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First published online December 16, 2008
Journal of Experimental Biology 212, 78-88 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.024612
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Multiplicity of expression of Na+,K+–ATPase {alpha}-subunit isoforms in the gill of Atlantic salmon (Salmo salar): cellular localisation and absolute quantification in response to salinity change

Steffen S. Madsen*, Pia Kiilerich and Christian K. Tipsmark

Institute of Biology, University of Southern Denmark, 5230 Odense M, Denmark


Figure 1
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  		Fig. 1. The effect of FW–SW (closed bars) or FW–FW (open bars) transfers on plasma [Na+] (A) and gill Na+,K+–ATPase activity (B) in Atlantic salmon. Different letters above bars indicate significant difference within FW (lowercase letters) or SW (uppercase letters). Asterisk indicates difference between FW and SW groups at a certain time point (P<0.05). FW, freshwater; SW, seawater.

 

Figure 2
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  		Fig. 2. The effect of FW–SW (closed symbols) or FW–FW (open symbols) transfers on {alpha}-subunit isoform mRNA levels (A, {alpha}1a; B, {alpha}1b; C, {alpha}1c; D, {alpha}3), the sum of {alpha}-subunit transcript levels (E) and the ratio between {alpha}1a and {alpha}1b in Atlantic salmon gill. Transcript levels are shown in amol 20 ng–1 total RNA. Asterisk indicate difference between FW and SW groups at a certain time point (P<0.05). FW, freshwater; SW, seawater.

 

Figure 3
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  		Fig. 3. The effect of FW–SW (closed symbols) or FW–FW (open symbols) transfers on β1-subunit mRNA levels in Atlantic salmon gill. Transcript levels are shown in amol 20 ng–1 total RNA. Asterisk indicates the difference between FW and SW groups at a certain time point (P<0.05). FW, freshwater; SW, seawater.

 

Figure 4
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  		Fig. 4. Diagrams showing the relative distribution in gill tissue of the four analysed {alpha}-subunit isoforms during acclimation from FW–SW or during sham-transfer from FW–FW. The four isoforms are indicated by different degree of shading ({alpha}1a, open; {alpha}1b, grey; {alpha}1c, hatched; {alpha}3, black). FW, freshwater; SW, seawater.

 

Figure 5
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  		Fig. 5. Representative gill sections probed with the {alpha}1a isoform AP-labelled cDNA probe. The insert in panel A shows orientation of sections: X-plane – frontal section; Y-plane – sagittal section. (A–C) Gills from FW salmon; (D,E) gills from SW salmon. A,B,D are sagittal sections, showing lamellae emerging perpendicularly from the filament. C,D are frontal sections at the base of the lamellae, showing cells in the interlamellar region. Solid arrows show labelled cells on lamellae, open arrows show labelled cells in filament. Gill sections were 10 µm thick. Bars indicate 100 µm. AP, alkaline phosphatase; FW, freshwater; SW, seawater.

 

Figure 6
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  		Fig. 6. Representative gill sections probed with the {alpha}1b isoform with AP-labelled cDNA probe. (A) Gill from FW salmon; (B–D) gills from SW salmon; (E) gill section probed with β-actin cDNA probe. For orientation of sections refer to Fig. 5.A,B,C,E are sagital sections, showing lamellae emerging perpendicularly from the filament. D is a frontal section at the base of the lamellae, showing cells in the interlamellar region. Solid arrows show labelled cells on lamellae, broken arrows show labelled cells deep in the filament, open arrows show large labelled cells in filament with apical contact. Gill sections were 10 µm thick. Bars indicate 100 µm. AP, alkaline phosphatase; FW, freshwater; SW, seawater.

 

Figure 7
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  		Fig. 7. Validation of probe specificity. Pair wise, adjacent FW gill sections were probed with AP-labelled {alpha}1a (A,B) or {alpha}1b (C,D) cDNA probes either alone (A,C) or in combination with 10-fold excess of the corresponding unlabelled cDNA sequence of the alternate isoform (i.e. {alpha}1b in B and {alpha}1a in D). No significant displacement of labelled probe takes place in either experiment, showing that the two probes are specific for their respective mRNA targets. (E,F) The sections were probed with the AP-labelled {alpha}1b probe either alone, E, or in combination with 5-fold excess of the corresponding unlabelled {alpha}1b cDNA sequence, F. The labelled probe was fully displaced under these conditions. Bars indicate 100 µm. AP, alkaline phosphatase; FW, freshwater.

 

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© The Company of Biologists Ltd 2009