Chloride cell subtypes in the gill epithelium of Japanese eel Anguilla japonica

The purpose of the present study was to characterize chloride cell subtypes in the fish gill and to monitor the kinetic change of cell division in the gill epithelia during seawater adaptation. Employing a three-step Percoll gradient method, the gill chloride cells and nonchloride cell population were isolated. The isolated cells were studied using multiparameter flow cytometry, recording the changes in 1) cell size, 2) cellular granularity, and 3) cell auto fluorescence. Two chloride cell subtypes were identified in the freshwater eels. Within 2-4 days after entering seawater, new subtypes of transitory chloride cell, with bigger cell size and more intense mitochondria autofluorescence, appeared. After full adaptation, two major seawater chloride cell subtypes were again discerned; their sizes were the largest and their mitochondria autofluorescence was the highest. In the second part of the experiment, cell cycle analysis demonstrated a progressive increase in the percentage of gill cells entering the DNA synthesis phase during seawater adaptation, where a small population of mitotic cells was identified in the nonchloride cell population but not in chloride cells. We hypothesize that the mitotic cells identified are stem cells, which will ultimately differentiate into seawater chloride cells. Our results confirm the existence of heterogeneity of chloride cells. Individual subtypes could be isolated in high purity for further studies to elucidate their respective function in mediating ion transport.