CALCIUM HOMEOSTATIC MECHANISMS OPERATING IN CULTURED POSTNATAL RAT HIPPOCAMPAL-NEURONS FOLLOWING FLASH-PHOTOLYSIS OF NITROPHENYL-EGTA

1. We examined Ca2+ homeostatic mechanisms in cultured postnatal rat h ippocampal neurones by monitoring the recovery of background-subtracte d fluo-3 fluorescence levels at 20-22 degrees C immediately following a rapid increase in Ca2+ levels induced by flash photolysis of the cag ed Ca2+ compound nitrophenyl-EGTA (NP-EGTA). 2. A variety of methods o r drugs were used in an attempt to block specifically efflux of Ca2+ b y the plasmalemmal Na+-Ca2+ exchanger or uptake of Ca2+ into mitochond ria. 3. Many of the experimental manipulations produced a decrease in intracellular pH (pH(1)) measured in sister cultures using the pH-sens itive dye -bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). Accordingly, in each case, we determined the appropriate amount of the weak base trimethylamine (TMA) required to restore baseline pH(1) pri or to flash photolysis. 4. Blockade of the plasmalemmal Na+-Ca2+ excha nger by replacement of external Na+ with either Li+ or iv-methyl-D-glu camine (NMDG) markedly reduced pH(1) but did not affect the rate of re covery of fluo-3 fluorescence intensities once pH(1) was restored. 5. Inhibition of mitochondrial Ca2+ uptake, using the protonophore carbon yl cyanide m-chloro-phenylhydrazone (CCCP), resulted in a reduction in pH(1), which could be restored by the addition of 2 mM TMA. Under the se conditions the rate of recovery of Ca2+ levels was significantly sl ower than in the controls. Similar results were found using the respir atory chain inhibitor rotenone. 6. We conclude that, when the potentia l effects of changes in pH(1) are taken into account, mitochondria app ear to sequester significant amounts of Ca2+ in the neuronal preparati ons used.