Effects of extracellular pH reductions on [³H]d-aspartate and [³H]noradrenaline release by presynaptic nerve terminals isolated from rat cerebral cortex

We analyzed the effects of extracellular pH reductions on the release of [³H]d-aspartate ([³H]d-ASP) and [³H]noradrenaline ([³H]NA) from cerebrocortical synaptosomes isolated from rats. Synaptosomes were superfused with standard medium at a physiologic pH of 7.4 and with acidified medium with a pH of 6.00, 5.50, or 5.0. Medium acidification produced pH-dependent stimulation of [³H]d-ASP release. The increase amounted to 202 ± 12.6% when the pH was reduced to 5.5. The [³H]d-ASP release evoked by low pH (5.50) was still observed in the absence of Ca²⁺ ions, but it was abolished by dl-threo-β-benzyloxyaspartate (dl-TBOA) (100 μM), which inhibits neuronal glutamate/aspartate transport. Exposure to 5-(N,N-hexamethylene)-amiloride (EIPA) (30–100 μM), a selective inhibitor of Na⁺/H⁺ exchange, caused concentration-dependent stimulation of [³H]d-ASP release; the increase observed with EIPA 30 μM was 160 ± 12%. The EIPA-induced release was not dependent on the presence of Ca²⁺ ions in the medium, but it was abolished when synaptosomes were pretreated with 100 μM dl-TBOA. Reduction of the extracellular pH (5.50–5.0) also stimulated the release of [³H]NA from rat cortical synaptosomes. Exposure to medium with a pH of 5.50 increased basal release by 136 ± 9.5%. The release-stimulating effect of this medium was calcium-independent and abolished by 3 μM desipramine (DMI). [³H]NA release was also stimulated by EIPA. The increase induced by a concentration of 30 μM amounted to 136 ± 9.50%, and this effect was calcium-independent and abolished by pretreatment with DMI (3 μM). These findings suggest that reduction of the extracellular pH stimulates release of [³H]D-ASP and [³H]NA by reversing neurotransmitter transport in the nerve terminal. This reversal might be activated by increased cytosolic concentrations of the transmitters caused by reduction of the pH gradient between the cytoplasm and the synaptic vesicles that take up the transmitters. This hypothesis is confirmed by the results of experiments conducted with EIPA. Selective blockade of Na⁺/H⁺ exchange with this compound induces accumulation of H⁺ in the nerve terminals and intracellular acidification, which leads to calcium-independent, transporter-mediated release of [³H]d-ASP and [³H]NA.