In situ hybridization was performed on sections of rainbow trout (Oncorhynchus mykiss) gill tissue using oligonucleotide probes complementary to the mRNA of the 3 1-kilodalton subunit of the bovine renal V-type H+-ATPase or rat kidney Band 3 anion exchanger (C1-IHCO, exchanger). This was conducted in conjunction with measurements of whole-body net acid fluxes and blood acid-base status during imposed conditions of respiratory acidosis (external hypercapnia) or metabolic alkalosis (NaHCO, infusion). A positive hybridization signal for the H+-ATPase mRNA was localized predominantly in lamellar epithelial cells and was less apparent in cells associated with the filament or interlamellar regioqs. The Hf -ATPase hybridization signal was enhanced during hypercapnic acidosis concurrently with a marked increase in whole-body net acid excretion. A positive hybridization signal for the C1-IHCOj exchanger mRNA was observed in epithelial cells on both the filament and lamella. During metabolic alkalosis induced by intra-arterial infusion of NaHCO,, there was a marked increase in the C1-/HCO; exchanger mRNA hybridization signal in cells on both the filament and lamella that occurred concurrently with a decrease in net acid excretion. The results of this study support the existence of a V-type Hf -ATPase and a Cl-/HCO; exchanger in rainbow trout gill epithelial cells and demonstrate that alterations in gene expression for the pump-exchanger may be a significant mechanism underlying the altered rates of net acid equivalent excretion during acid-base disturbances.

The expression of the V-type proton ATPase (H+-ATPase) was examined in the gill of the freshwater rainbow trout (Oncorhynchus mykiss) using immunocytochemistry in concert with laser scanning confocal or electron microscopy. A synthetic peptide consisting of the carboxy-terminal region of the 31 kDa subunit of the bovine renal H+-ATPase was used to generate an antiserum in rabbits, and its suitability for use in trout gill was confirmed by western blotting. Gill epithelial cells demonstrated specific immunoreactivity, the intensity of which was increased markedly after 18 h of exposure to hypercapnia (1 % CO2 in air). The increased intensity of H+-ATPase immunoreactivity was associated with elevated branchial net acid excretion. In the hypercapnic fish, the specific immunoreactivity was associated with both the apical membrane and cytoplasm. Electron microscopy revealed that specific immunoreactivity was localized to the pavement cells and was particularly associated with the apical membrane and subapical cytoplasmic vesicles. The increased H+-ATPase immunoreactivity in the epithelial cells of hypercapnic fish and the increased intensity of the immunoreactive bands in western blots from hypercapnic fish demonstrate an 'up-regulation' of this protein in response to respiratory acidosis. The results are discussed with reference to current models of acid­base and ion regulation in the gill of freshwater fish.

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