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Ip Y.K.,National University of Singapore | Wilson J.M.,Ecofisiologia CIMAR | Loong A.M.,National University of Singapore | Chen X.L.,National University of Singapore | And 4 more authors.
Journal of Comparative Physiology B: Biochemical, Systemic, and Environmental Physiology | Year: 2012

This study aimed to clone and sequence the cystic fibrosis transmembrane conductance regulator (cftr) from, and to determine the effects of seawater acclimation or exposure to 100 mmol l-1 NH4Cl in freshwater on its mRNA and protein expressions in, the gills of Anabas testudineus. There were 4,530 bp coding for 1,510 amino acids in the cftr cDNA sequence from A. testudineus. The branchial mRNA expression of cftr in fish kept in freshwater was low (<50 copies of transcript per ng cDNA), but significant increases were observed in fish acclimated to seawater for 1 day (92-fold) or 6 days (219-fold). Branchial Cftr expression was detected in fish acclimated to seawater but not in the freshwater control, indicating that Cl- excretion through the apical Cftr of the branchial epithelium was essential to seawater acclimation. More importantly, fish exposed to ammonia also exhibited a significant increase (12-fold) in branchial mRNA expression of cftr, with Cftr being expressed in a type of Na+/K+-ATPase-immunoreactive cells that was apparently different from the type involved in seawater acclimation. It is probable that Cl- excretion through Cftr generated a favorable electrical potential across the apical membrane to drive the excretion of NH4+ against a concentration gradient through a yet to be determined transporter, but it led to a slight loss of endogenous Cl-. Since ammonia exposure also resulted in significant decreases in blood pH, [HCO3-] and [total CO2] in A. testudineus, it can be deduced that active NH4+ excretion could also be driven by the exit of HCO3- through the apical Cftr. Furthermore, A. testudineus uniquely responded to ammonia exposure by increasing the ambient pH and decreasing the branchial bafilomycin-sensitive V-type H+-ATPase activity, which suggests that its gills might have low NH3 permeability. © 2012 Springer-Verlag. Source


Chew S.F.,Nanyang Technological University | Tng Y.Y.M.,National University of Singapore | Wee N.L.J.,National University of Singapore | Tok C.Y.,National University of Singapore | And 2 more authors.
Journal of Comparative Physiology B: Biochemical, Systemic, and Environmental Physiology | Year: 2010

The objective of this study was to elucidate the role of the intestine from juveniles of the marble goby, Oxyeleotris marmorata, during seawater (SW) exposure. It has been reported elsewhere that SW-exposed juvenile O. marmorata exhibits hypoosmotic and hypoionic regulation, with the induction of branchial Na+/K+-ATPase (NKA), Na+:K+:2Cl- cotransporter (NKCC), and cystic fibrosis transmembrane receptor-like chloride channels. Here, we report that SW exposure also led to significant increases in the activity and protein abundance of NKA in, and probably an increase in Na+ uptake through, its intestine. Additionally, there was an increase in apical NKCC immunoreactivity in the intestinal epithelium, indicating that there could be increased Cl- uptake through the intestine. These results suggest that absorption of ions, and hence water, from the intestinal lumen could be an essential part of the osmoregulatory process in juvenile O. marmorata during exposure to SW. Furthermore, there were significant increases in the glutamate content, and the aminating activity and protein abundance of glutamate dehydrogenase (GDH) in the intestine of fish exposed to SW. Since the intestinal glutamine synthetase activity and protein abundance decreased significantly, and the intestinal glutamine content remained unchanged, in the SW-exposed fish, excess glutamate formed via increased GDH activity in the intestine could be channeled to other organs to facilitate the increased synthesis of amino acids. Taken together, our results indicate for the first time that, besides absorbing ions and water during SW exposure, the intestine of juvenile O. marmorata also participated in altered nitrogen metabolism in response to salinity changes. © 2009 Springer-Verlag. Source


Ip Y.K.,National University of Singapore | Hiong K.C.,National University of Singapore | Wong S.Z.,National University of Singapore | Ching B.,National University of Singapore | And 6 more authors.
Frontiers in Physiology | Year: 2013

Himantura signifer is a freshwater stingray which inhabits rivers in Southeast Asia. It can survive in brackish water but not seawater. In brackish water, it becomes partially ureosmotic, but how it maintains its plasma hypoionic to the external medium is enigmatic because of the lack of a rectal gland. Here, we report for the first time the expression of Na+:K+:2Cl- cotransporter 1 (nkcc1) in the gills of freshwaterH. signifer, and its moderate up-regulation (~2-fold) in response to brackish water (salinity 20) acclimation. The absence of the Ste20-related proline-alanine-rich kinase and oxidation stress response kinase 1 interaction site from the N-terminus of H. signifer Nkcc1 suggested that it might not be effectively activated by stress kinases in response to salinity changes as in more euryhaline teleosts. The increased activity of Nkcc1 during salt excretion in brackish water would lead to an influx of Na+ into ionocytes, and the maintenance of intracellular Na+ homeostasis would need the cooperation of Na+/K+-ATPase (Nka). We demonstrated for the first time the expression of nkaa1, nkaa2 and nkaa3 in the gills of H. signifer, and the up-regulation of the mRNA expression of nkaa3 and the overall protein abundance of Nkaa in response to acclimation to brackish water. Immunofluorescence microscopy revealed the presence of a sub-type of ionocyte, co-expressing Nkcc1 and Nkaa, near the base of the secondary lamellae in the gills of H. signifer acclimated to brackish water, but this type of ionocyte was absent from the gills of fish kept in fresh water. Hence, there could be a change in the function of the gills of H. signifer from salt absorption to salt excretion during brackish water acclimation in the absence of a functioning rectal gland. © 2013 Ip, Hiong, Wong, Ching, Chen, Soh, Chng, Ong, Wilson and Chew. Source


Ching B.,National University of Singapore | Chen X.L.,National University of Singapore | Yong J.H.A.,National University of Singapore | Wilson J.M.,Ecofisiologia CIMAR | And 6 more authors.
Frontiers in Physiology | Year: 2013

This study aimed to test the hypothesis that branchial osmoregulatory acclimation involved increased apoptosis and replacement of mitochdonrion-rich cells (MRCs) in the climbing perch, Anabas testudineus, during a progressive acclimation from freshwater to seawater. A significant increase in branchial caspase-3/-7 activity was observed on day 4 (salinity 20), and an extensive TUNEL-positive apoptosis was detected on day 5 (salinity 25), indicating salinity-induced apoptosis had occurred. This was further supported by an up-regulation of branchial mRNA expression of p53, a key regulator of cell cycle arrest and apoptosis, between day 2 (salinity 10) and day 6 (seawater), and an increase in branchial p53 protein abundance on day 6. Seawater acclimation apparently activated both the extrinsic and intrinsic pathways, as reflected by significant increases in branchial caspase-8 and caspase-9 activities. The involvement of the intrinsic pathway was confirmed by the significant increase in branchial mRNA expression of bax between day 4 (salinity 20) and day 6 (seawater). Western blotting results revealed the presence of a freshwater Na+/K+-ATPase (Nka) α-isoform, Nka α1a, and a seawater isoform, Nka α1b, the protein abundance of which decreased and increased, respectively, during seawater acclimation. Immunofluorescence microscopy revealed the presence of two types of MRCs distinctly different in sizes, and confirmed that the reduction in Nka α1a expression, and the prominent increases in expression of Nka α1b, Na+:K+:2Cl- cotransporter 1, and cystic fibrosis transmembrane conductance regulator Cl- channel coincided with the salinity-induced apoptotic event. Since modulation of existing MRCs alone could not have led to extensive salinity-induced apoptosis, it is probable that some, if not all, freshwater-type MRCs could have been removed through increased apoptosis and subsequently replaced by seawater-type MRCs in the gills of A. testudineus during seawater acclimation. © 2013 Ching, Chen, Yong, Wilson, Hiong, Sim, Wong, Lam, Chew and Ip. Source

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