An investigation into acid-base balance at the gills of rainbow trout (Oncorhynchus mykiss) using in situ hybridization and immunocytochemistry for proton-ATPase and band 3.

Abstract

This thesis examines changes that occur in the epithelial cells of rainbow trout (Oncorhynchus mykiss) gills with respect to the expression of Na$\sp+$ and Cl$\sp-$ ion-transporting proteins during periods of acid-base disturbance. In the first part of the study, the expression of the V-type proton ATP-ase (H$\sp+$-ATPase) was examined in the gill of trout to provide direct evidence for its existence, determine its cellular distribution, and assess its response to acid-base disturbance. Gill epithelial cells demonstrated specific immunoreactivity, the intensity of which was increased markedly after 18 h of exposure to hypercapnia (1% CO$\sb2$ in air). The specificity of the antiserum for the 31-kDa subunit of the H$\sp+$-ATPase was supported by Western blotting with the presence of an immunoreactive band at 31-kDa. The increased H$\sp+$-ATPase immunoreactivity in the epithelial cells of hypercapnic fish demonstrates an "up-regulation" of this protein in response to respiratory acidosis. Oligonucleotide probes, complementary to the mRNA of the 31- and 56-kDa subunits of the bovine renal H$\sp+$-ATPase were constructed. The H$\sp+$-ATPase mRNA expression was increased markedly in the gills of acidotic fish and was accompanied by a pronounced elevation in branchial H$\sp+$ excretion. The probe for the Band 3 mRNA also demonstrated a specific hybridization signal in gill epithelial cells which was greatly increased in the gills of alkalotic fish, which were excreting large amounts of HCO$\sb3\sp-$. The results of this thesis provide direct evidence for the presence of an electrogenic H$\sp+$ pump located on the apical surface of the pavement cell which is actively up-regulated during periods of acidosis through increased transcription and translation of the protein as well as increased membrane association. Further evidence is provided for the presence of Band 3 or a Band 3-like protein which is responsible for Cl$\sp-$/HCO$\sb3\sp-$ exchange across the gill. (Abstract shortened by UMI.)