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Proton-atpase in fish gills

University of British Columbia

The cellular mechanisms responsible for branchial proton excretion and sodium absorption in freshwater rainbow trout, Oncorhynchus mykiss (Walbaum), were investigated by monitoring the proton excretion rate and determining the proton ATPase activity in gill tissue under different inhibitor treatments and environmental conditions. Evidence for the existence of anelectrogenic proton pump in fish gills was accumulated. Branchial proton excretion rate was estimated by measuring the total CO2, total ammonia, pH and buffer capacity of the inspired and expired water of the fish. The net proton excretion across fish gills was resistant to 0.1 mM amiloride which could completely abolish the branchial sodium uptake, indicating that aNa+/H+ exchanger was not responsible for this proton transport. Branchial proton excretion, however, was sensitive to vanadate and acetazolamide, suppressed by low external water pH and sodiumlevels, and stimulated by elevated ambient Pco2. All these characteristics are typical for proton transport mediated by anelectrogenic proton pump, as demonstrated in frog skin, turtle bladder and mammalian kidney. N-ethymaleimide-sensitive ATPase activity was measured incrude homogenates of gill tissue from rainbow trout using a coupled-enzyme ATPase assay in the presence of EGTA, ouabain andazide. This NEM-sensitive ATPase activity, determined to be about1.5 umol/mg.pr./h. at 15°C for freshwater trout, is also inhibited by other proton-ATPase blockers such as DCCD, DES,PCMBS and Bafilomycins. It was concluded, therefore, that the NEM-sensitive ATPase activity was generated by a proton-translocating ATPase. Since this NEM-sensitive ATPase was also sensitive to the plasma membrane ATPase inhibitor vanadate, the11+-ATPase in fish gill was speculated to be a plasma membrane type. Sodium concentration in the external media was the primary regulator of the H+-ATPase in fish gills, with low water sodium levels associated with high H+-ATPase activity. High external calcium concentration and plasma cortisol levels had a marked stimulating effect on H+-ATPase activity in fish gills only when the water sodium level was low. Thus the major role of the H+-ATPase in the gill epithelium is to facilitate Na+ uptake from fresh water. The H+-ATPase in the gills also plays a role inacid-base regulation. It was concluded that an electrogenic proton pump (H+-ATPase) indirectly coupled to a sodium conductive channel in the gill epithelium is the ion transport pathway which mediates proton excretion and energies sodium absorption in fresh waterfish.

Thesis/Dissertation

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2008-09-11

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http://hdl.handle.net/2429/1855

Zoology

University of British Columbia

UBCV

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