Characterization of Nucleotide Transport into Rat Brain Synaptic Vesicles

Journal of Neurochemistry Lippincott Williams & Wilkins, Inc., Philadelphia © 1999 International Society for Neurochemistry

Characterization of Nucleotide Transport into Rat Brain Synaptic Vesicles Javier Gualix, Jesu´s Pintor, and Maria Teresa Miras-Portugal Departamento de Bioquı´mica, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain

Abstract: ATP transport to synaptic vesicles from rat brain has been studied using the fluorescent substrate analogue 1,N 6 -ethenoadenosine 5⬘-triphosphate (⑀-ATP). The increase in intravesicular concentration was time dependent for the first 30 min, ⑀-ATP being the most abundant nucleotide. The complexity of the saturation curve indicates the existence of kinetic and allosteric cooperativity in the nucleotide transport, which exhibits various affinity states with K0.5 values of 0.39 ⫾ 0.06 and 3.8 ⫾ 0.1 mM with ⑀-ATP as substrate. The Vmax values obtained were 13.5 ⫾ 1.4 pmol 䡠 min⫺1 䡠 mg of protein⫺1 for the first curve and 28.3 ⫾ 1.6 pmol 䡠 min⫺1 䡠 mg of protein⫺1 considering both components. This kinetic behavior can be explained on the basis of a mnemonic model. The nonhydrolyzable adenine nucleotide analogues adenosine 5⬘-O-3-(thiotriphosphate), adenosine 5⬘-O-2-(thiodiphosphate), and adenosine 5⬘-(␤,␥-imino)triphosphate and the diadenosine polyphosphates P1,P3-di(adenosine)triphosphate, P1,P4-di(adenosine)tetraphosphate, and P1,P5di(adenosine)pentaphosphate inhibited the nucleotide transport. The mitochondrial ATP/ADP exchange inhibitor atractyloside, N-ethylmaleimide, and polysulfonic aromatic compounds such as Evans blue and 4,4⬘diisothiocyanostilbene-2,2⬘-disulfonic acid also inhibit ⑀-ATP vesicular transport. Key Words: ATP—Mnemonic kinetic—Nucleotide transporter—Purinergic transmission— Synaptic vesicles. J. Neurochem. 73, 1098 –1104 (1999).

tides act via P2 receptors that are divided into two groups depending on whether they are ligand-gated ion channels (P2X receptors) or G protein-coupled receptors (P2Y receptors) (Ralevic and Burnstock, 1998). ApnA can interact with some P2 receptors, but specific dinucleotide receptors have been described in both heart cells and brain synaptic terminals (Hildermann et al., 1991; Pintor and Miras-Portugal, 1995). The extracellular effects of the released nucleotides are terminated by the action of ecto-nucleotidases (Zimmermann, 1996b; Mateo et al., 1997a,b). Nevertheless, despite the increasing importance of nucleotides at the extracellular level, there is a lack of information about their vesicular transport, a necessary step for these compounds to be able to play a role as signaling molecules in the synaptic cleft. Nucleotide vesicular transport has been characterized only in the catecholaminergic chromaffin granules from adrenal medulla and the cholinergic vesicles from the Torpedo electric organ. Chromaffin granules from bovine adrenal medulla are the most widely employed experimental model. A wide variety of nucleotides such as ATP, ADP, AMP, GTP, UTP, and the ApnA can be internalized to the granules (Weber and Winkler, 1981; Bankston and Guidotti, 1996; Gualix et al., 1996, 1997). A similar lack of substrate specificity has been demonstrated in the synaptic vesicles of the cholinergic model of the T. marmorata electric organ (Luqmani, 1981). Despite this lack of specificity, the granular nucleotide transport is

The presence of ATP and other nucleotides and dinucleotides [diadenosine polyphosphates (ApnA)] has been described in secretory vesicles containing aminergic or cholinergic compounds such as the catecholaminergic chromaffin granules from adrenal medulla, the serotoninergic dense granules of platelets, and the acetylcholinergic synaptic vesicles from the Torpedo marmorata electric organ (Winkler and Carmichael, 1982; Lu¨thje and Ogilvie, 1983; Rodriguez del Castillo et al., 1988; Pintor et al., 1992b). The stored vesicular nucleotides are released to the extracellular medium on stimulation (Pintor et al., 1992a; Zimmermann, 1994), and their actions through a widely distributed family of receptors, called purinoceptors, have been described. Extracellular nucleo-

Received April 19, 1999; revised manuscript received May 24, 1999; accepted May 24, 1999. Address correspondence and reprint requests to Dr. M. T. MirasPortugal at Departamento de Bioquı´mica, Facultad de Veterinaria UCM, Ciudad Universitaria, 28040 Madrid, Spain. Abbreviations used: ⑀-ADP, 1,N 6-ethenoadenosine 5⬘-diphosphate (etheno-ADP); ADP␤S, adenosine 5⬘-O-2-(thiodiphosphate); ⑀-AMP, 1,N 6-ethenoadenosine 5⬘-monophosphate; AMPPNP, adenosine 5⬘(␤,␥-imino)triphosphate; Ap3A, P1,P3-di(adenosine)triphosphate (diadenosine triphosphate); Ap4A, P1,P4-di(adenosine)tetraphosphate (diadenosine tetraphosphate); Ap5A, P1,P5-di(adenosine)pentaphosphate (diadenosine pentaphosphate); ApnA, diadenosine polyphosphates in general; ⑀-ATP, 1,N 6-ethenoadenosine 5⬘-triphosphate (etheno-ATP); ATP␥S, adenosine 5⬘-O-3-(thiotriphosphate); DIDS, 4,4⬘-diisothiocyanostilbene-2,2⬘-disulfonic acid; NEM, N-ethylmaleimide; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid.

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NUCLEOTIDE TRANSPORT INTO SYNAPTIC VESICLES highly regulated by the cytosolic concentration of nucleotides, mainly ATP and ADP. These transport substrates are able to induce slow conformational changes between different forms of the transporter protein, resulting in very significant changes in transport capacity. This kinetic behavior, known as mnemonic, allows a security threshold in the cellular energetic metabolism (Gualix et al., 1996, 1997). The approach to nucleotide transport into brain synaptic vesicles has remained elusive. In this study, the transport of adenine nucleotides to synaptic vesicles from rat brain has been characterized with the help of fluorescent nucleotide analogues. The data reported here confirm the complex kinetic behavior of the vesicular nucleotide transporter and contribute to increase the significance of nucleotides as neurotransmitters in the central nervous system. MATERIALS AND METHODS Materials

1-N 6-Ethenoadenosine 5⬘-triphosphate (etheno-ATP; ⑀-ATP), 1-N 6-ethenoadenosine 5⬘-diphosphate (etheno-ADP; ⑀-ADP), adenosine 5⬘-O-2-(thiodiphosphate) (ADP␤S), adenosine 5⬘-O-3(thiotriphosphate) (ATP␥S), adenosine 5⬘-(␤,␥-imino)triphosphate (AMPPNP), P1,P3-di(adenosine)triphosphate (diadenosine triphosphate; Ap3A), P1,P 4-di(adenosine)tetraphosphate (diadenosine tetraphosphate; Ap4A), Basilen blue, Cibacron blue, Evans blue, 4,4⬘-diisothiocyanostilbene-2,2⬘-disulfonic acid (DIDS), atractyloside, N-ethylmaleimide (NEM), noradrenaline, and acetylthiocholine were all purchased from Sigma Chemical (St. Louis, MO, U.S.A.). P1,P5-Di(adenosine)pentaphosphate (diadenosine pentaphosphate; Ap5A) was purchased from BoehringerMannheim Biochemica (Mannheim, Germany). Other analytical-grade reagents were purchased from Merck (Darmstadt, Germany).

Preparation of synaptic vesicles Synaptic vesicles were isolated from whole brain of cervically dislocated and decapitated male Wistar rats, according to the procedure of Roseth et al. (1995), with some modifications. In a typical preparation, the brains of six rats were homogenized (700 rpm, seven homogenization steps) in 3% (wt/vol) medium containing 0.32 M sucrose, 0.5 mM EDTA, and 5 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES; pH 7.4). Homogenates were centrifuged at 800 g for 10 min. Pellets were removed and supernatants centrifuged at 20,000 g for 30 min to obtain crude synaptosomal fractions. To isolate the synaptic vesicles, crude synaptosomal fractions were osmotically shocked by resuspension in 60 ml of 0.5 mM EDTA and 5 mM TES (pH 7.4) and centrifuged at 17,000 g for 30 min. Synaptic vesicles were purified by subjecting the remaining supernatant to 0.4 and 0.6 M sucrose density gradient centrifugation at 100,000 g for 1 h. Vesicles fractions were isolated from the 0.4 M sucrose band and protein measured using the biuret method. Samples of synaptic vesicles were finally stored at ⫺80°C until use in transport experiments.

Transport experiments The nucleotide transport experiments were performed with samples of synaptic vesicles containing 0.45 mg of protein in 400 ␮l of a buffer medium containing 0.3 M sucrose, 5 mM MgCl2, and 10 mM Tris-HCl, (pH 7.4). Transport substrates

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and/or inhibitors were also present in this medium, at the accurate concentrations. Transport was stopped by adding 10 ml of ice-cold 0.15 M KCl, immediately followed by filtration through Millipore HAWP filters (25-mm diameter, 0.45-␮m pore size). To avoid unspecific binding of extravesicular fluorescent ⑀-ATP to the filters, these were maintained in a 2 mM ATP solution for almost 10 min before use. After sample passing, filters were washed twice with 10 ml of 0.15 M KCl and then removed and placed in Eppendorf tubes containing 500 ␮l of 1% sodium dodecyl sulfate. After vigorous agitation, the filters were discarded, and the samples were centrifuged at 13,000 rpm for 5 min and supernatant aliquots injected into HPLC. Experiments were carried out at 25°C beginning with the addition of the vesicular preparation, except when Basilen blue, Cibacron blue, Evans blue, or DIDS was used, in which case the vesicular preparation was preincubated for 10 min, at room temperature, in the presence of the inhibitor and the transport started by the addition of the ⑀-ATP. When necessary, an aliquot of the incubation medium was taken to follow the extravesicular metabolism of the ⑀-nucleotides. Substrate concentrations and incubation times used are specified where necessary.

HPLC procedures The HPLC equipment was supplied by Waters (Milford, MA, U.S.A.) and consists of a model 515 pump, an injector autosampler 717 Plus, a model 474 scanning fluorescence detector, and a Millennium 2010 chromatography manager system. Analysis was performed under ion-pair chromatography conditions by equilibrating the chromatographic system with the following mobile phase: 10 mM KH2PO4, 2 mM tetrabutylammonium hydrogen sulfate, and 17% acetonitrile (pH 7.5), with a flow rate of 2 ml/min. The column used was a Novapak C18 (3.9-mm internal diameter, 15-cm length) from Waters. The eluent from the column was excited at 306 nm, and the emission was recorded at 410 nm to detect fluorescent ⑀-adenine nucleotides (Secrist et al., 1972).

Data analysis

All results were expressed as means ⫾ SD, the number of experiments in triplicate being n. Significant differences were determined by the unpaired Student’s t test. The kinetic analysis of the complex experimental data was performed according to the following equation: V ⫽ ¥ [(V 䡠 S n)/ (K n ⫹ S n)], because the V/S representation was not hyperbolic and appeared to be the result of the addition of various Hilltype curves. A similar kinetic analysis has been carried out on the nucleotide transporter present in chromaffin granules (Gualix et al., 1996, 1997).

RESULTS

⑀-ATP transport to synaptic vesicles Purified synaptic vesicles from the whole rat brain were employed to study the nucleotide vesicular transport of ⑀-ATP. Figure 1 shows the extravesicular and intravesicular distribution of ⑀-adenine nucleotides as a function of experimental time. The nucleotide distribution in the extravesicular medium is shown in Fig. 1A. Small amounts of 1,N 6-ethenoadenosine 5⬘-monophosphate (⑀-AMP) and ⑀-ADP are present in the HPLC chromatogram at the outset as a result of their presence in commercially available ⑀-ATP. No significant hydroJ. Neurochem., Vol. 73, No. 3, 1999

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J. GUALIX ET AL. chromatograms of intravesicular etheno-nucleotides, both at the start (which can be taken as the control) and after 30 min, where a considerable increase in the intravesicular ⑀-ATP can be observed. To exclude the possibility of an exchange with intravesicular nucleotides, samples were maintained at 0°C during the whole experimental time and the rate of transport was nonsignificant compared with that obtained at 25°C. The distribution and content of ⑀-nucleotides inside the granule are different from outside and vary with the incubation time, as is shown in Fig. 1C. The transport appears to be linear for the first 30 min in our experimental conditions. Saturation studies of ⑀-ATP transport Experimental knowledge of the stability of ⑀-ATP in the synaptic vesicle incubation medium was used to establish the best conditions under which to undertake the kinetic studies. A 30-min incubation period was chosen for the transport experiments. Figure 2 shows the chromatographic profile of the concentration dependence

FIG. 1. ⑀-ATP transport to synaptic vesicles as a function of time. Samples of synaptic vesicles were incubated in the presence of 2 mM ⑀-ATP. At different time intervals, the transport was stopped and samples processed as described in Materials and Methods. A: Chromatographic profiles of the ⑀-nucleotides in the extravesicular medium at 0- and 30-min incubation times. B: Chromatographic profiles of the ⑀-nucleotides in the intravesicular medium at 0- and 30-min incubation times. C: Intravesicular ⑀-nucleotide content at different incubation times. Values are means ⫾ SD (n ⫽ 3). (}), ⑀-AMP; (Œ), ⑀-ADP; (■), ⑀-ATP; (F), total ⑀-nucleotides.

lysis of ⑀-ATP was observed during the transport experiments in which the incubation period was even 1 h. The percentage distribution of the nucleotides after 30-min incubation was 0.7, 10.1, and 89.2%, respectively, for ⑀-AMP, ⑀-ADP, and ⑀-ATP. Figure 1B shows the HPLC J. Neurochem., Vol. 73, No. 3, 1999

FIG. 2. Concentration dependence of ⑀-ATP transport to synaptic vesicles. Synaptic vesicles were incubated with ⑀-ATP concentrations ranging from 250 ␮M to 10 mM for 30 min, as described in Materials and Methods, and the vesicular ⑀-nucleotide content was measured by HPLC techniques. A: Consecutive chromatograms corresponding to the vesicular nucleotide content after incubation with 2, 2.5, 3, 3.5, 4, and 4.5 mM ⑀-ATP. Chromatograms of other ⑀-ATP concentrations were omitted to avoid a crowded figure. These chromatograms represent a typical experiment. B: Saturation dependence curve for the ⑀-ATP transport. The total ⑀-nucleotide vesicular content was used to calculate V values, which are the means ⫾ SD of five experiments in duplicate. C: The two sigmoidal curves, which additionally account for the experimental curve (B), are represented in dotted lines.

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transport studies for ⑀-ATP. As Fig. 2A reveals, ⑀-ATP was the most abundant of the nucleotides present inside the vesicles, but a significant fraction comprised ⑀-ADP and also ⑀-AMP. To approach the kinetic parameters for ⑀-ATP transport, the total granular ⑀-nucleotide content was taken into account when calculating the transport velocity at each ⑀-ATP concentration used. A saturation curve was plotted from these intravesicular ⑀-nucleotide amounts as shown in Fig. 2B. Under our experimental conditions, a nonhyperbolic saturation isotherm was observed. The complex dependence of transport velocity on extravesicular ⑀-ATP concentration made it necessary to interpret the saturation curve as the superposition of various sigmoidal kinetics. The experimental data were therefore processed using the equation described in Materials and Methods. The addition of two sigmoidal curves was necessary, in this case, to process the experimental data; the K0.5 values of each individual curve were 0.39 ⫾ 0.06 and 3.8 ⫾ 0.1 mM, the corresponding Hill numbers being 2.3 and 12.7. The Vmax value for the first sigmoidal curve was 13.5 ⫾ 1.4 and 28.3 ⫾ 1.6 pmol 䡠 min⫺1 䡠 mg of protein⫺1 considering the addition of the two curves.

⑀-ADP transport to purified synaptic vesicles The ⑀-ADP transport to rat brain synaptic vesicles was studied, and the HPLC chromatographic profiles were as shown in Fig. 3. At the extravesicular level, a small increase in ⑀-AMP can be observed, but the ⑀-ATP is present only in very small quantities and does not increase with incubation time (Fig. 3A). This rules out the action of adenylate kinase on the ⑀-ADP to produce ⑀-AMP and ⑀-ATP. The intravesicular content of ⑀-nucleotides when ⑀-ADP was the substrate to be transported is shown in Fig. 3B. In this case, the ⑀-ADP constituted the overwhelming majority of intravesicular nucleotides, but ⑀-ATP was also present and appeared at a higher ratio than outside the granule, together with ⑀-AMP. The percentage distribution of the nucleotides was ⬃16.3, 76.0, and 7.7% for ⑀-AMP, ⑀-ADP, and ⑀-ATP, respectively, for 30-min transport experiments. The ⑀-ADP transport velocity at every concentration used is about four times lower than that for ⑀-ATP at the same concentration, which means that studying the saturation of ⑀-ADP transport is beyond the limits of accuracy of our experimental approach. Inhibition of ⑀-ATP transport to synaptic vesicles The inhibition studies were performed with ⑀-ATP as the substrate and are summarized in Fig. 4. Different compounds were used: nucleotide analogues, polysulfonic aromatic derivatives, compounds reported to be co-stored with nucleotides, and others previously reported as inhibitors of the nucleotide vesicular transport. Diadenosine polyphosphates Ap3A, Ap4A, and Ap5A and the nucleotide nonhydrolyzable analogues ATP␥S, ADP␤S, and AMPPNP all proved to be inhibitors of

FIG. 3. ⑀-ADP transport to synaptic vesicles. Samples of synaptic vesicles were incubated in the presence of 1 mM ⑀-ADP. A: Chromatographic profiles of the ⑀-nucleotides in the extravesicular medium after the indicated incubation times. B: Chromatographic profiles of the intravesicular ⑀-nucleotides after the indicated periods of incubation.

⑀-ATP transport over a limited range of concentrations (Fig. 4A). The total nucleotide concentration considering ⑀-ATP and the other nucleotides studied should not exceed 2.7 mM in the assay; otherwise, activation of ⑀-ATP transport occurs. These results agree with the large plateau and the cooperativity exhibited by the saturation curve (Fig. 2A). The polysulfonic aromatic compounds Cibacron blue, Basilen blue, Evans blue, and DIDS (Fig. 4B) also inhibited ⑀-ATP transport to synaptic vesicles. In our model, Evans blue was the most effective inhibitor, ⑀-ATP transport being 51.5% of the control value at an Evans blue concentration of 100 ␮M. As vesicular nucleotides are usually co-stored with aminergic and cholinergic compounds, the effect of noradrenaline and acetylthiocholine on nucleotide transport was studied. Noradrenaline was ineffective in our model. Nevertheless, acetylthiocholine at 1 mM concentration showed a slight inhibitory effect on the ⑀-ATP transport, which was reduced to 80% of the control value (Fig. 4C). NEM and atractyloside were also capable of inhibiting ⑀-ATP transport to synaptic vesicles, showing 30 and 52% inhibition, respectively (Fig. 4C). J. Neurochem., Vol. 73, No. 3, 1999

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FIG. 4. Modulation of ⑀-ATP transport by several effectors. Synaptic vesicles were incubated with 1 mM ⑀-ATP for 30 min in the presence or in the absence of several putative nucleotide transport modulators. The values of transport velocity in the presence of each type of effector are represented as percentages of control (100%). Values are the means ⫾ SD of three experiments performed in triplicate. Significant differences from control experiments are indicated by asterisks (*p ⬍ 0.05, **p ⬍ 0.01). A: Effect of 1 mM concentration each of ATP␥S, AMPPNP, ADP␤S, Ap3A, Ap4A, and Ap5A on ⑀-ATP transport. B: Effect of 100 ␮M concentration each of Basilen blue, Cibacron blue, Evans blue, and DIDS on ⑀-ATP transport. C: Effect of 1 mM concentration each of noradrenaline, acetylthiocholine, NEM, and atractyloside on ⑀-ATP transport.

DISCUSSION The broad heterogeneity of the brain synaptic vesicles has been the main obstacle to carrying out studies on their nucleotide transport. In this study, the fluorescent derivatives ⑀-ATP and ⑀-ADP have been employed as substrate analogues, allowing the use of very sensitive fluorescent techniques. In the chromaffin granule model, which exhibits poor substrate selectivity, the ⑀-derivatives of adenine nucleotides have proved to be good transport substrate analogues (Gualix et al., 1996), and they have also proved to be useful tools when studying J. Neurochem., Vol. 73, No. 3, 1999

the rat brain vesicular model. In this model, the absence of transport at 0°C together with the time dependence of accumulation and the saturation kinetics clearly confirm that the vesicular transport of ⑀-ATP is a mediated transport process. The presence of a V-ATPase has been described in all the secretory vesicles studied so far. As this enzyme shows low substrate specificity, hydrolyzing a large variety of nucleotide triphosphates (Zimmermann, 1996a), it is reasonable to suppose that ⑀-ATP can be a substrate for this enzyme, and small amounts of ⑀-ADP can be observed in the incubation medium. Moreover, the low rate of extravesicular ⑀-ATP degradation excludes the presence, in the vesicular preparation, of high levels of nonspecific phosphatase activities of cytosolic or plasma membrane origin. On the other hand, it does not account for the significant quantities of ⑀-ADP and ⑀-AMP present inside the vesicles. Besides, the presence of ⑀-ATP, after ⑀-ADP transport, suggests the existence of intravesicular enzymatic activities that equilibrate the levels of the different nucleotides present inside these organelles. The interchange of phosphate groups among intravesicular nucleotides has already been reported in the chromaffin granule model (Aberer et al., 1978; Gualix et al., 1996). The linear uptake period reported here for ⑀-ATP is similar to that described in cholinergic vesicles from T. marmorata using [3H]ATP as substrate (Luqmani, 1981). This also agrees with the linear period for ⑀-ATP transport in chromaffin granules (Gualix et al., 1996). Further saturation studies were performed, taking into account this linear transport period. The two-step curve obtained for ⑀-ATP transport indicates a complex kinetic behavior. Enzymes with similar kinetic complexities and saturation curves, exhibiting various intermediate plateaus, have already been reported (Somero and Hochachka, 1969; Irving and Williams, 1973), and such kinetic behavior is known as hysteretic or mnemonic. The existence of membrane transporters with mnemonic kinetic behavior has already been reported for the equilibrative nucleoside transporter from neural cells (Casillas et al., 1993) and the vesicular nucleotide transporter from chromaffin granules (Gualix et al., 1996, 1997). The high values obtained for the Hill numbers indicate the existence of a multimeric form of the transporter. However, the existence of two different nucleotide transporters in brain synaptic vesicles cannot be ruled out. Concerning the maximal value that V can reach in rat brain synaptic vesicles, it is 1 order of magnitude lower than that reported for ⑀-ATP and [3H]ATP transport in chromaffin granules (Aberer et al., 1978; Gualix et al., 1996). When compared with the more closely related model of cholinergic synaptic vesicles isolated from the T. marmorata electric organ, transport capacity is found to be 40 times lower (Luqmani, 1981). This can be explained by the heterogeneity of synaptic vesicles when obtained from whole rat brain. This possibility is strengthened by comparing the nucleotide vesicular

NUCLEOTIDE TRANSPORT INTO SYNAPTIC VESICLES transporter with that of glutamate, which has been reported to be ⬃400 times more effective in the same model (Roseth et al., 1995). The nonhydrolyzable nucleotide analogues ATP␥S, ADP␤S, and AMPPNP behave as poor inhibitors of ⑀-ATP transport. These results agree with those obtained in chromaffin granules where these compounds act as activators at concentrations up to 1 mM, being poor inhibitors at higher concentrations, in close agreement with the cooperative kinetic model (Gualix et al., 1996). A similar situation exists with the structurally related diadenosine polyphosphates, which are substrates of the same transporter in the chromaffin granule model (Gualix et al., 1997). No good inhibitors have so far been found for nucleotide vesicular transport, although a large series of compounds with effects on the mitochondrial ATP/ADP exchanger or binding capabilities to nucleotide-recognizing sites (including both enzymes or receptors) have been assayed. This is the case for the polysulfonic aromatic compounds, among which are found the most potent antagonists of nucleotide receptors, such as suramin and pyridoxalphosphate-6-azophenyl-2⬘,4⬘-disulfonic acid (PPADS). Cibacron blue has been reported to be an inhibitor of nucleotide transport to chromaffin granule “ghosts” (Gru¨ninger et al., 1983), but it exhibits a reduced effect on the synaptic vesicle model. It is also worth emphasizing that, in the same vesicular model, Evans blue is able to inhibit glutamate vesicular transport in the nanomolar range (Roseth et al., 1995), but it is scarcely effective in inhibiting the nucleotide vesicular transport, even though it is the most potent of the polysulfonic derivatives assayed. NEM and atractyloside were also capable of inhibiting ⑀-ATP transport to synaptic vesicles to a similar extent and at similar concentrations as described for chromaffin granules and Torpedo synaptic vesicles (Luqmani, 1981; Gualix et al., 1996). As ATP is expected to be co-stored with aminergic and cholinergic compounds, the effects of noradrenaline and acetylthiocholine, a more hydrolysis-resistant analogue of acetylcholine, were studied. Noradrenaline, reported to be an activator of the nucleotide transport to chromaffin granule “ghosts” (Bankston and Guidotti, 1996), was ineffective in our model, and a very slight inhibitory effect was observed for acetylthiocholine. From the results presented here, ⑀-ATP appears to be a suitable substrate to characterize the nucleotide transport to synaptic vesicles from brain. This transport exhibits the same kinetic complexity as chromaffin granules. On the other hand, it could be a valuable tool that opens the possibility of a systematic scrutiny of specific and high-affinity inhibitors. Acknowledgment: This work was supported by research grants from the CAM (no. 8.5/18/98) and the Spanish Ministry of Education and Culture (DGCYT PM98-0089). J.G. is a recipient of a fellowship from the Spanish Ministry of Educa-

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tion and Culture. We thank Duncan Gilson for his help in preparing the manuscript.

REFERENCES Aberer W., Kostron H., Huber E., and Winkler H. (1978) A characterization of the nucleotide uptake by chromaffin granules of bovine adrenal medulla. Biochem. J. 172, 353–360. Bankston L. A. and Guidotti G. (1996) Characterization of ATP transport into chromaffin granule ghosts. Synergy of ATP and serotonin accumulation in chromaffin granule ghosts. J. Biol. Chem. 271, 17132–17138. Casillas T., Delicado E. G., Garcia-Carmona F., and Miras-Portugal M. T. (1993) Kinetic and allosteric cooperativity in L-adenosine transport in chromaffin cells. A mnemonical transporter. Biochemistry 32, 14203–14209. Gru¨ninger H. A., Apps D. K., and Phillips J. H. (1983) Adenine nucleotide and phosphoenolpyruvate transport by bovine chromaffin granule “ghosts.” Neuroscience 9, 917–924. Gualix J., Abal M., Pintor J., Garcı´a-Carmona F., and Miras-Portugal M. T. (1996) Nucleotide vesicular transporter of bovine chromaffin granules. Evidence for a mnemonic regulation. J. Biol. Chem. 271, 1957–1965. Gualix J., Fideu M. D., Pintor J., Rotlla´n P., Garcı´a-Carmona F., and Miras-Portugal M. T. (1997) Characterization of diadenosine polyphosphate transport into chromaffin granules from adrenal medulla. FASEB J. 11, 981–990. Hilderman R. H., Martin M., Zimmerman J. K., and Pivorun E. B. (1991) Identification of a unique membrane receptor for adenosine 5⬘,5⵮-P1,P4-tetraphosphate. J. Biol. Chem. 266, 6915– 6918. Irving M. G. and Williams J. F. (1973) Kinetic studies on the regulation of rabbit liver pyruvate kinase. Biochem. J. 131, 287–301. Luqmani Y. A. (1981) Nucleotide uptake by isolated cholinergic synaptic vesicles: evidence for a carrier of adenosine 5⬘-triphosphate. Neuroscience 6, 1011–1021. Lu¨thje J. and Ogilvie A. (1983) The presence of diadenosine 5⬘,5⵮P1,P3-triphosphate (Ap3A) in human platelets. Biochem. Biophys. Res. Commun. 115, 253–260. Mateo J., Miras-Portugal M. T., and Rotlla´n P. (1997a) Ectoenzymatic hydrolysis of diadenosine polyphosphates by cultured adrenomedullary vascular endothelial cells. Am. J. Physiol. 273, C918 –C927. Mateo J., Rotlla´n P., Martı´ E., Gomez de Aranda I., Solsona C., and Miras-Portugal M. T. (1997b) Diadenosine polyphosphate hydrolase from presynaptic plasma membranes of Torpedo electric organ. Biochem. J. 323, 677– 684. Pintor J. and Miras-Portugal M. T. (1995) A novel receptor for diadenosine polyphosphates coupled to calcium increase in rat midbrain synaptosomes. Br. J. Pharmacol. 115, 895–902. Pintor J., Diaz-Rey M. A., Torres M., and Miras-Portugal M. T. (1992a) Presence of diadenosine polyphosphates—Ap4A and Ap5A—in rat brain synaptic terminals. Ca2⫹ dependent release evoked by 4-aminopyridine and veratridine. Neurosci. Lett. 136, 141–144. Pintor J., Kowalewski H. J., Torres M., Miras-Portugal M. T., and Zimmermann H. (1992b) Synaptic vesicle storage of diadenosine polyphosphates in the Torpedo electric organ. Neurosci. Res. Commun. 10, 9 –14. Ralevic V. and Burnstock G. (1998) Receptors for purines and pyrimidines. Pharmacol. Rev. 50, 413– 492. Rodriguez del Castillo A., Torres M., Delicado E. G., and MirasPortugal M. T. (1988) Subcellular distribution studies of diadenosine polyphosphates—Ap4A and Ap5A—in bovine adrenal medulla: presence in chromaffin granules. J. Neurochem. 51, 1696 – 1703. Roseth S., Fykse E. M., and Fonnum F. (1995) Uptake of L-glutamate into rat brain synaptic vesicles: effect of inhibitors that bind specifically to the glutamate transporter. J. Neurochem. 65, 96 – 103.

J. Neurochem., Vol. 73, No. 3, 1999

1104

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Secrist J. A., Barrio J. R., Leonard N. J., and Weber G. (1972) Fluorescent modification of adenosine-containing coenzymes. Biological activities and spectroscopic properties. Biochemistry 11, 3499 –3506. Somero G. N. and Hochachka P. W. (1969) Isoenzymes and short-term temperature compensation in poikilotherms: activation of lactate dehydrogenase isoenzymes by temperature decreases. Nature 223, 194 –195. Weber A. and Winkler H. (1981) Specificity and mechanism of nucleotide uptake by adrenal chromaffin granules. Neuroscience 6, 2269 –2276.

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Winkler H. and Carmichael S. W. (1982) The secretory granule, in The Secretory Process (Poisner A. M. and Trifaro´ J. M., eds), pp. 3–79. Elsevier, Amsterdam. Zimmermann H. (1994) Signalling via ATP in the nervous system. Trends Neurosci. 17, 420 – 426. Zimmermann H. (1996a) Biochemistry, localization and functional roles of ecto-nucleotidases in the nervous system. Prog. Neurobiol. 49, 589 – 618. Zimmermann H. (1996b) Extracellular purine metabolism. Drug Dev. Res. 39, 337–352.