The regulation of cytosolic pH in isolated presynaptic nerve terminals from rat brain.

Abstract

A B S T R A C T Cytosolic pH (pH0 was measured in presynaptic nerve terminals isolated from rat brain (synaptosomes) using a fluorescent pH indicator, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The synaptosomes were loaded with BCECF by incubation with the membrane-permanent acetoxymethyl ester derivative of BCECF, which is hydrolyzed by intracellular esterases to the parent compound, pHi was estimated by calibrating the fluorescence signal after permeabilizing the synaptosomal membrane by two different methods. Synaptosomes loaded with 15-90 #M BCECF were estimated to have a pHi of 6.94 + 0.02 (mean + standard error; n = 54) if the fluorescence signal was calibrated after permeabilizing with digitonin; a similar value was obtained using synaptosomes loaded with 10 times less BCECF (6.9 3= 0.1; n = 5). When the fluorescence signal was calibrated by permeabilizing the synaptosomal membrane to H § with gramicidin and nigericin, pHi was estimated to be 7.19 3= 0.03 (n = 12). With the latter method, pHi = 6.95 3= 0.09 (n = 14) when the synaptosomes were loaded with 10 times less BCECF. Thus, pH~ in synaptosomes was -7.0 and could be more precisely monitored using the digitonin calibration method at higher BCECF concentrations. When synaptosomes were incubated in medium containing 20 mM NH4CI and then diluted into NH4Cl-free medium, pH~ immediately acidified to a level of ~6.6. After the acidification, phi recovered over a period of a few minutes. The buffering capacity of the synaptosomes was estimated to be ~50 mM/pH unit. Recovery was substantially slowed by incubation in an Na-free medium, by the addition of amiloride (K~ = 3 #M), and by abolition of the Nao/Nal gradient, pH~ and its recovery after acidification were not affected by incubation in an HCOs-containing medium; disulfonic stilbene anion transport inhibitors (SITS and DIDS, 1 mM) and replacement of CI with methylsulfonate did not affect the rate of recovery of pHi. It appears that an Na+/H + antiporter is the primary regulator of pH~ in mammalian brain nerve terminals.

Changes in intracellular pH associated with glutamate excitotoxicity

“…Two different transport processes have been identified in hippocampal neurons recovering from an NH,Cl-induced acid load: a DIDSsensitive HCO, --dependent acid extruder, and an amilorideinsensitive, harmaline-sensitive Na+ -H+ antiporter (Raley- Susman et al, 1991). Similar exchange mechanisms have been characterized in sympathetic neurons, synaptosomes, and leech neurons (Deitmer and Schlue, 1987;Tolkovsky and Richards, 1987;Nachshen and Drapeau, 1988). In agreement with this previous work, DIDS, harmaline, and removal of external sodium reduced the rate of recovery from GLU-induced acid loads among hippocampal cells.…”

Regulation of Intracellular pH in Single Rat Cortical Neurons in vitro: A Microspectrofluorometric Study

“…Furthermore, the introduc tion of C02/HC03 -following an acidification in the presence of harmaline did not enhance the recovery of pHi' Based on these observations, we conclude that rat cortical neurons did not utilize Na + dependent HC03 -IC1-exchange for regulation of their pHi after an acid load. Our results are thus in accordance with those previously reported for syn aptosomes (Nachsen and Drapeau, 1988) and cere bellar Purkinje cells (Gaillard and Dupont, 1990) (see introductory section).…”

“…We report an average steady-state pHj of 7.00 and 7.09 in cultured rat neurons main tained at 37°C (pHe 7.35) in nominally bicarbonate free and in C02/HC03 --containing media, respec tively. This steady-state pHj compares well with values previously reported for vertebrate neurons maintained under similar conditions (Tolkovsky and Richards, 1987;Nachsen and Drapeau, 1988;Gaillard and Dupont, 1990;Nedergaard et aI., 1991;Raley-Susman et aI., 1991).…”

“…Furthermore, neu rons failed to recover from an acid load when pHe was simultaneously decreased, maintaining a lower than normal pHi until pHe was returned to normal. Although not examined in any detail, similar results were reported in rat superior cervical ganglion cells (Tolkovsky and Richards, 1987) and rat brain syn aptosomes (Nachsen and Drapeau, 1988). Similar observations have also been made in other cells (L' Allemain et aI., 1984;Moolenaar and Tertoolen, 1984;Kettenmann and Schlue, 1988) .…”

“…Although different type of neurons have been the subject of subsequent stud ies (Chesler, 1990), there have been comparatively few reports of pH regulation in vertebrate neurons. Some authors have concluded that an Na + IH+ ex changer is solely responsible for acid extrusion in vertebrate neurons (Nachsen and Drapeau, 1988;Gaillard and Dupont, 1990), while others have found evidence for additional HC03 --dependent acid extrusion mechanisms (Chesler, 1986;Pocock and Richards, 1989;Raley-Susman et aI., 1991). There are no previous studies of pH regulation in vertebrate neurons following alkaline transients or concerning the effect of pHe on pHi-regulating mechanisms.…”