Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology
Distribution and dynamics of branchial ionocytes in houndshark reared in full-strength and diluted seawater environments
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.
References (47)
- et al.
The putative mechanism of Na+ absorption in euryhaline elasmobranchs exists in the gills of a stenohaline marine elasmobranch, Squalus acanthias
Comp. Biochem. Physiol.
(2007) - et al.
Structure and function of ionocytes in the freshwater fish gill
Respir. Physiol. Neurobiol.
(2012) - et al.
Immunolocalisation of sodium/proton exchanger-like proteins in the gills of elasmobranchs
Comp. Biochem. Physiol.
(2002) - et al.
Urea based osmoregulation and endocrine control in elasmobranch fish with special reference to euryhalinity
Comp. Biochem. Physiol.
(2003) - et al.
New insights into fish ion regulation and mitochondrion-rich cells
Comp. Biochem. Physiol.
(2007) - et al.
Neurohypophysial hormones of dogfish, Triakis scyllium: structures and salinity-dependent secretion
Gen. Comp. Endocrinol.
(2004) - et al.
The ClC-3 Cl−/H+ antiporter becomes uncoupled at low extracellular pH
J. Biol. Chem.
(2010) - et al.
Localization of the thiazide sensitive Na–Cl cotransporter, rTSC1 in the rat kidney
Kidney Int.
(1996) - et al.
Branchial mitochondria-rich cells in the dogfish Squalus acanthias
Comp. Biochem. Physiol.
(2002) - et al.
A simplified in situ hybridization protocol using non-radioactively labeled probes to detect abundant and rare mRNAs on tissue sections
Biochemica
(1998)
Function of the rectal gland in the spiny dogfish
Science
Roles of the rectal gland and kidneys in salt and water excretion in the spiny dogfish
Physiol. Zool.
NHE3 in an ancestral vertebrate: primary sequence, distribution, localization, and function in gills
Am. J. Phys. Regul. Integr. Comp. Phys.
Sharks of the World
The multifunctional fish gill: dominant site of gas excahange, osmoregulation, acid base regulation, and excretion of nitrogenous waste
Physiol. Rev.
Diluting segment in kidney of dogfish shark I. Localization and characterization of chloride absorption
Am. J. Physiol. Regul. Integ. Comp. Physiol.
Evidence for an apical Na–Cl cotransporter involved in ion uptake in a teleost fish
J. Exp. Biol.
A new model for fish ion regulation: identification ofionocytes in freshwater- and seawater-acclimated medaka (Oryzias latipes)
Cell Tissue Res.
Ion regulation in fish gills: recent progress in the cellular and molecular mechanisms
Am. J. Phys. Regul. Integr. Comp. Phys.
Osmoregulation in elephant fish Callorhinchus milii (Holocephali), with special reference to the rectal gland
J. Exp. Biol.
Morphological and functional classification of ion-absorbing mitochondria-rich cells in the gills of Mozambique tilapia
J. Exp. Biol.
Functional morphology of mitochondrion-rich cells in euryhaline and stenohaline teleosts
Aqua BioSci. Monogr.
Differential expression of Na+–Cl− cotransporter and Na+–K+–Cl− cotransporter 2 in the distal nephrons of euryhaline and seawater pufferfishes
Am. J. Phys. Regul. Integr. Comp. Phys.
Cited by (5)
Molecular mechanisms of Cl<sup>−</sup> transport in fishes: New insights and their evolutionary context
2021, Journal of Experimental Zoology Part A: Ecological and Integrative Physiology