Stimulation of Cl- uptake and morphological changes in gill mitochondria-rich cells in freshwater tilapia (Oreochromis mossambicus)

The purpose of the present article is to examine the relationships between ion uptakes and morphologies of gill mitochondria-rich (MR) cells in freshwater tilapia. Tilapia were acclimated to three different artificial freshwaters (high Na [10 mM], high Cl [7.5 mM]; high Na, low Cl [0.02-0.07 mM], and low Na [0.5 mM], low Cl) for 1 wk, and then morphological measurements of gill MR cells were made and ion influxes were determined. The number and the apical size of wavy-convex MR cells positively associated with the level of Cl(-) influx. Conversely, Na(+) influx showed no positive correlation with the morphologies of MR cells. The dominant MR cell type in tilapia gills changed from deep-hole to wavy-convex within 6 h after acute transfer from a high-Cl(-) to a low-Cl(-) environment. Deep-hole MR cells became dominant 24-96 h after acute transfer from a low-Cl(-) to a high-Cl(-) environment. We conclude that wavy-convex MR cells associate with Cl(-) uptake but not Na(+) uptake, and the rapid formation of wavy-convex MR cells reflects the timely stimulation of Cl(-) uptake to recover the homeostasis of internal Cl(-) levels on acute challenge with low environmental Cl(-).     

Differential design for hopping in two species of wallabies

We explored molecular and morphological alteration in gill mitochondria-rich (MR) cells of Mozambique tilapia, Oreochromis mossambicus, acclimated to deionized freshwater (DFW), freshwater (FW), 1/3-diluted seawater (1/3 SW) and seawater (SW). Scanning electron microscopic observations revealed that the apical membrane of MR cells appeared as a flat or slightly projecting disk in DFW and FW, being larger in DFW than in FW. In contrast, the apical membrane typically formed a pit structure in 1/3 SW and SW. The mRNA expression levels of Na(+)/H(+) exchanger-3 (NHE3) and Na(+)/Cl(-) cotransporter (NCC) in the gills were increased with decreasing environmental salinity, whereas Na(+)/K(+)/2Cl(-) cotransporter-1a (NKCC1a) expression was upregulated by increasing salinity. Immunofluorescence staining showed that the MR cell population of DFW- and FW-acclimated tilapia consisted mostly of MR cells with apical NHE3 and those with apical-NCC; MR cells with basolateral NKCC1a dominated in SW-acclimated tilapia. These results indicated that apical-NHE3 and apical-NCC MR cells were ion-absorbing cells, and that basolateral-NKCC1a MR cells were ion-secreting cells. In fish acclimated to 1/3 SW, both ion-absorbing and secreting cells existed in the gills, suggesting that fish in near-isotonic water were equipped with mechanisms of both hyper- and hypoosmoregulation to prepare for environmental salinity changes.     

Ion-deficient environment induces the expression of basolateral chloride channel, ClC-3-like protein, in gill mitochondrion-rich cells for chloride uptake of the tilapia Oreochromis mossambicus

Gill mitochondrion-rich (MR) cells contain different molecules to carry out functionally distinct mechanisms. To date, the putative mechanism of Cl(-) uptake through the basolateral chloride channel, however, is less understood. To clarify the Cl(-)-absorbing mechanism, this study explored the molecular and morphological alterations in branchial MR cells of tilapia acclimated to seawater (SW), freshwater (FW), and deionized water (DW). Scanning electron microscopic observations revealed that three subtypes of MR cells were exhibited in gill filament epithelia of tilapia. Furthermore, in DW-acclimated tilapia, the subtype I (ion-absorbing subtype) of MR cells predominantly occurred in gill filament as well as lamellar epithelia. Whole-mount double immunofluorescent staining revealed that branchial ClC-3-like protein and Na(+)/K(+)-ATPase (NKA), the basolateral marker of MR cells, were colocalized in tilapia. In SW-acclimated tilapia, all MR cells of gill filament epithelia exhibited faint fluorescence of ClC-3-like protein. In contrast, only some MR cells in gill filament epithelia of FW and DW tilapia expressed basolateral ClC-3-like protein; however, the fluorescence was more intense in FW and DW tilapia than in SW fish. In hyposmotic groups, the number of MR cells immunopositive for ClC-3-like protein was significantly higher in DW-exposed tilapia. Meanwhile, in gill lamellar epithelia of DW tilapia, all MR cells (subtype I) were ClC-3-like protein immunopositive. Double immunostaining of ClC-3-like protein and Na(+)/Cl(-) cotransporter (NCC) revealed that basolateral ClC-3-like protein and apical NCC were colocalized in some MR cells in FW and DW tilapia. Moreover, both mRNA and protein amounts of branchial ClC-3-like protein were significantly higher in DW-acclimated tilapia. To identify whether the expression of branchial ClC-3-like protein responded to changes in environmental [Cl(-)], tilapia were acclimated to artificial waters with normal [Na(+)]/[Cl(-)] (control), lower [Na(+)] (low Na), or lower [Cl(-)] (low Cl). Immunoblotting of crude membrane fractions for gill ClC-3-like protein showed that the protein abundance was evidently enhanced in tilapia acclimated to the low-Cl environment compared with the other groups. Our findings integrated morphological and functional classifications of ion-absorbing MR cells and indicated that ion-deficient water elevated the numbers of subtype I MR cells in both filament and lamellar epithelia of gills with positive ClC-3-like protein immunostaining and increased the expression levels of ClC-3-like protein. This study is the first to illustrate the exhibition of a basolateral chloride channel potentially responsible for Cl(-) absorption in the ion-absorbing subtype of gill MR cells of tilapia.     

Modulation of ion transporter expression in gill mitochondrion-rich cells of eels acclimated to low-Na(+) or-Cl(-) freshwater

In this study, we aimed to establish an experimental model to study the role of the gill mitochondrion-rich cells (MRCs) of freshwater fish in Na(+) uptake and to examine the effect of adjusting external Na(+) and Cl(-) ions on selected ion transporters in gill MRCs. Japanese eels (Anguilla japonica) acclimated to deionized (DI) water for 2 weeks were transferred directly to (a) ion-supplemented artificial freshwater (AF), (b) Na(+) -deficient AF, or (c) Cl(-) -deficient AF for 2 days. The effects of the transfer on the expression levels of ion transporters in isolated gill cells were investigated. Our data demonstrated that the 2-day acclimation in ion-supplemented AF, Na(+) -deficient AF, or Cl(-) -deficient AF led to a significant increase in serum osmolarity attributed mainly to an increase in serum Na(+) and/or Cl(-) levels when compared with DI-acclimated eel. Significant inductions of V-type H(+) -ATPase (V-H(+) -ATPase) and cotransporter (NBC1) mRNA expression in gill MRCs were detected in AF-acclimated fish. In fish acclimated to Na(+) -deficient AF, mRNA expression levels of V-H(+) -ATPase, NBC1, and Na(+) /H(+) -exchanger-3 (NHE3) were significantly increased in MRCs. Fish acclimated to Cl(-) -deficient AF showed no observable change in expression levels of ion transporters in gill MRCs. In addition, expression levels of ion transporters in pavement cells were stable throughout the 2-day experiments. These data indicate that the level of Na(+) in freshwater is important for altering the mRNA expression of ion transporters in gill MRCs, which supports the notion that gill MRCs play important roles in freshwater Na(+) uptake.     

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.     

Ionic mechanisms of cardiac cell swelling induced by blocking Na+/K+ pump as revealed by experiments and simulation

Although the Na(+)/K(+) pump is one of the key mechanisms responsible for maintaining cell volume, we have observed experimentally that cell volume remained almost constant during 90 min exposure of guinea pig ventricular myocytes to ouabain. Simulation of this finding using a comprehensive cardiac cell model (Kyoto model incorporating Cl(-) and water fluxes) predicted roles for the plasma membrane Ca(2+)-ATPase (PMCA) and Na(+)/Ca(2+) exchanger, in addition to low membrane permeabilities for Na(+) and Cl(-), in maintaining cell volume. PMCA might help maintain the [Ca(2+)] gradient across the membrane though compromised, and thereby promote reverse Na(+)/Ca(2+) exchange stimulated by the increased [Na(+)](i) as well as the membrane depolarization. Na(+) extrusion via Na(+)/Ca(2+) exchange delayed cell swelling during Na(+)/K(+) pump block. Supporting these model predictions, we observed ventricular cell swelling after blocking Na(+)/Ca(2+) exchange with KB-R7943 or SEA0400 in the presence of ouabain. When Cl(-) conductance via the cystic fibrosis transmembrane conductance regulator (CFTR) was activated with isoproterenol during the ouabain treatment, cells showed an initial shrinkage to 94.2 +/- 0.5%, followed by a marked swelling 52.0 +/- 4.9 min after drug application. Concomitantly with the onset of swelling, a rapid jump of membrane potential was observed. These experimental observations could be reproduced well by the model simulations. Namely, the Cl(-) efflux via CFTR accompanied by a concomitant cation efflux caused the initial volume decrease. Then, the gradual membrane depolarization induced by the Na(+)/K(+) pump block activated the window current of the L-type Ca(2+) current, which increased [Ca(2+)](i). Finally, the activation of Ca(2+)-dependent cation conductance induced the jump of membrane potential, and the rapid accumulation of intracellular Na(+) accompanied by the Cl(-) influx via CFTR, resulting in the cell swelling. The pivotal role of L-type Ca(2+) channels predicted in the simulation was demonstrated in experiments, where blocking Ca(2+) channels resulted in a much delayed cell swelling.     

Modification of morphology and function of integument mitochondria-rich cells in tilapia larvae (Oreochromis mossambicus) acclimated to ambient chloride levels.

Similar to those of the gills of adults, three types of mitochondria-rich (MR) cells with different morphologies of apical surfaces (wavy convex, shallow basin, and deep hole) were identified on the integument of freshwater-acclimated tilapia larvae (Oreochromis mossambicus). The object of this study is to test the hypothesis that these subtype cells may represent MR cells equipped with variable efficiencies in Cl(-) uptake. Larvae acclimated to low-Cl(-) =0.001-0.007 mM) water developed higher densities of MR cells than those acclimated to high-Cl(-) =7.3-7.9 mM) water. The percentage of wavy-convex-type cells in total MR cells was higher in low-Cl(-)-acclimated larvae than in high-Cl(-)-acclimated larvae, which displayed only deep-hole type. In addition, Cl(-) influx rates of whole larva measured with (36)Cl(-) showed a coincident correlation with MR cell densities, that is, low-Cl(-) larvae displayed higher Cl(-) influx rates than did high-Cl(-) larva, suggesting that tilapia larvae develop a higher density of MR cells with larger apical surfaces (wavy-convex type) to boost Cl(-) uptake in Cl(-)-deficient water. The distinct types of apical surfaces may represent different phases of MR cells that possess different efficiencies of Cl(-) uptake. Increased apical membrane surface areas of MR cells may provide larvae with rapid regulation of Cl(-) before new MR cells differentiate.     

Proton pump-driven cutaneous chloride uptake in anuran amphibia

Krogh introduced the concept of active ion uptake across surface epithelia of freshwater animals, and proved independent transports of Na(+) and Cl(-) in anuran skin and fish gill. He suggested that the fluxes of Na(+) and Cl(-) involve exchanges with ions of similar charge. In the so-called Krogh model, Cl(-)/HCO(3)(-) and Na(+)/H(+) antiporters are located in the apical membrane of the osmoregulatory epithelium. More recent studies have shown that H(+) excretion in anuran skin is due to a V-ATPase in mitochondria-rich (MR) cells. The pump has been localized by immunostaining and H(+) fluxes estimated by pH-stat titration and mathematical modelling of pH-profiles in the unstirred layer on the external side of the epithelium. H(+) secretion is voltage-dependent, sensitive to carbonic-anhydrase inhibitors, and rheogenic with a charge/ion-flux ratio of unity. Cl(-) uptake from freshwater is saturating, voltage independent, and sensitive to DIDS and carbonic-anhydrase inhibitors. Depending on anuran species and probably on acid/base balance of the animal, apical exit of protons is coupled to an exchange of Cl(-) with base (HCO(3)(-)) either in the apical membrane (gamma-type of MR cell) or in the basolateral membrane (alpha-type MR cell). The gamma-cell model accounts for the rheogenic active uptake of Cl(-) observed in several anuran species. There is indirect evidence also for non-rheogenic active uptake accomplished by a beta-type MR cell with apical base secretion and basolateral proton pumping. Several studies have indicated that the transport modes of MR cells are regulated via ion- and acid/base balance of the animal, but the signalling mechanisms have not been investigated. Estimates of energy consumption by the H(+)-ATPase and the Na(+)/K(+)-ATPase indicate that the gamma-cell accomplishes uptake of NaCl in normal and diluted freshwater. Under common freshwater conditions with serosa-positive or zero V(t), the K(+) conductance of the basolateral membrane would have to maintain the inward driving force for Na(+) uptake across the apical membrane. With the K(+) equilibrium potential across the basolateral membrane estimated to -105 mV, this would apply to external Na(+) concentrations down to 40-120 micromol/l. NaCl uptake from concentrations down to 10 micromol/l, as observed by Krogh, presupposes that the H(+) pump hyperpolarizes the apical membrane, which would then have to be associated with serosa-negative V(t). In diluted freshwater, exchange of cellular HCO(3)(-) with external Cl(-) seems to be possible only if the proton pump has the additional function of keeping the external concentration of HCO(3)(-) low. Quantitative considerations also lead to the conclusion that with the above extreme demand, at physiological intracellular pH of 7.2, the influx of Cl(-) via the apical antiporter and the passive exit of Cl(-) via basolateral channels would be possible within a common range of intracellular Cl(-) concentrations.     

Sodium channels are required during in vivo sodium chloride hyperosmolarity to stimulate increase in intestinal endothelial nitric oxide production

NaCl hyperosmolarity increases intestinal blood flow during food absorption due in large part to increased NO production. We hypothesized that in vivo, sodium ions enter endothelial cells during NaCl hyperosmolarity as the first step to stimulate an increase in intestinal endothelial NO production. Perivascular NO concentration ([NO]) and blood flow were determined in the in vivo rat intestinal microvasculature at rest and under hyperosmotic conditions, 330 and 380 mosM, respectively, before and after application of bumetanide (Na(+)-K(+)-2Cl(-) cotransporter inhibitor) or amiloride (Na(+)/H(+) exchange channel inhibitor). Suppressing amiloride-sensitive Na(+)/H(+) exchange channels diminished hypertonicity-linked increases in vascular [NO], whereas blockade of Na(+)-K(+)-2Cl(-) channels greatly suppressed increases in vascular [NO] and intestinal blood flow. In additional experiments we examined the effect of sodium ion entry into endothelial cells. We proposed that the Na(+)/Ca(2+) exchanger extrudes Na(+) in exchange for Ca(2+), thereby leading to the calcium-dependent activation of endothelial nitric oxide synthase (eNOS). We blocked the activity of the Na(+)/Ca(2+) exchanger during 360 mosM NaCl hyperosmolarity with KB-R7943; complete blockade of increased vascular [NO] and intestinal blood flow to hyperosmolarity occurred. These results indicate that during NaCl hyperosmolarity, sodium ions enter endothelial cells predominantly through Na(+)-K(+)-2Cl(-) channels. The Na(+)/Ca(2+) exchanger then extrudes Na(+) and increases endothelial Ca(2+). The increase in endothelial Ca(2+) causes an increase in eNOS activity, and the resultant increase in NO increases intestinal arteriolar diameter and blood flow during NaCl hyperosmolarity. This appears to be the major mechanism by which intestinal nutrient absorption is coupled to increased blood flow.     

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.     

Mode-specific inhibition of sodium-calcium exchange during protein phosphatase blockade

The effects of the protein phosphatase inhibitors calyculin A and okadaic acid on Na(+)/Ca(2+) exchange activity were examined in transfected Chinese hamster ovary cells expressing the bovine cardiac Na(+)/Ca(2+) exchanger. Incubating the cells for 5-10 min with 100 nM calyculin A reduced exchange-mediated (45)Ca(2+) uptake or Ba(2+) influx by 50-75%. Half-maximal inhibition of (45)Ca(2+) uptake was observed at 15 nM calyculin A. The nonselective protein kinase inhibitors K252a and staurosporine provided partial protection against the effects of calyculin A. Okadaic acid, another protein phosphatase inhibitor, nearly completely blocked exchange-mediated Ba(2+) influx. Chinese hamster ovary cells expressing a mutant exchanger in which 420 out of 520 amino acid residues were deleted from the central hydrophilic domain of the exchanger remained sensitive to the inhibitory effects of calyculin A and okadaic acid. Surprisingly, Na(o)(+)-dependent Ca(2+) efflux appeared to be only modestly inhibited, if at all, by calyculin A or okadaic acid. We conclude that protein hyperphosphorylation during protein phosphatase blockade selectively inhibits the Ca(2+) influx mode of Na(+)/Ca(2+) exchange, probably by an indirect mechanism that does not involve phosphorylation of the exchanger itself.     

Cerebral microvascular nNOS responds to lowered oxygen tension through a bumetanide-sensitive cotransporter and sodium-calcium exchanger

Na(+) cotransporters have a substantial role in neuronal damage during brain hypoxia. We proposed these cotransporters have beneficial roles in oxygen-sensing mechanisms that increase periarteriolar nitric oxide (NO) concentration ([NO]) during mild to moderate oxygen deprivation. Our prior studies have shown that cerebral neuronal NO synthase (nNOS) is essential for [NO] responses to decreased oxygen tension and that endothelial NO synthase (eNOS) is of little consequence. In this study, we explored the mechanisms of three specific cotransporters known to play a role in the hypoxic state: KB-R7943 for blockade of the Na(+)/Ca(2+) exchanger, bumetanide for the Na(+)-K(+)-2Cl(-) cotransporter, and amiloride for Na(+)/H(+) cotransporters. In vivo measurements of arteriolar diameter and [NO] at normal and locally reduced oxygen tension in the rat parietal cortex provided the functional analysis. As previously found for intestinal arterioles, bumetanide-sensitive cotransporters are primarily responsible for sensing reduced oxygen because the increased [NO] and dilation were suppressed. The Na(+)/Ca(2+) exchanger facilitated increased NO formation because blockade also suppressed [NO] and dilatory responses to decreased oxygen. Amiloride-sensitive Na(+)/H(+) cotransporters did not significantly contribute to the microvascular regulation. To confirm that nNOS rather than eNOS was primarily responsible for NO generation, eNOS was suppressed with the fusion protein cavtratin for the caveolae domain of eNOS. Although the resting [NO] decreased and arterioles constricted as eNOS was suppressed, most of the increased NO and dilatory response to oxygen were preserved because nNOS was functional. Therefore, nNOS activation secondary to Na(+)-K(+)-2Cl(-) cotransporter and Na(+)/Ca(2+) exchanger functions are key to cerebral vascular oxygen responses.     

A pharmacological examination of Na+ and Cl- transport in two species of freshwater fish

We examined branchial Na(+) and Cl(-) uptake in two species of stenohaline, freshwater fish (goldfish and the Amazonian neon tetra). Kinetic analysis revealed that the two species had similar uptake capacities and affinities for Na(+) and Cl(-). However, while uptakes of Na(+) and Cl(-) (JNain and JClin, respectively) by goldfish were completely inhibited at pH 4.5 and below, uptake in tetras was unaffected by pH down to 3.25. Examination of Cl(-) transport with blockers indicated that goldfish and neon tetras utilize Cl(-)/HCO-3 exchange; SITS and SCN(-) inhibited Cl(-) uptake in both species. In contrast, large differences in Na(+) transport were indicated between the species. In goldfish, exposure to four Na(+)/H(+) exchange blockers, as well as the Na(+) channel blocker phenamil, strongly inhibited JNain. Further, Na(+) and Cl(-) uptake were strongly inhibited by the Na(+)/K(+)/Cl(-) cotransport inhibitor furosemide, as was JNain in "Cl(-)-free" water and JClin in "Na(+)-free" water. This suggests the presence of multiple transporters and possibly even a direct linkage between the transport of Na(+) and Cl(-) in goldfish. In contrast, none of these drugs strongly reduced Na(+) transport in neon tetras, which raises the possibility of a significantly different Na(+) transport mechanism in this acid-tolerant species.     

Role of the choline exchanger in Na(+)-independent Mg(2+) efflux from rat erythrocytes

Two types of Na(+)-independent Mg(2+) efflux exist in erythrocytes: (1) Mg(2+) efflux in sucrose medium and (2) Mg(2+) efflux in high Cl(-) media such as KCl-, LiCl- or choline Cl-medium. The mechanism of Na(+)-independent Mg(2+) efflux in choline Cl medium was investigated in this study. Non-selective transport by the following transport mechanisms has been excluded: K(+),Cl(-)- and Na(+),K(+),Cl(-)-symport, Na(+)/H(+)-, Na(+)/Mg(2+)-, Na(+)/Ca(2+)- and K(+)(Na(+))/H(+) antiport, Ca(2+)-activated K(+) channel and Mg(2+) leak flux. We suggest that, in choline Cl medium, Na(+)-independent Mg(2+) efflux can be performed by non-selective transport via the choline exchanger. This was supported through inhibition of Mg(2+) efflux by hemicholinum-3 (HC-3), dodecyltrimethylammonium bromide (DoTMA) and cinchona alkaloids, which are inhibitors of the choline exchanger. Increasing concentrations of HC-3 inhibited the efflux of choline and efflux of Mg(2+) to the same degree. The K(d) value for inhibition of [(14)C]choline efflux and for inhibition of Mg(2+) efflux by HC-3 were the same within the experimental error. Inhibition of choline efflux and of Mg(2+) efflux in choline medium occurred as follows: quinine>cinchonine>HC-3>DoTMA. Mg(2+) efflux was reduced to the same degree by these inhibitors as was the [(14)C]choline efflux.     

Sodium and chloride transport in soft water and hard water acclimated zebrafish (Danio rerio)

While the zebrafish is commonly used for studies of developmental biology and toxicology, very little is known about their osmoregulatory physiology. The present investigation of Na(+) and Cl(-) transport revealed that the zebrafish is able to tolerate extremely low ambient ion concentrations and that this is achieved at least in part by a greatly enhanced apparent uptake capacity and affinity for both ions. Zebrafish maintain plasma and whole body electrolyte concentrations similar to most other freshwater teleosts even in deionized water containing only 35 microM NaCl, i.e soft water. We recorded an extremely low transport affinity constant (K(m)) of 8+/-1 microM for the active uptake of Cl(-) in soft water acclimated fish, while other transport kinetic parameters were in agreement with reports for other freshwater organisms. While both Na(+) and Cl(-) uptake in soft water clearly depends on apical proton pump activity, changes in abundance and possibly localization of this protein did not appear to contribute to soft water acclimation. Active Cl(-) uptake was strongly dependent on branchial carbonic anhydrase (CA) activity regardless of water type, while the response of Na(+) transport to a CA inhibitor was more variable. Differential response of Na(+) uptake to amiloride depending on acclimation medium suggests that different Na(+) transport mechanisms are employed by zebrafish acclimated to soft and hard water.     

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.     

Stimulation of Na+/Mg2+ antiport in rat erythrocytes by intracellular Cl-

Mg(2+) efflux from rat erythrocytes was measured in NaCl, NaNO(3), NaSCN and Na gluconate medium. Substitution of extracellular and intracellular Cl(-) with the permeant anions NO(3)(-) and SCN(-) reduced Mg(2+) efflux via Na(+)/Mg(2+) antiport. After substitution of extracellular Cl(-) with the non-permeant anion gluconate, Mg(2+) efflux was not significantly reduced. In Na gluconate medium, an influence of the changed membrane potential and intracellular pH on Mg(2+) efflux could be excluded. The results indicate the existence of Cl(-)-independent Na(+)/Mg(2+) antiport and of Na(+)/Mg(2+) antiport stimulated by intracellular Cl(-). Intracellular Cl(-), as determined by means of (36)Cl(-), was found to stimulate Na(+)/Mg(2+) antiport through a cooperative effect according to a sigmoidal kinetics. The Hill coefficient for intracellular Cl(-) amounted to 1.4-1.8, indicating that two intracellular Cl(-) may be simultaneously active. With respect to specificity, Cl(-) was most effective, followed by Br(-), J(-), and F(-). Stimulation of Na(+)/Mg(2+) antiport by intracellular Cl(-) together with intracellular Mg(2+) may play a role during deoxygenation of erythrocytes and in essential hypertension.     

In dialyzed squid axons oxidative stress inhibits the Na+/Ca2+ exchanger by impairing the Cai2+-regulatory site

The Na(+)/Ca(2+) exchanger, a major mechanism by which cells extrude calcium, is involved in several physiological and physiopathological interactions. In this work we have used the dialyzed squid giant axon to study the effects of two oxidants, SIN-1-buffered peroxynitrite and hydrogen peroxide (H(2)O(2)), on the Na(+)/Ca(2+) exchanger in the absence and presence of MgATP upregulation. The results show that oxidative stress induced by peroxynitrite and hydrogen peroxide inhibits the Na(+)/Ca(2+) exchanger by impairing the intracellular Ca(2+) (Ca(i)(2+))-regulatory sites, leaving unharmed the intracellular Na(+)- and Ca(2+)-transporting sites. This effect is efficiently counteracted by the presence of MgATP and by intracellular alkalinization, conditions that also protect H(i)(+) and (H(i)(+) + Na(i)(+)) inhibition of Ca(i)(2+)-regulatory sites. In addition, 1 mM intracellular EGTA reduces oxidant inhibition. However, once the effects of oxidants are installed they cannot be reversed by either MgATP or EGTA. These results have significant implications regarding the role of the Na(+)/Ca(2+) exchanger in response to pathological conditions leading to tissue ischemia-reperfusion and anoxia/reoxygenation; they concur with a marked reduction in ATP concentration, an increase in oxidant production, and a rise in intracellular Ca(2+) concentration that seems to be the main factor responsible for cell damage.     

Effect of temperature on osmotic and ionic regulation in goldfish,Carassius auratus L.

Summary Urine flow increased with acute temperature increases and showed temperature acclimation. When measured at 20 °C the urine flow of 10 °C acclimated fish was 3.2 times greater than the urine flow of 30 °C acclimated fish. In fish acclimated to 24 °C renal reabsorption of Na and Cl was independent of temperature over an intermediate range of temperatures (14–24 °C) but near the lower lethal temperature (6.5 °C) renal Na and Cl reabsorption was inhibited. Water permeability of the renal tubules was not affected by acute temperature change between 6.5 and 24 °C. Urine osmolality and urine Na, K and Cl concentrations showed nearly perfect temperature compensation in fish acclimated to 10 °C and 30 °C. The rate of renal excretion of Na and Cl showed temperature acclimation in that Na and Cl ecxretion measured at 20 °C was 7 to 8 times greater in 10 °C acclimated fish than in 30 °C acclimated fish. The rate of excretion of Na and Cl measured at 30 °C in 30 °C acclimated fish was approximately 1.7 times the rate of excretion measured at 10 °C in 10 °C acclimated fish.The branchial uptake of Na, measured in tap water, of fish acclimated to 10, 20 and 30 °C in demineralized water increased with acute increases in temperature. When the three acclimation groups were compared at an intermediate temperature (20 °C), the 10 °C acclimated group showed the highest rate of net uptake, and the 30 °C group the lowest rate of uptake. This apparent temperature acclimation of Na uptake was correlated with differences in the plasma Na concentration of the three acclimation groups. Plasma Cl concentrations were also correlated with acclimation temperature in fish acclimated in demineralized water, but the rate of net Cl uptake was considerably less than that for Na. Sodium and Cl uptake in fish which had been acclimated in tap water was very variable and was not clearly affected by acute changes in temperature. Uptake of Na and Cl by fish held in tap water did not show temperature acclimation. The difference between uptake and excretion of fish acclimated in tap water was not significantly different from zero, indicating that the fish were in salt balance.The study was supported by National Institutes of Health Grant GM 16932-02 to Dr. Bodil Schmidt-Nielsen. I am grateful to Dr. Schmidt-Nielsen for many useful discussions during the course of this work.     

1α,25-Dihydroxyvitamin D(3) signaling pathways on calcium uptake in 30-day-old rat Sertoli cells

1α,25-Dihydroxyvitamin D(3) (1,25D(3)) is the active metabolite of vitamin D(3) and the major calcium regulatory hormone in tissues. The aim of this work was to investigate the mechanism of action of 1,25D(3) on (45)Ca(2+) uptake in Sertoli cells from 30-day-old rats. Results showed that 10(-9) and 10(-12) M 1,25D(3) increased the rate of (45)Ca(2+) uptake 5 and 15 min after hormone exposure and that 1α,25(OH)(2) lumisterol(3) (JN) produced a similar effect suggesting that 1,25D(3) action occurs via a putative membrane receptor. The involvement of voltage-dependent calcium channels (VDCC) in 1,25D(3) action was evidenced by using nifedipine, while the use of Bapta-AM demonstrated that intracellular calcium was not implicated. Moreover, the incubation with ouabain and digoxin increased the rate of (45)Ca(2+) uptake, indicating that the effect of 1,25D(3) may also result from Na(+)/K(+)-ATPase inhibition. In addition, we demonstrated that the mechanism underlying the hormone action involved extracellular signal-regulated kinase (ERK) and protein kinase C (PKC) activation in a phospholipase C-independent way. Furthermore, a local elevation of the level of cAMP, as demonstrated by incubating cells with dibutyryl cAMP or a phosphodiesterase inhibitor, produced an effect similar to that of 1,25D(3), and the inhibition of protein kinase A (PKA) nullified the hormone action. In conclusion, the stimulatory effect of 1,25D(3) on (45)Ca(2+) uptake in Sertoli cells occurs via VDCC, as well as PKA, PKC, and ERK activation. These protein kinases seem to act by inhibiting Na(+)/K(+)-ATPase or directly phosphorylating calcium channels. The Na(+)/K(+)-ATPase inhibition may result in Na(+)/Ca(2+) exchanger activation in reverse mode and consequently induce the uptake of calcium into the cells.