spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online July 14, 2008
Journal of Experimental Biology 211, 2467-2477 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.017491
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ivanis, G.
Right arrow Articles by Perry, S. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ivanis, G.
Right arrow Articles by Perry, S. F.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Branchial expression and localization of SLC9A2 and SLC9A3 sodium/hydrogen exchangers and their possible role in acid–base regulation in freshwater rainbow trout (Oncorhynchus mykiss)

G. Ivanis, A. J. Esbaugh and S. F. Perry*

Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, Ontario, Canada K1N 6N5


Figure 1
View larger version (44K):
[in this window]
[in a new window]

  		Fig. 1. (A) Phylogenetic relationship of selected NHE1–3 proteins from different species and orthologous rainbow trout NHE2, NHE3a and NHE3b amino acid sequences. The grouping of each rainbow trout orthologue with its corresponding form is an indication that the rainbow trout clone is most closely related to that NHE isoform. (B) The existence of the two rainbow trout NHE3 transcripts is indicated in a representative Northern blot. K1–6, kidney samples 1–6. The tree in A was constructed using the neighbour-joining method, and numbers indicate bootstrap values for 1000 replicates. GenBank accession numbers are as follows. NHE1: Oncorhynchus mykiss (βNHE) Q01345, Pseudopleuronectes americanus AAO32340, Amphiuma tridactylum AAD33928, Takifugu obscurus BAE75800.1, Rattus norvegicus NP_036784.1, Anguilla anguilla CAB45085, Cyprinus carpio CAB45232, Homo sapiens NP_003038, Drosophila melanogaster AAF51559, Gallus gallus ABB82239. NHE2: Canis familiaris XP_531775.2, Gallus gallus XP_416918.2, Mus musculus NP_001028461 XP_129721, Oryctolagus cuniculus P50482, 708 amino acid (aa) Danio rerio XP_001336127.1, 795 aa Danio rerio XP_691364, Homo sapiens NP_003039, Myoxocephalus octodecemspinosus AAD46576, Rattus norvegicus P48763, partial 692 aa Oncorhynchus mykiss ABO32814, Squalus acanthias ABC54565, Tetraodon nigroviridis CAG05798. NHE3: 704 aa Danio rerio CAM47009.1, 829 aa Danio rerio XP_696670, 851 aa Danio rerio XP_696728, Gallus gallus XP_418895.2, Mus musculus NP_001074529.1, Dasyatis sabina AAT45738.2, Homo sapiens NP_004165.1, Rattus norvegicus NP_036786.1, NHE3a; 752 aa Oncorhynchus mykiss ABO32815.1 and NHE3b; 862 aa Oncorhynchus mykiss assembled sequence, Oreochromis mossambicus BAF80347, Tribolodon hakonensis AB055466.

 

Figure 2
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 2. Tissue distribution analysis of rainbow trout NHE2 (A) and NHE3 (B) mRNA as determined by real-time PCR and expressed relative to a reference tissue (brain) set to a relative value of 1. The inset (C) represents the relative mRNA levels of NHE2 and NHE3 in the gill. Values shown are means + 1 s.e.m.; asterisks indicate significant differences (P<0.05) from the reference tissue or gill NHE3 (inset). Ant., anterior; Post., posterior; W. muscle, white muscle.

 

Figure 3
View larger version (50K):
[in this window]
[in a new window]

  		Fig. 3. (A) Identification of PNA+ and PNA– negative mitochondria-rich cells (MRCs) in gill sections from adult rainbow trout. PNA+ MRCs are labelled with arrows; stars indicate PNA– MRCs. MRCs were identified on the basis of immunoreactivity against Na+/K+-ATPase (red); green indicates apical PNA and blue indicates nuclei (DAPI). (B) Higher magnification image of a PNA (green)-positive MRC. Negative controls included (C) omission of primary antibody against Na+/K+-ATPase and of biotinylated PNA. The image in C was captured using the same exposure and microscope settings as in A. Scale bar in A, 20 µm; and in B, 5 µm.

 

Figure 4
View larger version (89K):
[in this window]
[in a new window]

  		Fig. 4. Identification of NHE3-expressing cells as PNA+ MRCs in gill sections from adult rainbow trout. (A) NHE3 protein was detected in gill (G) and kidney (K) protein extracts using Western blots (N=6) to reveal a 98 kDa immunoreactive band (lanes 1 and 2) that was absent after the NHE3 antibody was incubated with excess peptide against which the antibody was raised (lanes 3 and 4). Size markers are given in kDa. (B) Co-localization of NHE3 (green) and Na+/K+-ATPase (red) in MRCs (nuclei are stained blue); NHE3-expressing MRCs are labelled with arrows and NHE3 negative MRCs are indicated by stars. (C) Localization of NHE3 to PNA+ MRCs; red indicates NHE3, green indicates PNA and nuclei are stained blue. (D) The NHE3 immunofluorescence disappeared with omission of primary NHE3 antibody. Scale bar in A, 40 µm; and in C, 10 µm.

 

Figure 5
View larger version (61K):
[in this window]
[in a new window]

  		Fig. 5. Detection of NHE2-expressing cells in rainbow trout gill by combined immunofluorescence and in situ hybridization. (A) PNA+ MRCs (labelled with arrows) were detected using a primary antibody against Na+/K+-ATPase (red) and biotinylated PNA (green). (B) In situ hybridization on the same section demonstrated that the PNA+ MRCs were enriched with NHE2 mRNA (labelled with arrows). Two PNA+ cells (labelled with asterisks) did not exhibit strong mRNA staining. No staining was observed when a sense probe was used (C) or when probe was omitted (D). Scale bar, 20 µm.

 

Figure 6
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 6. The effects of hypercapnia (water PCO2=7.5 mmHg) on trout gill NHE2 (A) and NHE3 (B) relative mRNA levels. Gill NHE2 mRNA levels were significantly increased at 3, 12 and 24 h of hypercapnia (P values of 0.014, 0.031 and 0.040, respectively). Gill NHE3 mRNA levels were unchanged throughout the period of hypercapnia exposure.

 

Figure 7
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 7. (A) The effects of exposure to hypercapnia for 24 h (water PCO2=7.5 mmHg) on trout gill NHE3 protein levels as revealed by Western blot analysis (N=6; each lane represents a different fish). Densitometric analysis of Western blots (B), normalized to β-actin intensities, revealed unaltered levels of NHE3 protein after 24 h of hypercapnia.

 

Figure 8
View larger version (9K):
[in this window]
[in a new window]

  		Fig. 8. The effects of elevated plasma cortisol levels on (A) gill relative NHE2 and (B) relative NHE3 mRNA expression after 24 (N=6), 48 (N=6) and 72 h (N=6) of administration of exogenous cortisol. NHE2 mRNA levels were significantly elevated at 48 (P=0.017) and 72 h (P=0.014) postinjection; NHE3 mRNA levels were unchanged.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2008