A critical analysis of transepithelial potential in intact killifish (Fundulus heteroclitus) subjected to acute and chronic changes in salinity

We investigated the in vivo salinity-dependent behavior of transepithelial potential (TEP) in Fundulus heteroclitus (3-9 g) using indwelling coelomic catheters, a technique which was validated against blood catheter measurements in a larger species (Opsanus beta; 35-70 g). In seawater (SW)-acclimated killifish, TEP was +23 mV (inside positive), but changed to -39 mV immediately after transfer to freshwater (FW). Acute transfer to dilute salinities produced a TEP profile, which rapidly attenuated as salinity increased (0, 2.5, 5 and 10% SW), with cross-over to positive values between 20 and 40% SW, and a linear increase thereafter (60, 80 and 100% SW). TEP response profiles were also recorded after acute transfer to comparable dilutions of 500 mmol L(-1) NaCl, NaNO3, Na gluconate, choline chloride, N-methyl-D-glutamate (NMDG) chloride, or 1,100 mosmol kg(-1) mannitol. These indicated high non-specific cation permeability and low non-specific anion permeability without influence of osmolality in SW-acclimated killifish. While there was a small electrogenic component in high salinity, a Na+ diffusion potential predominated at all salinities due to the low P Cl/P Na (0.23) of the gills. The very negative TEP in FW was attenuated in a linear fashion by log elevations in [Ca2+] such that P Cl/P Na increased to 0.73 at 10 mmol L(-1). SW levels of [K+] or [Mg2+] also increased the TEP, but none of these cations alone restored the positive TEP of SW-acclimated killifish. The very negative TEP in FW attenuated over the first 12 h of exposure and by 24-30 h reached +3 mV, representative of long-term FW-acclimated animals; this reflected a progressive increase in P Cl/P Na from 0.23 to 1.30, probably associated with closing of the paracellular shunt pathway. Thereafter, the TEP in FW-acclimated killifish was unresponsive to [Ca2+] (also to [K+], [Mg2+], or chloride salts of choline and NMDG), but became more positive at SW levels of [Na+]. Killifish live in a variable salinity environment and are incapable of gill Cl(-) uptake in FW. We conclude that the adaptive significance of the TEP patterns is that changeover to a very negative TEP in FW will immediately limit Na+ loss while not interfering with active Cl(-) uptake because there is none. Keeping the shunt permeability high for a few hours means that killifish can return to SW and instantaneously re-activate their NaCl excretion mechanism.     

Dilute culture media as an environmental or physiological simulant in cultured gill epithelia from freshwater rainbow trout

The electrophysiological and ion-transporting properties of cultured gill epithelia from freshwater (FW) rainbow trout were examined in the presence of dilute cell culture media as an environmental or physiological simulant. Gill epithelia were cultured on cell culture inserts under symmetrical conditions (L15 apical-L15 basolateral) for 6-7 d. The following experiments were then conducted. (1) To mimic a gradual lowering of environmental salinity, apical L15 medium was progressively diluted with FW (first to 2/3 L15 for 8 h and then to 1/3 L15 for 6 h) before the introduction of apical FW (FW apical-L15 basolateral, analogous to a fish in a natural FW environment). Dilute apical media had no significant effect on the electrophysiological properties of preparations compared with symmetrical culture conditions, and no evidence for active Na(+) or Cl(-) transport was observed. Preparations subsequently exposed to apical FW exhibited a negative transepithelial potential and evidence of active Cl(-) uptake and slight Na(+) extrusion. (2) To mimic the extracellular fluid dilution that occurs in euryhaline fish after abrupt transfer from saline to FW, the osmolality or ionic strength (or both) of basolateral media was reduced by 20-40% (using either FW or FW + mannitol) while simultaneously replacing apical media with FW. Under these conditions, Na(+) and Cl(-) influx rates were low compared with efflux rates, while the Ussing flux ratio analysis generally indicated active Cl(-) uptake and Na(+) extrusion. The Na(+)-K(+) adenosine triphosphatase activity was not affected by alterations in basolateral osmolality. Our studies indicate that cultured trout gill epithelia are tolerant of media dilution from both the apical and the basolateral direction; however, neither treatment alone appeared to increase ion influx rates or stimulate active Na(+) uptake in cultured trout gill epithelia.     

Osmoregulation and expression of ion transport proteins and putative claudins in the gill of southern flounder (Paralichthys lethostigma)

The southern flounder is a euryhaline teleost that inhabits ocean, estuarine, and riverine environments. We investigated the osmoregulatory strategy of juvenile flounder by examining the time-course of homeostatic responses, hormone levels, and gill Na(+),K(+)-ATPase and Na(+),K(+),2Cl(-) cotransporter protein expression after salinity challenge. Transfer of freshwater (FW)-acclimated flounder to sea water (SW) induced an increase in plasma osmolality and cortisol and a decrease in muscle water content, plasma insulin-like growth factor I (IGF-I) and hepatic IGF-I mRNA, all returning to control levels after 4 days. Gill Na(+),K(+)-ATPase and Na(+),K(+),2Cl(-) cotransporter protein levels were elevated in response to SW after 4 days. Transfer of SW-acclimated flounder to FW reduced gill Na(+),K(+)-ATPase and Na(+),K(+),2Cl(-) cotransporter protein, increased plasma IGF-I, but did not alter hepatic IGF-I mRNA or plasma cortisol levels. Gill claudin-3 and claudin-4 immunoreactive proteins were elevated in FW versus SW acclimated flounder. The study demonstrates that successful acclimation of southern flounder to SW or FW occurs after an initial crisis period and that the salinity adaptation process is associated with changes in branchial expression of ion transport and putative tight junction claudin proteins known to regulate epithelial permeability in mammalian vertebrates.     

Fatty acid and lipid class composition in eyes and brain from teleosts and elasmobranchs

The effect of 17beta-estradiol (E(2)) on osmoregulatory performance was examined in the euryhaline killifish, Fundulus heteroclitus. Fish were injected once with 1, 2 and 5 microg g(-1) E(2) and, 6 h after injection, transferred from 1 ppt seawater (SW) to full strength SW (40 ppt) or from SW to 1 ppt SW. In another set of experiments, fish were injected four times on alternate days with 2 microg g(-1) E(2) and then, 6 h after the last injection, transferred from 1 ppt SW to SW or from SW to 1 ppt SW. Fish were sampled 18 h after transfer (i.e., 24 h post-injection), and plasma osmolality, Na(+) and Cl(-) concentration and gill K(+)-pNPPase activity (a reflection of the sodium pump) were examined. Transfer from 1 ppt SW to SW resulted in significantly increased plasma osmolality, but did not affect gill K(+)-pNPPase activity. A single dose of E(2) (1, 2 and 5 microg g(-1)) prior to transfer from 1 ppt SW to SW increased plasma osmolality and decreased gill K(+)-pNPPase activity in a dose-dependent manner. Prolonged treatment with E(2) increased plasma osmolality and decreased gill K(+)-pNPPase activity in 1 ppt SW-adapted fish. Transfer of fish thus treated from 1 ppt SW to SW increased plasma osmolality and did not alter gill K(+)-pNPPase activity. Transfer from SW to 1 ppt SW had no significant effect on plasma osmolality or gill K(+)-pNPPase activity. Only the highest single dose of E(2) (5 microg g(-1)) prior to transfer from SW to 1 ppt SW decreased gill K(+)-pNPPase activity. Prolonged treatment with 2 microg g(-1) E(2) decreased gill K(+)-pNPPase activity only following transfer from SW to 1 ppt SW. The results substantiate an inhibitory action of E(2) on hypoosmoregulatory capacity in this euryhaline teleost.     

Acute changes in gill Na+-K+-ATPase and creatine kinase in response to salinity changes in the euryhaline teleost, tilapia (Oreochromis mossambicus)

Some freshwater (FW) teleosts are capable of acclimating to seawater (SW) when challenged; however, the related energetic and physiological consequences are still unclear. This study was conducted to examine the changes in expression of gill Na(+)-K(+)-ATPase and creatine kinase (CK) in tilapia (Oreochromis mossambicus) as the acute responses to transfer from FW to SW. After 24 h in 25 ppt SW, gill Na(+)-K(+)-ATPase activities were higher than those of fish in FW. Fish in 35 ppt SW did not increase gill Na(+)-K(+)-ATPase activities until 1.5 h after transfer, and then the activities were not significantly different from those of fish in 25 ppt SW. Compared to FW, the gill CK activities in 35 ppt SW declined within 1.5 h and afterward dramatically elevated at 2 h, as in 25 ppt SW, but the levels in 35 ppt SW were lower than those in 25 ppt SW. The Western blot of muscle-type CK (MM form) was in high association with the salinity change, showing a pattern of changes similar to that in CK activity; however, levels in 35 ppt SW were higher than those in 25 ppt SW. The activity of Na(+)-K(+)-ATPase highly correlated with that of CK in fish gill after transfer from FW to SW, suggesting that phosphocreatine acts as an energy source to meet the osmoregulatory demand during acute transfer.     

Ion fluxes and diffusion potentials in the Dungeness crab,Cancer magister

Summary Transepithelial potentials (TEP's) were measured in Dungeness crabs exposed to a variety of experimental media. The TEP's can be accounted for as diffusion potentials. In sea water (SW) theP _Na/P _Cl ratio (calculated by substitution in the Goldman equation) was 0.68, but in dilutions of SW the value increased, reaching a maximum of 3.33 in freshwater (FW). When the calcium and magnesium concentrations in the diluted media were maintained at SW levels theP _Na/P _Cl remained close to that in SW.The effluxes of Na and Cl were monitored in crabs exposed to the experimental media and the TEP's were measured simultaneously. After transfer from SW to FW the decrease in Na efflux was considerably less than expected from the change in potential alone, indicating an increased permeability to sodium, while transfer from SW to 500 mM NaCl caused a 3.4-fold increase in Na efflux without any associated change of potential. These results indicate an increase in the permeability of the gill epithelium to Na as the ambient concentrations of Ca and Mg decline. The Cl effluxes are not dependent on the external concentration of divalent ions, but about 30% of the Cl efflux may be due to exchange diffusion.Abbreviations FW freshwater - SW sea water - TEP transepithelial potential This project was in part supported by faculty research grants from Fordham University, Bronx, NY and Towson State University, Towson, MD to GDR     

Salinity regulates claudin mRNA and protein expression in the teleost gill

The teleost gill carries out NaCl uptake in freshwater (FW) and NaCl excretion in seawater (SW). This transformation with salinity requires close regulation of ion transporter capacity and epithelial permeability. This study investigates the regulation of tight-junctional claudins during salinity acclimation in fish. We identified claudin 3- and claudin 4-like immunoreactive proteins and examined their expression and that of select ion transporters by performing Western blot in tilapia (Oreochromis mossambicus) gill during FW and SW acclimation. Transfer of FW tilapia to SW increased plasma osmolality, which was corrected after 4 days, coinciding with increased gill Na+-K+-ATPase and Na+-K+-2Cl(-) cotransporter expression. Gill claudin 3- and claudin 4-like proteins were reduced with exposure to SW. Transfer to FW increased both claudin-like proteins. Immunohistochemistry shows that claudin 3-like protein was localized deep in the FW gill filament, whereas staining was found apically in SW gill. Claudin 4-like proteins are localized predominantly in the filament outer epithelial layer, and staining appears more intense in the gill of FW versus SW fish. In addition, tilapia claudin 28a and 30 genes were characterized, and mRNA expression was found to increase during FW acclimation. These studies are the first to detect putative claudin proteins in teleosts and show their localization and regulation with salinity in gill epithelium. The data indicate that claudins may be important in permeability changes associated with salinity acclimation and possibly the formation of deeper tight junctions in FW gill. This may reduce ion permeability, which is a critical facet of FW osmoregulation.     

Functional plasticity of mitochondrion-rich cells in the skin of euryhaline medaka larvae (Oryzias latipes) subjected to salinity changes

A noninvasive technique, the scanning ion-selective electrode technique (SIET) was applied to measure Na(+) and Cl(-) transport by the yolk-sac skin and individual mitochondrion-rich cells (MRCs) in intact medaka larvae (Oryzias latipes). In seawater (SW)-acclimated larvae, significant outward Na(+) and Cl(-) gradients were measured at the yolk-sac surface, indicating secretions of Na(+) and Cl(-) from the yolk-sac skin. With Na(+) pump immunostaining and microscopic observation, two groups of MRCs were identified on the yolk-sac skin of SW-larvae. These were single MRCs (s-MRCs), which do not have an accompanying accessory cell (AC), and multicellular complex MRCs (mc-MRCs), which usually consist of an MRC and an accompanying AC. The percentage of mc-MRC was ～60% in 30 parts per thousand of SW, and it decreased with the decrease of external salinity. By serial SIET probing over the surface of the MRCs and adjacent keratinocytes (KCs), significant outward fluxes of Na(+) and Cl(-) were detected at the apical opening (membrane) of mc-MRCs, whereas only outward Cl(-) flux, but not Na(+) flux, was detected at s-MRCs. Treatment with 100 μM ouabain or bumetanide effectively blocked the Na(+) and Cl(-) secretion. Following freshwater (FW) to SW transfer, Na(+) and Cl(-) secretions by the yolk-sac skin were fully developed in 5 h and 2 h, respectively. In contrast, both Na(+) and Cl(-) secretions downregulated rapidly after SW to FW transfer. Sequential probing at individual MRCs found that Na(+) and Cl(-) secretions declined dramatically after SW to FW transfer and Na(+)/Cl(-) uptake was detected at the same s-MRCs and mc-MRCs after 5 h. This study provides evidence demonstrating that ACs are required for Na(+) excretion and MRCs possess a functional plasticity in changing from a Na(+)/Cl(-)-secreting cell to a Na(+)/Cl(-)-absorbing cell.     

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     

Environmental salinity and sodium and chloride exchanges across the gill of Tilapia mossambica.

10 Freshwater-(FW)-adapted, one-third seawater (1/3 SW)-adapted and seawater (SW) adapted Tilapia mossambica were compared for their branchial Na+ influx and efflux as well as Cl- efflux. Na+ and Cl- effluxes were identical. Rates of effluxes were in 1/3 SW- and in SW-adapted fish 10 times and 200 times higher respectively than in FW specimens. 20 Shock due to handling and transfer to small experimental chambers induced, within 20 to 45 min., a considerable increase in Na+ efflux and a more discrete augmentation of the Na+ influx. 30 Branchial Mg++-and Na+-K+ activated ATPase activities increased significantly upon adaptation from FW to 1/3 SW. No significant increase was apparent upon adaptation from 1/3 SW to SW. 40 The trans-branchial potential observed in SW Tilapia resembled the pattern previously described in other species of teleosts.     

Responses of frog skin Na(+) and Cl(-) transport to guanylin

A possible role for the peptide hormone guanylin was investigated in frog skin (Rana pipiens) epithelium. Sodium and chloride fluxes in response to this peptide were evaluated in Ussing-type chambers. Net and unidirectional Na(+) fluxes were measured by using (22)Na(+) and atomic absorption analysis of total [Na(+)], whereas net Cl(-) fluxes were measured by using electrometric titration for [Cl(-)]. Mucosal application of guanylin (0.5-2.0 micromol/l) caused marked increases in serosal to mucosal net flux and efflux of Na(+). Serosal application of guanylin over the same dose range caused similar large increases in net serosal to mucosal (S-->M) Na(+) and Cl(-) flux as well as Na(+) efflux. Responses of Na(+) influx were small and inconsistent. When frog skin was bathed on the serosal side with Cl(-)-free Ringer's solution mucosal application of guanylin stimulated large efflux and S-->M net fluxes of Na(+). Serosal treatment yielded large Na(+) effluxes and S-->M Na(+) and Cl(-) net fluxes. When frog skin serosal surfaces were bathed with Na(+)- free Ringer's solution mucosal guanylin treatment had no effect but serosal treatment produced large S-->M Cl(-) net fluxes.     

A divergent CFTR homologue: highly regulated salt transport in the euryhaline teleost F.heteroclitus

The killifish,Fundulus heteroclitus, is a euryhalineteleost fish capable of adapting rapidly to transfer from freshwater (FW) to four times seawater (SW). To investigate osmoregulation at amolecular level, a 5.7-kilobase cDNA homologous to human cysticfibrosis transmembrane conductance regulator (hCFTR) was isolated froma gill cDNA library from SW-adapted killifish. This cDNA encodes aprotein product (kfCFTR) that is 59% identical to hCFTR,the most divergent form of CFTR characterized to date. Expression ofkfCFTR in Xenopus oocytes generatedadenosine 3',5'-cyclic monophosphate-activated,Cl-selective currentssimilar to those generated by hCFTR. In SW-adapted killifish,kfCFTR was expressed at high levels in the gill, opercular epithelium, and intestine. After abrupt exposure of FW-adapted killifish to SW, kfCFTR expression in the gill increasedseveralfold, suggesting a role for kfCFTR in salinity adaptation. Undersimilar conditions, plasma Na⁺levels rose significantly after 8 h and then fell, although it is notknown whether these changes are directly responsible for the changes inkfCFTR expression. The killifish provides a unique opportunity to understand teleost osmoregulation and the role of CFTR.

     

Decoupling the Na+-K+-ATPase in vivo: a possible new role in the gills of freshwater fishes

The literature suggests that when Na(+)-K(+)-ATPase has reduced access to its glycosphingolipid cofactor sulfogalactosyl ceramide (SGC), it is converted to a Na(+) uniporter. We recently showed that such segregation can occur within a single membrane when Na(+)-K(+)-ATPase is excluded from membrane microdomains or 'lipid rafts' enriched in SGC (D. Lingwood, G. Harauz, J.S. Ballantyne, J. Biol. Chem. 280, 36545-36550). Specifically we demonstrated that Na(+)-K(+)-ATPase localizes to SGC-enriched rafts in the gill basolateral membrane (BLM) of rainbow trout exposed to seawater (SW) but not freshwater (FW). We therefore proposed that since the freshwater gill Na(+)-K(+)-ATPase was separated from BLM SGC it should also transport Na(+) only, suggesting a new role for the pump in this epithelium. In this paper we discuss the biochemical evidence for SGC-based modulation of transport stoichiometry and highlight how a unique asparagine-lysine substitution in the FW pump isoform and FW gill transport energetics gear the Na(+)-K(+)-ATPase to perform Na(+) uniport.     

Mechanisms behind Pb-induced disruption of Na+ and Cl- balance in rainbow trout (Oncorhynchus mykiss)

The mechanism of Pb-induced disruption of Na(+) and Cl(-) balance was investigated in the freshwater rainbow trout (Oncorhynchus mykiss). Na(+) and Cl(-) influx rates were reduced immediately in the presence of 2.40 +/- 0.24 and 1.25 +/- 0.14 muM Pb, with a small increase in efflux rates occurring after 24-h exposure. Waterborne Pb caused a significant decrease in the maximal rate of Na(+) influx without a change in transporter affinity, suggesting a noncompetitive disruption of Na(+) uptake by Pb. Phenamil and bafilomycin markedly reduced Na(+) influx rate but did not affect Pb accumulation at the gill. Time-course analysis in rainbow trout exposed to 0, 0.48, 2.4, and 4.8 microM Pb revealed time- and concentration-dependent branchial Pb accumulation. Na(+)-K(+)-ATPase activity was significantly reduced, with 4.8 microM exposure resulting in immediate enzyme inhibition and 0.48 and 2.4 microM exposures inhibiting activity by 24 h. Reduced activity was weakly correlated with gill Pb accumulation after 3- and 8-h exposures; this relationship strengthened by 24 h. Reduced Na(+) uptake was correlated with gill Pb burden after exposures of 3, 8, and 24 h. Immediate inhibition of branchial carbonic anhydrase activity occurred after 3-h exposure to 0.82 +/- 0.05 or 4.30 +/- 0.05 microM Pb and continued for up to 24 h. We conclude that Pb-induced disruption of Na(+) and Cl(-) homeostasis is in part a result of rapid inhibition of carbonic anhydrase activity and of binding of Pb with Na(+)-K(+)-ATPase, causing noncompetitive inhibition of Na(+) and Cl(-) influx.     

Branchial Na(+)K(+)ATPase activity in brook charr (Salvelinus fontinalis): effect of gonadal development in hypo- and hyperosmotic environments

Changes in gill Na(+)K(+)ATPase activity were examined following the transfer of brook charr (Salvelinus fontinalis) from fresh water (FW) to seawater (SW). Gonadal development was altered at the hatching stage using three doses of ionizing radiation (IR): 6.2, 7.8, and 11.4 Gray (Gy). A non-irradiated control group was also included in the experimental set-up. Following 15 and 19 months of growth in FW, assessment of gill activity in regard to gonadal status (sterile vs. mature) and level of IR exposure was realized by conducting two estuarine challenge tests. A first introduction was performed during June (period of highest osmoregulatory capacities for this species) (summer experiment). A second introduction was conducted during October (period of diminished osmoregulatory capacities) (fall experiment). Gill Na(+)K(+)ATPase activity and water content were measured at different times and two FW control samplings were added in October and January. In the summer experiment (June-December), normal gonadal development of female brook charr was related to reduced gill Na(+)K(+)ATPase activity during the spawning period as compared to sterile fish (4.0+/-1.5 and 7.2+/-1.9 micromole Pi. mg protein(-1). hr(-1)) (P<0.0002). Similar results were not observed in FW conditions, implying that a lack of gonadal growth does not initiate a significant advantage when the osmoregulatory system including the gills are not highly in demand, i.e. in a FW environment. Ionizing radiation exposure of < or =11.4 Gy at the hatching stage had no significant negative or positive effect on Na(+)K(+)ATPase activity either in FW or SW conditions.     

Effects of environmental salinity on gill endothelin receptor expression in the killifish, Fundulus heteroclitus

We recently determined that rapid changes in environmental salinity alter endothelin-1 (EDN1) mRNA levels in the euryhaline killifish, Fundulus heteroclitus, so we hypothesized that EDN1 may be a local regulator of gill ion transport in teleost fishes. The purpose of the present study was to examine the effects of changes in environmental salinity on the gill endothelin receptors: EDNRA, EDNRB, and EDNRC. Using quantitative real-time PCR, we determined that after a fresh water (FW) to seawater (SW) transfer, there is a two to threefold increase in gill EDNRA and EDNRB mRNA levels. Likewise, we found a two to three fold increase in gill EDNRA and EDNRB protein concentration. In addition, killifish that have acclimated to FW for 30 days had significantly lower EDNRA mRNA and protein levels than SW killifish. ENDRA were immunolocalized to the mitochondrion-rich cells of the killifish gill, suggesting that EDN1 signaling cascades may affect MRC function. EDNRB were found throughout the gill vasculature and on lamellar pillar cells. We previously immunolocalized EDN1 to the pillar cell suggesting that EDN1 acts as an autocrine signaling molecule and potentially regulates pillar cell tone and lamellar perfusion. We conclude that EDN1 is physiologically active in the teleost gill, and regulated by environmental salinity. Future functional studies examining the physiological role of this system are necessary to completely understand EDN1 in the fish gill.     

Effect of salinity on expression of branchial ion transporters in striped bass (Morone saxatilis)

The time course of osmoregulatory adjustments and expressional changes of three key ion transporters in the gill were investigated in the striped bass during salinity acclimations. In three experiments, fish were transferred from fresh water (FW) to seawater (SW), from SW to FW, and from 15-ppt brackish water (BW) to either FW or SW, respectively. Each transfer induced minor deflections in serum [Na+] and muscle water content, both being corrected rapidly (24 hr). Transfer from FW to SW increased gill Na+,K+-ATPase activity and Na+,K+,2Cl- co-transporter expression after 3 days. Abundance of Na+,K+-ATPase alpha-subunit mRNA and protein was unchanged. Changes in Na+,K+,2Cl- co-transporter protein were preceded by increased mRNA expression after 24 hr. Expression of V-type H+-ATPase mRNA decreased after 3 days. Transfer from SW to FW induced no change in expression of gill Na+,K+-ATPase. However, Na+,K+,2Cl- co-transporter mRNA and protein levels decreased after 24 hr and 7 days, respectively. Expression of H+-ATPase mRNA increased in response to FW after 7 days. In BW fish transferred to FW and SW, gill Na+,K+-ATPase activity was stimulated by both challenges, suggesting both a hyper- and a hypo-osmoregulatory response of the enzyme. Acclimation of striped bass to SW occurs on a rapid time scale. This seems partly to rely on the relative high abundance of gill Na+,K+-ATPase and Na+,K+,2Cl- co-transporter in FW fish. In a separate study, we found a smaller response to SW in expression of these ion transport proteins in striped bass when compared with the less euryhaline brown trout. In both FW and SW, NEM-sensitive gill H+-ATPase activity was negligible in striped bass and approximately 10-fold higher in brown trout. This suggests that in striped bass Na+-uptake in FW may rely more on a relatively high abundance/activity of Na+,K+-ATPase compared to trout, where H+-ATPase is critical for establishing a thermodynamically favorable gradient for Na+-uptake.     

A fish out of water: gill and skin remodeling promotes osmo- and ionoregulation in the mangrove killifish Kryptolebias marmoratus

The euryhaline, amphibious mangrove killifish Kryptolebias marmoratus is known to survive weeks out of water in moist environments. We tested the hypothesis that the skin is a site of osmo- and ionoregulation in K. marmoratus. We predicted that under terrestrial conditions, gill and skin remodeling would result in an enhanced role for skin and a diminished role for the gills in osmo- and ionoregulation. Fish were exposed to water-either freshwater (FW, 1‰) or hypersaline water (saltwater [SW], 45‰)-or air over a moist surface of FW or SW for 9 d and then recovered in water. When fish were emersed for 9 d, (22)Na and (3)H-H(2)O were exchanged across the cutaneous surface. Homeostasis of whole-body Cl(-) and water levels but not of Na(+) levels was maintained over 9 d in air. In air-exposed fish, there was a significant increase in the size of skin ionocytes (in SW), a decrease in the number of skin mucous cells (in SW), and an increase in the gill interlamellar cell mass relative to those of fish in water. Gill ionocytes were mostly embedded away from the external surface in air-exposed fish, but the number and size of ionocytes increased (in FW). Interestingly, skin ionocytes formed distinct clusters of 20-30 cells. The estimated number of ionocytes over the whole skin surface was comparable to that in the gills. Overall, the findings support the hypothesis that the skin is a site of osmo- and ionoregulation in K. marmoratus in aquatic and terrestrial environments. Reversible cellular and morphological changes to the skin and gills during air exposure probably enhanced the cutaneous contribution to ion and water balance.