| Summary: | In neurons, calcium (Ca²⁺) is used for a variety of processes and, as a result, its
intracellular regulation is an important factor in determining which messenger systems are activated and the extent of that activation. To date two principal methods have been used to induce increases in the free intracellular Ca²⁺ concentration ([Ca²⁺]i) in populations of neurons. These are the activation of either voltage or ligand gated Ca²⁺-channels by supervisions with either high K⁺-solutions or solutions containing ligand gated Ca²⁺-channel agonists.
Unfortunately, with the use of either of these methods, substantial Ca²⁺-gradients are formed and decay kinetics are difficult to measure as a result of the lag time between agonist application and peak [Ca²⁺]i. In order to circumvent these problems a novel method has been developed to examine potential Ca²⁺-homeostatic mechanisms in cultured post-natal rat hippocampal neurons. The method requires the monitoring of the recovery of background subtracted fluorescence levels of the Ca²⁺-indicator dye fluo-3 at 20-22 °C immediately
following a rapid increase in [Ca²⁺]i induced by flash photolysis of the caged Ca²⁺-compound nitrophenyl-EGTA (NP-EGTA).
A variety of methods or drugs were used in an attempt to block efflux of Ca²⁺ by the
plasma membrane Na⁺/Ca²⁺ exchanger (PM-Na⁺/Ca²⁺) or uptake of Ca²⁺ into mitochondria. We found that many of the experimental manipulations produced a decrease in intracellular pH
(pH[sub i]) measured in sister cultures using the pH sensitive dye 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). These changes in pH[sub i] are likely to influence neuronal Ca²⁺-homeostatic mechanisms directly by decreasing mitochondrial Ca²⁺-uptake, decreasing the affinity of Ca²⁺-binding proteins for Ca²⁺, and inhibition of the PM-ATPase. Accordingly, for each experimental situation we determined the appropriate amount of the weak base
trimethylamine (TMA) required to restore baseline pH[sub i] prior to flash photolysis. When the Na⁺/Ca⁺ exchanger was inhibited by replacement of all extracellular Na⁺ with
Af-methyl-D-glucamine (NMDG) we observed a significant prolongation in the rate of recovery to baseline Ca²⁺-levels. However, this treatment markedly reduced pHi and when this effect was corrected with 5 mM TMA, the resulting recovery rates of fluo-3 fluorescent intensities were virtually identical to those seen in control situations. Similar results were found when all external Na⁺ was replaced by Li⁺. These experiments were particularly revealing since the effects of Li⁺ on pH[sub i] were time dependent. At early time intervals pH[sub i] was reduced and there was an apparent reduction in the rate of recovery of fluo-3 fluorescent intensities. At later
times pH[sub i] was restored towards normal values and no effect of blockade of the Na⁺/Ca²⁺
exchanger on Ca²⁺ recovery rates was observed. It is concluded therefore that, in our neuronal preparations, the Na⁺/Ca²⁺ exchanger is relatively unimportant in the removal of Ca²⁺-loads induced by the caged Ca²⁺-compound NP-EGTA. Inhibition of mitochondrial Ca²⁺-uptake, using the protonophore carbonyl cyanide chlorophenylhydrazone (CCCP), resulted in a reduction in pH; which could be restored with 2 mM TMA. Under these conditions the rate of recovery of Ca²⁺-levels was significantly slower than controls. Similar results were found using the respiratory chain inhibitor rotenone. In order to avoid the potentially confounding effects that mitochondrial Ca²⁺-uptake inhibitors
such as CCCP have on ATP-levels, oligomycin was added to the superfusate to block reverse
activity of the ATP-synthase in order to sustain ATP levels, over the short term. Under these conditions we still observed a significant prolongation in the rates of recovery to baseline values. We conclude therefore that the uptake of Ca²⁺ into mitochondria is an important homeostatic mechanism in cultured post-natal rat hippocampal neurons following a Ca²⁺-load
induced by photolysis of NP-EGTA. === Medicine, Faculty of === Cellular and Physiological Sciences, Department of === Graduate
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