Uptake of GABA by rat brain synaptic vesicles isolated by a new procedure.

Uptake of GABA was demonstrated in rat brain synaptic vesicles which were prepared by a new and efficient procedure. The uptake activity co-purified with the synaptic vesicles during the isolation procedure. The purity of the vesicle fraction was rigorously examined by analysis of marker enzymes and marker proteins and also by immunogold electron microscopy using antibodies against p38 (synaptophysin). Contamination by other cellular components was negligible, indicating that GABA uptake by the synaptic vesicle fraction is specific for synaptic vesicles and not due to the presence of other structure possessing GABA uptake or binding activities. GABA uptake was ATP dependent and similar to the uptake of glutamate, which was assayed for a comparison. Both uptake activities were independent of sodium. They were inhibited by the uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, indicating that the energy for the uptake is provided by an electrochemical proton gradient. This gradient is generated by a proton ATPase of the vacuolar type as suggested by the effects of various ATPase inhibitors on neurotransmitter uptake and proton pumping. Competition experiments revealed that the transporters for GABA and glutamate are selective for the respective neurotransmitters.     

Glutamate uptake by brain synaptic vesicles. Energy dependence of transport and functional reconstitution in proteoliposomes

The dependence of glutamate uptake on ATP-generated proton electrochemical potential was studied in a highly purified preparation of synaptic vesicles from rat brain. At low chloride concentration (4 mM), the proton pump present in synaptic vesicles generated a large membrane potential (inside-positive), associated with only minor acidification. Under these conditions, the rate of L-[3H]glutamate uptake was maximal. In addition, L-glutamate induced acidification of the vesicle interior. D-Glutamate produced only 40% of the effect, and L-aspartate or gamma-aminobutyric acid produced less than 5%. The initial rate of glutamate-induced acidification increased with increasing glutamate concentration. It was saturable and showed first-order kinetics (KM = 0.32 mM). Correspondingly, L-glutamate induced a small reduction in the membrane potential. The rate of ATP hydrolysis was unaffected. In comparison, glutamate had no effect on acidification or membrane potential in resealed membranes of chromaffin granules. At high chloride concentration (150 mM), the vesicular proton pump generated a large pH difference, associated with a small change in membrane potential. Under these conditions, uptake of L-[3H]glutamate by synaptic vesicles was low. For reconstitution, vesicle proteins were solubilized with the detergent sodium cholate, supplemented with brain phospholipids, and incorporated into liposomes. Proton pump and glutamate uptake activities of the proteoliposomes showed properties similar to those of intact vesicles indicating that the carrier was reconstituted in a functionally active form. It is concluded that glutamate uptake by synaptic vesicles is dependent on the membrane potential and that all components required for uptake are integral parts of the vesicle membrane.     

Effective Mechanism for Synthesis of Neurotransmitter Glutamate and its Loading into Synaptic Vesicles

Glutamate accumulation into synaptic vesicles is a pivotal step in glutamate transmission. This process is achieved by a vesicular glutamate transporter (VGLUT) coupled to v-type proton ATPase. Normal synaptic transmission, in particular during intensive neuronal firing, would demand rapid transmitter re-filling of emptied synaptic vesicles. We have previously shown that isolated synaptic vesicles are capable of synthesizing glutamate from α-ketoglutarate (not from glutamine) by vesicle-bound aspartate aminotransferase for immediate uptake, in addition to ATP required for uptake by vesicle-bound glycolytic enzymes. This suggests that local synthesis of these substances, essential for glutamate transmission, could occur at the synaptic vesicle. Here we provide evidence that synaptosomes (pinched-off nerve terminals) also accumulate α-ketoglutarate-derived glutamate into synaptic vesicles within, at the expense of ATP generated through glycolysis. Glutamine-derived glutamate is also accumulated into synaptic vesicles in synaptosomes. The underlying mechanism is discussed. It is suggested that local synthesis of both glutamate and ATP at the presynaptic synaptic vesicle would represent an efficient mechanism for swift glutamate loading into synaptic vesicles, supporting maintenance of normal synaptic transmission.     

Guanine derivatives modulate L-glutamate uptake into rat brain synaptic vesicles

Glutamate uptake into synaptic vesicles is driven by a proton electrochemical gradient generated by a vacuolar H(+)-ATPase and stimulated by physiological concentrations of chloride. This uptake plays an important role in glutamatergic transmission. We show here that vesicular glutamate uptake is selectively inhibited by guanine derivatives, in a time- and concentration-dependent manner. Guanosine, GMP, GDP, guanosine-5'-O-2-thiodiphosphate, GTP, or 5'-guanylylimidodiphosphate (GppNHp) inhibited glutamate uptake in 1.5 and 3 min incubations, however, when incubating for 10 min, only GTP or GppNHp displayed such inhibition. By increasing ATP concentrations, the inhibitory effect of GTP was no longer observed, but GppNHp still inhibited glutamate uptake. In the absence of ATP, vesicular ATPase can hydrolyze GTP in order to drive glutamate uptake. However, 5mM GppNHp inhibited ATP hydrolysis by synaptic vesicle preparations. GTP or GppNHp decreased the proton electrochemical gradient, whereas the other guanine derivatives did not. Glutamate saturation curves were assayed in order to evaluate the specificity of inhibition of the vesicular glutamate carrier by the guanine derivatives. The maximum velocity of the initial rate of glutamate uptake was decreased by all guanine derivatives. These results indicate that, although GppNHp can inhibit ATPase activity, guanine derivatives are more likely to be acting through interaction with vesicular glutamate carrier.     

Characterization of the solubilized and reconstituted ATP-dependent vesicular glutamate uptake system

We have previously provided evidence for ATP-dependent glutamate uptake into synaptic vesicles, and, based upon the unique properties of the vesicular uptake system, we have proposed that the vesicular glutamate translocator plays a crucial role in selecting glutamate for neurotransmission. In this study, we have solubilized the vesicular glutamate uptake system, proposed to consist of at least a glutamate translocator and a proton pump Mg-ATPase, from rat brain synaptic vesicles, and reconstituted the functional ATP-dependent glutamate uptake system into liposomes. The glutamate uptake in the reconstituted system is dependent upon ATP, markedly potentiated by low millimolar concentrations of chloride and inhibited by agents known to dissipate electrochemical proton gradients. Moreover, it exhibited low affinity for glutamate (Km = 2 mM), yet high specificity for glutamate; thus, it did not recognize aspartate and other agents known to interact with glutamate receptors. These properties are indistinguishable from those observed in intact synaptic vesicles. The solubilized functional components of the glutamate uptake system, alone or as a complex, have been estimated to have a Stokes radius in the range of 69 to 84 A. The reconstitution experiments described here provide a functional assay for the solubilized vesicular glutamate uptake system and represent an initial step towards the purification of the glutamate translocator.     

Synaptic vesicles are capable of synthesizing the VGLUT substrate glutamate from α-ketoglutarate for vesicular loading

Synaptic vesicle loading of glutamate is a pivotal step in glutamate synaptic transmission. The molecular machinery responsible for this step is comprised of v-type proton-pump ATPase and a vesicular glutamate transporter. Recent evidence indicates that synaptic vesicles are endowed with glycolytic ATP-synthesizing enzymes, providing energy for immediate use by vesicle-bound proton-pump ATPase. In this study, we provide evidence that synaptic vesicles are also capable of synthesizing the vesicular glutamate transporter substrate glutamate, from α-ketoglutarate and l-aspartate (as the amino group donor); glutamate thus produced is taken up into vesicles. We also report a finding that α-ketoglutarate-derived glutamate uptake into synaptic vesicles and aspartate aminotransferase are inhibited by 2,3-pyrazinedicarboxylate. Evidence is given that this is a selective inhibitor for aspartate aminotransferase. These observations provide insight into understanding the nerve endings' mechanism for high efficiency in glutamate transmission. Finding this inhibitor may have implications for further experimentation on the role of α-ketoglutarate-derived glutamate in glutamate transmission.     

Energy coupling of L-glutamate transport and vacuolar H(+)-ATPase in brain synaptic vesicles

Energy coupling of L-glutamate transport in brain synaptic vesicles has been studied. ATP-dependent acidification of the bovine brain synaptic vesicles was shown to require CI-, to be accelerated by valinomycin and to be abolished by ammonium sulfate, nigericin or CCCP plus valinomycin, and K+. On the other hand, ATP-driven formation of a membrane potential (positive inside) was found to be stimulated by ammonium sulfate, not to be affected by nigericin and to be abolished by CCCP plus valinomycin and K+. Like formation of a membrane potential, ATP-dependent L-[3H]glutamate uptake into vesicles was stimulated by ammonium sulfate, not affected by nigericin and abolished by CCCP plus valinomycin and K+. The L-[3H]glutamate uptake differed in specificity from the transport system in synaptic plasma membranes. Both ATP-dependent H+ pump activity and L-glutamate uptake were inhibited by bafilomycin and cold treatment (common properties of vacuolar H(+)-ATPase). ATP-dependent acidification in the presence of L-glutamate was also observed, suggesting that L-glutamate uptake lowered the membrane potential to drive further entry of H+. These results were consistent with the notion that the vacuolar H(+)-ATPase of synpatic vesicles formed a membrane potential to drive L-glutamate uptake. ATPase activity of the vesicles was not affected by the addition of Cl-, glutamate or nigericin, indicating that an electrochemical H+ gradient had no effect on the ATPase activity.     

Characterization of Glutamate Uptake into Synaptic Vesicles

Recent evidence indicates that L-glutamate is taken up into synaptic vesicles in an ATP-dependent manner, supporting the notion that synaptic vesicles may be involved in glutamate synaptic transmission. In this study, we further characterized the ATP-dependent vesicular uptake of glutamate. Evidence is provided that a Mg-ATPase, not Ca-ATPase, is responsible for the ATP hydrolysis coupled to the glutamate uptake. The ATP-dependent glutamate uptake was inhibited by agents known to dissipate the electrochemical proton gradient across the membrane of chromaffin granules. Hence, it is suggested that the vesicular uptake of glutamate is driven by electrochemical proton gradients generated by the Mg-ATPase. Of particular interest is the finding that the ATP-dependent glutamate uptake is markedly stimulated by chloride over a physiologically relevant, millimolar concentration range, suggesting an important role of intranerve terminal chloride in the accumulation of glutamate in synaptic vesicles. The vesicular glutamate translocator is highly specific for L-glutamate, and failed to interact with aspartate, its related agents, and most of the glutamate analogs tested. It is proposed that this vesicular translocator plays a crucial role in determining the fate of glutamate as a neurotransmitter.     

Boar acrosin is a two-chain molecule. Isolation and primary structure of the light chain; homology with the pro-part of other serine proteinases

Cholinergic synaptic vesicles from the electric organ of Torpedo marmorata are associated with a Mg2+-ATPase insensitive to ouabain and oligomycin. Treatment of vesicle membranes with dichloromethane releases a Mg2+-ATPase with apparent molecular mass of around 250 kDa as determined by gel filtration. The vesicular ATPase resembles the mitochondrial F1-ATPase in these properties. Gel electrophoresis of the solubilized ATPase shows however that only a single 50-kDa band is present as compared to the alpha-subunit (52 kDa) and beta-subunit (50 kDa) of electric organ mitochondrial F1-ATPase present in this range of molecular mass range. In agreement, covalent photoaffinity labelling of isolated vesicles with azido-ATP shows a 50-kDa band. Vesicle ghosts were found to accumulate [14C]methylamine in an ATP-dependent manner indicating the presence of an inwardly directed proton pump. We conclude that cholinergic vesicles contain a proton pump probably driven by the Mg2+-ATPase here described, which generates an electrochemical gradient across the vesicle membrane and is necessary for uptake and storage of acetylcholine within the vesicles.     

Cholinergic Synaptic Vesicles Contain a V-Type and a P-Type ATPase

Fifty to eighty-five percent of the ATPase activity in different preparations of cholinergic synaptic vesicles isolated from Torpedo electric organ was half-inhibited by 7 microM vanadate. This activity is due to a recently purified phosphointermediate, or P-type, ATPase, Acetylcholine (ACh) active transport by the vesicles was stimulated about 35% by vanadate, demonstrating that the P-type enzyme is not the proton pump responsible for ACh active transport. Nearly all of the vesicle ATPase activity was inhibited by N-ethylmaleimide. The P-type ATPase could be protected from N-ethylmaleimide inactivation by vanadate, and subsequently reactivated by complexation of vanadate with deferoxamine. The inactivation-protection pattern suggests the presence of a vanadate-insensitive, N-ethylmaleimide-sensitive ATPase consistent with a vacuolar, or V-type, activity expected to drive ACh active transport. ACh active transport was half-inhibited by 5 microM N-ethylmaleimide, even in the presence of vanadate. The presence of a V-type ATPase was confirmed by Western blots using antisera raised against three separate subunits of chromaffin granule vacuolar ATPase I. Both ATPase activities, the P-type polypeptides, and the 38-kilodalton polypeptide of the V-type ATPase precisely copurify with the synaptic vesicles. Solubilization of synaptic vesicles in octaethyleneglycol dodecyl ether detergent results in several-fold stimulation of the P-type activity and inactivation of the V-type activity, thus explaining why the V-type activity was not detected previously during purification of the P-type ATPase. It is concluded that cholinergic vesicles contain a P-type ATPase of unknown function and a V-type ATPase which is the proton pump.     

ATP-Dependent Glutamate Uptake into Synaptic Vesicles from Cerebellar Mutant Mice

The ATP-dependent glutamate uptake system in synaptic vesicles prepared from mouse cerebellum was characterized, and the levels of glutamate uptake were investigated in the cerebellar mutant mice, staggerer and weaver, whose main defect is the loss of cerebellar granule cells, and the nervous mutant, whose main defect is the loss of Purkinje cells. The ATP-dependent glutamate uptake is stimulated by low concentrations of chloride, is insensitive to aspartate, and is inhibited by agents known to dissipate the electrochemical proton gradient. These properties are similar to those of the glutamate uptake system observed in the highly purified synaptic vesicles prepared from bovine cortex. The ATP-dependent glutamate uptake system is reduced by 68% in the staggerer and 57-67% in the weaver mutant; these reductions parallel the substantial loss of granule cells in those mutants. In contrast, the cerebellar levels of glutamate uptake are not altered significantly in the nervous mutant, which has lost Purkinje cells, but not granule cells. In view of evidence that granule cells are glutamatergic neurons and Purkinje cells are GABAergic neurons, these observations support the notion that the ATP-dependent glutamate uptake system is present in synaptic vesicles of glutamatergic neurons.     

Inhibition of vesicular glutamate storage and exocytotic release by Rose Bengal

It had been thought that quantal size in synaptic transmission is invariable. Evidence has been emerging, however, that quantal size can be varied under certain conditions. We present evidence that alteration in vesicular [(3)H]L-glutamate (Glu) content within the synaptosome (a pinched-off nerve ending preparation) leads to a change in the amount of exocytotically released [(3)H]Glu. We found that Rose Bengal, a polyhalogenated fluorescein derivative, is a quite potent membrane-permeant inhibitor (K(i) = 19 nM) of glutamate uptake into isolated synaptic vesicles. This vesicular Glu uptake inhibition was achieved largely without affecting H(+)-pump ATPase. We show that various degrees of reduction elicited by Rose Bengal in [(3)H]Glu in synaptic vesicles inside the synaptosome result in a corresponding decrease in the amount of [(3)H]Glu released in a depolarization- (induced by 4-aminopyridine) and Ca(2+)-dependent manner. In contrast, fluorescein, the halogen-free analog of Rose Bengal, which is devoid of inhibitory activity on vesicular [(3)H]Glu uptake, failed to change the amount of exocytotically released [(3)H]Glu. These observations suggest that glutamate synaptic transmission could be altered by pharmacological intervention of glutamate uptake into synaptic vesicles in the nerve terminal, a new mode of synaptic manipulation for glutamate transmission.     

Partial Purification and Characterization of the Vacuolar H+-ATPase of Mammalian Synaptic Vesicles

Several major proteins of synaptic vesicles from rat or cow brain sediment as a large complex on sucrose density gradients when solubilized in nonionic detergents. A vacuolar H(+)-ATPase identified by sensitivity to bafilomycin A1 appears to be associated with this oligomeric protein complex. Two subunits of this complex, synaptic vesicle proteins S and U, correspond to the 57-kDa (B) and 39-kDa accessory (Ac39) subunits, respectively, of bovine chromaffin granule vacuolar H(+)-ATPase as shown by Western immunoblot analysis. The five subunits of the oligomeric complex constitute approximately 20% of the total protein of rat brain synaptic vesicles. Taken together, these results strongly suggest that the abundant, multisubunit complex partially purified from brain synaptic vesicles by density gradient centrifugation is a vacuolar H(+)-ATPase. Bafilomycin A1 completely blocks proton pumping in rat brain synaptic vesicles as measured by [14C]methylamine uptake and also blocks catecholamine accumulation measured by [3H]dopamine uptake. Moreover, ATPase activity, [14C]methylamine uptake, and [3H]dopamine uptake are inhibited by bafilomycin A1 at similar I50 values of approximately 1.7 nmol/mg of protein. These findings indicate that the vacuolar H(+)-ATPase is essential for proton pumping as well as catecholamine uptake by mammalian synaptic vesicles.     

Golgi membranes contain an electrogenic H+ pump in parallel to a chloride conductance

Rat liver Golgi vesicles were isolated by differential and density gradient centrifugation. A fraction enriched in galactosyl transferase and depleted in plasma membrane, mitochondrial, endoplasmic reticulum, and lysosomal markers was found to contain an ATP-dependent H+ pump. This proton pump was not inhibited by oligomycin but was sensitive to N-ethyl maleimide, which distinguishes it from the F0-F1 ATPase of mitochondria. GTP did not induce transport, unlike the lysosomal H+ pump. The pump was not dependent on the presence of potassium nor was it inhibited by vanadate, two of the characteristics of the gastric H+ ATPase. Addition of ATP generated a membrane potential that drove chloride uptake into the vesicles, suggesting that Golgi membranes contain a chloride conductance in parallel to an electrogenic proton pump. These results demonstrate that Golgi vesicles can form a pH difference and a membrane potential through the action of an electrogenic proton translocating ATPase.     

Internal anion binding site and membrane potential dominate the regulation of proton pumping by the chromaffin granule ATPase

Effects of anions and membrane potential on the reconstituted proton pump from chromaffin granules were investigated. When acetate was present inside of the vesicles, ATP-dependent proton uptake was absolutely dependent on external chloride. Without external chloride, however, substantial proton uptake was observed when chloride or sulfate was present inside of the vesicles. Inside negative membrane potential drove ATP-dependent proton uptake regardless of the anion species present inside or outside of the vesicles. It is concluded that the internal anion binding site and membrane potential regulate the proton pumping activity of the ATPase.     

Amino acid neurotransmission: Dynamics of vesicular uptake

Glutamate, GABA and glycine, the major neurotransmitters in CNS, are taken up and stored in synaptic vesicles by a Mg²⁺-ATP dependent process. The main driving force for vesicular glutamate uptake is the membrane potential, whereas both the membrane potential and the proton gradient contribute to the uptake of GABA and glycine. Glutamate is taken up by a specific transporter with no affinity for aspartate. Evans blue and related dyes are competitive inhibitors of the uptake of glutamate. GABA, β-alanine, and glycine are taken up by the same family of transporter molecules. Aspartate, taurine, and proline are not taken up by any synaptic vesicle preparations. It is suggested that vesicular uptake and release are characteristics that identify these amino acids as neurotransmitters. We also discuss that “quanta” in the brain are not necessarily related the content of neurotransmitter in the synaptic vesicles, but rather to postsynaptic events. Special issue dedicated to Dr. Herman Bachelard.     

Glutamate uptake into synaptic vesicles of bovine cerebral cortex and electrochemical potential difference of proton across the membrane.

Measurements have been made of the ATP-dependent membrane potential (delta psi) and pH gradient (delta pH) across the membranes of the synaptic vesicles purified from bovine cerebral cortex, using the voltage-sensitive dye bis[3-propyl-5-oxoisoxazol-4-yl]pentamethine oxanol and the delta pH-sensitive fluorescent dye 9-aminoacridine respectively. A pre-existing small delta pH (inside acidic) was detected in the synaptic vesicles, but no additional significant contribution by MgATP to delta pH was observed. In contrast, delta psi (inside positive) increased substantially upon addition of MgATP. This ATP-dependent delta psi was reduced by thiocyanate anion (SCN-), a delta psi dissipator, or carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), a protonmotive-force dissipator. Correspondingly, a substantially larger glutamate uptake occurred in the presence of MgATP, which was inhibited by SCN- and FCCP. A nonhydrolysable analogue of ATP, adenosine 5'-[beta gamma-methylene]triphosphate, did not substitute for ATP in either delta psi generation or glutamate uptake. The results support the hypothesis that a H+-pumping ATPase generates a protonmotive force in the synaptic vesicles at the expense of ATP hydrolysis, and the protonmotive force thus formed provides a driving force for the vesicular glutamate uptake. The delta psi generation by ATP hydrolysis was not affected by orthovanadate, ouabain or oligomycin, but was inhibited by N-ethylmaleimide, quercetin, trimethyltin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid. These results indicate that the H+-pumping ATPase in the synaptic vesicle is similar to that in the chromaffin granule, platelet granule and lysosome.     

Functional reconstitution of the gamma-aminobutyric acid transporter from synaptic vesicles using artificial ion gradients.

The gamma-aminobutyric acid transporter of rat brain synaptic vesicles was reconstituted in proteoliposomes, and its activity was studied in response to artificially created membrane potentials or proton gradients. Changes of the membrane potential were monitored using the dyes oxonol VI and 3,3'-diisopropylthiodicarbocyanine iodide, and changes of the H+ gradient were followed using acridine orange. An inside positive membrane potential was generated by the creation of an inwardly directed K+ gradient and the subsequent addition of valinomycin. Under these conditions, valinomycin evoked uptake of [3H]GABA which was saturable. Similarly, [3H]glutamate uptake was stimulated by valinomycin, indicating that both transporters can be driven by the membrane potential. Proton gradients were generated by the incubation of K(+)-loaded proteoliposomes in a buffer free of K+ or Na+ ions and the subsequent addition of nigericin. Proton gradients were also generated via the endogenous H+ ATPase by incubation of K(+)-loaded proteoliposomes in equimolar K+ buffer in the presence of valinomycin. These proton gradients evoked nonspecific, nonsaturable uptake of GABA and beta-alanine but not of glycine in proteoliposomes as well as protein-free liposomes. Therefore, transporter activity was monitored using glycine as an alternative substrate. Proton gradients generated by both methods elicited saturable glycine uptake in proteoliposomes. Together, our data confirm that the vesicular GABA transporter can be energized by both the membrane potential and the pH gradient and show that transport can be achieved by artificial gradients independently of the endogenous proton ATPase.     

Ca2+ Sensitivity of Synaptic Vesicle Dopamine, γ-Aminobutyric Acid,and Glutamate Transport Systems

The effect of Ca2⁺ on the uptake of neurotransmitters by synaptic vesicles was investigated in a synaptic vesicle enriched fraction isolated from sheep brain cortex. We observed that dopamine uptake, which is driven at expenses of the proton concentration gradient generated across the membrane by the H⁺-ATPase activity, is strongly inhibited (70%) by 500 M Ca²⁺. Conversely, glutamate uptake, which essentially requires the electrical potential in the presence of low Cl^– concentrations, is not affected by Ca²⁺, even when the proton concentration gradient greatly contributes for the proton electrochemical gradient. These observations were checked by adding Ca²⁺ to dopamine or glutamate loaded vesicles, which promoted dopamine release, whereas glutamate remained inside the vesicles. Furthermore, similar effects were obtained by adding 150 M Zn²⁺ that, like Ca²⁺, dissipates the proton concentration gradient by exchanging with H⁺. With respect to -aminobutyric acid transport, which utilizes either the proton concentration gradient or the electrical potential as energy sources, we observed that Ca²⁺ or Zn²⁺ do not induce great alterations in the -aminobutyric acid accumulation by synaptic vesicles. These results clarify the nature of the energy source for accumulation of main neurotransmitters and suggest that stressing concentrations of Ca²⁺ or Zn²⁺ inhibit the proton concentration gradient-dependent neurotransmitter accumulation by inducing H⁺ pump uncoupling rather than by interacting with the neurotransmitter transporter molecules.     

Inhibition of Glutamate Uptake and Proton Pumping in Synaptic Vesicles by S-Nitrosylation

Abstract: Nitric oxide (NO; including NO^•, NO⁺, and NO⁻) was found to inhibit glutamate uptake by isolated synaptic vesicles of rat brain. This was observed when two unrelated NO donors, S -nitrosogluthathione and S -nitroso- N -acetylpenicillamine, were used. The primary target of NO is the H⁺-ATPase found in the synaptic vesicles, which leads to dissipation of the electrochemical proton gradient and inhibition of glutamate uptake. Oxyhemoglobin (12 µ M ) and, to a much lesser extent, methemoglobin protected the vacuolar H⁺-ATPase from inhibition. Inhibition of H⁺ pumping by NO was reversed by addition of 0.5 m M dithiothreitol. The results indicate that the vacuolar H⁺-ATPase from synaptic vesicles is inhibited by NO by a mechanism that involves S -nitrosylation of critical sulfhydryl groups in the enzyme. The interaction of NO with synaptic vesicles might be of importance for the understanding of the multiple effects of NO in neurotransmission.