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Abstract:We introduce the use of the pH-sensitive dye acridine orange (AO) to monitor exo/endocytosis of acidic neurotransmitter-containing vesicles in synaptosomes. AO is accumulated exclusively in acidic v-ATPase-dependent bafilomycin (Baf)-sensitive compartments. A fraction of the accumulated AO is rapidly released (fluorescence increase) upon depolarization with KCl in the presence of Ca 2ϩ . The release (completed in 5-6 s) is followed by reuptake to values below the predepolarization baseline. The reuptake, but not the release, is inhibited by Baf added 5 s prior to KCl. In a similar protocol, Baf does not affect the initial fast phase of glutamate release measured enzymatically, but it abolishes the subsequent slow phase. Thus, the fast AO release corresponds to the rapid phase of glutamate release and the slow phase depends on vesicle cycling. AO reuptake depends in part on the progressive accumulation of acidloaded vesicles during cycling. Stopping exocytosis at selected times after KCl by Ca 2ϩ removal with EGTA evidences endocytosis: Its T 1/2 was 12 Ϯ 0.6 s. The K A ϩ , channel inhibitors 4-aminopyridine (100 M) and ␣-dendrotoxin (10 -100 nM) are known to induce glutamate release by inducing the firing of Na ϩ channels; their action is potentiated by the activation of protein kinase C. Also these agents promote a Ca 2ϩ -dependent AO release, which is prevented by the Na ϩ channel inhibitor tetrodotoxin and potentiated by 4␤-phorbol 12-myristate 13-acetate (PMA). With ␣-dendrotoxin, endocytosis was monitored by stopping exocytosis at selected times with EGTA or alternatively with Cd 2ϩ or tetrodotoxin. The T 1/2 of endocytosis, which was unaffected by PMA, was 12 Ϯ 0.4 s with EGTA and Cd 2ϩ and 9.5 Ϯ 0.5 s with tetrodotoxin. Protein kinase C activation appeared to facilitate vesicle turnover. Key Words: Acridine orangeExocytosis-Endocytosis-Synaptosome-Rat cerebral cortex-Acidic neurotransmitter-containing vesicles. J. Neurochem. 72, 625-633 (1999).Synaptosomes retain all the machinery for the uptake, storage, and exocytosis of neurotransmitters. In situ the terminal functions largely autonomously from the relatively distant cell body, requiring only the electrical signal from the axonal action potential to trigger release, and replenishment of materials via axonal transport mechanisms for long-term survival. The cerebral cortical preparation contains a high proportion of glutamatergic terminals (Nicholls, 1993), thus making nonspecific systems to monitor exo/endocytosis likely representations of the turnover of glutamate-releasing synapses.Although much information is available on exocytosis, only limited information is presently available on the physiology and biochemistry of endocytosis and on vesicle turnover. Available methods are based on measurements of membrane capacitance (Alvarez de Toledo et al., 1993;Thomas et al., 1994) that are not readily applicable to typical fast synapses or on the distribution of fluorescent optical tracers as N-(3-triethylammoniumpropyl)-4-[4-(dibutylamino)styryl]py...

In the absence of extracellular Ca2+, extensive Ca2+ release from the platelet intracellular stores [monitored as an increase of intracellular Ca2+ concentration ([Ca2+]i)] is produced by the combined action of the endomembrane Ca(2+)-ATPase inhibitor thapsigargin and 2 nM ionomycin. The titration of Ca2+ unloading with thapsigargin (plus ionomycin) shows that a substantial fraction of the store-associated Ca2+ is released by 8-10 nM thapsigargin, but that 100-200 nM thapsigargin is required for the complete release. The store depletion obtained in similar conditions with a different endomembrane Ca(2+)-ATPase inhibitor, 2,5-di-(tert-butyl)-1,4-benzohydroquinone (TBHQ), is always incomplete. It is completed by thrombin or by 10 nM thapsigargin. We conclude that two different types of Ca2+ pumps exist in platelets, one sensitive to TBHQ and to high thapsigargin, the other insensitive to TBHQ and sensitive to low thapsigargin. They are distributed separately in discrete subpopulations of the agonist-sensitive stores. The influx of external Ca2+ is maximal when both types of stores are Ca(2+)-depleted, either by high thapsigargin or by the combined action of low thapsigargin and TBHQ.

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