Cl- uptake mechanism in freshwater-adapted tilapia (Oreochromis mossambicus)

In this study, the correlation between Cl(-) influx in freshwater tilapia and various transporters or enzymes, the Cl(-)/HCO(3)(-) exchanger, Na(+),K(+)-ATPase, V-type H(+)-ATPase, and carbonic anhydrase were examined. The inhibitors 2x10(-4) M ouabain (a Na(+),K(+)-ATPase inhibitor), 10(-5) M NEM (a V-type H(+)-ATPase inhibitor), 10(-2) M ACTZ (acetazolamide, a carbonic anhydrase inhibitor), and 6x10(-4) M DIDS (a Cl(-)/HCO(3)(-) exchanger inhibitor) caused 40%, 60%-80%, 40%-60%, and 40%-60% reduction in Cl(-) influx of freshwater tilapia, respectively. The inhibitor 2x10(-4) M ouabain also caused 50%-65% inhibition in gill Na(+),K(+)-ATPase activity. Western blot results showed that protein levels of gill Na(+),K(+)-ATPase, V-type H(+)-ATPase, and carbonic anhydrase in tilapia acclimated in low-Cl(-) freshwater were significantly higher than those acclimated to high-Cl(-) freshwater. Based on these data, we conclude that Na(+),K(+)-ATPase, V-H(+)-ATPase, the Cl(-)/HCO(3)(-) exchanger, and carbonic anhydrase may be involved in the active Cl(-) uptake mechanism in gills of freshwater-adapted tilapia.     

Acclimation of ion regulatory capacities in gills of marine fish under environmental hypercapnia

The preservation of ion balance and pH despite environmental fluctuations is essential for the maintenance of vital cellular functions. While several ion transporters contribute to acid-base regulation in fish, the involvement and expression of key transporters under hypercapnia remain to be established. Here, two members of the HCO(3)(-) transporter family (Na(+)/HCO(3)(-) cotransporter NBC1 and Cl(-)/HCO(3)(-) exchanger AE1) were described for the first time in gills of marine fish. Benthic eelpout Zoarces viviparus were acclimated to 10,000 ppm CO(2). Hypercapnia did not affect whole animal oxygen consumption over a period of 4 days. During a time series of 6 wk NBC1 mRNA levels first decreased by about 40% (8 to 24 h) but finally increased about threefold over control. mRNA expression of AE1 decreased transiently by 50% at day 4 but recovered to control levels only. Reduced mRNA levels were also found for two Na(+)/H(+) exchangers (NHE1A, NHE1B) during the first days (by 50-60% at days 1 and 2), followed by restoration of control levels. This pattern was mirrored in a slight decrease of NHE1 protein contents and its subsequent recovery. In contrast, Na(+)-K(+)-ATPase mRNA and protein contents, as well as maximum activity, rose steadily from the onset of hypercapnia, and reached up to twofold control levels at the end. These results indicate shifting acclimation patterns between short- and long-term CO(2) exposures. Overall, ion gradient-dependent transporter mRNA levels were transiently downregulated in the beginning of the disturbance. Upregulation of NBC1 on long timescales stresses the importance of this transporter in the hypercapnia response of marine teleosts. Long-term rearrangements include Na(+)-K(+)-ATPase at higher densities and capacities, indicating a shift to elevated rates of ion and acid-base regulation under environmental hypercapnia.     

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.     

Responses of gill mitochondria-rich cells in Mozambique tilapia exposed to acidic environments (pH 4.0) in combination with different salinities

On exposure to hyposmotic acidic water, teleost fish suffer from decreases in blood osmolality and pH, and consequently activate osmoregulatory and acid-base regulatory mechanisms to restore disturbed ion and acid-base balances. In Mozambique tilapia Oreochromis mossambicus exposed to acidic (pH 4.0) or neutral (pH 7.4-7.7) freshwater in combination with 0mM or 50mM NaCl, we examined functional and morphological changes in gill mitochondria-rich (MR) cells. We assessed gene expression of Na(+)/H(+) exchanger-3 (NHE3), Na(+)/Cl(-) cotransporter (NCC), vacuolar-type H(+)-ATPase (V-ATPase) and Na(+)/HCO(3)(-) cotransporter-1 (NBC1) in the gills. The mRNA expression of NHE3 and NCC in tilapia gills were higher in acidic freshwater than in that supplemented with 50mM NaCl, while there was no significant difference in mRNA levels of V-ATPase and NBC1. In addition, immunocytochemical observations showed that apical-NHE3 MR cells were enlarged, and frequently formed multicellular complexes with developed deep apical openings in acidic freshwater with 0mM and 50mM NaCl. These findings suggest that gill MR cells respond to external salinity and pH treatments, by parallel manipulation of osmoregulatory and acid-base regulatory mechanisms.     

Gill Na(+)-K(+)-2Cl(-) cotransporter abundance and location in Atlantic salmon: effects of seawater and smolting

Na(+)-K(+)-2Cl(-) cotransporter abundance and location was examined in the gills of Atlantic salmon (Salmo salar) during seawater acclimation and smolting. Western blots revealed three bands centered at 285, 160, and 120 kDa. The Na(+)-K(+)-2Cl(-) cotransporter was colocalized with Na(+)-K(+)-ATPase to chloride cells on both the primary filament and secondary lamellae. Parr acclimated to 30 parts per thousand seawater had increased gill Na(+)-K(+)-2Cl(-) cotransporter abundance, large and numerous Na(+)-K(+)-2Cl(-) cotransporter immunoreactive chloride cells on the primary filament, and reduced numbers on the secondary lamellae. Gill Na(+)-K(+)-2Cl(-) cotransporter levels were low in presmolts (February) and increased 3.3-fold in smolts (May), coincident with elevated seawater tolerance. Cotransporter levels decreased below presmolt values in postsmolts in freshwater (June). The size and number of immunoreactive chloride cells on the primary filament increased threefold during smolting and decreased in postsmolts. Gill Na(+)-K(+)-ATPase activity and Na(+)-K(+)-2Cl(-) cotransporter abundance increased in parallel during both seawater acclimation and smolting. These data indicate a direct role of the Na(+)-K(+)-2Cl(-) cotransporter in salt secretion by gill chloride cells of teleost fish.     

Theoretical considerations underlying Na(+) uptake mechanisms in freshwater fishes

Ion and acid-base regulating mechanisms have been studied at the fish gill for almost a century. Original models proposed for Na(+) and Cl(-) uptake, and their linkage with H(+) and HCO(3)(-) secretion have changed substantially with the development of more sophisticated physiological techniques. At the freshwater fish gill, two dominant mechanisms for Na(+) uptake from dilute environments have persisted in the literature. The use of an apical Na(+)/H(+) exchanger driven by a basolateral Na(+)/K(+)-ATPase versus an apical Na(+) channel electrogenically coupled to an apical H(+)-ATPase have been the source of debate for a number of years. Advances in molecular biology have greatly enhanced our understanding of the basic ion transport mechanisms at the fish gill. However, it is imperative to ensure that thermodynamic principles are followed in the development of new models for gill ion transport. This review will focus on the recent molecular advances for Na(+) uptake in freshwater fish. Emphasis will be placed on thermodynamic constraints that prevent electroneutral apical NHE function in most freshwater environments. By combining recent advances in molecular and functional physiology of fish gills with thermodynamic considerations of ion transport, our knowledge in the field should continue to grow in a logical manner.     

Na(+), Cl(-), Ca(2+) and Zn(2+) transport by fish gills: retrospective review and prospective synthesis

The secondary active Cl(-) secretion in seawater (SW) teleost fish gills and elasmobranch rectal gland involves basolateral Na(+),K(+)-ATPase and NKCC, apical membrane CFTR anion channels, and a paracellular Na(+)-selective conductance. In freshwater (FW) teleost gill, the mechanism of NaCl uptake is more controversial and involves apical V-type H(+)-ATPase linked to an apical Na(+) channel, apical Cl(-)-HCO-3 exchange and basolateral Na(+),K(+)-ATPase. Ca(2+) uptake (in FW and SW) is via Ca(2+) channels in the apical membrane and Ca(2+)-ATPase in the basolateral membrane. Mainly this transport occurs in mitochondria rich (MR) chloride cells, but there is a role for the pavement cells also. Future research will likely expand in two major directions, molded by methodology: first in physiological genomics of all the transporters, including their expression, trafficking, operation, and regulation at the molecular level, and second in biotelemetry to examine multivariable components in behavioral physiological ecology, thus widening the integration of physiology from the molecular to the environmental levels while deepening understanding at all levels.     

Wild Arctic char (Salvelinus alpinus) upregulate gill Na+,K+-ATPase during freshwater migration

The successful acclimation of eurhyhaline fishes from seawater to freshwater requires the gills to stop actively secreting ions and start actively absorbing ions. Gill Na(+),K(+)-ATPase is known to be an integral part of the active ion secretion model of marine fishes, but its importance in the active ion uptake model of freshwater fishes is less clear. This study, conducted in the high Arctic, examines gill Na(+),K(+)-ATPase regulation in wild anadromous arctic char returning to freshwater from the ocean. Gill Na(+),K(+)-ATPase activity, protein expression, and mRNA expression of Na(+),K(+)-ATPase isoforms alpha 1a and alpha 1b were monitored in arctic char at three points along their migration route to and from Somerset Island, Nunavut, Canada: out at sea (Whaler's Point), in seawater near the river mouth (Nat's Camp), and after entering the Union River. Arctic char collected from the Union River had more than twofold greater gill Na(+),K(+)-ATPase activity. This was associated with a significant increase (threefold) in Na(+),K(+)-ATPase isoform alpha 1a mRNA expression and a significant increase in plasma sodium and osmolality levels compared with seawater char. Compared with char sampled from Whaler's Point, Na(+),K(+)-ATPase isoform alpha 1b mRNA expression was decreased by approximately 50% in char sampled at Nat's Camp and the Union River. These results suggest that the upregulation of gill Na(+),K(+)-ATPase activity is involved in freshwater acclimation of arctic char and implicate a role for Na(+),K(+)-ATPase isoform alpha 1a in this process. In addition, we discuss evidence that arctic char go through a preparatory phase, or "reverse smoltification," before entering freshwater.     

The distribution of mitochondria-rich cells in the gills of air-breathing fishes

Respiration and ion regulation are the two principal functions of teleostean gills. Mainly found in the gill filaments of fish, mitochondria-rich cells (MRCs) proliferate to increase the ionoregulatory capacity of the gill in response to osmotic challenges. Gill lamellae consist mostly of pavement cells, which are the major site of gas exchange. Although lamellar MRCs have been reported in some fish species, there has been little discussion of which fish species are likely to have lamellar MRCs. In this study, we first compared the number of filament and lamellar MRCs in air-breathing and non-air-breathing fish species acclimated to freshwater and 5 g NaCl L(-1) conditions. An increase in filament MRCs was found in both air-breathing and non-air-breathing fish acclimated to freshwater. Lamellar MRCs were found only in air-breathing species, but the number of lamellar MRCs did not change significantly with water conditions, except in Periophthalmus cantonensis. Next, we surveyed the distribution of MRCs in the gills of 66 fish species (including 29 species from the previous literature) from 12 orders, 28 families, and 56 genera. Our hypothesis that lamellar MRCs are more likely to be found in air-breathing fishes was supported by a significant association between the presence of lamellar MRCs and the mode of breathing at three levels of systematic categories (species, genus, and family). Based on this integrative view of the multiple functions of fish gills, we should reexamine the role of MRCs in freshwater fish.     

V-H(+)-ATPase, Na(+)/K(+)-ATPase and NHE2 immunoreactivity in the gill epithelium of the Pacific hagfish (Epatretus stoutii)

We report the presence of the ion transporting proteins V-H(+)-ATPase, Na(+)/K(+)-ATPase and NHE2 in the gill epithelium of the Pacific hagfish Epatretus stoutii. Heterologous antibodies recognized single bands of the appropriate sizes for the three transporters in western blots. Immunohistochemical staining revealed that the distribution of labeled cells in the gill epithelium was identical for the three proteins. Immunopositive cells were most abundant in the primary filament from the afferent side of the gill pouch, and their number diminished towards the lamella. Na(+)/K(+)-ATPase-like immunoreactivity (L-IR) occurred throughout the cell cytoplasm, probably associated to the basolateral tubular system. V-H(+)-ATPase L-IR was similar to Na(+)/K(+)-ATPase, although some cells had slightly heavier staining in either the supra- or infra-nuclear region. NHE2 L-IR was also generally cytoplasmic, but a minority of the cells had stronger immunoreactivity in the apical region. In general, all three ion transporting proteins were localized in the same cells, as estimated from 4-microm immunostained consecutive sections. We hypothesize that these putative ion-transporting cells are involved in systemic acid/base regulation and discuss other possible roles. This is the first report of V-H(+)-ATPase in myxinoids, and the first NHE2 report in the Pacific hagfish.     

Branchial mitochondria-rich cells in the dogfish Squalus acanthias

In marine teleost fishes, the gill mitochondria-rich cells (MRCs) are responsible for NaCl elimination; however, in elasmobranch fishes, the specialized rectal gland is considered to be the most important site for salt secretion. The role of the gills in elasmobranch ion regulation, although clearly shown to be secondary, is not well characterized. In the present study, we investigated some morphological properties of the branchial MRCs and the localization, and activity of the important ionoregulatory enzyme Na(+)/K(+)-ATPase, under control conditions and following rectal gland removal (1 month) in the spiny dogfish, Squalus acanthias. A clear correlation can be made between MRC numbers and the levels of Na(+)/K(+)-ATPase activity in crude gill homogenates (r(2)=-0.69). Strong Na(+)/K(+)-ATPase immunoreactivity is also clearly associated with the basolateral membrane of these MRCs. In addition, the dogfish were able to maintain ionic balance after rectal gland removal. These results all suggest a possible role of the dogfish gill in salt secretion. MRCs were, however, unresponsive to rectal gland removal in terms of changes in number, fine structure and Na(+)/K(+)-ATPase activity, as might be expected if they were compensating for the loss of salt secretion by the rectal gland. Thus, the specific role that these MRCs play in ion regulation in the dogfish remains to be determined     

Gene expression of Na+/H+ exchanger in zebrafish H+ -ATPase-rich cells during acclimation to low-Na+ and acidic environments

In mammalian nephrons, most of the Na(+) and HCO(3)(-) is reabsorbed by proximal tubular cells in which the Na(+)/H(+) exchanger 3 (NHE3) is the major player. The roles of NHEs in Na(+) uptake/acid-base regulation in freshwater (FW) fish gills are still being debated. In the present study, functional genomic approaches were used to clone and sequence the full-length cDNAs of the nhe family from zebrafish (Danio rerio). A phylogenetic tree analysis of the deduced amino acid sequences showed that zNHE1-8 are homologous to their mammalian counterparts. By RT-PCR analysis and double/triple in situ hybridization/immunocytochemistry, only zebrafish NHE3b was expressed in zebrafish gills and was colocalized with V-H(+)-ATPase but not with Na(+)-K(+)-ATPase, indicating that H(+)-ATPase-rich (HR) cells specifically express NHE3b. A subsequent quantitative RT-PCR analysis demonstrated that acclimation to low-Na(+) FW caused upregulation and downregulation of the expressions of znhe3b and zatp6v0c (H(+)-ATPase C-subunit), respectively, in gill HR cells, whereas acclimation to acidic FW showed reversed effects on the expressions of these two genes. In conclusion, both NHE3b and H(+)-ATPase are probably involved in Na(+) uptake/acid-base regulation in zebrafish gills, like mammalian kidneys, but the partitioning of these two transporters may be differentially regulated depending on the environmental situation in which fish are acclimatized.     

V-H+ -ATPase translocation during blood alkalosis in dogfish gills: interaction with carbonic anhydrase and involvement in the postfeeding alkaline tide

We investigated the involvement of carbonic anhydrase (CA) in mediating V-H(+)-ATPase translocation into the basolateral membrane in gills of alkalotic Squalus acanthias. Immunolabeling revealed that CA is localized in the same cells as V-H(+)-ATPase. Blood plasma from dogfish injected with acetazolamide [30 mg/kg at time (t) = 0 and 6 h] and infused with NaHCO(3) for 12 h (1,000 microeq.kg(-1).h(-1)) had significantly higher plasma HCO(3)(-) concentration than fish that were infused with NaHCO(3) alone (28.72 +/- 0.41 vs. 6.57 +/- 2.47 mmol/l, n = 3), whereas blood pH was similar in both treatments (8.03 +/- 0.11 vs. 8.04 +/- 0.11 pH units at t = 12 h). CA inhibition impaired V-H(+)-ATPase translocation into the basolateral membrane, as estimated from immunolabeled gill sections and Western blotting on gill cell membranes (0.24 +/- 0.08 vs. 1.00 +/- 0.28 arbitrary units, n = 3; P < 0.05). We investigated V-H(+)-ATPase translocation during a postfeeding alkalosis ("alkaline tide"). Gill samples were taken 24-26 h after dogfish were fed to satiety in a natural-like feeding regime. Immunolabeled gill sections revealed that V-H(+)-ATPase translocated to the basolateral membrane in the postfed fish. Confirming this result, V-H(+)-ATPase abundance was twofold higher in gill cell membranes of the postfed fish than in fasted fish (n = 4-5; P < 0.05). These results indicate that 1) intracellular H(+) or HCO(3)(-) produced by CA (and not blood pH or HCO(3)(-)) is likely the stimulus that triggers the V-H(+)-ATPase translocation into the basolateral membrane in alkalotic fish and 2) V-H(+)-ATPase translocation is important for enhanced HCO(3)(-) secretion during a naturally occurring postfeeding alkalosis.     

Morphology and function of gill mitochondria-rich cells in fish acclimated to different environments

The objective of this study is to test the hypothesis that morphologically different mitochondria-rich (MR) cells may be responsible for the uptake of different ions in freshwater-adapted fish. Tilapia (Oreochromis mossambicus) were acclimated to high-Ca, mid-Ca, low-Ca, and low-NaCl artificial freshwater, respectively, for 2 wk. Cell densities of wavy-convex, shallow-basin, and deep-hole types of gill MR cells as well as whole-body Ca(2+), Na(+), and Cl(-) influxes were measured. Low-Ca fish developed more shallow-basin MR cells in the gills and a higher Ca(2+) influx than those acclimated to other media. However, fish acclimated to low-NaCl artificial freshwater predominantly developed wavy-convex cells, and this was accompanied by the highest Na(+) and Cl(-) influxes. Relative abundance of shallow-basin and wavy-convex MR cells appear to be associated with changes in Ca(2+) and Na(+)/Cl(-) influxes, suggesting that shallow-basin and wavy-convex MR cells are mainly responsible for the uptake of Ca(2+) and Na(+)/Cl(-), respectively.     

Cultured gill epithelia as models for the freshwater fish gill

We review recent progress in the development of models for the freshwater teleost gill based on reconstructed flat epithelia grown on permeable filter supports in primary culture. Methods are available for single-seeded insert (SSI) preparations consisting of pavement cells (PVCs) only from trout and tilapia, and double-seeded insert (DSI) preparations from trout, containing both PVCs (85%) and mitochondria-rich cells (MRCs, 15%), as in the intact gill. While there are some quantitative differences, both SSI and DSI epithelia manifest electrical and passive permeability characteristics typical of intact gills and representative of very tight epithelia. Both preparations withstand apical freshwater exposure, exhibiting large increases in transepithelial resistance (TER), negative transepithelial potential (TEP), and low rates of ion loss, but there is only a small active apical-to-basolateral "influx" of Cl(-) (and not of Na(+)). Responses to various hormonal treatments are described (thyroid hormone T3, prolactin, and cortisol). Cortisol has the most marked effects, stimulating Na(+),K(+)-ATPase activity and promoting active Na(+) and Cl(-) influxes in DSI preparations, and raising TER and reducing passive ion effluxes in both epithelia via reductions in paracellular permeability. Experiments using DSI epithelia lacking Na(+) uptake demonstrate that both NH(3) and NH(4)(+) diffusion occur, but are not large enough to account for normal rates of branchial ammonia excretion, suggesting that Na(+)-linked carrier-mediated processes are important for ammonia excretion in vivo. Future research goals are suggested.     

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.     

Regulation of the ion-transporting mitochondrion-rich cell during adaptation of teleost fishes to different salinities

The mitochondrion-rich cells (MRCs) in teleost gill and equivalent tissues are important osmoregulatory sites in maintaining ionic balance. These cells express a variety of ion pumps, transporters, and channels, which play central roles in ionic regulation. Recently, two types of MRCs have been identified in euryhaline fishes: seawater (SW)-type MRCs extrude Na and Cl ions in SW conditions; freshwater (FW)-type MRCs take up at least Cl-. Long-term development/differentiation of the two types of MRCs during adaptation to different salinities appears to be regulated mainly by endocrine factors. Osmolality, Ca2+, neurotransmitters, and fast-acting hormones rapidly regulate the SW MRCs. Recent information is assembled in this review and suggests the functional plasticity of highly specialized MRCs.     

Structures and immunolocalization of Na+, K+ -ATPase, Na+ /H+ exchanger 3 and vacuolar-type H+ -ATPase in the gills of blennies (Teleostei: Blenniidae) inhabiting rocky intertidal areas

The structure and immunolocalization of the ion transporters Na(+) ,K(+) -ATPase (NKA), Na(+) /H(+) exchanger (NHE3) and vacuolar-type H(+) -ATPase (VHA) were examined in the gills of teleosts of the family Blenniidae, which inhabit rocky shores with vertical zonation in subtropical seas. These features were compared among the following species with different ecologies: the amphibious rockskipper blenny Andamia tetradactylus, the intertidal white-finned blenny Praealticus tanegasimae and the purely marine yaeyama blenny Ecsenius yaeyamaensis. Light and electron microscopic observations indicated that thick gill filaments were arranged close to each other and alternately on two hemibranches of a gill arch in the opercular space of A. tetradactylus. Many mucous cells (MC) and mitochondrion-rich cells (MRC) were present in the interlamellar regions of the gill filament. An immunohistochemical study demonstrated that numerous NKA, NHE3 and some VHA were located predominantly on presumed MRCs of gill filaments and at the base of the lamellae. Analyses using serial (mirror image) sections of the gills indicated that only a few NKA immunoreactive cells (IRC) were colocalized with VHA on some MRCs in the filaments. In the gills of P. tanegasimae, NKA- and NHE3-IRCs were observed in the interlamellar region of the filaments and at the base of the lamellae. VHA-IRCs were located sparsely on the lamellae and filaments. In the gills of E. yaeyamaensis, the lamellae and filaments were thin and straight, respectively. MCs were located at the tip as well as found scattered in the interlamellar region of gill filaments. NKA-, NHE3- and VHA-IRCs were moderately frequently observed in the filaments and rarely on the lamellae. This study shows that the structure and distribution of ion transporters in the gills differ among the three blennid species, presumably reflecting their different ecologies.     

The osmorespiratory compromise in sculpins: impaired gas exchange is associated with freshwater tolerance

We acclimated two species of sculpin, the freshwater prickly sculpin (Cottus asper) and the closely related marine Pacific staghorn sculpin (Leptocottus armatus) to freshwater ( approximately 0 g/L), brackish water (15 g/L), and seawater (30 g/L) for at least 4 wk and examined the relationships between respiration, ion regulation, gill morphology, and freshwater tolerance. The prickly sculpin successfully acclimated to all three salinities and did not experience appreciable changes in plasma osmolality, [Cl-], or mortality. Gill Na+/K+-ATPase activity was lowest in prickly sculpins acclimated to freshwater, their native salinity, and increased during acclimation to seawater. Furthermore, prickly sculpins acclimated to freshwater had a 30% higher P(crit) than fish acclimated to brackish water or seawater; P(crit) is the environmental P(O2) below which an animal can no longer maintain a routine (.-)M(O2), and an increase in P(crit) represents a compromise of respiratory gas exchange. The higher P(crit) observed in prickly sculpins acclimated to freshwater is likely a consequence of their having small, relatively thick gills that increase in thickness (by approximately 1 microm) during freshwater exposure. In contrast, the marine Pacific staghorn sculpin successfully acclimated to brackish water and seawater, but high mortality (25%) was observed after 3 wk of exposure to freshwater. Pacific staghorn sculpins exposed to freshwater suffered significant, 15%-20%, reductions in plasma osmolality and [Cl-], and these losses in plasma ions resulted in a 1.4-fold increase in gill Na+/K+-ATPase activity. Pacific staghorn sculpins have large, thin gills that are not modified in response to salinity acclimation, and as a result, these animals show no respiratory compromise during freshwater acclimation, as evidenced by the lack of change in P(crit), but show significant ion regulatory disturbance. Overall, this study suggests that gill thickening and the resulting respiratory compromise are necessary for freshwater tolerance in sculpins.     

Acid-base regulation in fishes: cellular and molecular mechanisms

The mechanisms underlying acid-base transfers across the branchial epithelium of fishes have been studied for more than 70 years. These animals are able to compensate for changes to internal pH following a wide range of acid-base challenges, and the gill epithelium is the primary site of acid-base transfers to the water. This paper reviews recent molecular, immunohistochemical, and functional studies that have begun to define the protein transporters involved in the acid-base relevant ion transfers. Both Na(+)/H(+) exchange (NHE) and vacuolar-type H(+)-ATPase transport H(+) from the fish to the environment. While NHEs have been thought to carry out this function mainly in seawater-adapted animals, these proteins have now been localized to mitochondrial-rich cells in the gill epithelium of both fresh and saltwater-adapted fishes. NHEs have been found in the gill epithelium of elasmobranchs, teleosts, and an agnathan. In several species, apical isoforms (NHE2 and NHE3) appear to be up-regulated following acidosis. In freshwater teleosts, H(+)-ATPase drives H(+) excretion and is indirectly coupled to Na(+) uptake (via Na(+) channels). It has been localized to respiratory pavement cells and chloride cells of the gill epithelium. In the marine elasmobranch, both branchial NHE and H(+)-ATPase have been identified, suggesting that a combination of these mechanisms may be utilized by marine elasmobranchs for acid-base regulation. An apically located Cl(-)/HCO(3)(-) anion exchanger in chloride cells may be responsible for base excretion in fresh and seawater-adapted fishes. While only a few species have been examined to date, new molecular approaches applied to a wider range of fishes will continue to improve our understanding of the roles of the various gill membrane transport processes in acid-base balance.