2279
The Journal of Experimental Biology 203, 2279–2296 (2000) Printed in Great Britain © The Company of Biologists Limited 2000 JEB2668
NaCl UPTAKE BY THE BRANCHIAL EPITHELIUM IN FRESHWATER TELEOST FISH: AN IMMUNOLOGICAL APPROACH TO ION-TRANSPORT PROTEIN LOCALIZATION JONATHAN M. WILSON1,2,*, PIERRE LAURENT2, BRUCE L. TUFTS3, DALE J. BENOS4, MARK DONOWITZ5, A. WAYNE VOGL6 AND DAVID J. RANDALL1 1Department of Zoology, University of British Columbia, Vancouver, Canada V6T 1Z4, 2Centre Ecologie et Physiologie Energetique, Centre National Recherche Scientifique, Strasbourg 67037, France, 3Department of Biology, Queens University, Kingston, Ontario, Canada K7L 3N6, 4Departments of Biophysics and Physiology, University of Alabama at Birmingham, AL 35294-0005, USA, 5Departments of Medicine and Physiology, GI Unit, Johns Hopkins University, Baltimore, MD 21205-2195, USA and 6Department of Anatomy, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3 *Present address: Centro Interdisciplinar de Investigação Marinho e Ambiental (CIIMAR), Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal (e-mail:
[email protected])
Accepted 3 May; published on WWW 10 July 2000 Summary Teleost fishes, living in fresh water, engage in active ion mossambicus) and rainbow trout (Oncorhynchus mykiss), uptake to maintain ion homeostasis. Current models for V-ATPase and ENaC-like immunoreactivity co-localized to NaCl uptake involve Na+ uptake via an apical amiloridepavement cells, although apical labelling was also found in MR cells in the trout. In the freshwater tilapia, apical sensitive epithelial Na+ channel (ENaC), energized by an apical vacuolar-type proton pump (V-ATPase) or anion-exchanger-like immunoreactivity is found in the MR alternatively by an amiloride-sensitive Na+/H+ exchange cells. Thus, a freshwater-type MR chloride cell exists in (NHE) protein, and apical Cl− uptake mediated by an teleost fishes. The NHE-like immunoreactivity is associated electroneutral, SITS-sensitive Cl−/HCO3− anion-exchange with the accessory cell type and with a small population of protein. Using non-homologous antibodies, we have pavement cells in tilapia. determined the cellular distributions of these ion-transport proteins to test the predicted models. Na+/K+-ATPase was Key words: fish, gill, ion transport, H+-ATPase, Na+/K+-ATPase, epithelial Na+ channel, Na+/H+ exchanger, anion exchanger, used as a cellular marker for differentiating branchial pavement cell, mitochondria-rich cell, tilapia, Oreochromis epithelium mitochondria-rich (MR) cells from pavement mossambicus, rainbow trout, Oncorhynchus mykiss. cells. In both the freshwater tilapia (Oreochromis
Introduction In freshwater fish, the active uptake of Na+ and Cl− is necessary for ionic homeostasis. The consequence of living in a hypo-osmotic medium and having a large surface area (the gill) subject to large outward ion gradients is the continuous loss of salts by passive diffusion and gain of water by osmosis. The fish manages the water gain by producing copious amounts of urine; however, this also adds to ion loss problems, because the kidney is not capable of reabsorbing all the salts. Active branchial ion uptake is crucial for ion homeostasis. The uptake of Na+ and Cl− is achieved by electroneutral Na+/H+ and Cl−/HCO3− exchange mechanisms (Krogh, 1939). Na+ uptake models uptake is mediated by an amiloride-sensitive Na+/H+ exchange that is either directly or indirectly coupled and shows saturation kinetics (Wright, 1991; Potts, 1994). It is difficult to Na+
explain Na+ uptake using the directly coupled carrier-mediated Na+/H+ exchange (NHE) mechanism because of the absence of physiologically relevant gradients to drive the exchange process under many natural conditions (for a review, see Lin and Randall, 1995). The Na+ levels in the epithelium (6.4–16.5 mmol l−1 in isolated pavement cells and MR cells; Li et al., 1997) are typically greater than in the freshwater environment (
