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First published online August 17, 2006
Journal of Experimental Biology 209, 3440-3447 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02384

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Na⁺/H⁺ antiporter, V-H⁺-ATPase and Na⁺/K⁺-ATPase immunolocalization in a marine teleost (Myoxocephalus octodecemspinosus)

Justin S. Catches^1,2, Julie M. Burns², Susan L. Edwards^2,3 and James B. Claiborne^1,2,*

¹ Department of Biology, Georgia Southern University, Statesboro, GA 30460, USA
² Mount Desert Island Biological Laboratory, Salisbury Cove, ME 04672, USA
³ Department of Physiology and Pharmacology, School of Biomedical Sciences, James Cook University, Cairns, QLD, Australia

View larger version (149K): [in a new window]   Fig. 1. Longhorn sculpin gill sections from seawater controls with chromagenic and fluorescent staining against NHE2 and Na+/K+-ATPase. Gill sections stained with (A) DAB and (B) green fluorescein against NHE2 with sculpin-specific antibodies. Controls (lacking primary antibody, antigen-antibody competition and pre-immune serum) were all negative (not shown). Staining is punctate in the apical and subapical regions of large cells in the filament. The cell distribution is similar to the pattern for Na+/K+-ATPase-rich cells. Serial sections stained with (C) DAB and (D) red-rhodamine against Na+/K+-ATPase show intense immunoreactivity throughout the cell which is consistent with staining along the tubular infoldings of the basolateral membrane in cells that are probably mitochondria-rich chloride cells. Scale bars, 20 µm.

View larger version (78K): [in a new window]   Fig. 2. Confocal merged image of antibodies against NHE2 (rhodamine) and Na+/K+-ATPase (fluorescein) at higher magnification (60x; scale bar, 20 µm). Arrows indicate interlamellar cells expressing both NHE2 and Na+/K+-ATPase. Autofluorescent erythrocytes in the lamellae are indicated with asterisks. Scale bar, 20 µm.

View larger version (110K): [in a new window]   Fig. 3. Colocalization of NHE2, Na+/K+-ATPase and V-ATPase. Staining against both NHE2 (A94-APS: 1/10,000, Vector VIP, purple) and Na+/K+-ATPase (5: 1/5000, Vector SG, blue/grey) is evident in the interlamellar region of many gill epithelial cells. No lamellar cells were stained. NHE2 (A94-APS: 1/10,000, Vector VIP, purple) and V-ATPase (HAB: 1/5000, Vector SG, blue/grey) colocalized in some of the same cells. Tissue double-labeled for V-ATPase (HAB: 1/5000, Vector VIP, purple) and Na+/K+-ATPase (5: 1/5000, Vector SG, blue/grey) demonstrated diffuse staining of both. Most stained cells only stained for Na+/K+-ATPase, however, some cells stained for both transporters. This could indicate that V-ATPase is present in only a subpopulation of mitochondria-rich cells. *, colocalization in each picture; +, staining of NHE2 only; ^, staining of HAB only; #, staining of Na/K-ATPase only.

View larger version (58K): [in a new window]   Fig. 4. Relative NHE2 protein expression in control and acidotic fish. (A) This representative western blot, probed with anti-NHE2 antibodies, contains protein from two control (lanes 4 and 8) and two acid-infused (lanes 2 and 6) sculpin. Even-numbered lanes are the membrane fraction and odd-numbered lanes are the cytosolic fraction of homogenized longhorn sculpin gill tissue. An 85 kDa signal was detected when the immunoblot was probed with anti-NHE2 antibodies (541-AP). Preimmune and peptide-antibody competition controls were negative (not shown). Markers: 100 kDa, 75 kDa. (B) Graph showing differences in relative protein abundance of NHE2 in acid infused versus control fish. Values are mean ratios ± s.e.m. (N=4).