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The rate of spontaneous efflux of Ca from liver mitochondria incubated in the absence of ATP and Mg increases with time and is associated with a synchronous collapse of membrane potential and with Pi efflux. In the presence of Mg and ATP the ruthenium-red-induced Ca efflux does not change with time. The activity of the Ca efflux pathway in Pi-depleted mitochondria is 15-fold greater than in mitochondria equilibrated with 3.3 m M Pi. 50 % inhibition is caused by 0.3 mM Pi. The membrane potential is not affected by changes in Pi concentration, although the steadystate extra-mitochondria1 free Ca concentration reflects the alterations in efflux rate. In the presence of Pi, the ruthenium-red-induced efflux rate is independent of the total matrix Ca content; however in P,-depleted mitochondria, with acetate substituting as permeant anion, the efflux rate increases with total matrix Ca content. The lowered efflux rate in the presence of Pi is not due to a limitation in the rate of dissociation of the matrix Ca-phosphate complex. The efflux pathway is activated by a lowered membrane potential, but the relative effect of Pi is retained. Under the present conditions Na slightly inhibits the efflux rate. The lack of an effect of total matrix Ca content on the efflux rate in the presence of Pi is used as the basis of a highly accurate determination of the activity of the Ca uniporter as a function of external free Ca concentration.The role of Pi in liver mitochondrial Ca transport has been the subject of considerable debate [for reviews see 1-31. It is generally agreed that Pi crosses the inner membrane electroneutrally, by nominal proton symport [4], and that it therefore allows Ca to be accumulated in the matrix without alkalinization of the matrix [I -31. It is also generally [I -31 accepted that Ca and Pi form a complex in the matrix and that the extensive acidification of the medium which can be observed under these conditions [5] is a consequence of the expulsion by the respiratory chain of the protons generated in the matrix during the formation of this complex [6].In addition to these effects, however, Pi has variously been postulated to activate the Ca uniporter [7,8], to be involved in a Ca-Pi symport for the uptake [8] or release [9] of Ca and to activate the independent efflux pathway [lo], while we have reported the converse, namely that Pi inhibits the efflux pathway [Ill. Superimposed upon this complexity is the long established ability of Pi to potentiate the spontaneous efflux of Ca from mitochondria incubated in media lacking Mg and The purpose of the present paper is to resolve some of these complexities, firstly by determining the precise temporal sequence of Ca efflux, Pi release and All/ collapse during spontaneous Ca efflux. Secondly we investigate the interaction of Pi concentration, membrane potential and monovalent cations on the activity of the efflux pathway. Finally we confirm and attempt to explain why the Ca efflux rate appears not to depend upon the matrix Ca content in P,-containing mitocho...

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...

Complex I (NADH:ubiquinone oxidoreductase) is responsible for most of the mitochondrial H2O2 release, both during the oxidation of NAD-linked substrates and during succinate oxidation. The much faster succinate-dependent H2O2 production is ascribed to Complex I, being rotenone-sensitive. In the present paper, we report high-affinity succinate-supported H2O2 generation in the absence as well as in the presence of GM (glutamate/malate) (1 or 2 mM of each). In brain mitochondria, their only effect was to increase from 0.35 to 0.5 or to 0.65 mM the succinate concentration evoking the semi-maximal H2O2 release. GM are still oxidized in the presence of succinate, as indicated by the oxygen-consumption rates, which are intermediate between those of GM and of succinate alone when all substrates are present together. This effect is removed by rotenone, showing that it is not due to inhibition of succinate influx. Moreover, alpha-oxoglutarate production from GM, a measure of the activity of Complex I, is decreased, but not stopped, by succinate. It is concluded that succinate-induced H2O2 production occurs under conditions of regular downward electron flow in Complex I. Succinate concentration appears to modulate the rate of H2O2 release, probably by controlling the hydroquinone/quinone ratio.