Na+ and Ca2+ homeostatic mechanisms in isolated chloride cells of the teleost Oreochromis mossambicus analysed by confocal laser scanning microscopy

Abstract

Chloride cells (CCs; recognised by the vital mitochondrial stain DASPEI) and pavement cells (PCs) isolated from tilapia opercular epithelium were adhered to Cell-Tak-coated glass coverslips and loaded with fluorescent probes for the measurement of intracellular concentrations of Na+ or Ca2+. Basal levels of cytosolic Na+ and Ca2+ ranged from 6.4 to 16.5mmol l-1 and from 76 to 110 nmol l-1, respectively, and did not differ between CCs and PCs. In CCs, inhibition of Na+/K+-ATPase by ouabain or Cu2+ increased intracellular [Na+]. Replacing extracellular Na+ with N-methyl-D-glucamine+ led to a rise in cytosolic [Ca2+] that was dependent on the extracellular [Ca2+], indicating that a Na+/Ca2+ exchanger was operating in reverse mode (importing Ca2+). The forward mode of this exchanger could be demonstrated by inhibition with bepridil. The CC has various pathways for passive Na+ influx: a tetrodotoxin-sensitive pathway, an amiloride-sensitive pathway and other as yet unidentified pathways.