the measurement of plasma and urine composition (urea, Na⁺, Cl^-, glucose, H₂O), glomerular filtration rate ([³H]PEG-4000), and urine flow rate, thereby allowing quantification of tubular reabsorption rates. The fractional reabsorption of urea (72%) was greater than that of H₂O (50%) but less than that of Na⁺, Cl^-, or glucose (95–100%) and occurred against an apparent concentration gradient, suggesting active reabsorptive transport; [urea] in the urine was only 59% of that in blood plasma. When fish were infused with exogenous urea loads, these patterns remained largely unchanged, and urea reabsorption increased in direct proportion to the filtered urea load. There was no evidence for saturation of the reabsorptive transport mechanism at urea filtration rates up to 4-fold above the normal range, representing urea loading rates that proved toxic. Extra-renal excretion, presumably through the gills, increased markedly, almost keeping pace with urea loading. This evidence suggests that carrier-mediated reabsorptive transport of urea occurs in the kidney and that plasma urea levels are normally subject to tight homeostatic control in freshwater trout.     

Osmoregulation in the mudskipper,Boleophthalmus boddaerti I. Responses of branchial cation activated and anion stimulated adenosine triphosphatases to changes in salinity

The mudskipperB. boddaerti, was able to survive in waters of intermediate salinities (4–27). Fish submerged in dechlorinated tap water suffered 60% mortality by the fifth day while 60% of those in 100% sea-water (sw) died after the third day of exposure. After being submerged in 50% or 80% sw for 7 days, the plasma osmolality, plasma Na⁺ and Cl^– concentrations and the branchial Na⁺ and K⁺ activated adenosine triphosphatase (Na⁺,K⁺-ATPase) activity were significantly higher than those of fish submerged in 10% sw for the same period. However, the activities of the branchial HCO₃ ^– and Cl^– stimulated adenosine triphosphatase (HCO₃ ^–,Cl^–-ATPase) and carbonic anhydrase of the latter fish were significantly greater than those of the former. Such correlation suggests that Na⁺,K⁺-ATPase is important for hyperosmotic adaptation in this fish while HCO₃ ^–-Cl^–-ATPase and carbonic anhydrase may be involved in hypoosmotic survival.     

β-adrenergic signal transduction in fish: interactive effects of catecholamines and cortisol

The elevation of plasma catecholamine levels during acute stress initiates a series of compensatory physiological and biochemical mechanisms to alleviate the disruptive effects of stress on blood oxygen transport. Of particular importance is the β-adrenergic activation of a Na⁺/H⁺ antiporter associated with the red blood cell (rbc) membrane. Upon activation, the Na⁺/H⁺ antiporter extrudes H⁺ from the rbc and the resultant alkalinization of the rbc interior serves to enhance both the affinity and the capacity of haemoglobin O₂ binding. The activation of the Na⁺/H⁺ exchanger is dependent upon the intracellular accumulation of cyclic AMP. The extent of cyclic AMP accumulation is determined, in part, by the number and/or affinities of cell surface β-adrenoreceptors. Recent studies have shown that the number of cell surface β-adrenoreceptors are rapidly increased during acute hypoxia and that this phenomenon may explain the enhanced responsiveness of hypoxic rbc's to exogenous catecholamines. In certain instances, plasma catecholamine and cortisol levels rise concurrently. We recently have shown that chronic (10 day) elevation of cortisol levels, in vivo, or short-term (24h) elevation, in vitro, caused significant elevation of internalized β-adrenoreceptors. Upon exposure of the rbc's to hypoxia, these additional receptors are rapidly recruited to the cell surface where they become functionally coupled to adenylate cyclase. Ultimately, therefore, chronic elevation of plasma cortisol levels increases the responsiveness of the rbc to circulating catecholamines. We recently have identified similar enhancement of cell surface β-adrenoreceptors by cortisol and increased physiological responsiveness (glycogenolysis) to catecholamines in trout hepatocytes. Thus, chronic elevation of cortisol levels appears to be generally adaptive for increasing the sensitivity of the β-adrenergic signal transduction system of at least two cell types (rbc's, hepatocytes) involved in the amelioration of acute stress when plasma catecholamine levels rise.

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Résumé L'élévation des niveaux de cathécolamines plasmatiques pendant un stress aigu déclenche une série de mécanismes physiologiques et biochimiques de compensation destinés à suprimer les effets destructeurs du stress sur les transport d'oxygène par le sang. L'activation β-adrénergique de l'antipore Na⁺/H⁺ associé à la membrane des globules rouges du sang (rbc) est particulièrement importante. Après activation, l'antipore Na⁺/H⁺ excrète du globule rouge H⁺ et l'alcalinisation du milieu intérieur du globule qui en résulte sert à stimuler l'affinité et la capacité de liaison hémoglobine-O₂. L'activation de l'échangeur Na⁺/H⁺ est dépendante de l'accumulation intracellulaire d'AMP cyclique. La quantité d'AMP cyclique accumulée est déterminée en partie par le nombre et/ou l'affinité des β-adrénorécepteurs situés à la surface des cellules. Des études récentes montrent que le nombre de β-adrénorécepteurs de surface augmente rapidement pendant une hypoxie aigue et que ce phénomène pourrait expliquer la stimulation de la réponse des globules rouges hypoxiques à des cathécolamines exogènes. Dans certains cas, les niveaux plasmatiques de cortisol et de cathécolamine augmentent simultanément. Nous avons récemment montré qu'une augmentation chronique (10 jours) des niveaux de cortisol in vivo ou une élévation de courte durée (24h) in vitro conduisent à une augmentation des β-adrénorécepteurs internalisés. Suite à l'exposition de globules rouges à une hypoxie, ces nouveaux récepteurs sont rapidement recrutés à la surface de la cellule où ils deviennent fonctionnellement couplés à l'adénylate cyclase. En dernier lieu donc, une élévation chronique des niveaux de cortisol augmente la réponse des globules rouges aux cathécolamines circulants. Nous avons récemment identifié une augmentation similaire des β-adrénorécepteurs de surface cellulaire par le cortisol et une réponse physiologique (glycogénolyse) stimulée par les cathécolamines dans les hépatocytes de truite. Ainsi, une élévation chronique des niveaux de cortisol semble être en général adaptative et augmentant la sensibilité du système de transduction du signal β-adrénergique dans au moins 2 types de cellules (les globules rouges et les hépatocytes) impliquées dans l'amélioration du stress aigu quand les niveaux de cathécolamine plasmatique augmentent.

     

Characterization of transport Na+-ATPases in gills of freshwater tilapia

Branchial plasma membranes from the freshwater cichlid teleostOreochromis mossambicus (tilapia) contain two Na⁺-dependent ATPases: Na⁺/K⁺ ATPase, and an amiloride-sensitive ATPase which is postulated to operate as a Na⁺/H⁺ (–NH₄ ⁺) ATPase. It is suggested that both enzyme activities are located in the basolateral membrane system of the chloride cells. K⁺ has opposing effects on the two enzymes: it stimulates Na⁺/K⁺ ATPase and inhibits Na⁺/H⁺ (–NH₄ ⁺) ATPase activity. Na⁺/H⁺ ATPase appears more sensitive to NH₄ ⁺ at low concentrations than Na⁺/K⁺ ATPase and the stimulatory effect by NH₄ ⁺ ions on the first enzyme could be important in facilitating NH₄ ⁺ excretion by tilapia gills under physiological conditions.In vitro maximum stimulation by NH₄ ⁺ is similar for the two enzymes (200%). In contrast to Na⁺/K⁺ ATPase, Na⁺/H⁺ ATPase activity is inhibited by supra-physiological (>20 mM) concentrations of NH₄ ⁺.     

Effects of sulfide on K+ flux pathways in red blood cells of crucian carp and rainbow trout

The effect of sulfide on K⁺ influx pathways was measured in red blood cells (RBCs) of sulfide-sensitive rainbow trout (Oncorhynchus mykiss) and sulfide-tolerant crucian carp (Carassius carassius). In trout RBCs, maximal inhibition of Na⁺, K⁺-ATPase was attained at 10 mol l^–1 sulfide and amounted to 32% without being influenced by pH between 6.7 and 8.3. Ouabain-resistant K⁺ influx in the absence and presence of sulfide was insignificant at pH values between 6.7 and 7.7. At higher pH values ouabain-resistant K⁺ influx increased, but was inhibited to about 15% by 30 mol l^–1 sulfide. In RBCs of crucian carp neither Na⁺, K⁺-ATPase nor ouabain-resistant K⁺ influx were affected by sulfide concentrations up to 850 mol l^–1. Differences in sulfide-sensitivity of K⁺ influx between both species can be based upon different properties of the membrane transporter themselves. The reduced Na⁺, K⁺-ATPase activity in trout RBCs may also result from a slightly reduced (by 9%) ATP level after sulfide exposure. In addition, intracellular sulfide concentrations were higher in trout RBCs as compared to crucian carp. In trout, intracellular sulfide concentrations reached extracellular levels within 5 min of incubation whereas sulfide concentrations in crucian carp RBCs remained about 2-fold lower than extracellular concentrations. Although the physiological basis of sulfide-insensitive K⁺ influx in crucian carp RBCs is currently unknown it may contribute to the extremely high sulfide-tolerance of this species.     

Morphological responses of the rainbow trout (Oncorhynchus mykiss) gill to hyperoxia,base (NaHCO3) and acid (HCl) infusions

Marked morphological responses occur in the gills of freshwater rainbow trout in response to experimental acid-base disturbance and these responses play an important role in acid-base correction. Compensated respiratory acidosis induced by 70h exposure to environmental hyperoxia (elevated water PO₂) caused a 33% decrease in branchial chloride cell fractional surface area (CCFA). Metabolic alkalosis induced by normoxic recovery (6h) from hyperoxia (72h) caused a 50% increase in CCFA, whereas metabolic alkalosis induced by infusion (19h) of NaHCO₃ caused a 70% rise. However, the largest increase (135%) in CCFA was seen in response to infusion (19h) of HCl. NaCl infusion had no effect. A particular goal was to assess the relative importance of changes in CCFA vs. changes in internal substrate (HCO₃ ^–) availability in regulating the activity of the branchial Cl^–/HCO₃ ^– exchange system. For each of the experimental treatments, the accompanying blood acid-base status and branchial transport kinetics (K_m, J_max) for Cl^– uptake had been determined in earlier studies. In the present study, a positive linear relationship was established between CCFA and J^Cl– _max in individual control fish in the absence of an acid-base disturbance. By reference to this relationship, observed changes in J^Cl– _max during metabolic acid-base disturbances were clearly due to changes in both CCFA and internal substrate levels (plasma [HCO₃ ^–]) with the two factors having approximately equal influence.     

Ionoregulatory responses to temperature change in two species of freshwater fish

We examined the ionoregulatory responses to temperature changes in two species of freshwater fish that differ in thermal preferences; the stenothermal, cold-water rainbow trout (Oncorhynchus mykiss) and the more eurythermal, warm-water common shiner (Notropis cornutus). We found that rainbow trout maintained constant plasma Na⁺ levels over the entire temperature regime (5–20 °C). Upon transfer from 15 °C (holding temperature) to 5 and 10 °C, rainbow trout experienced a significant drop in Na⁺ uptake (J_in ^Na), but after two weeks J_in ^Na had upregulated to warm temperature levels. Further, Na⁺ efflux (J_out ^Na) fell significantly at the colder temperatures. As a result, trout at the lowest temperatures were still in ion balance. When trout were exercised to exhaustion both O₂ consumption (MO₂) and J_out ^Na rose significantly at all temperatures, but while MO₂ continued to be dependent upon temperature, J_out ^Na was high and constant. In contrast to the trout, common shiners experienced a 20% drop in plasma Na⁺ at 5 °C. Upon exposure to cold temperatures they experienced a reduced J_in ^Na, and showed no signs of acclimation during the subsequent two weeks. Likewise J_out ^Na was constant at all temperatures. These findings raise questions regarding the degree to which fish employ homeostatic mechanisms designed to defend a set- point (i.e., steady-state) osmolarity and ionic composition.     

The carbonic anhydrase inhibitor in trout plasma: purification and its effect on carbonic anhydrase activity and the Root effect

The plasma inhibitor of carbonic anhydrase (CA) from rainbow trout was purified using ion exchange chromatography. The inhibitor has a high isoelectric point value (pI>10). SDS-PAGE electrophoresis and gel filtration demonstrated that the inhibitor is a low molecular weight compound of about 6,000 daltons. The plasma inhibitor was more effective against gill CA than against blood CA in vitro, probably reflecting the presence of various CA-isoenzymes in red blood cells and gill tissue. The apparent Root effect, i.e., the impairment of the oxygen binding capacity of hemoglobin in red blood associated with increased blood P_{CO 2}was counteracted by the plasma inhibitor, probably by acting on membrane-bound and/or cytosolic blood CA. This interaction may be of importance in adaptive mechanisms, e.g., during the acidemic phase, when the fish is being acclimated to hypercapnic conditions.     

Basal H2-receptor stimulated and pH-dependent acid secretion from an isolated stomach mucosa preparation of the cod,Gadus morhua,studied using a modified pH-static titration method

Gastric acid secretion from isolated cod stomach mucosa was measured using a pH-static titration method. A basal acid secretion rate (BASR) of 6.0±0.6 nEqH⁺min^–1cm^–1 was measured when using 0.9% NaCl as luminal solution. There was a dose-dependent increase in response to histamine between 0.12 and 0.20 M (EC₅₀=0.15 M), above which gastric acid secretion plateaued at 13.5±1.8 nEqH⁺min^–1cm^–1. Ranitidine, a H₂-receptor antagonist, completely blocked the stimulatory effect of histamine and reduced the BASR. The H₁-receptor antagonist, clemastine, did not inhibit the response to histamine. Acid secretion rates decreased significantly when the pH of the luminal side of the mucosa was lowered from pH 5.75 to pH 4.50, indicating that a negative feedback mechanism was operating. Histological staining showed that oxynticopeptic cells were uniformly distributed throughout the cardiac stomach.It is concluded that the acid secretion in the isolated stomach mucosa of cod can be measuredin vitro with a pH-static titration method. The method was used to demonstrate that the BASR is downregulated by a decrease in pH. Furthermore, we conclude that the histamine receptor in the cod stomach mucosa resembles the mammalian H₂-receptor and that histamine is secreted under basal conditions.     

Interactions among dietary minerals,arginine and lysine in rainbow trout (Salmo gairdneri)

Studies were conducted to determine whether interactions occur among dietary lysine, arginine and monovalent minerals in rainbow trout. In one experiment, rainbow trout fingerlings were fed diets containing three levels of lysine (2.4, 3.1 and 3.8 g per 100 g diet), two levels of arginine (1.7 and 2.5 g per 100 g diet) and two mixtures of Na⁺ K⁺ and Cl in a 3×2×2 factorial design. The mixtures varied in the proportions of cations to anions such that Cl equalled the sum of Na⁺ and K⁺ (cations – anions = 0 mEq/kg diet) in one mixture and exceeded the sum of Na⁺ and K⁺ (cations – anions = –200 mEq/kg diet) in the second mixture. Growth and efficiency of feed conversion were not affected by dietary lysine and arginine in fish fed diets containing – 200 mEq/kg balance, but when fish were fed diets containing a 0 mEq/kg balance, 3.8% lysine and a combination of 3.1% lysine and 2.5% arginine depressed both measures of response. Trout receiving the 0 mEq/kg cation-anion balance had significantly higher free histidine concentrations and lower free lysine concentrations in muscle and higher hepatic arginase activity (P0.01) than those receiving –200 mEq/kg. In another experiment, trout were fed diets containing three levels of K⁺ (21, 191 and 360 mEq/kg), two levels of Na⁺ (21 and 191 mEq/kg) and two levels of Cl^– (179 and 347 mEq/kg) in a 3×2×2 factorial design. Growth and efficiency of feed conversion were depressed and hepatosomatic index increased with higher levels of dietary K⁺ (P0.01), Na⁺ (P0.05) and Cl (P0.01), with significant K⁺ x Cl⁺ (P0.01) and K⁺ x Na⁺ x Cl (P0.05) interactions. Increasing dietary K⁺ resulted in increased levels of muscle free histidine and decreased levels of muscle free lysine and arginine (P0.01), while increasing dietary Cl increased muscle free lysine, the effect of which was dependent on dietary potassium (K⁺ x Cl^–, P0.01). It is concluded that dietary levels of K⁺, Na⁺ and Cl^–, irrespective of overall cation-anion balance of these minerals, affects growth rate, efficiency of feed utilization and the metabolism of basic amino acids in tissues of trout. Excess lysine causes depressed growth and efficiency of feed utilization. These effects were due to a lysine toxicity rather than a lysine-arginine antagonism, as they were not prevented by supplemental dietary arginine.