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Viable chloride and pavement cells were isolated from the gill epithelium of Japanese eel, Anguilla japonica, by a 3-step percoll gradient centrifugation at low speed. Viability of the isolated cells were tested by the trypan blue exclusion test, rhodamine-123, or pre-labelling the cells with fluo-3 Ca2+ dye and examined by laser confocal microscopy. Isolated chloride cells responded to ionomycin with a rapid increase in Ca2+ fluorescence, which was abolished by chelating external Ca2+ with EGTA. Peptide hormones, including arginine vasotocin, isotocin, insulin-like growth factor I and II, and urotensin I increased Ca2+ entry, urotensin II had no effect, and eel corpuscles of Stannius extract reduced residual Ca2+ fluorescence. Isolated chloride cells and pavement cells from eels were analyzed for their enzymatic activities involved in intermediary and nitrogen metabolism. Chloride cells had high levels of glutaminase I, glutamate dehydrogenase, glutamate oxalacetate transaminase, HCO3-ATPase and carbamoyl phosphate synthetase II. Pavement cells had highly active glucose-6-phosphate dehydrogenase and AMP deaminase. Both had high levels of lactate dehydrogenase compared with other tissues. |
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