Synaptic vesicles isolated from sheep brain cortex exhibit an ATP-dependent Ca 2+ accumulation that is inhibited by the protonophore uncoupler carbonyl cyanide m-chorophenylhydrazone (CCCP) and completely released by the Ca 2+ ionophore ionomycin. This transport activity was sensitive to the V-type ATPase inhibitor, bafilomycin, but not to the P-type ATPase inhibitor, vanadate. We also observed that the proton gradient, established across the synaptic vesicle membranes in the presence of ATP, is partially dissipated by the addition of Ca 2+ (100-860 mM) in correlation to an increase of ATP hydrolysis by the H + -pumping ATPase. In contrast, the activity of the H

As we previously reported, synaptic vesicles isolated from sheep brain cortex contain a Ca 2q rH q antiport that permits Ca 2qŽ .q accumulation inside the vesicles ; 5 nmolrmg protein at expenses of the pH gradient generated by the H -pumping ATPase. We observed that the system associates Ca 2q influx to H q release and operates with low affinity for Ca 2q . In the present work, we found that Ca 2q rH q antiport mediates exchange of protons with other cations such as Zn 2q and Cd 2q , suggesting that these cations and Ca 2q share the same transporter molecules to enter the intravesicular space. Zn 2q and Cd 2q induce H q release in a concentration-dependent manner Ž .2q Ž . fluorimetrically evaluated and they inhibit the antiport-mediated Ca uptake by the vesicles isotopically measured . In contrast, large cations such as Ba 2q and Cs q do not alter Ca 2q influx and they are unable to induce proton release from the vesicles. With respect to Sr 2q , which has an intermediary size relatively to the other groups of cations, we found that it does not induce H q liberation from the vesicles, but it has a concentration-dependent inhibitory effect on the Ca 2q -induced H q release and Ca 2q uptake by the vesicles. These results indicate that the cation selectivity of the synaptic vesicles Ca 2q rH q antiport is essentially determined by the size of the cation transported into the vesicles. q

Synaptic vesicles isolated from sheep brain cortex accumulate Ca2+ by a mechanism of secondary active transport associated to the H(+)-pump activity. The process can be visualized either by measuring Ca(2+)-induced H+ release or DeltapH-dependent Ca2+ accumulation. We observed that the amount of Ca2+ taken up by the vesicles increases with the magnitude of the DeltapH across the membrane, particularly at Ca2+ concentrations (approximately 500 microM) found optimal for the antiporter activity. Similarly, H+ release induced by Ca2+ increased with the magnitude of DeltapH. However, above 60% DeltapH (high H(+)-pump activity), the net H+ release from the vesicles decreased as the pump-mediated H+ influx exceeded the Ca(2+)-induced H+ efflux. We also observed that the Ca2+/H+ antiport activity depends, essentially, on the DeltapH component of the electrochemical gradient (approximately 3 nmol Ca2+ taken up/mg protein), although the Deltaphi component may also support some Ca2+ accumulation by the vesicles (approximately 1 nmol/mg protein) in the absence of DeltapH. Both Ca(2+)-induced H+ release and DeltapH-dependent Ca2+ uptake could be driven by an artificially imposed proton motive force. Under normal conditions (H+ pump-induced DeltapH), the electrochemical gradient dependence of Ca2+ uptake by the vesicles was checked by inhibition of the process with specific inhibitors (bafilomycin A(1), ergocryptin, folymicin, DCCD) of the H(+)-pump activity. These results indicate that synaptic vesicles Ca2+/H+ antiport is indirectly linked to ATP hydrolysis and it is essentially dependent on the chemical component (DeltapH) of the electrochemical gradient generated by the H(+)-pump activity.

Synaptic vesicles, isolated from a sheep brain cortex, accumulate Ca(2+) in a manner that depends on the pH and pCa values. In the presence of 100 microM CaCl(2), most of the Ca(2+) taken up by the vesicles was vanadate-inhibited (86%) at pH 7.4, whereas at pH 8.5, part of the Ca(2+) accumulated (36%) was DeltapH-dependent (bafilomycin and CCCP inhibited) and part was insensitive to those drugs (31%). We also observed that both vanadate-sensitive and bafilomycin-sensitive Ca(2+) accumulations were completely released by the Ca(2+) ionophore, ionomycin, and that these processes work with high (K(0.5)=0.6 microM) and low (K(0.5)=217 microM) affinity for Ca(2+), respectively. The DeltapH-dependent Ca(2+) transport appears to be largely operative at Ca(2+) concentrations (>100 microM) which completely inhibited the vanadate-sensitive Ca(2+) uptake. These Ca(2+) effects on the Ca(2+) accumulation were well correlated with those observed on the vanadate-inhibited Ca(2+)-ATPase and bafilomycin-inhibited H(+)-ATPase, respectively. The Ca(2+)-ATPase activity reached a maximum at about 25 microM (pH 7.4) and sharply declined at higher Ca(2+) concentrations. In contrast, Ca(2+) had a significant stimulatory effect on the H(+)-ATPase between 250 and 500 microM Ca(2+) concentration. Furthermore, we found that DeltapH-sensitive Ca(2+) transport was associated with proton release from the vesicles. About 21% of the maximal proton gradient was dissipated by addition of 607.7 microM CaCl(2) to the reaction medium and, if CaCl(2) was present before the proton accumulation, lower pH gradients were reached. Both vanadate-inhibited and bafilomycin-inhibited systems transported Ca(2+) into the same vesicle pool of our preparation, suggesting that they belong to the same cellular compartment. These results indicate that synaptic vesicles of the sheep brain cortex contain two distinct mechanisms of Ca(2+) transport: a high Ca(2+) affinity, proton gradient-independent Ca(2+) pump that has an optimal activity at pH 7.4, and a low Ca(2+) affinity, proton gradient-dependent Ca(2+)/H(+) antiport that works maximally at pH 8.5.

The involvement of Ca(2+)-storage organelles in the modulation of synaptic transmission is well-established [M.K. Bennett, Ca(2+) and the regulation of neurotransmitter secretion, Curr. Opin. Neurobiol. 7 (1997) 316-322 [1]; M.J. Berridge, Neuronal calcium signaling, Neuron 21 (1998) 13-26 [2]; Ph. Fossier, L. Tauc, G. Baux, Calcium transients and neurotransmitter release at an identified synapse, Trends Neurosci. 22 (1999) 161-166 [7] ]. Various Ca(2+) sequestering reservoirs (mitochondria, endoplasmic reticulum and synaptic vesicles) have been reported at the level of brain nerve terminals [P. Kostyuk, A. Verkhratsky, Calcium stores in neurons and glia, Neuroscience 63 (1994) 381-404 [18]; V. Mizuhira, H. Hasegawa, Microwave fixation and localization of calcium in synaptic terminals using X-ray microanalysis and electron energy loss spectroscopy imaging, Brain Res. Bull. 43 (1997) 53-58 [21]; A. Parducz, Y. Dunant, Transient increase of calcium in synaptic vesicles after stimulation, Neuroscience 52 (1993) 27-33 [23]; O.H. Petersen, Can Ca(2+) be released from secretory granules or synaptic vesicles?, Trends Neurosci. 19 (1996) 411-413 [24] ]. However, the knowledge of the specific contribution of each compartment for spatial and temporal control of the cytoplasmic Ca(2+) concentration requires detailed characterization of the Ca(2+) uptake and Ca(2+) release mechanisms by the distinct intracellular stores. In this work, we described rapid and simple experimental procedures for analysis of a Ca(2+)/H(+) antiport system that transport Ca(2+) into synaptic vesicles at expenses of the energy of a DeltapH generated either by activity of the proton pump or by a pH jumping of the vesicles passively loaded with protons. This secondary active Ca(2+) transport system requires high Ca(2+)100 microM) for activation, it is dependent on the chemical component (DeltapH) of the proton electrochemical gradient across the synaptic vesicle membrane and its selectivity is essentially determined by the size of the transported cation [P.P. Gonçalves, S.M. Meireles, C. Gravato, M.G. P. Vale, Ca(2+)-H(+)-Antiport activity in synaptic vesicles isolated from sheep brain cortex, Neurosci. Lett. 247 (1998) 87-90 [10]; P.P. Gonçalves, S.M. Meireles, P. Neves, M.G.P. Vale, Ionic selectivity of the Ca(2+)/H(+) antiport in synaptic vesicles of sheep brain cortex, Mol. Brain Res. 67 (1999) 283-291 [11]; P.P. Gonçalves, S.M. Meireles, P. Neves, M.G.P. Vale, Synaptic vesicle Ca(2+)/H(+) antiport: dependence on the proton electrochemical gradient, Mol. Brain Res. 71 (1999) 178-184 [12] ]. The protocols described here allow to ascertain the characteristics of the Ca(2+)/H(+) antiport in synaptic vesicles and, therefore, may be useful for clarification of the physiological role of synaptic vesicles in fast buffering of Ca(2+) at various sites of the neurotransmission machinery.