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Calcium homeostatic mechanisms operating in cultured postnatal rat hippocampal neurones following flash photolysis of nitrophenyl‐EGTA
Author(s) -
Sidky Adam O.,
Baimbridge Kenneth G.
Publication year - 1997
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1111/j.1469-7793.1997.579bd.x
Subject(s) - chemistry , egta , protonophore , flash photolysis , biophysics , ionophore , oligomycin , calcium , membrane potential , intracellular ph , biochemistry , intracellular , atpase , membrane , kinetics , biology , physics , enzyme , organic chemistry , quantum mechanics , reaction rate constant
1 We examined Ca 2+ homeostatic mechanisms in cultured postnatal rat hippocampal neurones by monitoring the recovery of background‐subtracted fluo‐3 fluorescence levels at 20–22°C immediately following a rapid increase in Ca 2+ levels induced by flash photolysis of the caged Ca 2+ compound nitrophenyl‐EGTA (NP‐EGTA). 2 A variety of methods or drugs were used in an attempt to block specifically efflux of Ca 2+ by the plasmalemmal Na + –Ca 2+ exchanger or uptake of Ca 2+ into mitochondria. 3 Many of the experimental manipulations produced a decrease in intracellular pH (pH i ) 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 pH i prior to flash photolysis. 4 Blockade of the plasmalemmal Na + ‐Ca 2+ exchanger by replacement of external Na + with either Li + or N ‐methyl‐ d ‐glucamine (NMDG) markedly reduced pH i , but did not affect the rate of recovery of fluo‐3 fluorescence intensities once pH i was restored. 5 Inhibition of mitochondrial Ca 2+ uptake, using the protonophore carbonyl cyanide m ‐chlorophenylhydrazone (CCCP), resulted in a reduction in pH i , which could be restored by the addition of 2 m m TMA. Under these conditions the rate of recovery of Ca 2+ 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 pH i are taken into account, mitochondria appear to sequester significant amounts of Ca 2+ in the neuronal preparations used.