Yusuke Kumai

Ammonia excretion via Rhcg1 facilitates Na+ uptake in larval zebrafish, Danio rerio, in acidic water

The involvement of a Na(+)/H(+) exchanger (NHE) in mediating Na(+) uptake by freshwater fish is currently debated. Although supported indirectly by empirical molecular and pharmacological data, theoretically its operation should be constrained thermodynamically, owing to unfavorable chemical gradients. Recently, there has been an increasing focus on ammonia channels (Rh proteins) as potentially contributing to Na(+) uptake across the freshwater fish gill. In this study, we tested the hypothesis that Rhcg1, a specific apical isoform of Rh protein, is critically important in facilitating Na(+) uptake in zebrafish larvae via its interaction with NHE. Treating larvae (4 days postfertilization) with 5-(N-ethyl-N-isopropyl) amiloride (EIPA), an inhibitor of NHE, caused a significant reduction in Na(+) uptake in fish reared in acidic water (pH ∼ 4.0). A role for NHE in Na(+) uptake was further confirmed by translational knockdown of NHE3b, an isoform of NHE thought to be responsible for Na(+)/H(+) exchange in zebrafish larvae. Exposing the larvae reared in acidic water to 5 mM external ammonium sulfate or increasing the buffering capacity of the water with 10 mM HEPES caused concurrent reductions in ammonia excretion and Na(+) uptake. Furthermore, translational knockdown of Rhcg1 significantly reduced ammonia excretion and Na(+) uptake in larvae chronically (4 days) or acutely (24 h) exposed to acidic water. Unlike in sham-injected larvae, EIPA did not affect Na(+) uptake in fish experiencing Rhcg1 knockdown. Additionally, exposure of larvae to bafilomycin A1 (an inhibitor of H(+)-ATPase) significantly reduced Na(+) uptake in fish reared in acidic water. These observations suggest the existence of multiple mechanisms of Na(+) uptake in larval zebrafish in acidic water: one in which Na(+) uptake via NHE3b is linked to ammonia excretion via Rhcg1, and another facilitated by H(+)-ATPase.

Strategies for maintaining Na+ balance in zebrafish (Danio rerio) during prolonged exposure to acidic water

The objective of the present study was to characterize the capacity of zebrafish (Danio rerio) to regulate whole body Na⁺ levels during exposure to acidic (pH 3.8-4.0) water. Exposure to acidic water significantly affected the mRNA levels of 14 claudin and two occludin isoforms, tight junction proteins thought to be involved in regulating paracellular efflux. Despite these changes, Na⁺ efflux as well as uptake of polyethylene glycol (PEG), a marker for paracellular pathway, was persistently elevated during the 2-week period of acid exposure, although there was a transient recovery between 12- and 72-h. Pre-exposing fish to acidic water for 2 weeks failed to attenuate the increase in Na⁺ efflux associated with acute exposure to acidic water of low [Ca²⁺]. However, during recovery in water of circumneutral pH following exposure to acidic water, normal rates of Na⁺ efflux were restored within 5h. The rate of Na⁺ uptake was significantly elevated between 4 and 7 days of exposure to acidic water; the increase was associated with significant increases in maximal Na⁺ uptake capacity (J(MAX)Na⁺) and affinity constant (K(M)). These results demonstrate that in acidic water, zebrafish maintain their whole body Na⁺ balance primarily by regulating Na⁺ uptake, rather than Na⁺ efflux.

The physiology of fish at low pH: the zebrafish as a model system

Ionic regulation and acid–base balance are fundamental to the physiology of vertebrates including fish. Acidification of freshwater ecosystems is recognized as a global environmental problem, and the physiological responses to acid exposure in a few fish species are well characterized. However, the underlying mechanisms promoting ionic and acid–base balance for most fish species that have been investigated remain unclear. Zebrafish (Danio rerio) has emerged as a powerful model system to elucidate the molecular basis of ionic and acid–base regulation. The utility of zebrafish is related to the ease with which it can be genetically manipulated, its suitability for state-of-the-art molecular and cellular approaches, and its tolerance to diverse environmental conditions. Recent studies have identified several key regulatory mechanisms enabling acclimation of zebrafish to acidic environments, including activation of the sodium/hydrogen exchanger (NHE) and H+-ATPase for acid secretion and Na+ uptake, cortisol-mediated regulation of transcellular and paracellular Na+ movements, and ionocyte proliferation controlled by specific cell-fate transcription factors. These integrated physiological responses ultimately contribute to ionic and acid–base homeostasis in zebrafish exposed to acidic water. In the present review, we provide an overview of the general effects of acid exposure on freshwater fish, the adaptive mechanisms promoting extreme acid tolerance in fishes native to acidic environments, and the mechanisms regulating ionic and acid–base balance during acid exposure in zebrafish.

Mechanisms and regulation of Na(+) uptake by freshwater fish.

Mechanisms of ion uptake by freshwater (FW) fish have received considerable attention over the past 80 years. Through an assortment of techniques incorporating whole animal physiology, electrophysiology and molecular biological approaches, three models have been proposed to account for Na(+) uptake. (1) Direct exchange of Na(+) and H(+) via one or more types of Na(+)/H(+) exchanger (slc9), (2) uptake of Na(+) through epithelial Na(+) channels energized by an electrical gradient created by H(+)-ATPase and (3) Na(+)/Cl(-) co-transport (slc12). While each mechanism is supported at least in part by theoretical or experimental data, there are several outstanding questions that have not yet been fully resolved. Furthermore, there are few details concerning how these Na(+) uptake mechanisms are fine tuned in response to the fluctuating FW environments. In this review, we summarize the current understanding of these three Na(+) uptake mechanisms and discuss their regulation by endocrine (cortisol and prolactin) and neurohumoral (catecholamines) factors.

The role of hydrogen sulphide in the control of breathing in hypoxic zebrafish (Danio rerio)

Hydrogen sulphide (H2S), a gaseous neurotransmitter, is involved in oxygen sensing in glomus cells, which are oxygen‐sensing cells found in the mammalian carotid body. Neuroepithelial cells (NECs) are oxygen‐sensing cells of fish and are thought to be phylogenetic precursors of mammalian glomus cells; however, the oxygen‐sensing mechanisms of these cells remain largely unknown. Both adult and larval zebrafish responded to exogenous H2S by increasing ventilation in a dose‐dependent manner; H2S increased intracellular [Ca2+] in NECs. Inhibiting endogenous H2S production decreased or abolished the ventilatory response to hypoxia in both adult and larval zebrafish. The results demonstrate an important role for H2S in oxygen sensing in zebrafish.

The effects of gill remodeling on transepithelial sodium fluxes and the distribution of presumptive sodium-transporting ionocytes in goldfish (Carassius auratus)

Goldfish, Carassius auratus, adaptively remodel their gills in response to changes in ambient oxygen and temperature, altering the functional lamellar surface area to balance the opposing requirements for respiration and osmoregulation. In this study, the effects of thermal- and hypoxia-mediated gill remodeling on branchial Na+ fluxes and the distribution of putative Na+-transporting ionocytes in goldfish were assessed. When assessed either in vitro (isolated gill arches) or in vivo at a common water temperature, the presence of an interlamellar cell mass (ILCM) in fish acclimated to 7°C clearly decreased Na+ efflux across the gill relative to fish maintained at 25°C and lacking an ILCM. However, loss of the ILCM in 7°C-acclimated fish exposed to hypoxia led to a decrease in Na+ efflux (assessed under hypoxic conditions) despite the apparent large increases in functional lamellar surface area. Goldfish possessing an ILCM were able to sustain Na+ uptake, albeit at a lower rate matched to efflux, owing to the re-distribution of ionocytes expressing genes thought to be involved in Na+ uptake [Na+/H+ exchanger isoform 3 (NHE3) and V- type H+-ATPase] to the edge of the ILCM where they can establish contact with the surrounding environment. NHE-expressing cells co-localized with Na+/K+-ATPase expression, suggesting a role for NHE in Na+-uptake in the goldfish. Implications of the ILCM on ion fluxes in the goldfish are discussed.

The consequences of reversible gill remodelling on ammonia excretion in goldfish (Carassius auratus)

SUMMARY Goldfish acclimated to cold water (e.g. 7°C) experience a marked reduction in functional lamellar surface area owing to the proliferation of an interlamellar cell mass (ILCM), a phenomenon termed gill remodelling. The goal of the present study was to assess the consequences of the reduced functional surface area on the capacity of goldfish to excrete ammonia. Despite the expected impact of ambient temperature on functional surface area, fish acclimated to 7°C and 25°C exhibited similar rates of ammonia excretion (Jnet,amm); the Q10 values for fed and starved fish were 1.07 and 1.20, respectively. To control for possible temperature-related differences in rates of endogenous ammonia production, Jnet,amm was determined at the two acclimation temperatures after loading fish with 1.12 μmol g–1 of NH4Cl. In the 3 h post-injection period, Jnet,amm was elevated to a greater extent in the 25°C fish. To estimate the potential contribution of increased ventilation and cardiac output to ammonia clearance in the warmer fish, the ammonia loading experiment was repeated on the 7°C fish immediately after they were exercised to exhaustion. The rate of excretion of ammonia was significantly increased in the exercised 7°C fish (presumably experiencing increased ventilation and cardiac output for at least some of the measurement period) suggesting that differences in external and internal convection may at least partially explain the enhanced capacity of the 25°C fish to clear the ammonia load. To more specifically assess the contribution of the different functional surface areas on the differing rates of ammonia clearance at the two acclimation temperatures, the 7°C fish were exposed for 7 days to hypoxia (PO2=10 mmHg=1.33 kPa), a treatment known to cause the disappearance of the ILCM. The results demonstrated that the hypoxia-associated loss of the ILCM was accompanied by a significant increase in the rate of ammonia clearance in the 7°C fish when returned to normoxic conditions. To determine whether compensatory changes in the ammonia transporting proteins might be contributing to sustaining Jnet,amm under conditions of reduced functional lamellar surface area, the relative expression and branchial distribution of four Rh proteins were assessed by western blotting and immunocytochemistry. Although the relative expression of the Rh proteins was unaffected by acclimation temperature, there did appear to be a change in the spatial distribution of Rhag, Rhbg and Rhcg1. Specifically, these three Rh proteins (and to a lesser extent Rhcg2) appeared to localize in cells on the outer edge of the ILCM that were enriched with Na+/K+-ATPase. Thus, we suggest that despite the impediment to ammonia excretion imposed by the ILCM, goldfish acclimated to 7°C are able to sustain normal rates of excretion owing to the redistribution of ammonia transporting cells.

A role for nitric oxide in the control of breathing in zebrafish (Danio rerio).

ABSTRACT Nitric oxide (NO) is a gaseous neurotransmitter, which, in adult mammals, modulates the acute hypoxic ventilatory response; its role in the control of breathing in fish during development is unknown. We addressed the interactive effects of developmental age and NO in the control of piscine breathing by measuring the ventilatory response of zebrafish (Danio rerio) adults and larvae to NO donors and by inhibiting endogenous production of NO. In adults, sodium nitroprusside (SNP), a NO donor, inhibited ventilation; the extent of the ventilatory inhibition was related to the pre-existing ventilatory drive, with the greatest inhibition exhibited during exposure to hypoxia (PO2=5.6 kPa). Inhibition of endogenous NO production using l-NAME suppressed the hypoventilatory response to hyperoxia, supporting an inhibitory role of NO in adult zebrafish. Neuroepithelial cells (NECs), the putative oxygen chemoreceptors of fish, contain neuronal nitric oxide synthase (nNOS). In zebrafish larvae at 4 days post-fertilization, SNP increased ventilation in a concentration-dependent manner. Inhibition of NOS activity with l-NAME or knockdown of nNOS inhibited the hypoxic (PO2=3.5 kPa) ventilatory response. Immunohistochemistry revealed the presence of nNOS in the NECs of larvae. Taken together, these data suggest that NO plays an inhibitory role in the control of ventilation in adult zebrafish, but an excitatory role in larvae. Summary: Nitric oxide, a gaseous neurotransmitter, plays a modulatory role in controlling breathing in zebrafish during acute changes in environmental oxygen levels, and its role changes throughout development.

Evidence for a role of tight junctions in regulating sodium permeability in zebrafish ( Danio rerio ) acclimated to ion-poor water

Freshwater teleosts are challenged by diffusive ion loss across permeable epithelia including gills and skin. Although the mechanisms regulating ion loss are poorly understood, a significant component is thought to involve paracellular efflux through pathways formed via tight junction proteins. The mammalian orthologue (claudin-4) of zebrafish (Danio rerio) tight junction protein, claudin-b, has been proposed to form a cation-selective barrier regulating the paracellular loss of Na+. The present study investigated the cellular localization and regulation of claudin-b, as well as its potential contribution to Na+ homeostasis in adult zebrafish acclimated to ion-poor water. Using a green fluorescent protein-expressing line of transgenic zebrafish, we found that claudin-b was expressed along the lamellar epithelium as well as on the filament in the inter-lamellar regions. Co-localization of claudin-b and Na+/K+-ATPase was observed, suggesting its interaction with mitochondrion-rich cells. Claudin-b also appeared to be associated with other cell types, including the pavement cells. In the kidney, claudin-b was expressed predominantly in the collecting tubules. In addition, exposure to ion-poor water caused a significant increase in claudin-b abundance as well as a decrease in Na+ efflux, suggesting a possible role for claudin-b in regulating paracellular Na+ loss. Interestingly, the whole-body uptake of a paracellular permeability marker, polyethylene glycol-400, increased significantly after prolonged exposure to ion-poor water, indicating that an increase in epithelial permeability is not necessarily coupled with an increase in passive Na+ loss. Overall, our study suggests that in ion-poor conditions, claudin-b may contribute to a selective reduction in passive Na+ loss in zebrafish.

β-Adrenergic regulation of Na+ uptake by larval zebrafish Danio rerio in acidic and ion-poor environments.

The potential role of adrenergic systems in regulating Na(+) uptake in zebrafish (Danio rerio) larvae was investigated. Treatment with isoproterenol (a generic β-adrenergic receptor agonist) stimulated Na(+) uptake, whereas treatment with phenylephrine (an α(1)-adrenergic receptor agonist) as well as clonidine (an α(2)-adrenergic receptor agonist) significantly reduced Na(+) uptake, suggesting opposing roles of α- and β-adrenergic receptors in Na(+) uptake regulation. The increase in Na(+) uptake associated with exposure to acidic water (pH = 4.0) was attenuated in the presence of the nonselective β-receptor antagonist propranolol or the β(1)-receptor blocker atenolol; the β(2)-receptor antagonist ICI-118551 was without effect. The stimulation of Na(+) uptake associated with ion-poor water (32-fold dilution of Ottawa tapwater) was unaffected by β-receptor blockade. Translational gene knockdown of β-receptors using antisense oligonucleotide morpholinos was used as a second method to assess the role of adrenergic systems in the regulation of Na(+) uptake. Whereas β(1)- or β(2B)-receptor knockdown led to significant decreases in Na(+) uptake during exposure to acidic water, only β(2A)-receptor morphants failed to increase Na(+) uptake in response to ion-poor water. In support of the pharmacology and knockdown experiments that demonstrated an involvement of β-adrenergic systems in the control of Na(+) uptake, we showed that the H(+)-ATPase-rich (HR) cell, a subtype of ionocyte known to be a site of Na(+) uptake, is innervated and appears to express β-adrenergic receptors (propranolol binding sites) at 4 days postfertilization. These data indicate an important role of adrenergic systems in regulating Na(+) uptake in developing zebrafish.