Subcellular distribution of cytosolic Ca 2+ in isolated rat hepatocyte couplets: Evaluation using confocal microscopy

Abstract

Ca 2+ agonists induce Ca 2+ waves and other non-uniform Ca 2+ patterns in the cytosol of epithelial cells. To define subcellular Ca 2+ transients in the cytosol of hepatocytes we examined Fluo-3-loaded isolated rat hepatocyte couplets using confocal microscopy. Optical sections of less than 1 micron in thickness were observed in couplets, and fluorescence from cytosolic Ca 2+ signals was readily distinguished from nuclear, mitochondrial, and lysosomal fluorescence. The nature of the noncytosolic components of the fluorescent images was verified by double labelling with the mitochondrial dye DIOC 6(3) and with the lysosomal marker acridine orange. Using the line scanning mode of confocal microscopy, measurements of cytosolic Ca 2+ made with a frequency of up to 250 Hz and without significant bleaching. It was found that phenylephrine-induced Ca 2+ signals generally began at the basal pole of the hepatocytes, then spread to the canallculus at average speeds of 80 μm/s. These findings demonstrate the utility of confocal line scanning microscopy for detecting rapid changes in the subcellular distribution of cytosolic Ca 2+ in hepatocyte couplets, and suggest that phenylephrine-induced Ca 2+ waves radiate in a basal-to-apical direction in this cell type.