On the Mechanism of Sodium Extrusion across the Irrigated Gill of Sea Water-Adapted Rainbow Trout (Salmo gairdneri)

Sodium efflux (J_out^Na) across the irrigated trout gill was rapid in sea water (SW), but only about 25 % as large in fresh water (FW). The difference correlated with a change in the potential difference across the gill (TEP). The latter was about +10 mV (blood positive) in SW, but –40 mV in FW. Both flux and electrical data indicated that gills in this fish are permeable to a variety of cations including Na⁺, K⁺, Mg²⁺, choline, and Tris. They are less permeable to anions; P_Na:P_K:P_Cl was estimated to be 1:10:0.3, and P_Cl > P_gluconate. The TEP was shown to be a diffusion potential determined by these permeabilities and the extant ionic gradients in SW, FW as well as in other media. J_out^Na appeared to be diffusive in all of the experiments undertaken. Exchange diffusion need not be posited, and the question of whether there is an active component remains open.     

TEP on the tide in killifish (<Emphasis Type="Italic">Fundulus heteroclitus</Emphasis>): effects of progressively changing salinity and prior acclimation to intermediate or cycling salinity

Transepithelial potentials (TEP) were measured in killifish, acclimated to freshwater (FW), seawater (SW), 33% SW or cycling salinities relevant to tidal cycles in an estuary, and subsequently subjected to salinity changes in progressive or random order. Random compared to progressive salinity changes in an upward or downward direction in FW- and SW-acclimated fish, respectively, did not greatly influence responses to salinity change. Fish acclimated to SW or 33% SW as well as those acclimated to cycling salinities behaved similarly (TEP more positive than +15 mV in 100% SW, decreasing to ~0 mV at 20–40% SW, and more negative than −30 mV in FW). In contrast, FW-acclimated fish displayed a less pronounced TEP response to salinity (0 mV in FW through 20% SW, increasing thereafter to values more positive than +10 mV at 100% SW). We conclude that when evaluated under estuarine tidal conditions, the killifish gill exhibits adaptive electrical characteristics, opposing Na⁺ loss at low salinity and favouring Na⁺ extrusion at high salinity, changes explained at least in part by the Cl⁻ to Na⁺ permeability ratio. Thus animals living in the estuaries can move to lower and higher salinities for short periods with little physiological disturbance, but this ability is lost after acclimation to FW.     

Na+ uptake through the brush border membranes of intestine from fresh water and sea-water adapted trout (Salmo gairdneri,R.)

Summary A comparative study of the mechanisms of Na⁺ absorption through brush border membranes of enterocytes from freshwater (FW) and seawater (SW) adapted trout were carried out using purified vesicle preparations. In contrast to FW trout, SW trout were found to possess a Na⁺–K⁺–Cl^– cotransport process. This finding is regarded as a major adaptation to SW since this cotransport allows an increase of ions and water absorption. Both FW and SW trout were equipped with a Na⁺–H⁺ exchange. In FW, the intestine of the trout had both a Na⁺–Na⁺ exchange and a Na⁺ conductance which may be responsible for enterocyte Na⁺ uptake along the potential gradient.     

Changes in branchial and intestinal osmoregulatory mechanisms and growth hormone levels during smolting in hatchery-reared and wild brown trout

Evidence of smolting was studied in Danish hatchery-reared brown trout Salmo trutta L. Twenty-four hour seawater (SW) challenge tests (28‰, 10°C) at regular intervals showed that maximal hypo-osmoregulatory ability developed within a 3–4-week period in March and April. The improved ability to regulate plasma osmolality, muscle water content and plasma total [Mg] developed asynchronously, indicating that developmental changes in the gill, the gastrointestinal system and the kidney may not necessarily concur during smolting. Gill Na⁺, K⁺-ATPase activity peaked in April at the time of optimal hypo-osmoregulatory ability. Na⁺, K⁺-ATPase a -subunit mRNA level in gills was unchanged from January until April, but decreased in May in parallel with a decrease in the activity of the enzyme. In the middle region of the intestine, Na⁺, K⁺-ATPase activity increased in February and remained high until April. In the posterior region of the intestine, the activity was stable from January until April after which it decreased. In vitro fluid transport capacitity, J_v, in the middle intestine fluctuated throughout the spring. In the posterior intestine, J_v was low until late March, when it increased fivefold until early May. Drinking rate in fish transferred to SW for 24 h surged during spring. Na⁺, K⁺-ATPase activity in the pyloric caeca was elevated from March until May, and increased in response to SW transfer in June, suggesting a hypo-osmoregulatory function of the pyloric caeca. Plasma GH levels surged in FW trout during spring, concurring with the increase in gill Na⁺, K⁺-ATPase activity and SW tolerance, but peaked in May when gill Na⁺, K⁺-ATPase activity and SW tolerance were regressing. GH levels were generally low in SW-challenged fish, and there was no consistent effect of 24-h SW exposure on GH levels. In wild anadromous trout, gill Na⁺, K⁺-ATPase activity varied seasonally as in hatchery-reared fish, but peaked at higher levels suggesting a more intense smolting in fish living in their natural environment.     

Electrogenic action of calcium on crayfish gill

When intact crayfish are in an ion-poor medium (KCl, 0.1 mmol·l^-1+KHCO₃, 0.1 mmol·l^-1) there is a large potential difference (transepithelial potential difference),-20 to-40 mV (hemolymph negative), across the gills. Addition of Ca²⁺ to the medium is followed by a rapid change in transepithelial potential difference to near 0 mV. The transepithelial potential difference showed a non-linear dependence on [Ca²⁺]_out with a limiting value of+2 to+10 mV at>1 mmol·l^-1. The concentration generating a half-maximum transepithelial potential difference change (15–20 mV) was 0.1 to 0.2 mmol·l^-1. Three other alkaline earth ions were also electrogenic; Ba²⁺ caused slightly larger transepithelial potential difference changes, Sr²⁺ and Mg²⁺ were a little less effective. It has been suggested that the transepithelial potential difference in ion-poor medium (in fish) is due to the diffusive efflux of NaCl across the gills, with a Cl^-/Na⁺ permeability ratio of <1. Evidence is presented that this might be the case in crayfish. The electrogenic effect of Ca²⁺ might then be due to its effect on gill permeability to Na⁺ and Cl^- such that the permeability ratio increased and approached unity as the transepithelial potential difference approached 0. However, this was shown to be unlikely. An alternative explanation for Ca²⁺ dependence of the transepithelial potential difference is that active inward Ca²⁺ transport is electrogenic.Abbreviations FW fresh water - I _out ion efflux - IP ion-poor solution - P _c Cl-permeability - P _Na Na⁺ permeability - R electrical resistance - SW sea water - TEP transepithelial potential difference     

Contribution of Na/Na and Cl/Cl exchanges to sodium and chloride fluxes in the crabGoniopsis cruentata

Summary Sodium or chloride efflux and transepithelial potentials (TEP) were measured in crabs exposed to seawater concentrations ranging from 150 to 25% SW. In crabs acclimated to 150% SW the Na⁺ efflux (3.8 mmol/h·100 g) was significantly higher than the Cl^– efflux (2.1 mmol/h·100 g), but both fluxes decreased to about 0.6 mmol/h·100 g in crabs from 50 or 25% SW. The TEP varied linearly from –1 mV (blood negative) in 150% SW, to –11 mV in 25% SW. In 150 and 100% SW the calculated components of the ion fluxes (i.e., diffusive, urinary, active uptake or extrusion) added up to less than one-half of the isotopically measured values. In 50 and 25% SW the measured effluxes were fully accounted for by their calculated components. In crabs transferred from 150% SW to low-Na 150% SW (=TRIS ASW), the Na⁺ efflux decreased abruptly, from 3.7 to 0.6 mmol/h; the Cl^– efflux decreased much less, from 1.9 to 1.5 mmol/h. A large fraction of the Na⁺ (or Cl^–) fluxes in crabs from concentrated SW meets the criteria for exchange diffusion, which decreases or disappears as the external concentration of each ion is lowered. This suggests that changes of the permeability to ions, in response to alterations of environmental salinity, may not constitute an important adaptive strategy in this species.Abbreviations SW seawater - TEP transepithelial potential - TRIS ASW artificial seawater 150%     

Two-substrate kinetic analysis: a novel approach linking ion and acid-base transport at the gills of freshwater trout,Oncorhynchus mykiss

Summary The novel application of a two-substrate model (Florini and Vestling 1957) from enzymology to transport kinetics at the gills of freshwater trout indicated that Na⁺/acidic equivalent and Cl^-/basic equivalent flux rates are normally limited by the availability of the internal acidic and basic counterions, as well as by external Na⁺ and Cl^- levels. Adult rainbow trout fitted with dorsal aortic and bladder catheters were chronically infused (10–16 h) with isosmotic HCl to induce a persistent metabolic acidosis. Acid-base neutral infusions of isosmotic NaCl and non-infused controls were also performed. Results were compared to previous data on metabolic alkalosis in trout induced by either isosmotic NaHCO₃ infusion or recovery from environmental hyperoxia (Goss and Wood 1990a, b). Metabolic acidosis resulted in a marked stimulation of Na⁺ influx, no change in Cl^- influx, positive Na⁺ balance, negative Cl^- balance, and net H⁺ excretion at the gills. Metabolic alkalosis caused a marked inhibition of Na⁺ influx and stimulation of Cl^- influx, negative Na⁺ balance, positive Cl^- balance, and net H⁺ uptake (=base excretion). Mean gill intracellular pH qualitatively followed extracellular pH. Classical one-substrate Michaelis-Menten analysis of kinetic data indicated that changes in Na⁺ and Cl^- transport during acid-base disturbance are achieved by large increases and decreases in J_max, and by increases in K_m. However, one-substrate analysis considers only external substrate concentration and cannot account for transport limitations by the internal substrate. The kinetic data were fitted successfully to a two-substrate model, using extracellular acid-base data as a measure of internal HCO ₃ ^- and H⁺ availability. This analysis indicated that true J_max values for Na⁺/acidic equivalent and Cl^-/basic equivalent transport are 4–5 times higher than apparent J_max values by one-substrate analysis. Flux rates are limited by the availability of the internal counterions; transport K_m values for HCO ₃ ^- and H⁺ are far above their normal internal concentrations. Therefore, small changes in acid-base status will have large effects on transport rates, and on apparent J_max values, without alterations in the number of transport sites. This system provides an automatic, negative feedback control for clearance or retention of acidic/basic equivalents when acid-base status is changing.Abbreviations Amm total ammonia in water - DMO 55-dimethyl-24-oxyzolidine-dione - J_in unidirectional inward ion movement across the gill - J_out unidirectional outward ion movement across the gill - J_net net transfer of ions (sum of J_in and J_out) across the gill - J_max maximal transport rate for ion - K_m inverse of affinity of transporter for ion - PIO₂ partial pressure of oxygen in inspired water - P_aCO₂ partial pressure of carbon dixide in arterial blood - TAlk titratable alkalinity of the water - PEG polyethylene glycol - NEN New England Nuclear     

Distinct Na+/K+/2Cl- cotransporter localization in kidneys and gills of two euryhaline species, rainbow trout and killifish

The kidney is an organ playing an important role in ion regulation in both freshwater (FW) and seawater (SW) fish. The mechanisms of ion regulation in the fish kidney are less well studied than that of their gills, especially at the level of transporter proteins. We have found striking differences in the pattern of Na⁺/K⁺/2Cl^- cotransporter (NKCC) expression between species. In the killifish kidney, NKCC is apically localized in the distal and collecting tubules and basolaterally localized in the proximal tubules. However, in the SW killifish gill, NKCC is basolaterally co-localized with Na⁺/K⁺-ATPase, whereas in FW, NKCC immunoreactivity is primarily apical, although still colocalized within the same mitochondria-rich cell with basolateral Na⁺/K⁺-ATPase. Rainbow trout kidney has NKCC only in the apical membrane of the distal and collecting tubules in both environments, with no signal being detected in the proximal tubule. On the other hand, in the trout gill, NKCC is found basolaterally in both FW and SW environments. An important observation is that, in the gills of rainbow trout, the trailing edge of the filament possesses mostly Na⁺/K⁺-ATPase-positive but NKCC-negative mitochondria-rich cells, whereas in the region between and at the roots of the gill lamellae, most mitochondria-rich cells exhibit both Na⁺/K⁺-ATPase- and NKCC-positive immunoreactivity. These results suggest that the differential localization of transporters between the two species represents differences in function between these two euryhaline fishes with different life histories and strategies. Funding for this research was provided by NSERC Discovery Grants to G.G.G. and W.S.M., an Alberta Ingenuity Fund PDF, and a fellowship from the NSERC Research Capacity Development Grant to F.K.     

Freshwater to seawater acclimation of juvenile bull sharks (<Emphasis Type="Italic">Carcharhinus leucas</Emphasis>): plasma osmolytes and Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase activity in gill,rectal gland,kidney and intestine

This study examined the osmoregulatory status of the euryhaline elasmobranch Carcharhinus leucas acclimated to freshwater (FW) and seawater (SW). Juvenile C. leucas captured in FW (3 mOsm l^–1 kg^–1) were acclimated to SW (980–1,000 mOsm l^–1 kg^–1) over 16 days. A FW group was maintained in captivity over a similar time period. In FW, bull sharks were hyper-osmotic regulators, having a plasma osmolarity of 595 mOsm l^–1 kg^–1. In SW, bull sharks had significantly higher plasma osmolarities (940 mOsm l^–1 kg^–1) than FW-acclimated animals and were slightly hypo-osmotic to the environment. Plasma Na⁺, Cl^–, K⁺, Mg²⁺, Ca²⁺, urea and trimethylamine oxide (TMAO) concentrations were all significantly higher in bull sharks acclimated to SW, with urea and TMAO showing the greatest increase. Gill, rectal gland, kidney and intestinal tissue were taken from animals acclimated to FW and SW and analysed for maximal Na⁺/K⁺-ATPase activity. Na⁺/K⁺-ATPase activity in the gills and intestine was less than 1 mmol Pi mg^–1 protein h^–1 and there was no difference in activity between FW- and SW-acclimated animals. In contrast Na⁺/K⁺-ATPase activity in the rectal gland and kidney were significantly higher than gill and intestine and showed significant differences between the FW- and SW-acclimated groups. In FW and SW, rectal gland Na⁺/K⁺-ATPase activity was 5.6±0.8 and 9.2±0.6 mmol Pi mg^–1 protein h^–1, respectively. Na⁺/K⁺-ATPase activity in the kidney of FW and SW acclimated animals was 8.4±1.1 and 3.3±1.1 Pi mg^–1 protein h^–1, respectively. Thus juvenile bull sharks have the osmoregulatory plasticity to acclimate to SW; their preference for the upper reaches of rivers where salinity is low is therefore likely to be for predator avoidance and/or increased food abundance rather than because of a physiological constraint.     

The effect of Cu on gill and esophagus lipid metabolism in the rainbow trout Oncorhynchus mykiss

Rainbow trout Oncorhynchus mykiss were exposed to 0, 100, 300 and 800 μgl⁻¹ ambient Cu in brackish water (BW) for 4 days at 13 °C and subsequently transferred to either clean BW, clean fresh water (FW) or clean seawater (SW) at 16 °C. After incubation with ³²P-phosphate and ¹⁴C-acetate added as precursors to the water the fish showed a degradation, depending on previous [CU], of ³²P- and ¹⁴C-labelled gill membrane phospholipids if they had been transferred to SW or remained in BW. Corresponding experiments where the fish were exposed to Cu in BW for 12 days showed a similar subsequent degradation in SW and BW of both gill and esophagus membrane phospholipids, however to a much lesser degree in gill tissue than after 4 days. Plasma Na⁺ was similarly reduced by up to 8%, depending on previous ambient Cu, but in this case only after transfer to FW and only after 4 days of exposure. Both the effect on membrane lipid metabolism and plasma Na⁺ thus showed acclimation to ambient Cu but there was apparently no direct correlation between the two different types of observed changes in membrane function.     

Influence of Transepithelial Potential Difference on the Sodium Uptake at the Outer Surface of the Isolated Frog Skin

The unidirectional uptake of sodium across the outer surface of the isolated frog skin (J₁₂^Na) was measured in the presence of transepithelial potential difference (Δ_ψ) ranging from +100 to -100 mV. With a sodium concentration of 115 mM in the bathing solutions J₁₂^Na increases significantly when the spontaneous Δ_ψ is reduced to zero by short-circuiting the skin. With an Na concentration of 6 mM a progressive increase J₁₂^Na can be observed when Δ_ψ is decreased in several steps from +100 to -100 mV (serosal side positive and negative, respectively). The observed change J₁₂^Na amounts to a fraction only of that predicted from the shift in Δ_ψ. The results suggest that under open circuit conditions the potential step across the outside surface is at most one half of Δ_ψ and that the resistance across the outside and inside barrier of the skin is ohmic. This is in agreement with measurements of intracellular potentials in the frog skin and with resistance measurements carried out in the toad skin. The data strongly support the view that the saturating component of J_ψ proceeds via a charged carrier system. Exposure to negative values of Δ_ψ of 50 mV or more for times of 24 min or more result in a marked reduction of J₁₂^Na which shows only partial or no reversibility.     

Ion and acid–base regulation in the freshwater mummichog (Fundulus heteroclitus): a departure from the standard model for freshwater teleosts

Ion and acid–base balance were examined in the freshwater-adapted mummichog (Fundulus heteroclitus) using a series of treatments designed to perturb the coupling mechanisms. Unidirectional Cl⁻ uptake (J^Cl_in) was extremely low whereas J^Na_in was substantial (three- to sixfold higher); comparable differences occurred in unidirectional efflux rates (J^Cl_out, J^Na_out). J^Cl_in was refractory to all treatments, suggesting that Cl⁻/base exchange was unimportant or absent. Indeed, no base excretion or modulation of ion fluxes occurred for acid–base balance for up to 8 h after NaHCO₃⁻ loading (injections of 1000 or 3000 nequiv.·g⁻¹). Acute environmental low pH (4.5) and amiloride (10⁻⁴ M) treatments caused concurrent inhibition of J^Na_in and net H⁺ excretion (J^H+_net), indicating the presence of Na⁺/H⁺ exchange. J^Na_in was elevated and J^H+_net restored during recovery from both treatments, but this exchange did not appear to be dynamically adjusted for acid–base homeostasis. High external ammonia exposure (1 mmol·l⁻¹) initially blocked ammonia excretion (J^Amm_net) but had no effect on J^Na_in, whereas high pH (9.4) reduced both J^Amm_net and J^Na_in. Inhibition of J^Na_in by the low pH and amiloride treatments had no effect on J^Amm_net. These results indicate that ammonia excretion is entirely diffusive and independent of both Na⁺uptake and the protons that are transported via the Na⁺/H⁺ coupling. In addition, ureagenesis served as a compensatory mechanism during high external ammonia exposure, as a marked elevation in urea excretion partially replaced the inhibited J^Amm_net. In all treatments, changes in the Na⁺–Cl⁻ net flux differential were consistent with changes in J^H+_net measured by traditional water titration techniques, indicating that the former can be used as an estimate of the acid–base status of the fish. Overall, the results demonstrate that the freshwater-adapted F. heteroclitus does not conform to the ion/acid–base relationships described in the standard model based on commonly studied species such as trout, goldfish, and catfish.     

Dynamic gene expression of GH/PRL-family hormone receptors in gill and kidney during freshwater-acclimation of Mozambique tilapia

In teleosts, prolactin (PRL) and growth hormone (GH) act at key osmoregulatory tissues to regulate hydromineral balance. This study was aimed at characterizing patterns of expression for genes encoding receptors for the GH/PRL-family of hormones in the gill and kidney of Mozambique tilapia (Oreochromis mossambicus) during freshwater (FW)-acclimation. Transfer of seawater (SW)-acclimated tilapia to FW elicited rapid and sustained increases in plasma levels and pituitary gene expression of PRL₁₇₇ and PRL₁₈₈; plasma hormone and pituitary mRNA levels of GH were unchanged. In the gill, PRL receptor 1 (PRLR1) mRNA increased markedly after transfer to FW by 6 h, while increases in GH receptor (GHR) mRNA were observed 48 h and 14 d after the transfer. By contrast, neither PRLR2 nor the somatolactin receptor (SLR) was responsive to FW transfer. Paralleling these endocrine responses were marked increases in branchial gene expression of a Na⁺/Cl^? cotransporter and a Na⁺/H⁺ exchanger, indicators of FW-type mitochondrion-rich cells (MRCs), at 24 and 48 h after FW transfer, respectively. Expression of Na⁺/K⁺/2Cl^? cotransporter, an indicator of SW-type MRCs, was sharply down-regulated by 6 h after transfer to FW. In kidney, PRLR1, PRLR2 and SLR mRNA levels were unchanged, while GHR mRNA was up-regulated from 6 h after FW transfer to all points thereafter. Collectively, these results suggest that the modulation of the gene expression for PRL and GH receptors in osmoregulatory tissues represents an important aspect of FW-acclimation of tilapia.     

Appearance of cuboidal cells in relation to salinity in gills of Fundulus heteroclitus, a species exhibiting branchial Na+ but not Cl− uptake in freshwater

Fundulus heteroclitus (killifish) is a model organism for ionoregulatory studies, particularly because of its opercular epithelium, although the gills are the major sites of ion exchange. Whereas Na⁺ and Cl⁻ are excreted through the gills in seawater (SW), the killifish is unusual in taking up only Na⁺ and not Cl⁻ at the gills in freshwater (FW). We describe morphological changes in the branchial epithelium following transfer from an acclimation medium of 10% SW to 100% SW or FW. In 10% SW, mitochondria-rich cells resemble typical seawater chloride cells (SWCCs) with accessory cells. After transfer to 100% SW, no change occurs in pavement cell (PVC) morphology or mitotic rate (measured by bromo-deoxyuridine technique), although the density of SWCC apertures increases several fold because of the uncovering of buried SWCCs by PVCs, in accord with increased rates of Na⁺ and Cl⁻ efflux. After transfer to FW, PVC morphology remains unchanged, but SWCCs and accessory cells are quickly covered by PVCs, with many undergoing apoptosis or necrosis. The mitotic rate doubles by 10–14 h but typical freshwater chloride cells (FWCCs) do not appear. Instead, a wedge-shaped cell type that is moderately rich in apically oriented mitochondria, with a large ovoid nucleus, thin cytoplasmic layer, paucity of vesicular-tubular network, and variably villous surface rapidly (by 3 h) and progressively appears in the filament epithelium, by both uncovering and mitosis. This cell type is similar to that recently identified as the site of Na⁺ uptake in the FW trout gill. We propose the new term “cuboidal cell” for this cell, based on its morphology, to avoid confusion with traditional terminology (of PVC). We hypothesize that the cuboidal cells are the sites of active Na⁺ uptake in FW F. heteroclitus and suggest that the lack of Cl⁻ uptake is attributable to the absence of typical FWCCs previously described in teleosts.This work was supported by NSERC Discovery grants (to C.M.W.) and by an NSERC International Fellowship (to P.L.). C.M.W. is supported by the Canada Research Chair Program.     

Effects of copper on branchial ionoregulation in the rainbow trout,Salmo gairdneri Richardson

Summary Hard- and softwater acclimated adult rainbow trout were statically exposed to copper (12.5, 25, 50, 100, and 200 ppb) for two, 12 h periods at neutral and pH 5.0. Unidirectional Na⁺, and Cl⁻, and net Na⁺, Cl⁻, K⁺, and ammonia fluxes were monitored as a measure of branchial ionoregulatory disturbance. Copper concentrations as low as 12.5 ppb led to measurable ion losses. Net Na⁺, Cl⁻, and K⁺ losses were concentration dependent and unaffected by prior acclimation to either hard- or softwater at both neutral pH and pH 5. From 12.5 to 50 ppb net NaCl losses arose primarily as a result of the inhibition ofJ _in, and at higher concentrations,J _out was also stimulated. In softwater,J _in was more resistant to inhibition than in hardwater. However, in hardwater,J _out recovered to normal levels during the second 12 h period, but no such recovery was found in softwater. Plasma NaCl was inversely correlated with [copper], while plasma glucose and ammonia increased with [copper]. At pH 5.0 and [copper] from 12.5 to 50 ppb, H⁺ contributed significantly to the total ion loss, while at 100 and 200 ppb, ion losses were no greater at pH 5.0 than at neutral pH. In no case were the effects of copper and H⁺ strictly additive.     

Volume Reabsorption, Transepithelial Potential Differences, and Ionic Permeability Properties in Mammalian Superficial Proximal Straight Tubules

This paper describes experiments designed to evaluate Na⁺ and Cl^- transport in isolated proximal straight tubules from rabbit kidneys. When the perfusing solution was Krebs-Ringer buffer with 25 mM HCO₃^- (KRB) and the bath contained KRB plus 6% albumin, net volume reabsorption (J_v, nl min^-1 mm^-1 was -0.46 ± 0.03 (SEM); V_e, the spontaneous transepithelial potential difference, was -1.13 ± 0.05 mV, lumen negative. Both J_v, and V_e, were reduced to zero at 21°C or with 10^-4 M ouabain, but J_v, was not HCO₃^- dependent. Net Na⁺ reabsorption, measured as the difference between ²²Na⁺ fluxes, lumen to bath and bath to lumen, accounted quantitatively for volume reabsorption, assuming the latter to be an isotonic process, and was in agreement with the difference between lumen to bath ²²Na⁺ fluxes during volume reabsorption and at zero volume flow. The observed flux ratio for Na⁺ was 1.46, and that predicted for a passive process was 0.99; thus, Na⁺ reabsorption was rationalized in terms of an active transport process. The Cl^- concentration of tubular fluid rose from 113.6 to 132.3 mM during volume reabsorption. Since V_e, rose to +0.82 mV when tubules were perfused with 138.6 mM Cl^- solutions, V_e may become positive when tubular fluid Cl^- concentrations rise during volume reabsorption. The permeability coefficients P_Na and P_Cl computed from tracer fluxes were, respectively, 0.23 x 10^-4 and 0.73 x 10^-4 cm s^-1. A P_Na/P_Cl ratio of 0.3 described NaCl dilution potentials at zero volume flow. The magnitudes of the potentials were the same for a given NaCl gradient in either direction and P_Na/P_Cl was constant in the range 32–139 mM NaCl. We infer that the route of passive ion permeation was through symmetrical extracellular interfaces, presumably tight junctions, characterized by neutral polar sites in which electroneutrality is maintained by mobile counterions.     

Dietary lipid composition affects the gene expression of gill Na+/K+-ATPase α1b but not the α1a isoform in juvenile fall chinook salmon (Oncorhynchus tshawytscha)

We assessed the effects of dietary fatty acid composition on sodium–potassium ATPase (Na⁺/K⁺-ATPase) activity and isoform expression in the gills of juvenile fall chinook salmon, Oncorhynchus tshawytscha by supplementing diets with either anchovy oil (AO) or AO blended with canola oil (CO) so that CO comprised 0% (0CO), 11% (11CO), 22% (22CO), 33% (33CO), 43% (43CO), or 54% (54CO) of the measured dietary lipid content. The effects of diet were assessed in freshwater (FW) following 104 days of diet manipulation, in response to 24-h seawater (SW) transfer at this time, and following an additional 35 days of SW acclimation. Gill Na⁺/K⁺-ATPase activity was not significantly affected by diet at any sampling time, and there were no consistent effects of diet on the expression of the Na⁺/K⁺-ATPase α1a isoform. As dietary CO increased, Na⁺/K⁺-ATPase α1b mRNA decreased in fish held in FW, with the 43CO and 54CO diet groups having significantly lower levels than fish fed the 0CO and 11CO diets. Twenty-four-hour SW challenge did not affect the expression of the Na⁺/K⁺-ATPase α1a isoform in any diet group, but this isoform was down-regulated in all diet groups following 35 days of SW acclimation. Na⁺/K⁺-ATPase α1b expression levels increased in response to 24-h SW transfer and SW acclimation only in fish fed the 54CO diet. The effects of the two extreme diets (0CO and 54CO) were also assessed at various time points during 104 days of rearing in FW. Na⁺/K⁺-ATPase α1b mRNA levels were greater in fish fed diet 0CO versus those fed diet 54CO at all times during the FW culture period. These data demonstrate that dietary fatty acid composition can influence the gill Na⁺/K⁺-ATPase isoform physiology of juvenile fall-run chinook salmon prior to SW transfer.     

Unidirectional Na(+) and Ca (2+) fluxes in two euryhaline teleost fishes, Fundulus heteroclitus and Oncorhynchus mykiss, acutely submitted to a progressive salinity increase

Na⁺ and Ca²⁺ regulation were compared in two euryhaline species, killifish (normally estuarine-resident) and rainbow trout (normally freshwater-resident) during an incremental salinity increase. Whole-body unidirectional fluxes of Na⁺ and Ca²⁺, whole body Na⁺ and Ca²⁺, and plasma concentrations (trout only), were measured over 1-h periods throughout a total 6-h protocol of increasing salinity meant to simulate a natural tidal flow. Killifish exhibited significant increases in both Na⁺ influx and efflux rates, with efflux slightly lagging behind efflux up to 60% SW, but net Na⁺ balance was restored by the time killifish reached 100% SW. Whole body Na⁺ did not change, in agreement with the capacity of this species to tolerate daily salinity fluctuations in its natural habitat. In contrast, rainbow trout experienced a dramatic increase in Na⁺ influx (50-fold relative to FW values), but not Na⁺ efflux between 40 and 60% SW, resulting in a large net loading of Na⁺ at higher salinities (60–100% SW), and increases in plasma Na⁺ and whole body Na⁺ at 100% SW. Killifish were in negative Ca²⁺ balance at all salinities, whereas trout were in positive Ca²⁺ balance throughout. Ca²⁺ influx rate increased two- to threefold in killifish at 80 and 100% SW, but there were no concomitant changes in Ca²⁺ efflux. Ca²⁺ flux rates were affected to a larger degree in trout, with twofold increases in Ca²⁺ influx at 40% SW and sevenfold increases at 100% SW. Again, there was no change in Ca²⁺ efflux with salinity, so plasma Ca²⁺ concentration increased in 100% SW. As the killifish is regularly submitted to increased salinity in its natural environment, it is able to rapidly activate changes in unidirectional fluxes in order to ensure ionic homeostasis, in contrast to the trout.     

Na+-dependent Ca2+ uptake in isolated opercular epithelium of Fundulus heteroclitus

It is concluded that Ca²⁺ transport across the basolateral membranes of the ionocytes in killifish skin is mediated for the major part by a Na⁺/Ca²⁺-exchange mechanism that is driven by the (transmembrane) Na⁺ gradient established by Na⁺/K⁺-ATPase. The conclusion is based, firstly, on the biochemical evidence for the presence of a Na⁺/Ca²⁺-exchanger next to the Ca²⁺-ATPase in the basolateral membranes of killifish gill cells. Secondly, the transcellular Ca²⁺ uptake measured in an Ussing chamber setup was 85% and 80% reduced in freshwater (FW) and SW (SW) opercular membranes, respectively, as the Na⁺ gradient across the basolateral membrane was directly or indirectly (by ouabain) reduced. Thapsigargin or dibutyryl-cAMP/IBMX in SW opercular membranes reduced Ca²⁺ influx to 46%, comparable to the effects seen in FW membranes [reduction to 56%; Marshall et al. 1995a]. Basal Ca²⁺ influx across the opercular membrane was 48% lower in membranes from fish adapted to SW than in membranes from fish adaptated to FW. Branchial Na⁺/K⁺-ATPase activity was two times higher in SW adapted fish. Accepted: 29 October 1996