A DIGITIZED FLUORESCENCE IMAGING STUDY OF INTRACELLULAR FREE CALCIUM,MITOCHONDRIAL INTEGRITY AND CYTOTOXICITY IN RAT RENAL-CELLS EXPOSED TO IONOMYCIN, A CALCIUM IONOPHORE

The objective of this study was to explore the role of extracellular C a2+ and mitochondrial integrity in ionomycin-induced cytotoxicity in p rimary cultures of rat kidney cortical epithelial cells using digitize d fluorescence imaging (DFI), which is a powerful tool for continuousl y observing the dynamic intracellular biochemistry of single living ce lls. Using DFI, intracellular free calcium ion concentration ([Ca2+](i )), mitochondrial membrane potential and loss of cell viability in ind ividual rat renal cortical epithelial cells were examined temporally b y fura-2, rhodamine 123 (Rh-123) and propidium iodide (PI), respective ly. Images were taken within 10 min after exposure to 5 and 10 mu M io nomycin. These three parameters, [Ca2+](i),chondrial membrane potentia l and cell viability, were also measured in populations of cells by a multiwell fluorescence scanner with fluo-3, Rh-123 and PI, respectivel y. Cytotoxicity was also assessed by two colorimetric cytotoxicity tes ts (LDH leakage and mitochondriar MTT reduction). Using DFI, the fluor escence scanner and the colorimetric cytotoxicity tests, we found that exposure of primary cultures of rat kidney cortical epithelial cells to high concentrations of ionomycin (5 and 10 mu M) caused a rapid and sustained rise in [Ca2+](i), which preceded dissipation of the mitoch ondrial membrane potential and loss of cell viability and that chelati on of extracellular Ca2+ with EGTA attenuated these responses. We demo nstrated the value of using DFI to continuously observe the dynamic in tracellular biochemistry of single living cells by establishing a sequ ence of elevated [Ca2+ ](i), dissipation of mitochondrial membrane pot ential and cytotoxicity. We conclude that a combination of the influx of extracellular Ca2+ and loss of mitochondrial integrity may be respo nsible for the cytotoxicity observed in individual renal cells and pop ulations of renal cells after treatment with ionomycin.