In vivo experiments were conducted on spiny dogfish (Squalus acanthias) and rainbow trout (Oncorhynchus mykiss) in sea water to determine the potential role of externally oriented or gill boundary layer carbonic anhydrase in carbon dioxide excretion. This was accomplished by assessing pH changes in expired water using a stopped-flow apparatus. In dogfish, expired water was in acid-base disequilibrium as indicated by a pronounced acidification (delta pH=−0.11+/−0.01; N=22; mean +/− s.e.m.) during the period of stopped flow; inspired water, however, was in acid-base equilibrium (delta pH=−0.002+/−0.01; N=22). The acid-base disequilibrium in expired water was abolished (delta pH=−0.005+/−0.01; N=6) by the addition of bovine carbonic anhydrase (5 mg l-1) to the external medium. Addition of the carbonic anhydrase inhibitor acetazolamide (1 mmol l-1) to the water significantly reduced the magnitude of the pH disequilibrium (from −0.133+/−0.03 to −0.063+/−0.02; N=4). However, after correcting for the increased buffering capacity of the water caused by acetazolamide, the acid-base disequilibrium during stopped flow was unaffected by this treatment (control delta [H+]=99.8+/−22.8 micromol l-1; acetazolamide delta [H+]=81.3+/−21.5 micromol l-1). In rainbow trout, expired water displayed an acid-base disequilibrium (delta pH=0.09+/−0.01; N=6) that also was abolished by the application of external carbonic anhydrase (delta pH=0.02+/−0.01).The origin of the expired water acid-base disequilibrium was investigated further in dogfish. Intravascular injection of acetazolamide (40 mg kg-1) to inhibit internal carbonic anhydrase activity non-specifically and thus CO2 excretion significantly diminished the extent of the expired water disequilibrium pH after 30 min (from −0.123+/−0.01 to −0.065+/−0.01; N=6). Selective inhibition of extracellular carbonic anhydrase activity using a low intravascular dose (1.3 mg kg-1) of the inhibitor benzolamide caused a significant reduction in the acid-base disequilibrium after 5 min (from −0.11+/−0.01 to −0.07+/−0. 01; N=14). These results demonstrate that the expired water acid-base disequilibrium originates, at least in part, from excretory CO2 and that extracellular carbonic anhydrase in dogfish may have a significant role in carbon dioxide excretion. However, externally oriented carbonic anhydrase (if present in dogfish) plays no role in catalysing the hydration of the excretory CO2 in water flowing over the gills and thus is unlikely to facilitate CO2 excretion.

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