title: Both seawater acclimation and environmental ammonia exposure lead to increases in mRNA expression and protein abundance of Na⁺:K⁺:2Cl⁻ cotransporter in the gills of the climbing perch, Anabas testudineus.
abstract: The freshwater climbing perch, Anabas testudineus, is an obligatory air-breathing teleost which can acclimate to seawater, survive long period of emersion, and actively excrete ammonia against high concentrations of environmental ammonia. This study aimed to clone and sequence the Na⁺:K⁺:2Cl⁻ cotransporter (nkcc) from the gills of A. testudineus, and to determine the effects of seawater acclimation or exposure to 100 mmol l⁻¹ NH₄Cl in freshwater on its branchial mRNA expression. The complete coding cDNA sequence of nkcc from the gills of A. testudineus consisted of 3,495 bp, which was translated into a protein with 1,165 amino acid residues and an estimated molecular mass of 127.4 kDa. A phylogenetic analysis revealed that the translated Nkcc of A. testudineus was closer to fish Nkcc1a than to fish Nkcc1b or Nkcc2. After a progressive increase in salinity, there were significant increases in the mRNA expression and protein abundance of nkcc1a in the gills of fish acclimated to seawater as compared with that of the freshwater control. Hence, it can be concluded that similar to marine teleosts, Cl⁻ excretion through basolateral Nkcc1 of mitochondrion-rich cells (MRCs) was essential to seawater acclimation in A. testudineus. Exposure of A. testudineus to 100 mmol l⁻¹ NH₄Cl for 1 or 6 days also resulted in significant increases in the mRNA expression of nkcc1a in the gills, indicating a functional role of Nkcc1a in active ammonia excretion. It is probable that NH₄⁺ enter MRCs through basolateral Nkcc1a before being actively transported across the apical membrane. Since the operation of Nkcc1a would lead to an increase in the intracellular Na⁺ concentration, it can be deduced that an upregulation of basolateral Na⁺/K⁺-ATPase (Nka) activity would be necessary to compensate for the increased influx of Na⁺ into MRCs during active NH₄⁺ excretion. This would imply that the main function of Nka in active NH₄⁺ excretion is to maintain intracellular Na⁺ and K⁺ homeostasis instead of transporting NH₄⁺ directly into MRCs as proposed previously. In conclusion, active salt secretion during seawater acclimation and active NH₄⁺ excretion during exposure to ammonia in freshwater could involve similar transport mechanisms in the gills of A. testudineus.
authors.affiliation: Department of Biological Science, National University of Singapore, Kent Ridge, Singapore, Republic of Singapore.
authors.forename: Ai M
authors.initials: AM
authors.lastname: Loong
authors.forename: Shit F
authors.initials: SF
authors.lastname: Chew
authors.forename: Wai P
authors.initials: WP
authors.lastname: Wong
authors.forename: Siew H
authors.initials: SH
authors.lastname: Lam
authors.forename: Yuen K
authors.initials: YK
authors.lastname: Ip
mesh_terms: D000064:Acclimatization / Q000187:drug effects; D000595:Amino Acid Sequence; D000643:Ammonium Chloride / Q000494:pharmacology*; D000818:Animals; D029941:Fish Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005880:Gills / Q000187:drug effects / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010471:Perches / Q000502:physiology*; D010802:Phylogeny; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D054712:Salinity; D012623:Seawater / Q000737:chemistry; D016415:Sequence Alignment; D028021:Sodium-Potassium-Chloride Symporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D064506:Solute Carrier Family 12, Member 2; D013312:Stress, Physiological; D015854:Up-Regulation / Q000187:drug effects; D014874:Water Pollutants, Chemical / Q000494:pharmacology; D014882:Water-Electrolyte Balance / Q000187:drug effects
chemical_list: D029941:Fish Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D011994:Recombinant Proteins; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D014874:Water Pollutants, Chemical; D000643:Ammonium Chloride