Studies of Some Molecular Properties of the Vacuolar H+-ATPase in Rainbow Trout (Oncorhynchus mykiss)

Abstract

A cloned cDNA fragment of the rainbow trout gill vacuolar H +-ATPase (H+ VATPase; proton pump) B subunit was used as a probe to examine: i) its interspecific distribution among marine and freshwater species and ii) its expression during a variety of acid-base and ionic disturbances. Northern blots of gill total RNA, performed under conditions of high stringency, revealed cross hybridisation between the trout probe and 11 of 16 species that were examined. Cross hybridisation was not observed in the pacific hagfish (E. stoutit), Lake Magadi tilapia (Oroechromis alcalicus Grahami), bigfin eelpout (Lycodes cortezianus), blackfin poacher (Bathyagonus nigripinnus) or freshwater American eel (Anguilla rostrata). Acute (3 h) exposure of trout to external hypercapnia (PwCO2 = ∼7 mm Hg) was associated with a transient increase after 1 h in gill H +-ATPaSe mRNA levels. Thus, the increase in gill H+-ATPase activity that is known to accompany hypercapnic acidosis in trout may reflect, at least in part, its transcriptional or post- transcriptional regulation. Plasma Cortisol levels were elevated in the hypercapnic fish (45±15 to 83±4 ng mL-1) and because 30 Cortisol was previously implicated as a regulator of H+-ATPase activity, mRNA levels were quantified in fish subjected to chronic Cortisol elevation. An increase in plasma Cortisol concentration from 90±10 (sham implants) to 300±60 ng mL-1 (Cortisol implants) for 4 days was associated with an approximate doubling of gill H+-ATPase steady-state mRNA levels. Exposure of trout for 72 h to ion-poor water caused a persistent reduction in the concentration of gill H+-ATPase steady-state mRNA. The functional significance of this response is unclear but may reflect a reduced rate OfNa+ uptake across the gill. These results are discussed with reference to the physiological role of the branchial H+-ATPase in both acid-base and ionic regulation.