Abstract
The role of aquaporin water channels such as aquaporin 4 (Aqp4) in elasmobranchs such as the dogfish Squalus acanthias is completely unknown. This investigation set out to determine the expression and cellular and sub-cellular localization of Aqp4 protein in dogfish tissues. Two polyclonal antibodies were generated (AQP4/1 and AQP4/2) and these showed somewhat different characteristics in Western blotting and immunohistochemistry. Western blots using the AQP4/1 antibody showed two bands (35.5 and 49.5 kDa) in most tissues in a similar fashion to mammals. Liver had an additional band of 57 kDa and rectal gland two further faint bands of 37.5 and 38.5 kDa. However, unlike in mammals, Aqp4 protein was ubiquitously expressed in all tissues including gill and liver. The AQP4/2 antibody appeared much less specific in Western blots. Both antibodies were used in immunohistochemistry and showed similar cellular localizations, although the AQP4/2 antibody had a more restricted sub-cellular distribution compared to AQP4/1 and therefore appeared to be more specific for Aqp4. In kidney a sub-set of tubules were stained which may represent intermediate tubule segments (In-III–In-VI). AQP4/1 and AQP4/2 antibodies localized to the same tubules segments in serial sections although the intensity and sub-cellular distribution were different. AQP4/2 showed a basal or basolateral membrane distribution whereas AQP4/1 was often distributed throughout the whole cell including the nuclear region. In rectal gland and cardiac stomach Aqp4 was localized to secretory tubules but again AQP/1 and AQP/2 exhibited different sub-cellular distributions. In gill, both antibodies stained large cells in the primary filament and secondary lamellae. Again AQP4/1 antibody stained most or all the cell including the nucleus, whereas AQP4/2 had a plasma membrane or plasma membrane and cytoplasmic distribution. Two types of large mitochondrial rich transport cells are known to exist in elasmobranchs, that express either Na, K-ATPase, or V-type ATPase ion transporters. Using Na, K-ATPase, and V-type ATPase antibodies, Aqp4 was colocalized with these proteins using the AQP4/1 antibody. Results show Aqp4 is expressed in both (and all) branchial Na, K-ATPase, and V-type ATPase expressing cells.
Highlights
Aquaporin 4 (AQP4) is a member of the water-selective sub-group of aquaporin water channel cell-membrane proteins found in all organisms so far investigated
Western blotting of crude membrane protein extracts from a variety of different dogfish tissues and using the AQP4/1 antibody showed that aquaporin 4 (Aqp4) is expressed ubiquitously in all the tissues studied (Figure 1A)
The second AQP4/2 antibody was used in Western blotting of tissue crude membrane extracts (Figure 1B)
Summary
Aquaporin 4 (AQP4) is a member of the water-selective sub-group of aquaporin water channel cell-membrane proteins found in all organisms so far investigated. AQP4 is widely expressed in a variety of tissues including the brain (Zelanina, 2010), retina (Hirrlinger et al, 2011), salivary gland (Delporte and Steinfeld, 2006), trachea (Borok and Verkman, 2002), heart and muscle (Butler et al, 2006; Wakayama, 2010), gastrointestinal tract (Ma and Verkman, 1999; Xu et al, 2009), and kidney (Nejsum, 2005), but is not expressed in the lung itself or liver tissues (Ishibashi et al, 2009) While aquaporins such as AQP4 have been studied in a wide variety of (mostly) higher vertebrates, no complete studies have yet been published on the role of aquaporins in elasmobranch fish such as the dogfish shark (Cutler et al, 2005; Cutler, 2006, 2007).
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