Distribution and dynamics of branchial ionocytes in houndshark reared in full-strength and diluted seawater environments

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Abstract

In teleost fishes, it is well-established that the gill serves as an important ionoregulatory organ in addition to its primary function of respiratory gas exchange. In elasmobranchs, however, the ionoregulatory function of the gills is still incompletely understood. Although two types of ionocytes, Na+/K+-ATPase (NKA)-rich (type-A) cell and vacuolar-type H+-ATPase (V-ATPase)-rich (type-B) cell, have been found in elasmobranch fishes, these cells were considered to function primarily in acid–base regulation. In the present study, we examined ion-transporting proteins expressed in ionocytes of Japanese-banded houndshark, Triakis scyllium, reared in full-strength seawater (SW) and transferred to diluted (30%) SW. In addition to the upregulation of NKA and Na+/H+ exchanger type 3 (NHE3) mRNAs in the type-A ionocytes, we found that Na+, Cl cotransporter (NCC, Slc12a3) is expressed in a subpopulation of the type-B ionocytes, and that the expression level of NCC mRNA was enhanced in houndsharks transferred to a low-salinity environment. These results suggest that elasmobranch gill ionocytes contribute to NaCl uptake in addition to the already described function of acid–base regulation, and that NCC is most probably one of the key molecules for hyper-osmoregulatory function of elasmobranch gills. The existence of two types of ionocytes (NHE3- and NCC-expressing cells) that are responsible for NaCl absorption seems to be a common feature in both teleosts and elasmobranchs for adaptation to a low salinity environment. A possible driving mechanism for NCC in type-B ionocytes is discussed.

Introduction

Ion regulation is one of the most important issues in the maintenance of body-fluid homeostasis. This is particularly vital for fishes in aquatic environments where they are surrounded by waters where the concentrations of ions can change. It is well-established that the teleost fish gill contributes importantly to ion regulation, in addition to the primary function of respiratory gas exchange. In teleost fish, ionocytes, also known as chloride cells or mitochondrion-rich cells, in the branchial epithelium are responsible for ionoregulation (see review, Evans et al., 2005, Kaneko et al., 2008). Ionocytes of seawater (SW) teleost fish have a well-developed tubular system with numerous mitochondria and form a multicellular complex with an accessory cell to provide a paracellular pathway through which Na+ is thought to be secreted. A set of ion-transporting proteins, namely basolaterally-located Na+/K+-ATPase (NKA) and Na+, K+, 2Cl cotransporter isoform 1 (NKCC1) and apically-located cystic fibrosis transmembrane conductance regulator (CFTR), are expressed in ionocytes of SW teleost fish to secrete Cl transcellularly (Marshall, 2002). Contributions of multiple membrane proteins, including NKA, vacuolar-type H+-ATPase (V-ATPase), Na+/H+ exchangers (NHEs) and Na+, Cl cotransporter (NCC, slc12a10), to ion uptake have also been described in freshwater (FW) teleosts; however, the molecular mechanisms for ion uptake seem to vary among species (Hwang and Lee, 2007, Hwang et al., 2011, Dymowska et al., 2012). In Mozambique tilapia and zebrafish, multiple types of cells (cell types I to IV for tilapia; and NCC, NaR and HR cells for zebrafish) have been identified according to the membrane transporters expressed in those cells, and on their possible function (Hiroi et al., 2008, Inokuchi et al., 2009, Hwang et al., 2011).

In contrast to teleost fish, the iono-regulatory function of the elasmobranch gill is less well understood. Although ionocytes have also been found in elasmobranch gill epithelia, their function has been considered to be different from that of teleost ionocytes (Wilson and Laurent, 2002, Evans et al., 2005). Marine elasmobranchs have a specialized salt-secreting gland, the rectal gland (Burger, 1965), and the gill ionocytes have consequently been proposed to be involved in acid–base regulation (Edwards et al., 2002, Tresguerres et al., 2006, Choe et al., 2007, Tresguerres et al., 2007). Molecular and histochemical investigations of ion-transporting proteins have revealed that there are two types of ionocytes in elasmobranch gills: type-A ionocytes (NKA-rich ionocytes) and type-B ionocytes (V-ATPase-rich ionocytes) in spiny dogfish Squalus acanthias and Atlantic stingray Dasyatis sabina (Choe et al., 2005, Choe et al., 2007, Piermarini and Evans, 2001). The type-A ionocytes express NHE isoform 3 (NHE3) on the apical membrane, suggesting that low intracellular Na+ created by the basolaterally-located NKA promotes H+ excretion to the environment concomitantly with Na+ uptake (Choe et al., 2005). On the other hand, HCO3 excretion is proposed in the type-B ionocytes, since the pendrin-like Cl/HCO3 exchanger (PDN, slc26a4) is located on the apical membrane (Piermarini et al., 2002). These molecular investigations supported the idea that elasmobranch gill ionocytes are involved in acid–base regulation rather than NaCl excretion in the SW environment.

Recently, we discovered a novel aggregate structure made up of cells with basolaterally-expressed NKA in the inter-filamental space of the gill septum (Takabe et al., 2012). The cell aggregates, named follicularly-arranged NKA-rich cells, express NHE3 and Ca2 + transporter 1 (CaT), and thus are most likely involved in Ca2 + homeostasis. During the course of this investigation, we also found expression of CaT mRNA in a small number of ionocytes in the branchial filament. These observations imply that elasmobranch ionocytes still have unidentified functions for ion homeostasis. In the present study, to expose the roles of ionocytes of elasmobranchs, we examined expression of ion-transporting proteins in ionocytes of Japanese-banded houndshark Triakis scyllium. We found that, in addition to NKA, NHE3, V-ATPase and PDN already found in the gills of spiny dogfish and Atlantic stingray, CaT and NCC (Slc12a3) were expressed in a certain portion of type-A and type-B cells, respectively. Acclimation to a low-salinity environment induced increases in the numbers of NKA- and NHE3-expressing type-A cells and of V-ATPase-expressing type-B cells. After transfer, the proportion of NCC-expressing type-B cells (type-B-II cell) to total type-B cells (V-ATPase expressing cell) rose, implying that elasmobranch branchial ionocytes contribute importantly to hyper-osmoregulatory ability.

Section snippets

Fish

All animal experiments were conducted according to the Guidelines for Care and Use of Animals approved by the committee of the University of Tokyo. Japanese banded houndshark, T. scyllium, (750–1550 g) were collected in Koajiro Bay, Kanagawa, Japan. They were transported to the Atmosphere and Ocean Research Institute and kept in 2 × 103L holding tanks (20–22 °C, aerated) under a constant photoperiod (12 h:12 h, L:D). The fish were fed on squid for at least 2 weeks before experiments. For sampling,

Cloning of pump/transporter/channel cDNAs expressed in the houndshark gill

In preliminary work, we comprehensively searched cDNA sequences encoding putative ion-transporting molecules using the genome database of the holocephalan elephant fish, Callorhinchus milii, a species which has attracted attention as a model for molecular studies of cartilaginous fish (Hyodo et al., 2007). Subsequently, partial sequences of orthologous cDNAs encoding NKA, NKCC1, NKCC2, NCC, CFTR, NHE3, V-ATPase, PDN, CaT and CLC3 were cloned from the houndshark. The NKA, NKCC1, CFTR, NHE3 and

Discussion

In elasmobranch gills, two types of ionocytes have been reported: type-A (NKA-rich) cell and type-B (V-ATPase-rich) cell (Choe et al., 2005, Piermarini et al., 2002, Reilly et al., 2011). The presence of two types of ionocytes was confirmed in the present study using in situ hybridization with specific probes for houndshark mRNAs encoding NKA α1-subunit, V-ATPase, NHE3, PDN, NCC, CLC3 and CaT. In situ hybridization is advantageous for comprehensive identification of transporters that are

Acknowledgments

We sincerely thank Prof. Christopher A Loretz of Univ. of Buffalo for critical reading of the manuscript. This study was supported by Grant-in-Aid for Scientific Research (B) (Grant No. 26291065) and Challenging Exploratory Research (Grant No. 26650110) from the Japan Society for the Promotion of Science (JSPS) to S.H. and JSPS Research Fellowships for Young Scientists (Grant No. 15J07266) to M.I.

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