Intracellular acidification of the leech giant glial cell evoked by glutamate and aspartate

Glutamate is an excitatory receptor agonist in both neurones and glial cells, and, in addition, glutamate is also a substrate for glutamate transporter in glial cells. We have measured intracellular and extracellular pH changes induced by bath application of glutamate, its receptor agonist kainate, and its transporter agonist aspartate, in the giant neuropile glial cell in the central nervous system of the leech Hirudo medicinalis, using double-barrelled pH-sensitive microelectrodes. The giant glial cells responded to glutamate and aspartate (100-500 microM), and kainate (5-20 microM) with a membrane depolarization or an inward current and with a distinct intracellular acidification. Glutamate and aspartate (both 500 microM) evoked a decrease in intracellular pH (pHi) by 0.187 +/- 0.081 (n = 88) and 0.198 +/- 0.067 (n = 86) pH units, respectively. With a resting pHi of 7.1 or 80 nM H+, these acidifications correspond to a mean increase of the intracellular H+ activity by 42 nM and 45 nM. Kainate caused a decrease of pHi by 0.1-0.35 pH units (n = 15). The glutamate/aspartate-induced decrease in pHi was not significantly affected by the glutamate receptor blockers kynurenic acid (1 mM) and 6-cyano-7-dinitroquinoxaline-2,3-dione (CNQX, 50-100 microM), which greatly reduced the kainate-induced change in pHi. Extracellular alkalinizations produced by glutamate and aspartate were not affected by CNQX. Reduction of the external Na+ concentration gradually decreased the intracellular pH change induced by glutamate/aspartate, indicating half maximal activation of the acidifying process at 5-10 mM external Na+ concentration. When all external Na+ was replaced by NMDG+, the pHi responses were completely suppressed (glutamate) or reduced to 10% (aspartate). When Na+ was replaced by Li+, the glutamate- and aspartate-evoked pHi responses were reduced to 18% and 14%, respectively. Removal of external Ca2+ reduced the glutamate- and aspartate-induced pHi responses to 93 and 72%, respectively. The glutamate/aspartate-induced intracellular acidifications were not affected by the putative glutamate uptake inhibitor amino-adipidic acid (1 mM). DL-aspartate-beta-hydroxamate (1 mM), and dihydrokainate (2 mM), which caused some pHi decrease on its own, reduced the glutamate/aspartate-induced pHi responses by 40 and 69%, respectively. The putative uptake inhibitor DL-threo-beta-hydroxyaspartate (THA, 1 mM) induced a prominent intracellular acidification (0.36 +/- 0.05 pH units, n = 9), and the pHi change evoked by glutamate or aspartate in the presence of THA was reduced to less than 10%. The results indicate that glutamate, aspartate, and kainate produce substantial intracellular acidifications, which are mediated by at least two independent mechanisms: 1) via activation of non-NMDA glutamate receptors and 2) via uptake of the excitatory amino acids into the leech glial cell.