Extracellular protons differentially potentiate the responses of native AMPA receptor subtypes regulating neurotransmitter release

Abstract

1. The effects of pH changes on the basal and evoked release of [(3)H]noradrenaline ([(3)H]NA) and [(3)H]5-hydrohytryptamine ([(3)H]5-HT) from hippocampal synaptosomes and of [(3)H]dopamine ([(3)H]DA) and [(3)H]acetylcholine ([(3)H]ACh) from striatal and cortical synaptosomes were investigated in rat brain. 2. Changing pH between 6.4 and 8.0 did not affect the spontaneous release of the four [(3)H]neurotransmitters; alkalinization to pH 8.8 significantly enhanced release. Acidification to pH 6.4 augmented the AMPA-evoked overflows of [(3)H]NA, [(3)H]5-HT and [(3)H]DA, but not that of [(3)H]ACh. In contrast, lowering pH to 6.4 decreased the K(+)-evoked overflows of [(3)H]NA, [(3)H]5-HT, [(3)H]DA and [(3)H]ACh. 3. AMPA released transmitters in a Ca(2+)-dependent, exocytotic manner since its effects, at pH 7.4 or 6.4, were abolished by omitting external Ca(2+) or by depleting vesicular transmitter stores with bafilomycin A1. AMPA did not evoke carrier-mediated release because the uptake blockers nisoxetine, 6-nitroquipazine, GBR12909 and hemicholinium-3 could not inhibit the AMPA-induced release of [(3)H]NA, [(3)H]5-HT, [(3)H]DA and [(3)H]ACh. 4. Extraterminal acidification to pH 6.4 prevented the potentiating effect of cyclothiazide on the AMPA-evoked release of [(3)H]NA, [(3)H]DA and [(3)H]5-HT, whereas the proton-insensitive AMPA-evoked release of [(3)H]ACh, previously found to be cyclothiazide-insensitive at pH 7.4 was cyclothiazide-resistant also at pH 6.4. 5. To conclude, the cyclothiazide-sensitive AMPA receptors mediating release of NA, 5-HT and DA, but not the cyclothiazide-insensitive AMPA receptors mediating the release of ACh, become more responsive when external pH is lowered to pathophysiologically relevant values. The results with cyclothiazide suggest that H(+) ions may prevent desensitization of some AMPA receptor subtypes.

Figure 1

Effects of pH on the spontaneous release of neurotransmitters from superfused synaptosomes. Hippocampal synaptosomes were prelabeled with [³H]NA or [³H]5-HT, striatal synaptosomes with [³H]DA and cortical synaptosomes with [³H]choline. Synaptosomes were exposed to superfusion media of different pH, for 90 s. Superfusion was then continued with standard medium (pH 7.4) till the end of the experiment. Results are expressed as induced overflow. Data are mean±s.e.m. of four experiments run in triplicate (three superfusion chambers for each experimental condition). ^*P<0.05 versus control.

Figure 2

Effects of pH on the AMPA-evoked release of transmitters from superfused synaptosomes. Synaptosomes were exposed to 100 μM AMPA for 90 s concomitantly with media of different pH. Superfusion was then continued with standard medium (pH 7.4) till the end of the experiment. Results are expressed as induced overflow. Data are mean±s.e.m. of three to seven experiments run in triplicate. ^*P< 0.05 versus control.

Figure 3

Effects of pH on the KCl-evoked release of transmitters from superfused synaptosomes. Synaptosomes were exposed for 90 s to K⁺-enriched (15 mM) media of different pH. Superfusion was then continued till the end of the experiment with standard medium (pH 7.4). Results are expressed as induced overflow. Data are mean±s.e.m. of three to four experiments run in triplicate. ^*P<0.05 versus control.

Figure 4

Effects of protons and cyclothiazide on the AMPA-evoked release of neurotransmitters from superfused synaptosomes. Synaptosomes were exposed for 90 s to AMPA alone or to AMPA plus cyclothiazide concomitantly with media of different pH. Superfusion was then continued with standard medium (pH 7.4) till the end of the experiment. Results are expressed as induced overflow. Data are mean±s.e.m. of three to five experiments run in triplicate. ^*P< 0.05 versus control.