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Estuaries and lagoons are characterized by fluctuating salinity and significant amounts of microplastics (MPs) and are increasingly subjected to various anthropogenic pressures. We investigated whether the accumulation of MPs in the gills of fish inhabiting these fragile ecosystems alters osmoregulation and, consequently, their ability to tolerate fluctuating salinity. The effects of a 15-day exposure to an environmentally relevant concentration (20 μg/L) of spherical polystyrene microplastics (PS-MPs) with a diameter of 5 μm were assessed in the Mediterranean killifish Aphanius fasciatus, focusing on tissue and gene expression changes related to factors of paracellular and transcellular permeability of the gill epithelium during the transition from seawater to freshwater. Our results revealed that PS-MPs indirectly impaired osmoregulation, particularly in fresh water, through their toxic effects on the gill tissue. Toxicity was evidenced by epithelial lifting, a decrease in the proportion of secondary lamellae available for gas exchange, and upregulation of superoxide dismutase and heat shock protein genes. Furthermore, exposure to PS-MPs directly affected gill epithelial permeability by maintaining relatively high paracellular permeability through the downregulation of claudin 3 and by modifying the expression of the transcellular transporter Na + /K + -ATPase and cystic fibrosis transmembrane conductance regulator in the gill epithelium. Overall, these findings confirm the toxic effects of PS-MPs on gill tissue and demonstrate, for the first time, that environmentally relevant concentrations of MPs adversely affect gill epithelium permeability during decreased salinity acclimation in the euryhaline fish A. fasciatus., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

To maintain internal ion balance in marine environments, teleost fishes leverage seawater (SW)-type ionocytes to actively secrete Na + and Cl - into the environment. It is well established that SW-type ionocytes use apically expressed cystic fibrosis transmembrane conductance regulator 1 (Cftr1) as a conduit for Cl - to exit the gill. Here, we investigated whether the Ca 2+ -activated Cl - channel, anoctamin 1 (Ano1), provides an additional path for Cl - -secretion in euryhaline mummichogs ( Fundulus heteroclitus ). Two ano1 gene isoforms, denoted ano1.1a and -1.1b , exhibited higher expression in the gill and opercular epithelium of mummichogs long-term acclimated to SW versus fresh water (FW). Branchial ano1.1b and cftr1 expression was increased in mummichogs sampled 24 h after transfer from FW to SW; ano1.1a and -1.1b were upregulated in the gill and opercular epithelium following transfer from SW to hypersaline SW. Alternatively, the expression of ano1.1a , -1.1b , and cftr1 in the gill and opercular epithelium was markedly decreased after transfer from SW to FW. Given its role in attenuating ion secretion, we probed whether prolactin downregulates ano1 isoforms. In addition to attenuating cftr1 expression, a prolactin injection reduced branchial ano1.1a and -1.1b levels. Given how Ano1 mediates Cl - secretion by mammalian epithelial cells, the salinity- and prolactin-sensitive nature of ano1 expression reported here indicates that Ano1 may constitute a novel Cl - -secretion pathway in ionocytes. This study encourages a wider evaluation of this putative Cl - -secretion pathway and its regulation by hormones in teleost fishes. NEW & NOTEWORTHY In this study, we provide evidence in a teleost fish that the Ca 2+ -activated Cl - channel, anoctamin 1 may provide an additional path for Cl - secretion by seawater-type ionocytes. Not only is this the first report of a Cftr-independent Cl - -secreting pathway conferring survival in seawater but also the first description of its regulation by the pituitary hormone prolactin.

Mummichogs (Fundulus heteroclitus) can tolerate abrupt changes in environmental salinity because of their ability to rapidly adjust the activities of ionocytes in branchial and opercular epithelia. In turn, the concerted expression of sub-cellular effectors of ion transport underlies adaptive responses to fluctuating salinities. Exposure to seawater (SW) stimulates the expression of Na + /K + /2Cl - cotransporter 1 (nkcc1) and cystic fibrosis transmembrane regulator (cftr) mRNAs in support of ion extrusion by SW-type ionocytes. Given the incomplete understanding of how freshwater (FW)-type ionocytes actually operate in mummichogs, the transcriptional responses essential for ion absorption in FW environments remain unresolved. In a subset of species, a 'fish-specific' Na + /Cl - cotransporter denoted Ncc2 (Slc12a10) is responsible for the uptake of Na + and Cl - across the apical surface of FW-type ionocytes. In the current study, we identified an ncc2 transcript that is highly expressed in gill filaments and opercular epithelium of FW-acclimated mummichogs. Within 1 day of transfer from SW to FW, ncc2 levels in both tissues increased in parallel with reductions in nkcc1 and cftr. Conversely, mummichogs transferred from FW to SW exhibited marked reductions in ncc2 concurrent with increases in nkcc1 and cftr. Immunohistochemical analyses employing a homologous antibody revealed apical Ncc2-immunoreactivity in Na + /K + -ATPase-immunoreactive ionocytes of FW-acclimated animals. Our combined observations suggest that Ncc2/ncc2-expressing ionocytes support the capacity of mummichogs to inhabit FW environments.

This study aims to investigate the influence of high temperature on cadmium (Cd) toxicity in Aphanius fasciatus (Pisces: Cyprinodontidae). For this reason, Cd, mineral, and organic content in the vertebral column as well as the histological structure of gills and bone were compared in fishes exposed for 30 days to Cd (2 mg/L CdCl2) and/or high temperature (26 °C). Cd exposure caused a negative correlation between Cd and Ca concentrations (r = 0.98, p < 0.05), as well as a significant decrease in inorganic components (p < 0.05) and ash weight/dry weight ratio (p < 0.05) in the vertebral column. These changes were accompanied by an increased frequency of histological alterations in gills and bone. Concomitant treatment with Cd and high temperature increases Cd accumulation and Ca depletion in the skeletal tissue and increases the frequency and the severity of histological alterations. These results confirm that temperature increases Cd toxicity and needs to be taken into account for the accurate prediction and assessment of Cd-induced spinal deformities in fish.

The Gulf killifish, Fundulus grandis, is a euryhaline teleost which has important ecological roles in the brackish-water marshes of its native range as well as commercial value as live bait for saltwater anglers. Effects of osmoregulation on growth, survival, and body condition at 0.5, 5.0, 8.0 and 12.0‰ salinity were studied in F. grandis juveniles during a 12-week trial. Relative expression of genes encoding the ion transport proteins Na(+)/K(+)-ATPase (NKA), Na(+)/K(+)/2Cl(-) cotransporter(NKCC1), and cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel was analyzed. At 0.5‰, F. grandis showed depressed growth, body condition, and survival relative to higher salinities. NKA relative expression was elevated at 7 days post-transfer but decreased at later time points in fish held at 0.5‰ while other salinities produced no such increase. NKCC1, the isoform associated with expulsion of ions in saltwater, was downregulated from week 1 to week 3 at 0.5‰ while CFTR relative expression produced no significant results across time or salinity. Our results suggest that Gulf killifish have physiological difficulties with osmoregulation at a salinity of 0.5‰ and that this leads to reduced growth performance and survival while salinities in the 5.0-12.0‰ are adequate for normal function., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Estuarine fish, such as the Atlantic killifish (Fundulus heteroclitus), are constantly and rapidly exposed to changes in salinity. Although ion transport in killifish gills during acclimation to increased salinity has been studied extensively, no studies have examined the role of aquaglyceroporin 3 (AQP3), a water, glycerol, urea, and ammonia transporter, during acclimation to increased salinity in this sentinel environmental model organism. The goal of this study was to test the hypothesis that transfer from freshwater to seawater decreases AQP3 gene and protein expression in the gill of killifish. Transfer from freshwater to seawater decreased AQP3 mRNA in the gill after 1 day, but had no effect on total gill AQP3 protein abundance as determined by western blot. Quantitative confocal immunocytochemistry confirmed western blot studies that transfer from freshwater to seawater did not change total AQP3 abundance in the gill; however, immunocytochemistry revealed that the amount of AQP3 in pillar cells of secondary lamellae decreased in seawater fish, whereas the amount of AQP3 in mitochondrion rich cells (MRC) in primary filaments of the gill increased in seawater fish. This response of AQP3 expression is unique to killifish compared to other teleosts. Although the role of AQP3 in the gill of killifish has not been completely elucidated, these results suggest that AQP3 may play an important role in the ability of killifish to acclimate to increased salinity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The peptide urotensin II (UII) and its receptor (UT) mediate cardiovascular and renal effects in both mammals and fishes. In both groups, vasopressor and diuretic responses predominate, although, in mammals, some secondary vasodilatation is found, mediated by secondary release of nitric oxide or prostacyclin. In fishes, gill extrusion of NaCl is inhibited by UII, but a single study has determined that UT is expressed in gill vasculature, not on the epithelium that mediates the transport. To begin to clarify the pathways involved in UII inhibition of gill transport, we have cloned the cDNA encoding UII and UT from the euryhaline killifish (Fundulus heteroclitus L.) gill and spinal cord, quantified UT mRNA expression in various tissues and measured relative expression in gill tissue from fish acclimated to seawater (SW) vs fresh water (FW). We have also localized UT in the gill epithelium, and measured the effect of UII on ion transport across the opercular epithelium. We found that both UII and UT are synthesized in the gill of F. heteroclitus and that gill UT mRNA levels are ~80% higher in SW- vs FW-acclimated individuals. In addition, UII inhibits NaCl transport across the opercular epithelium in a concentration-dependent manner, and this inhibition is at least partially mediated by both nitric oxide and a prostanoid.

Euryhaline teleosts such as Atlantic killifish (Fundulus heteroclitus) are able to acclimate to changing environmental salinity by tightly regulating NaCl absorption and secretion across their gills. Many studies have examined the mechanisms responsible for long-term (days) salinity acclimation; however, much remains unknown about the mechanisms of acute (hours) salinity acclimation. In this study, we tested the hypotheses that phosphorylation of the Na(+)-K(+)-Cl(-) cotransporter (NKCC1) located in the basolateral membrane of the gill plays a role in acute salinity acclimation and that changes in NKCC1 phosphorylation are mediated by a cAMP-protein kinase A (cAMP-PKA) pathway. Using a phospho-specific antibody, we determined the time course of changes in total and phosphorylated NKCC1 protein during acclimation to water of various salinities. Long-term (>or=14 days) acclimation of killifish to seawater (SW) and 2x SW resulted in 4- to 6-fold and 5- to 8-fold increases, respectively, in total gill NKCC1 protein relative to fish maintained in freshwater (FW). NKCC1 was found to be between 20% and 70% activated in fish, with lower average activation in fish acclimated to SW and 2x SW compared with FW fish. Increases and decreases in the fractional level of NKCC1 phosphorylation were seen within 1 h of transfer of fish to water of higher and lower salinity, respectively, consistent with a regulatory role of phosphorylation prior to an increase in the biosynthesis of NKCC1; large changes in protein expression of NKCC1 were observed over periods of hours to days. We found that NKCC1 phosphorylation is acutely regulated in the killifish gill in response to changing environmental salinity and that phosphorylation in excised gills increases in response to forskolin stimulation of the cAMP-PKA pathway. The role of phosphorylation is further underscored by the observation that mRNA expression of sterile 20 (Ste20)-related proline-alanine-rich kinase (SPAK) changes with salinity acclimation, being 2.7-fold greater in SW-acclimated killifish relative to FW fish. Overall, these results demonstrate an important role of NKCC1 phosphorylation in the gill of Atlantic killifish during acute salinity acclimation.

In the current study, reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) PCR were used to clone full-length putative Na(+)-H(+) exchanger isoforms (NHE2a) cDNA from the gills of Fundulus heteroclitus. The 2480 bp cDNA includes a coding region for a protein that shows a 57% amino acid homology to rabbit NHE2. These sequences allowed data mining of available fish genome data, which revealed at least three NHE2 subtypes in some teleost species.

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.

Here, we describe a laboratory experiment as part of an upper-level vertebrate physiology course for biology majors to investigate the physiological response of vertebrates to osmoregulatory challenges. The experiment involves measuring plasma osmolality and Na+-K+-ATPase activity in gill tissue of teleost fish acclimated to water of differing salinity. We describe results obtained using the widely available goldfish (Carassius auratus) and a common baitfish, the Gulf killifish (Fundulus grandis). The procedures described are generally applicable to other fish species, and they provide an alternative to the experimental use of humans or other mammalian species to investigate osmoregulation mechanisms. In addition to reenforcing the conceptual material covered in lecture, this laboratory exercise trains students in a wide range of laboratory and analytical skills, such as calculating and performing dilutions, pipetting, tissue sampling and homogenizing, preparing standard curves, conducting enzymatic assays, and analyzing and interpreting results. Typical student results are presented and discussed, as are common experimental and conceptual mistakes made by students.

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.

Neuronal NOS (nNOS) is a constitutively expressed enzyme that catalyzes the oxidation of L-arginine and water to L-citrulline and the gas nitric oxide (NO). Nitric oxide is involved in regulation of a variety of processes, including: vascular tone, neurotransmission, and ion balance in mammals and fishes. In this study, we have cloned and characterized a putative NOS homologue from the brain of the euryhaline killifish, Fundulus heteroclitus. Killifish NOS has 75% amino acid identity to human nNOS, and phylogenetic analysis groups the killifish sequence with the mammalian nNOS, suggesting that it is a mammalian orthologue. Relative quantitative reverse transcriptase-PCR demonstrated that killifish nNOS mRNA is highly expressed in the brain and gill followed by the stomach, kidney, opercular epithelium, intestine and heart. Immunohistochemistry localized nNOS to nerve fibers and epithelial cells adjacent to mitochondrion-rich cells (ion transporting cell) in the gill, suggesting that nNOS production of NO may contribute to regulation of vascular tone and/or MRC function in the teleost gill.

We have explored the molecular basis for differences in physiological function between the gills and opercular epithelium of the euryhaline killifish Fundulus heteroclitus. These tissues are functionally similar in seawater, but in freshwater the gills actively absorb Na+ but not Cl-, whereas the opercular epithelium actively absorbs Cl- but not Na+. These differences in freshwater physiology are likely due to differences in absolute levels of gene expression (measured using real-time PCR), as several proteins important for Na+ transport, namely Na+,H+-exchanger 2 (NHE2), carbonic anhydrase 2 (CA2), Na+,HCO3- cotransporter 1, and V-type H+-ATPase, were expressed at 3- to over 30-fold higher absolute levels in the gills. In gills, transfer from 10% seawater to freshwater increased the activity of Na+,K+-ATPase by twofold (from 12 h to 7 days), increased the expression of NHE2 (at 12 h) and CA2 (from 12 h to 7 days), and decreased the expression of NHE3 (from 12 h to 3 days). In opercular epithelium, NHE2 was not expressed; furthermore, Na+,K+-ATPase activity was unchanged after transfer to freshwater, CA2 mRNA levels decreased, and NHE3 levels increased. Consistent with their functional similarities in seawater, killifish gills and opercular epithelium expressed Na+,K+-ATPase alpha 1a, Na+,K+,2Cl- cotransporter 1 (NKCC1), cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel and the signalling protein 14-3-3a at similar absolute levels. Furthermore, NKCC1 and CFTR were suppressed equally in each tissue after freshwater transfer, and 14-3-3a mRNA increased in both. These results provide insight into the mechanisms of ion transport by killifish gills and opercular epithelia, and demonstrate a potential molecular basis for the differences in physiological function between these two organs.

The current models for branchial acid excretion in fishes include Na(+)/H(+) exchange and the electrogenic excretion of H+ via H+-ATPase. The predominant route of acid excretion in some freshwater fishes is thought to be via the H+-ATPase/Na+ channel system. The euryhaline Fundulus heteroclitus may not fit this profile even when adapted to freshwater (FW). We hypothesize that the Na+/H+ exchanger (NHE) in this species may play a predominant role in acid-base regulation for both marine and FW adapted animals. Acidosis induced by ambient hypercapnia (1% CO2 in air), resulted in an increase in net H+ excretion to the water in F. heteroclitus pre-adapted to FW, brackish (isoosmotic; BW) and seawater (SW). Both FW and SW adapted mummichogs were tested for NHE protein expression using mammalian NHE antibodies, and we identified NHE-like immunoreactive proteins in gill membrane preparations from both groups. Hypercapnia induced a approximately three-fold elevation in gill NHE2-like protein in FW animals but SW adapted fish showed inconsistent NHE3-like protein expression. There was no change in NHE-1 levels in FW fish. In contrast, SW animals demonstrated a significant increase in both NHE1 and NHE3-like proteins following hypercapnia but limited expression of the NHE2 protein. We hypothesize that different isoforms of NHE may be preferentially expressed depending on the salinity to which the animals are adapted. Net H+ transfers during acidosis may be driven, at least in part by the action of these transporters., (Copyright 2005 Wiley-Liss, Inc.)

The effects of salinity on chloride cells (CC) and Na(+)/K(+)-ATPase content in gill epithelium of euryhaline killifish Fundulus heteroclitus were analyzed using laser scanning cytometry (LSC) and tissue microarrays (TMAs). Salinity acclimations consisted of acute transfer from freshwater (FW) to 1x seawater (SW) and gradual transfer from FW to 2.4x SW. Suspensions of dissociated gill epithelial cells were stained with DASPMI and evaluated using LSC. CC number and volume are proportional to external salinity, being lower in FW (0.5+/-0.2 x 10(5) and 405+/-32 micro m(3), respectively) and higher after 5 weeks in 2.4x SW (3.7+/-0.9 x 10(5) and 2697+/-146 micro m(3), respectively). TMAs were constructed from fixed gill tissues and developed using antibody for Na(+)/K(+)-ATPase to visualize CCs in situ and compare their characteristics with isolated CCs. Na(+)/K(+)-ATPase content per CC increases transiently (from 2.2+/-0.5 x 10(6) to 4.8+/-1.1 x 10(6) relative fluorescence units, RFU) after 1 week of acute acclimation to 1x SW but returns to baseline values (2.4+/-0.5 x 10(6) RFU) within 5 weeks. In contrast, gradual acclimation to 2.4x SW permanently increases Na(+)/K(+)-ATPase content per CC (from 2.0+/-0.8 x 10(6) to 6.7+/-2.7 x 10(6) RFU after 5 weeks). CC size in situ did not correlate well to salinity because of basolateral membrane infoldings. Taken together, these data suggest that euryhaline fishes are capable of sensing environmental salinity to utilize transient short-term and permanent long-term adaptations for coping with salinity changes. These results also demonstrate the power of LSC and TMA for comparative biology.

Much of the early research elucidating the general mechanisms of euryhalinity was performed on the common killifish. More recently, its opercular epithelium with abundant mitochondria-rich cells has proven to be a powerful model for analyzing the mechanisms of active NaCl transport under Ussing conditions in vitro (i.e., with isotonic saline on both surfaces, at short-circuit). However, it is unclear whether this preparation duplicates the gill under real world conditions-i.e., at open-circuit, with real seawater (SW) or freshwater (FW) on the mucosal surface. There have been only limited studies, mostly about 35 years ago, on ion transport in the intact killifish. Therefore, using radioisotopes (22Na, 36Cl), we developed and evaluated methods for the independent measurement of unidirectional Na(+) and Cl(-) influx and efflux rates and internal pools in intact killifish acclimated to 10% SW and abruptly transferred to either 100% SW or FW. Internal Na(+) pools were disturbed less than internal Cl(-) pools by transfer, and were corrected after 3 days in 100% SW or 7 days in FW. Influx and efflux rates in 10% SW were about 3000 micromol kg(-1) h(-1) and increased to 15,000-18,000 micromol kg(-1) h(-1) after transfer to 100% SW, remaining approximately equal and equimolar for Na(+) and Cl(-), and stable from 0.5 to 7 days post-transfer. After transfer to FW, Na(+) influx and efflux rates dropped to 1000-1500 micromol kg(-1) h(-1), with efflux slightly exceeding influx, and remained approximately stable from 0.5 to 7 days. However, while Cl(-) efflux responded similarly, Cl(-) influx rate dropped immediately to negligible values (20-50 micromol kg(-1) h(-1)) without recovery through 7 days. These results differ from early ion transport data in 100% SW, and demonstrate that fluxes stabilize quickly after salinity transfer. They also show that the intact animal responds more quickly than the epithelium, provide qualitative but not quantitative support for the opercular epithelium as a model for the gill under real world SW conditions, and no support for its use as a gill model under real world FW conditions, where branchial Cl(-) uptake is negligible.

We examined the involvement of mitochondria-rich (MR) cells in ion uptake through gill epithelia in freshwater-adapted killifish Fundulus heteroclitus, by morphological observation of MR cells and molecular identification of the vacuolar-type proton pump (V-ATPase). MR cell morphology was compared in fish acclimated to defined freshwaters with different NaCl concentrations: low (0.1 mmol l(-1))-, mid (1 mmol l(-1))- and high (10 mmol l(-1))-NaCl environments. MR cells, mostly located on the afferent-vascular side of the gill filaments, were larger in low- and mid-NaCl environments than in the high-NaCl environment. Electron-microscopic observation revealed that the apical membrane of well-developed MR cells in low- and mid-NaCl environments was flat or slightly projecting, and equipped with microvilli to expand the surface area exposed to these environments. On the other hand, in the high-NaCl environment, the apical membrane was invaginated to form a pit, and MR cells often formed multicellular complexes with accessory cells, although the NaCl concentration was much lower than that in plasma. We cloned and sequenced a cDNA encoding the A-subunit of killifish V-ATPase. The deduced amino acid sequence showed high identity with V-ATPase A-subunits from other vertebrate species. Light-microscopic immunocytochemistry, using a homologous antibody, revealed V-ATPase-immunoreactivity in Na(+)/K(+)-ATPase-immunoreactive MR cells in low-NaCl freshwater, whereas the immunoreactivity was much weaker in higher NaCl environments. Furthermore, immuno-electron microscopy revealed V-ATPase to be located in the basolateral membrane of MR cells. These findings indicate that MR cells are the site responsible for active ion uptake in freshwater-adapted killifish, and that basolaterally located V-ATPase is involved in the Na(+) and/or Cl(-) absorbing mechanism of MR cells.

The eurohaline fish, Fundulus heteroclitus, adapts rapidly to enhanced salinity by increasing the ion secretion by gill chloride cells. An increase of approximately 70 mOsm in plasma osmolarity was previously found during the transition. To mimic this in vitro, isolated opercular epithelia of seawater-adapted Fundulus mounted in a modified Ussing chamber were exposed to an increase in NaCl and/or osmolarity on the basolateral side, which immediately increased I(SC). Various Cl(-) channel blockers as well as the K(+) channel blocker Ba(2+) added to the basolateral side all inhibited the steady-state as well as the hypertonic stimulation of I(SC). The exists -agonist isoproterenol stimulates I(SC) in standard Ringer solutions. In contrast, when cell volume was kept at the larger value by simultaneous addition of water, the stimulation with isoproterenol was abolished, suggesting that the key process for activation of the Na(+), K(+), 2Cl(-) cotransporter is cell shrinkage. The protein kinase C (PKC) inhibitor chelerythrine and the myosin light chain kinase (MLCK) inhibitor ML-7 had strong inhibitory effects on the mannitol activation of I(SC), thus both MLCK and PKC are involved. The two specific protein kinase A (PKA) inhibitors H-89 and KT 5720 had no effect after mannitol addition whereas isoproterenol stimulation was completely blocked by H-89. This indicates that PKA is involved in the activation of the apical Cl(-) channel via c-AMP whereas the shrinkage activation of the Na(+), K(+), 2Cl(-) cotransporter is independent of PKA activation. The steady-state Cl(-) secretion was stimulated by an inhibitor of serine/threonine phosphatases of the PP-1 and PP-2A type and inhibited by a PKC inhibitor but not by a PKA inhibitor. Thus, it seems to be determined by continuous phosphorylation and dephosphorylation involving PKC but not PKA. The steady-state Cl(-) secretion and the maximal obtainable Cl(-) secretion were measured in freshwater-adapted fish and in fish retransferred to saltwater. No I(SC) could be measured in freshwater-adapted fish or in the fish within the first 18 h after transfer to saltwater. As evidenced from Western blot analysis using antiserine-antibodies, a heavily serine phosphorylated protein of about 190 kDa was consistently observed in the saltwater-acclimated fish, but was only weakly present in freshwater-acclimated fish. This observation indicates that acclimatization to saltwater stimulates the expression of this 190-kDa protein and/or a serine/threonine kinase, which subsequently phosphorylates the protein.