Effect of lysine acetylsalicylate on aluminium accumulation and (Na(+)/K(+))ATPase activity in rat brain cortex synaptosomes after aluminium ingestion.

Aluminium is neurotoxic in humans and has been implicated in several neurological disorders. Chronic use of buffered aspirins, as aspegic, would likely constitute the major human aluminium uptake source. Low-dose aspirin is beneficial in secondary prevention of cardiovascular events, so it is widely used for long periods of time. We studied if oral administration of aspegic to rats modified the aluminium inhibitory effect on brain (Na(+)/K(+))ATPase due to alteration in synaptosomal membrane aluminium content. Adult male Wistar rats were submitted to sub-acute (1.00g/day during 10 days) and chronic (0.03g/day during 4 months) dietary AlCl3 exposure and/or to aspegic (0.11g/day). The exposure protocol increased the synaptosomal aluminium content especially after a long-term exposure to aluminium and aspegic. Although no alterations were observed in rat body weight gain and adenylate energy charge, the (Na(+)/K(+))ATPase activity was significantly reduced when aluminium was orally administered to rats. The oral administration of aspegic increased the synaptosomal aluminium content and concomitantly enhanced the (Na(+)/K(+))ATPase inhibition. In our exposure protocol the increase in synaptosomal aluminium content correlates with the reduction of the (Na(+)/K(+))ATPase activity.

Alteration of aluminium inhibition of synaptosomal (Na(+)/K(+))ATPase by colestipol administration.

The ability of aluminium to inhibit the (Na(+)/K(+))ATPase activity has been observed by several authors. During chronic dietary exposure to AlCl3, brain (Na(+)/K(+))ATPase activity drops, even if no alterations of catalytic subunit protein expression and of energy charge potential are observed. The aluminium effect on (Na(+)/K(+))ATPase activity seems to implicate the reduction of interacting protomers within the oligomeric ensemble of the membrane-bound (Na(+)/K(+))ATPase. The activity of (Na(+)/K(+))ATPase is altered by the microviscosity of lipid environment. We studied if aluminium inhibitory effect on (Na(+)/K(+))ATPase is modified by alterations in synaptosomal membrane cholesterol content. Adult male Wistar rats were submitted to chronic dietary AlCl3 exposure (0.03 g/day of AlCl3) and/or to colestipol, a hypolidaemic drug (0.31 g/day) during 4 months. The activity of (Na(+)/K(+))ATPase was studied in brain cortex synaptosomes with different cholesterol contents. Additionally, we incubate synaptosomes with methyl-ß-cyclodextrin for both enrichment and depletion of membrane cholesterol content, with or without 300 µM AlCl3. This enzyme activity was significantly reduced by micromolar AlCl3 added in vitro and when aluminium was orally administered to rats. The oral administration of colestipol reduced the cholesterol content and concomitantly inhibited the (Na(+)/K(+))ATPase. The aluminium inhibitory effect on synaptosomal (Na(+)/K(+))ATPase was reduced by cholesterol depletion both in vitro and in vivo.

Aluminium-induced changes of fusion pore properties attenuate prolactin secretion in rat pituitary lactotrophs.

Hormone secretion is mediated by Ca(2+)-regulated exocytosis. The key step of this process consists of the merger of the vesicle and the plasma membranes, leading to the formation of a fusion pore. This is an aqueous channel through which molecules stored in the vesicle lumen exit into the extracellular space on stimulation. Here we studied the effect of sub-lethal dose of aluminium on prolactin secretion in isolated rat pituitary lactotrophs with an enzyme immunoassay and by monitoring electrophysiologically the interaction of a single vesicle with the plasma membrane in real time, by monitoring membrane capacitance. After 24-h exposure to sub-lethal AlCl(3) (30 µM), the secretion of prolactin was reduced by 14±8% and 46±11% under spontaneous and K(+)-stimulated conditions, respectively. The frequency of unitary exocytotic events, recorded by the high-resolution patch-clamp monitoring of membrane capacitance, a parameter linearly related to the membrane area, under spontaneous and stimulated conditions, was decreased in aluminium-treated cells. Moreover, while the fusion pore dwell-time was increased in the presence of aluminium, the fusion pore conductance, a measure of fusion pore diameter, was reduced, both under spontaneous and stimulated conditions. These results suggest that sub-lethal aluminium concentrations reduce prolactin secretion downstream of the stimulus secretion coupling by decreasing the frequency of unitary exocytotic events and by stabilizing the fusion pore diameter to a value smaller than prolactin molecule, thus preventing its discharge into the extracellular space.

Screening for pulmonary tuberculosis in Teresópolis, RJ, Brazil: the search for respiratory symptomatic patients in emergency service of "Hospital das Clínicas de Teresópolis Costantino Ottaviano, Fundação Educacional Serra dos Orgãos".

The aim of the present study was to investigate the detection percentage of tuberculosis among patients that are respiratory symptomatic (TB suspects). In this work, we present the preliminary results of research carried out at "Hospital das Clínicas de Teresópolis Costantino Ottaviano da Fundacao Educacional Serra dos Orgãos (FESO)" from November 2003 to April 2004. Among the 40 respiratory symptomatic individuals identified and referred to the Tuberculosis Control Program in Teresópolis , two (5.0%) were characterized as smear-positive. These results confirm reports in the literature and underscore the need for and importance of this strategy.

Screening for pulmonary tuberculosis in Teresópolis, RJ, Brazil: the search for respiratory symptomatic patients in emergency service of "Hospital das Clínicas de Teresópolis Costantino Ottaviano, Fundação Educacional Serra dos Orgãos"

Investigar o percentual de detecção de tuberculose entre sintomáticos respiratórios é o objetivo do presente estudo. Nesta nota prévia, apresentam-se os resultados preliminares da pesquisa desenvolvida no Hospital das Clinicas de Teresópolis Costantino Ottaviano da Fundação Educacional Serra dos Orgãos (FESO), de novembro de 2003 a abril de 2004. Dos 40 sintomáticos respiratórios identificados e encaminhados ao Programa de Controle da Tuberculose do município de Teresópolis, dois (5.0%) foram caracterizados como bacilíferos. Esses resultados corroboram com os relatos da literatura e confirmam a necessidade e importância desta estratégia.

Aluminium-induced impairment of Ca2+ modulatory action on GABA transport in brain cortex nerve terminals.

The gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in vertebrate CNS. At GABAergic synapses, a high-affinity transporter exists, which is responsible for GABA reuptake and release during neurotransmission. GABA transporter activity depends on the phosphorylation/dephosphorylation state, being modulated by Ca(2+)/calmodulin-dependent protein phosphatase 2B (calcineurin). Aluminium is known to interfere with the Ca(2+)/calmodulin signalling pathway. In this work, we investigate the action of aluminium on GABA translocation mediated by the high-affinity transporter, using synaptic plasma membrane (SPM) vesicles and synaptosomes isolated from brain cortex. Aluminium completely relieved Ca(2+) downregulation of GABA transporter, when mediating uptake or release. Accordingly, aluminium inhibited Ca(2+)/calmodulin-dependent calcineurin activity present in SPM, in a concentration-dependent manner. The deleterious action of aluminium on the modulation of GABA transport was ascertained by comparative analysis of the aluminium effect on GABA uptake and release, under conditions favouring SPM dephosphorylation (presence of intracellular micromolar Ca(2+)) or phosphorylation (absence of Ca(2+) and/or presence of W-7, a selective calmodulin antagonist). In conclusion, aluminium-induced relief of Ca(2+) modulatory action on GABA transporter may contribute significantly to modify GABAergic signalling during neurotoxic events in response to aluminium exposure.

Ca2+ sensitivity of synaptic vesicle dopamine, gamma-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 microM 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 microM Zn2+ that, like Ca2+, dissipates the proton concentration gradient by exchanging with H+. With respect to gamma-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 gamma-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.

Distinction between Ca(2+) pump and Ca(2+)/H(+) antiport activities in synaptic vesicles of sheep brain cortex.

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.

Methods for analysis of Ca(2+)/H(+) antiport activity in synaptic vesicles isolated from sheep brain cortex.

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.

Ca(2+) regulation of the carrier-mediated gamma-aminobutyric acid release from isolated synaptic plasma membrane vesicles.

The regulation of the carrier-mediated gamma-aminobutyric acid (GABA) efflux was studied in isolated synaptic plasma membrane (SPM) vesicles, which are particularly useful to study neurotransmitter release without interference of the exocytotic machinery. We investigated the effect of micromolar intravesicular Ca(2+) on the GABA release from SPM vesicles under conditions of basal release (superfusion with 150 mM NaCl), homoexchange (superfusion with 500 microM GABA) and K(+) depolarization-induced release (superfusion with 150 mM KCl). We observed that, in the presence of intravesicular Ca(2+) (10 microM), the maximal velocity (J(max)) of K(+) depolarization-induced GABA release is decreased by about 64%, and this effect was abolished in the presence of the channel blocker, La(3+). In contrast, the other mechanisms were not significantly altered by these cations. In agreement with our earlier results, inhibition of GABA uptake by intravesicular Ca(2+) was also observed by determining the kinetic parameters (K(0.5) and J(max)) of influx into the SPM vesicles. Under these conditions, the J(max) of GABA uptake was 17.4 pmol/s per mg protein, whereas in control experiments (absence of Ca(2+)), this value achieved 25.5 pmol/s per mg protein. The inhibitory effect of Ca(2+) on translocation of GABA across SPM appears to be mediated by calcium/calmodulin activation of protein phosphatase 2B (calcineurin), since it was completely relieved by W7 (calmodulin antagonist) and by cyclosporin A (calcineurin inhibitor). These results show that the GABA transport system, operating either in forward or backward directions, requires phosphorylation of internally localized calcineurin-sensitive sites to achieve maximal net translocation velocity.

Synaptic vesicle Ca2+/H+ antiport: dependence on the proton electrochemical gradient.

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.

Ionic selectivity of the Ca2+/H+ antiport in synaptic vesicles of sheep brain cortex.

As we previously reported, synaptic vesicles isolated from sheep brain cortex contain a Ca2+/H+ antiport that permits Ca2+ accumulation inside the vesicles ( approximately 5 nmol/mg protein) at expenses of the pH gradient generated by the H+-pumping ATPase. We observed that the system associates Ca2+ influx to H+ release and operates with low affinity for Ca2+. In the present work, we found that Ca2+/H+ antiport mediates exchange of protons with other cations such as Zn2+ and Cd2+, suggesting that these cations and Ca2+ share the same transporter molecules to enter the intravesicular space. Zn2+ and Cd2+ induce H+ release in a concentration-dependent manner (fluorimetrically evaluated) and they inhibit the antiport-mediated Ca2+ uptake by the vesicles (isotopically measured). In contrast, large cations such as Ba2+ and Cs+ do not alter Ca2+ influx and they are unable to induce proton release from the vesicles. With respect to Sr2+, which has an intermediary size relatively to the other groups of cations, we found that it does not induce H+ liberation from the vesicles, but it has a concentration-dependent inhibitory effect on the Ca2+-induced H+ release and Ca2+ uptake by the vesicles. These results indicate that the cation selectivity of the synaptic vesicles Ca2+/H+ antiport is essentially determined by the size of the cation transported into the vesicles.

Regulation of the gamma-aminobutyric acid transporter activity by protein phosphatases in synaptic plasma membranes.

The influence of the phosphorylation dephosphorylation states on the gamma-aminobutyric acid (GABA) transporter activity of synaptic plasma membranes (SPM) was studied by using either specific phosphatase inhibitors or activators. Calyculin A and okadaic acid (phosphatase 1 and phosphatase 2A inhibitors) inhibited the GABA uptake by isolated SPM vesicles, whereas cyclosporin A (phosphatase 2B inhibitor) had a stimulatory effect (approximately 10%) which was higher (approximately 38%) when all these drugs were present in the reaction medium. On the other hand, intravesicular Ca2+, up to about 10 microM, inhibited the GABA uptake (approximately 50%) in a manner which appeared to be facilitated in the presence of PP1 and PP2A inhibitors and this inhibition was relieved by the calmodulin antagonist W-7. We also observed that isolated SPM vesicles contain both Ca(2+)-independent phosphatase activity that is significantly inhibited by PP1 and PP2A inhibitors, and Ca(2+)-dependent phosphatase activity that is abolished in the presence of the PP2B inhibitor, cyclosporin A. These results indicate that regulation of the SPM GABA transporter is determined by the internally localized Ca-calmodulin-dependent phosphatase activity (calcineurin), and that other phosphorylated sites, sensitive to PP1 and PP2A inhibitors, potentiate either the positive or negative effects exerted by those internal sites when they are in their phosphorylated or dephosphorylated states, respectively.

Ca2+-H+ antiport activity in synaptic vesicles isolated from sheep brain cortex.

Synaptic vesicles isolated from sheep brain cortex exhibit an ATP-dependent Ca2+ accumulation that is inhibited by the protonophore uncoupler carbonyl cyanide m-chorophenylhydrazone (CCCP) and completely released by the Ca2+ 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 Ca2+ (100-860 microM) in correlation to an increase of ATP hydrolysis by the H+-pumping ATPase. In contrast, the activity of the H+-ATPase, measured under uncoupling conditions (presence of CCCP), appears to be unaltered by the calcium ion. The Ca2+-induced H+ release visualized by fluorescence quenching of acridine orange correlates well with the Ca2+ uptake determined isotopically. These results indicate that synaptic vesicles accumulate Ca2+, via a low affinity Ca2+-H+ antiport system energized by the protonmotive force originated from the H+-pumping ATPase activity.

Membrane potential manipulation in synaptic plasma membrane vesicles for studying neurotransmitter uptake and release.

Synaptic plasma membrane (SPM) vesicles represent a membrane fraction very useful in studying non-vesicular neurotransmitter release. The procedure described here to isolate SPM vesicles from a crude synaptosomal fraction of sheep brain cortex is quick, simple (ultracentrifugation in a discontinuous density gradient of dextran T110), and combines a high yield (130 micrograms/g brain) with a satisfactory grade of purification. The preparation of SPM vesicles consists of vesicles (approximately 0.54 +/- 0.8 micron diameter) delimited by a single membrane with the native orientation. We are able to ascertain these characteristics on the basis of morphology studies (electron microscopy observations), enzyme activities (Na+/K(+)-ATPase, Ca2+/Mg(2+)-ATPase, acetylcholinesterase and glucose-6-phosphatase), biochemical composition (lipid and protein analysis) and the tetrodotoxin sensitivity of the veratridine-induced gamma-aminobutyric acid (GABA) release. Isolating the SPM vesicles by the proposed procedure permits manipulating the ionic gradients across the membrane by changing the ion concentrations on either side or by utilizing specific ionophores. The vesicles retain their various activities, including their capacity for neurotransmitter uptake and release assays for at least 3 months, when preserved at -70 degrees C. Furthermore, the vesicles permit depicting the electrochemical gradients across the membranes into chemical and electrical components. We describe the use of the tetraphenylphosphonium cation (TPP+) to dissipate the membrane potential (delta psi) of the vesicles, while preserving ionic gradients. The characteristics of the lipid-soluble cation TPP+ allows a massive inflow of this cation into vesicular compartments and a consequent depolarization.

Regulation of [gamma-3H]aminobutyric acid transport by Ca2+ in isolated synaptic plasma membrane vesicles.

We studied the effect of Ca2+ on the transport of the gamma-aminobutyric acid (GABA) by synaptic plasma membrane (SPM) vesicles isolated from sheep brain cortex and observed that intravesicular Ca2+ inhibits the [3H]GABA accumulation in a concentration-dependent manner. This inhibitory effect of Ca2+ exhibited two distinct components: one in the micromolar range of Ca2+ concentration, and the other in the millimolar range. Previous EGTA washing of the membranes, or incorporation of trifluoperazine into the vesicular space reduced the inhibitory action of Ca2+, particularly at low Ca2+ (1-5 microM). Okadaic acid (1 microM) also relieved the Ca2+ inhibition at low, but not at high Ca2+ concentrations (1 mM), whereas the calpain inhibitor I did not alter the effect of the low Ca2+, but it partially reduced (approximately 28%) the effect of Ca2+ in the millimolar range. The results indicate that the GABA transporter is regulated by low Ca2+ concentration (microM) and probably its effect is mediated by the (Ca2+ x calmodulin)-stimulated phosphatase 2B (calcineurin). In contrast, the GABA uptake inhibition observed at high Ca2+ concentrations (1 mM) is less specific, and probably it is partially related to the proteolytic activity of membrane bound calpain II.

Dual role of K+ and Na+ on the transport of [3H]-gamma-aminobutyric acid by synaptic plasma membrane vesicles.

The influence of the monovalent cations (Na+ and K+) and of the electrical gradient on the high-affinity [3H]-gamma-aminobutyric acid ([3H]GABA) transport was investigated in synaptic plasma membrane (SPM) vesicles isolated from sheep brain cortex. This process specifically requires internal K+, since when it is replaced by Li+, the delta psi remains of the same order of magnitude, but no uptake of [3H]GABA occurs. The influence of the external Na+ concentration on the rate of [3H]GABA uptake suggests that this mechanism exhibits two components, whose characteristics are determined by the delta psi. Depolarization reduces the Jmax of [3H]GABA influx and enhances the binding of Na+ associated to [3H]GABA transport. Nevertheless, depolarization does not affect the K0.5 of binding sites for Na+ and the stoichiometry of translocation. These results suggest that intravesicular K+ and external Na+ have a dual role on the mechanism of [3H]GABA uptake: K+ acts directly on the carrier and determines the membrane polarization; Na+ is cotransported with GABA and, according to the polarization state of the membrane, it modulates the operation of the carrier in its inward GABA translocation.

Characterization of the carrier-mediated [3H]GABA release from isolated synaptic plasma membrane vesicles.

Synaptic plasma membrane (SPM) vesicles were isolated under conditions which preserve most of their biochemical properties. Therefore, they appeared particularly useful to study the cytoplasmic GABA release mechanism through its neuronal transporter without interference of the exocytotic mechanism. In this work, we utilized SPM vesicles isolated from sheep brain cortex to investigate the process of [3H]GABA release induced by ouabain, veratridine and Na+ substitution by other monovalent cations (K+, Rb+, Li+, and choline). We observed that ouabain is unable to release [3H]GABA previously accumulated in the vesicles and, in our experimental conditions, it does not act as a depolarizing agent. In contrast, synaptic plasma membrane vesicles release [3H]GABA when veratridine is present in the external medium, and this process is sensitive to extravesicular Na+ and it is inhibited by extravesicular Ca2+ (1mM) under conditions which appear to permit its entry. However, veratridine-induced [3H]GABA release does not require membrane depolarization, since this drug does not induce any significant alteration in the membrane potential, which is determined by the magnitude of the ionic gradients artificially imposed to the vesicles. The substitution of Na+ by other monovalent cations promotes [3H]GABA release by altering the Na+ concentration gradient and the membrane potential of SPM vesicles. In the case of choline and Li+, we observed that the fraction of [3H]GABA released relatively to the total amount of neurotransmitter released by K+ or Rb+ is about 28% and 68%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of anions on the uptake and release of gamma-aminobutyric acid by isolated synaptic plasma membranes.

The influence of various anions on the uptake and release of gamma-aminobutyric acid (GABA) was investigated in synaptic plasma membrane (SPM) vesicles isolated from sheep brain cortex. We observed that substitution of Cl- by various anions greatly reduces [3H]GABA accumulation by SPM vesicles. The magnitude of the effect is about 30, 85, 95 and 100% when Cl- is replaced by Br-, NO3-, CH3COO- and SO4(2-), respectively. However, no effect was observed when these anions were added together with Cl-, which indicates that they do not inhibit the [3H]GABA uptake mechanism by SPM vesicles. On the other hand, we observed that [3H]GABA release, either by homoexchange or induced by K+ depolarization, is maximal in the presence of Cl- or Br-, whereas the other anions (NO3-, CH3COO- and SO4(2-)) caused a 50% reduction in the two processes of [3H]GABA release. We also observed that the basal release of [3H]GABA is not greatly altered by Br- and NO3-, but it is greatly enhanced by CH3COO- and SO4(2-) in substitution of Cl-. In contrast to these alterations in [3H]GABA movements, the membrane potential is not significantly affected by any of the anions tested. The results confirm the idea that GABA uptake implies Cl- co-transport and they demonstrate that the maximal release of [3H]GABA through its carrier (homoexchange or K+ depolarization-induced release) requires the presence of small anions (Cl- or Br-) at the opposite side of the membrane from where the neurotransmitter is translocated. Furthermore, it appears that CH3COO- and SO4(2-) uncouple the system by inducing basal release, whereas it remains coupled in the presence of Cl-, Br- and NO3-.

Release of gamma-[3H]aminobutyric acid from synaptosomes: effect of external cations and of ouabain.

In the present study we have investigated the effect of cations and ouabain on Ca(2+)-independent and Ca(2+)-dependent release of gamma-[3H]aminobutyric acid ([3H]GABA) from sheep brain synaptosomes. The presence of Na+ in the external medium is essential for the Ca(2+)-independent release induced by K+ or ouabain. Thus, in the absence of Ca2+, ouabain or K+ causes the release of [3H]GABA provided that Na+ is present in the external medium. Under K(+)-depolarizing conditions, in a Na+ medium, either ouabain or Ca2+ further increases the [3H]GABA release induced by depolarization, but their effects are not additive. The presence of external Na+ is not required for the Ca(2+)-dependent release of [3H]GABA due to K+ depolarization, and this release, which occurs in a choline medium, is not modified by ouabain. Under these conditions (choline medium) K(+)-depolarization dependent release is absolutely dependent on external Ca2+, which suggests that this release of [3H]GABA occurs only by exocytosis, without the carrier-mediated efflux which normally co-exists with exocytosis due to K(+)-depolarization in a Na+ medium. It is likely that the release induced by ouabain or K+ involves the membrane carrier which responds to changes in membrane potential.