INTRACELLULAR ACIDIFICATION AND CA2+ TRANSIENTS IN CULTURED RAT CEREBELLAR ASTROCYTES EVOKED BY GLUTAMATE AGONISTS AND NORADRENALINE

The effect of different neurotransmitters on the intracellular pH (pH(i)) and intracellular calcium (Ca-i(2+)) was studied in cultured astrocytes from neonatal rat cerebellum, using the fluorescent dyes 2,7'-bis(carboxyethyl)-5,6-carboxy-fluorescein (BCECF) and Fura-2. Application of glutamate or kainate (100 mu M) in a HEPES-buffered, CO2/HCO3--free saline induced a decrease in pH(i) and an increase in Ca-i(2+). Amplitude and time course of the pH(i) and Ca-i(2+) transients were different. Glutamate and kainate evoked a mean acidification of 0.22 +/- 0.05 (n = 29) and 0.20 +/- 0.04 (n = 12) pH units, respectively. The changes in pH(i) and Ca-i(2+) induced by kainate, but not by glutamate were inhibited by 6-cyano-7-dinitroquinozalin-2,3-dion (CNQX; 50 mu M). In order to elucidate the mechanism of the agonist-induced acidification, whether the pH(i) changes were secondary to the Ca2+ rises was tested. In the absence of extracellular Ca2+, the kainate-induced Ca-i(2+) transient was suppressed, while the intracellular acidification was only reduced by 13%. Removal of extracellular Ca2+ reduced the glutamate-induced pH(i) change by 8%, while the second component of the Ca-i(2+) transient was abolished. Application of trans-(+)-1-amino-(1S,3R)-cyclopentadicarboxylic acid (t-ACPD, 100 mu M), a metabotropic glutamate receptor agonist, and of noradrenaline (20 mu M) evoked a Ca-i(2+) increase, but no change of pH(i). D-aspartate, which has a low affinity to glutamate receptors, but is known to be transported by the glutamate uptake system in some astrocytes, evoked an intracellular acidification, similar to that induced by glutamate, but no Ca-i(2+) transient. The results suggest that the kainate-induced acidification is only partly due to the concomitant Ca-i(2+) rise, while the glutamate/aspartate-induced acidification is mainly due to the activation of the glutamate uptake system. (C) 1995 Wiley-Liss, Inc.