Calcium homeostatic mechanisms operating in cultured postnatal rat hippocampal neurones following flash photolysis of nitrophenyl-EGTA

1. We examined Ca2+ homeostatic mechanisms in cultured postnatal rat hippocampal neurones by monitoring the recovery of background-subtracted fluo-3 fluorescence levels at 20-22 degrees C immediately following a rapid increase in Ca2+ levels induced by flash photolysis of the caged Ca2+ compound nitrophenyl-EGTA (NP-EGTA). 2. A variety of methods or drugs were used in attempt to block specifically efflux of Ca2+ by the plasmalemmal Na(+)-Ca2+ exchanger or uptake of Ca2+ into mitochondria. 3. Many of the experimental manipulations produced a decrease in intracellular pH (pHi) measured in sister cultures using the pH-sensitive dye 2',7'-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 pHi prior to flash photolysis. 4. Blockade of the plasmalemmal Na(+)-Ca2+ exchanger by replacement of external Na+ with either Li+ or N-methyl-D-glucamine (NMDG) markedly reduced pHi but did not affect the rate of recovery of fluo-3 fluorescence intensities once pHi was restored. 5. Inhibition of mitochondrial Ca2+ uptake, using the protonophore carbonyl cyanide m-chloro-phenylhydrazone (CCCP), resulted in a reduction in pHi, which could be restored by the addition of 2 mM TMA. Under these conditions the rate of recovery of Ca2+ levels was significantly slower than in the controls. Similar results were found using the respiratory chain inhibitor rotenone. 6. We conclude that, when the potential effects of changes in pHi are taken into account, mitochondria appear to sequester significant amounts of Ca2+ in the neuronal preparations used.