Neurochemistry International 50 (2007) 581–590 www.elsevier.com/locate/neuint
Biochemical analysis of ecto-nucleotide pyrophosphatase phosphodiesterase activity in brain membranes indicates involvement of NPP1 isoenzyme in extracellular hydrolysis of diadenosine polyphosphates in central nervous system§ Aaron C. Asensio a, Carmen R. Rodrı´guez-Ferrer a, Agustı´n Castan˜eyra-Perdomo b,c, Sol Oaknin a, Pedro Rotlla´n a,c,* a
Departamentos de Bioquı´mica y Biol. Molecular, Universidad de La Laguna, 38206 La Laguna, Tenerife, Spain b Anatomı´a, Anat. Patolo´gica e Histologı´a, Universidad de La Laguna, Spain c ICIC, Tenerife, Canary Islands, Spain Received 18 July 2006; received in revised form 14 November 2006; accepted 17 November 2006 Available online 21 December 2006
Abstract Synaptosomes and plasma membranes obtained from rat brain display ectoenzymatic hydrolytic activity responsible for hydrolysis of the neurotransmitter/neuroregulatory nucleotides diadenosine polyphosphates. Intact synaptosomes and plasma and synaptic membranes isolated by sucrose-gradient ultracentrifugation from several brain regions (hypothalamus, hippocampus, temporal cortex, frontal cortex striatum and cerebellum) degraded the fluorogenic substrates diethenoadenosine polyphosphates up to ethenoadenosine as by-product. Purified ectoenzyme cleaved substrates always releasing the mononucleotide moieties ethenoadenosine 50 -monophosphate and the corresponding ethenoadenosine (n 1) 50 -phosphate. Ectoenzymatic hydrolysis reached maximal activity at pH 9.0 (pH range 6.5–9.0) and was activated by Ca2+ and Mg2+ ions, with maximal effects around 2.0 mM cation. EDTA drastically reduced activity and Zn2+ was required for enzyme reactivation. Hydrolysis of substrates followed hyperbolic kinetics with Km values in the 3–10 mM range. Diadenosine polyphosphates and heparin behaved as competitive inhibitors in the enzymatic hydrolysis of diethenoadenosine polyphosphates and AMP, ATP, a,b-methyleneADP, ADPbS ATPgS, b,gmethyleneATP, suramin and diethyl pyrocarbonate were also inhibitors. Ectoenzymatic activity shared the typical characteristics of members of the ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) family and inhibition data suggest that NPP1 ectoenzyme is involved in the cleavage of extracellular diadenosine polyphosphates in brain. Synaptic membranes from cerebellum, hypothalamus and hippocampus presented the highest activities and no activity differences were observed between young and aged animals. However, plasma membranes showed a more homogeneous distribution of ectoenzymatic activity but a general increase was detected in aged animals. Enhancement of ectoenzymatic diadenosine polyphosphate cleaving activity found in plasma membranes from old animals could play a deleterious role in aged brain by limiting neuroprotective effects reported for extracellular diadenosine tetraphosphate. # 2006 Elsevier Ltd. All rights reserved. Keywords: Diadenosine polyphosphates; E-NPP enzymes; Plasma membranes; Synaptic membranes; Ageing
Abbreviations: ApnA, diadenosine 50 ,5000 -P1,Pn-polyphosphates; Ap2A, diadenosine 50 ,5000 -P1,P2-pyrophosphate; Ap3A, diadenosine 50 ,5000 -P1,P3-triphosphate; Ap4A, diadenosine 50 ,5000 -P1,P4-tetraphosphate; Ap5A, diadenosine 50 ,5000 -P1,P5-pentaphosphate; TMPpnp, thymidine 50 -monophosphate p-nitrophenyl ester; e(ApnA), di(1,N6-ethenoadenosine) 50 ,5000 -P1,Pn-polyphosphates; e-(Ap2A), di(1,N6-ethenoadenosine) 50 ,5000 -P1,P2-pyrophosphate; e-(Ap3A), di(1,N6-ethenoadenosine) 50 ,5000 -P1,P3-triphosphate; e-(Ap4A), di(1,N6-ethenoadenosine) 50 ,5000 -P1,P4-tetraphosphate; DEPC, diethyl pyrocarbonate; LPA, 1-oleyl lysophosphatidic acid; Ap3Aase, diadenosine triphosphatase (EC 3.6.1.29); Ap4Aase, asymmetrical diadenosine tetraphosphatase (EC 3.6.1.17); E-NPP, ecto-nucleotide pyrophosphatase/ phosphodiesterase (EC 3.1.4.1; EC 3.6.1.9); FU, fluorescence units; HPLC, high performance liquid chromatography § This paper is dedicated to the memory of our recently deceased friend and colleague, Dr. Javier Corzo Varillas. * Corresponding author. Tel.: +34 922 318357; fax: +34 922 318354. E-mail address:
[email protected] (P. Rotlla´n). 0197-0186/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2006.11.006
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A.C. Asensio et al. / Neurochemistry International 50 (2007) 581–590
Diadenosine polyphosphates (ApnA) contain two adenosine moieties joined through the 50 position of the ribose rings by a polyphosphate chain composed of n 2 members. They are the most investigated members of the large family of dinucleoside polyphosphates, a group of dinucleotides with intriguing biological functions (McLennan, 2000). Several of these have been identified in the releasable content of storage granules in specialized neurosecretory cells, endothelial cells and platelets and are thought to function as extracellular signalling molecules in the vascular and nervous systems. They therefore bind to plasma membrane receptors and display physiological actions as blood pressure regulators, neurotransmitters and/or neuromodulators (Pintor et al., 2000; Hilderman et al., 2001; Hoyle et al., 2001; Jankowski et al., 2005). In the nervous system, ApnA (n = 2–6) and other dinucleoside polyphosphates have been found in the catecholaminergic and cholinergic vesicles from adrenomedullary chromaffin cells and neurons co-stored with mononucleotides, mainly ATP and ADP (Pintor et al., 2000; Jankowski et al., 2003). Exocytotically released ApnA interact with specific dinucleotide receptors but the ionotropic P2X, metabotropic P2Y and P1 adenosine receptors may also function as ApnA targets (Pintor and Miras-Portugal, 1995; Dı´az-Herna´ndez et al., 2001; Hoyle et al., 2001). On addition, results obtained using very different experimental approaches support the idea that extracellular ApnA can effectively modulate neural functions (Jime´nez et al., 1998; Pereira et al., 2000; Hoyle et al., 2001; Oaknin et al., 2001). More recently, neuroprotective effects against injuries induced by ischemia and 6-hydroxydopamine in rat brain have been reported for extracellular Ap4A (Wang et al., 2003). The inactivation of ApnA by ectoenzymes at the cell surface provides a necessary mechanism to regulate the receptormediated actions of ApnA (Zimmermann, 2001; Rotlla´n et al., 2002). Available data indicate that these widely distributed membrane-bound enzymes catalyze the hydrolytic cleavage at the polyphosphate chain of these dinucleotides to produce the mononucleotidic moieties AMP + adenosine 50 (n 1) phosphate. Most of these display biochemical characteristics typical of members of the ecto-nucleotide pyrophosphatase phosphodiesterase (E-NPP) family formerly known as ecto-phosphodiesterase/pyrophosphatase I, PC-1, or phosphodiesterase/ nucleotide pyrophosphatase family (Zimmermann, 2001). This family contains seven members, three of which, NPP1, NPP2 and NPP3, cleave mono- and dinucleotides (Zimmermann, 2001; Stefan et al., 2005). These activities may be considered as the first step of an ectoenzymatic cascade responsible for the catabolism of extracellular dinucleotides producing mainly adenosine, and other nucleosides, which may be salvaged by transport systems inside cells. Other enzymes that cleave ApnA, although located inside cells and main regulators of intracellular ApnA concentrations, are the two specific hydrolases Ap4Aase (20 kDa) and Ap3Aase (32 kDa) the latter recently identified with the tumour suppressor Fhit protein (Guranowski and Sillero, 1992; Asensio et al., 2006). While ectoenzymatic hydrolysis of ApnA has been analysed in adrenomedullary chromaffin cells (Rodrı´guez-Pascual et al., 1992; Ramos et al., 1995) and synaptic membranes of the Torpedo electric organ
(Mateo et al., 1997b), there are scarce data available on the ecto-nucleotidases inactivating exocytotically released ApnA in central nervous system. In this paper we present a systematic biochemical characterization of the ectoenzymatic ApnAcleaving activity in plasma and synaptic membranes from rat brain, together with its regional distribution and ageing-related changes.
1. Experimental procedures 1.1. Materials ApnA n = 2–5, e-Ado1, e-adenosine 50 -mononucleotides, e-NAD+, TMPpnp and other nucleotide analogues, heparin (average 3 kDa) and its derived disaccharides, DEPC, LPA, Hepes and molecular weight markers were purchased from Sigma. Suramin was from RBI, C. durissus phosphodiesterase from Boehringer and Tris from Merck. DEAE-Sephacel and Hi-prep column packed with Sephacryl S-200 HR were from Pharmacia Biotech. All other reagents were of analytical grade. The fluorogenic substrates e-(ApnA) n = 2–5 were prepared from ApnA, purified and their properties verified as previously described (Ramos et al., 1995).
1.2. Animals Wistar male rats, 3 and 24 months old (young adults and aged, respectively) were maintained in the research facilities centre in our University under controlled light and temperature, receiving standard laboratory food and water ad libitum. The present investigation conformed to the ‘‘Guide for the care and use of laboratory animals’’ (U.S. National Institutes of Health, NIH publication No. 85-23, revised in 1996). Efforts were made to minimize the number of animals and their suffering. Animals were killed by decapitation after previous anaesthesia (Equithesin, 2 ml/kg, i.p.) and brains rapidly removed for immediate processing.
1.3. Preparation of membranes and synaptosomes All operations were performed at 0–4 8C. For purification and biochemical characterization of ectoenzymatic ApnA-cleaving activity, forebrain and cerebellum were separated and independently processed to obtain plasma and synaptic membranes and synaptosomes. To determine regional distribution of ectoenzymatic ApnA-cleaving activity in brain, the hypothalamus, hippocampus, temporal cortex, frontal cortex, striatum and cerebellum were dissected from brain and processed to obtain plasma and synaptic membranes. Membranes and synaptosomes were obtained according to a published protocol (Scha¨fer and Reiser, 1997) which involves ultracentrifugation in a sucrose step gradient (1.2, 1.0, 0.8 and 0.32 M sucrose) of a particulate fraction obtained from fresh brain tissue homogenized in isotonic Hepes buffer. Plasma membranes and synaptosomes were collected from the interfaces 0.32/0.8 M sucrose and 1.0/1.2 M, respectively. Collected synaptosomes were resuspended in 5 mM Tris–HCl, pH 7.4, 0.32 M sucrose, washed twice (10,000 g, 45 min) and the resulting pellet carefully resuspended in the same buffer to obtain a final synaptosome suspension to be immediately used in enzymatic assays. To obtain synaptic membranes, synaptosomes were further processed to be lysed and ultracentrifuged in a second sucrose step gradient and synaptic membranes collected from the interface 1.0/1.2 M sucrose. Plasma and synaptic membranes collected from sucrose gradients were each resuspended in 5 mM Tris–HCl, pH 7.4, washed twice (10,000 g, 45 min) and final suspensions stored at 40 8C. These membrane preparations were used in enzyme assays and as starting material in ectoenzyme purification. An L8-M ultracentrifuge
1
The symbol ‘‘e’’ stands for 1,N6-etheno bridge. e-AMP, e-ADP, e-ATP, eAp4, e-Ado and e-NAD+ refer to the 1,N6-etheno derivatives of AMP, ADP, ATP, adenosine 50 -tetraphosphate, adenosine and NAD+, respectively.
A.C. Asensio et al. / Neurochemistry International 50 (2007) 581–590 with an SW41Ti rotor (Beckman) and a Sorvall RC-5B with a SS-34 rotor (DuPont) were used throughout.
1.4. Fluorimetric enzyme assays 1.4.1. Continuous (on-line) fluorimetric assays These were used to record the time-dependent fluorescence increase associated with the hydrolysis of e-(ApnA) to e-mononucleotide moieties by membrane preparations and were performed in quartz microcuvettes using a Hitachi F-2000 spectrofluorimeter set at lex 305 nm and lem 410 nm. The usual assay mixtures (250 ml) contained Tris–HCl 50 mM pH 7.5 or 9.0 as required, CaCl2 4 mM, Zn2+ 200 mM, the appropriate concentration of substrate and enzyme and were incubated at 37 8C under stirring for up to 5 min. To establish equivalence between fluorescence units and substrate moles, fluorescence was read again after total substrate hydrolysis, which was ensured by addition of C. durissus phosphodiesterase (0.5 units, 0.5 ml). For pH and cation dependence studies, the assay mixture was accordingly modified. We found (see Section 2) that in addition to the ectoenzymatic ApnA-cleaving activity of broad substrate specificity, membrane preparations displayed Ap3Aase and Ap4Aase activities which, respectively, act on Ap3A and Ap4A but not on Ap2A. To avoid interference from these specific hydrolases, leading to activity overestimations when using the substrates e-(Ap3A) or e-(Ap4A), Zn2+ (acetate) was included in assay mixtures as it is an inhibitor of both Ap3Aase and Ap4Aase but an essential cation for the ectoenzyme. F (1 mM NaF) may also be included to selectively inhibit Ap4Aase. See an extensive review by Guranowski and Sillero (1992) for useful information on molecular and kinetic aspects of ApnAcleaving enzymes. Use of pH 9.0, optimal for ectoenzymatic activity, and the substrate e-(Ap2A) provides maximal sensitivity for ectoenzymatic activity measurements. One unit (U) of enzyme activity is the amount of enzyme hydrolysing 1 mmol substrate/min at 20 mM substrate. To determine Km values for e-(ApnA), initial reaction rates were measured at fixed substrate concentrations ranging between 0.5 and 20 mM. Enzyme activity was adjusted to obtain initial linear time-dependent fluorescence increases and substrate consumptions below 10%. When using intact synaptosomes, the assay mixture was composed of Tris–HCl 25 mM pH 7.4, CaCl2 4 mM, glucose 5 mM and sucrose 0.25 M. Kinetic parameters were determined using the FigP software (Biosoft). 1.4.2. Non-continuous fluorimetric assays These were used to detect ApnA-cleaving activities in chromatographic eluates. The substrates e-(Ap2A), e-(Ap3A), e-(Ap4A), e-(Ap5A) and e-NAD+ were used to detect dinucleotide-cleaving activity. Reaction mixtures (250 ml) containing Tris–HCl 50 mM pH 9.0, CaCl2 4 mM, 0.2% Triton X-100, substrate 5 mM and protein eluate (50 ml) were prepared in eppendorf tubes and incubated at 37 8C for the required time (10–60 min). The substrates e(Ap3A) and e-(Ap4A) were used to detect specific Ap3Aase and asymmetrical Ap4Aase, respectively. Reaction mixtures (250 ml) contained Hepes-KOH 50 mM pH 7.2, MgCl2 4 mM, 0.2% Triton X-100, substrate 5 mM and protein eluate (50 ml). Reactions were stopped by cooling in an ice-water bath and fluorescence was immediately measured.
1.5. Spectrophotometric assays These were performed in a Shimadzu UV-160 instrument. The typical artificial substrate TMPpnp was used to detect ecto-nucleotide pyrophosphatase phosphodiesterase activity. Assay mixtures contained Tris–HCl 50 mM pH 9.0, MgCl2 4 mM, 0.2% Triton X-100, TMPpnp 200 mM in a final volume of 100 ml. Absorbance due to the p-nitrophenol released was measured in 0.1 M NaOH at 400 nm. Protein concentration was spectrophotometrically determined by the Coomassie binding method using BSA as a standard. Protein elution profiles in chromatographic eluates were detected by measuring A295 instead of A280 to avoid Triton X-100 interference.
1.6. Purification of ectoenzymatic ApnA-cleaving activity Preparations of purified plasma and synaptic membranes isolated from forebrain and cerebellum of young animals by sucrose gradient ultracentrifugation were treated with 2% Triton X-100 in Tris–HCl 20 mM, pH 7.5 with
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shaking overnight at 4 8C. Insoluble material was eliminated by centrifugation (10,000 g, 30 min) and the clear supernatant sequentially fractionated by ionexchange and gel-filtration chromatography. Enzyme activity profiles were detected by non-continuous fluorimetric assays using the substrates e-(ApnA) and e-NAD+ or spectrophotometrically using the substrate TMPpnp. 1.6.1. Anion-exchange chromatography Solubilised protein was applied to a DEAE-Sephacel column (16 mm 400 mm) equilibrated in 20 mM Tris–HCl pH 7.5, 0.2% Triton X-100 and eluted with the same buffer until eluent A295 was null. ApnA-cleaving activity was eluted by application of a linear NaCl gradient (0–0.3 M, 400 ml) in the equilibrating buffer at 0.8 ml/min and 8 ml fractions were collected; then the column was washed with 100 ml of 2 M NaCl and re-equilibrated with starting buffer. Fractions containing enzymatic activity were pooled and concentrated using Centricon concentration devices (Millipore) for further fractionation by gel filtration chromatography. 1.6.2. Gel filtration chromatography The concentrated active fraction collected in ion-exchange chromatography was applied to a Hi-prep column (26 600) packed with Sephacryl S-200, equilibrated and eluted with 20 mM Tris–HCl, pH 7.5, 0.1 M NaCl, 0.2% Triton X-100 at a flow rate of 2 ml/min, collecting 4 ml fractions. The column was calibrated with the markers blue dextran, b-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa) and cytochrome c (12.4 kDa).
1.7. HPLC assays A Waters instrument composed of a 470 fluorescence detector set at lex 305 nm, lem 410 nm, an M-510 pump and a U6K injector was used to detect substrates and reaction products. The column, Nova-Pak C18 (Waters), was eluted with 10 mM KH2PO4, 25% acetonitrile and 4 mM tetrabutylammonium bromide pH 7.0 at 1.0 ml/min. Incubation mixtures were prepared in eppendorf tubes, final volume 100 ml (Tris–HCl 50 mM pH 9.0, CaCl2 4 mM, 0.2% Triton X-100) and incubated at 37 8C for the required time. The enzymatic reaction was stopped by placing tubes in an ice-water bath with immediate addition of 9 vol ice-cold mobile phase, filtered to eliminate proteins and 20–50 ml were injected into the chromatograph.
2. Results 2.1. General biochemical features of e-(ApnA) hydrolysis by plasma and synaptic plasma membranes and intact synaptosomes HPLC analysis demonstrated that the substrates e-(ApnA) n = 2–5 were all converted into e-Ado by both plasma and synaptic membranes derived from forebrain and cerebellum. All possible intermediate e-mononucleotide moieties in the conversion of e-(ApnA) into e-Ado were observed in chromatograms (Fig. 1). Continuous on-line fluorimetric assays, which specifically detect the hydrolytic cleavage of e-(ApnA) into e-mononucleotides were used throughout as they are particularly well suited for kinetic studies. These assays demonstrated that hydrolysis of e-(ApnA) n = 2–5 by both plasma and synaptic membranes was greatly stimulated at alkaline pH; in the pH range 6.5–9.0, maximal activity was observed at pH 9.0 (Fig. 2). Hydrolysis by membranes was stimulated by Ca2+ and Mg2+ ions and similar activity plateaux for each cation were achieved at around 2 mM and maintained up to at least 5 mM. About half the plateau activity was measured in the absence of divalent cations and this residual
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Fig. 1. Representative HPLC profile showing degradation of e-(Ap4A) by rat brain plasma membranes. Preparations of plasma and synaptic membranes from forebrain and cerebellum were incubated at 37 8C with 5 mM e-(ApnA), n = 2– 5, in Tris–HCl 50 mM pH 9.0, CaCl2 4 mM (final volume 100 ml) for 0, 10 and 20 min. Aliquots of 20 ml filtered reaction mixtures were injected into the chromatograph and peaks were detected by fluorescence detection. Similar profiles were obtained for degradation of the substrates e-(ApnA) n = 2, 3 and 5 by both plasma and synaptic membranes.
activity was further decreased by 1 mM EDTA. Addition of 4– 5 mM Mg2+ or Ca2+ to EDTA-treated membranes produced only a slight reactivation; full reactivation required 200 mM Zn2+ in addition to Mg2+ or Ca2+, but Zn2+ alone had only slight activating effect (not shown). Heparin and suramin, but not LPA, were inhibitors of ectoenzymatic ApnA-cleaving activity in both plasma and synaptic membranes as shown in Fig. 3. Hydrolysis of e-(Ap3A) and e-(Ap4A), but not of e-(Ap2A), by plasma and synaptic membranes was partially inhibited (30– 40%) by typical inhibitors of the highly specific intracellular soluble enzymes Ap3Aase (Zn2+ 200 mM) and Ap4Aase (F 1 mM, Zn2+ 200 mM or Ca2+ 5 mM) (Guranowski and Sillero,
Fig. 2. Effect of pH on ectoenzymatic ApnA-cleaving activity of plasma membranes and synaptosomes. Preparations of plasma membranes and intact synaptosomes (inset) from forebrain were incubated at 37 8C in the presence of e-(ApnA) n = 2–5, 20 mM, and hydrolysis rates at different pH values were measured by continuous fluorimetric assays. Plasma membranes were incubated in Tris–HCl 50 mM, CaCl2 4 mM and synaptosomes in Tris–HCl 25 mM, CaCl2 4 mM, glucose 5 mM and sucrose 0.25 M in 250 ml final volume.
Fig. 3. Inhibition of ectoenzymatic ApnA-cleaving activity in membranes by heparin and suramin. Plasma and synaptic membranes from forebrain and cerebellum were assayed by continuous fluorimetric assays (except in the case of suramin, see below). Reaction mixtures incubated at 37 8C contained Tris– HCl 50 mM pH 9.0, CaCl2 4 mM and e-(Ap2A) 10 mM at varying concentrations of heparin and suramin (0–100 mM) and of LPA (0–10 mM, data on LPA are shown in red). Activities are expressed as percentage of linear fluorescence trace slopes in the presence of inhibitor relative to control slopes without inhibitor. Graphs represent the means of two experiments, each performed in triplicate. To avoid quenching of fluorescence by suramin (negligible at concentrations lower than 5 mM), experimental protocol was slightly modified as follows: Reaction mixtures of 100 ml were prepared in eppendorf tubes and incubated at 37 8C. Aliquots of 10 ml (up to 4 for each reaction mixture) were taken each 4 min and immediately diluted in 1 ml of 10 mM Hepes pH 7.5, to read fluorescence. Activities were calculated from slopes of generated graphs as in continuous assays.
1992; Ramos and Rotlla´n, 1995). These Ap3Aase and Ap4Aase activities could not be eliminated from membrane preparations by washing but were released by Triton X-100 treatment, together with e-(Ap2A) ectoenzymatic cleaving activity (Fig. 4). The presence of Ap3Aase and Ap4Aase associated to membranes was later confirmed by gel-filtration chromatography of detergent-solubilised membrane proteins (next section). These data indicated that preparations of highly purified plasma and synaptic membranes contain an ectoenzymatic activity which cleaves e-(ApnA) n = 2–5 with the characteristics of members of the E-NPP family, but also other accompanying activities displaying properties of the soluble intracellular enzymes Ap3Aase and Ap4Aase. Interference by these two specific hydrolases was usually avoided by including 200 mM zinc acetate in assay mixtures. Lysis of intact synaptosomes by Triton X-100 dramatically increased hydrolytic activity on e-(Ap3A) and e-(Ap4A) but not on e-(Ap2A), as shown in Fig. 5. Detergent-released activities hydrolysing e(Ap3A) and e-(Ap4A) were very sensitive to inhibitors of Ap3Aase and Ap4Aase, indicating release of these enzymes from intrasynaptosomal space into milieu. Ectoenzymatic hydrolysis of e-(ApnA) n = 2–4 by plasma membranes from forebrain and cerebellum, and by forebrain synaptosomes, followed hyperbolic kinetics with Km and Vmax values presented in Table 1 and Fig. 6. No significant
A.C. Asensio et al. / Neurochemistry International 50 (2007) 581–590
Fig. 4. Diversity of ApnA-cleaving activities in isolated rat brain plasma membranes. Suspensions of forebrain plasma membranes (500 ml in 5 mM Tris–HCl pH 7.5) were assayed for ApnA-cleaving activity by continuous fluorimetric assays using the substrates e-(Ap2A), e-(Ap3A) or e-(Ap4A) at 20 mM in Tris–HCl 50 mM pH 7.5, MgCl2 4 mM in the absence (control) or presence of 100 mM Zn2+. (A) Activities measured in membrane suspensions. (B) Activities measured in clear supernatants after centrifugation (17,000 g, 30 min). (C) Activities measured in membrane suspensions resulting from replacing supernatants (obtained in B) with fresh suspension buffer and resuspension on membrane pellet. (D) Activities measured in membrane suspensions after treatment by 2% Triton X-100. Bars represent the means of two experiments, each performed in duplicate. Solubilisation of membrane pellets (obtained in B) in 2% Triton X-100 in suspension buffer, yielded very similar activity profiles to those shown in (D). Enzymatic activities are associated with membranes, since very low activity was detected in supernatants after centrifugation. Hydrolysis of e-(Ap2A) by the ectoenzyme was unaffected by Zn2+, but this cation partially inhibited hydrolysis of e-(Ap3A) and e-(Ap4A), suggesting that membrane-associated Ap3Aase and Ap4Aase contribute, respectively, to e-(Ap3A) and e-(Ap4A) cleavage by membrane suspensions.
differences were observed in Km values determined using synaptic membranes or intact synaptosomes. 2.2. Purification of ectoenzymatic ApnA-cleaving activity present in plasma and synaptic membranes and further enzymatic characterization Plasma and synaptic membranes derived from forebrain and cerebellum were solubilised by Triton X-100 treatment and fractionated by ion-exchange chromatography. Hydrolytic activities on e-(ApnA), n = 2–5, and e-NAD+ yielded very similar superimposed profiles, completely overlapping with the peak originated from TMPpnp, a typical marker substrate for nucleotide pyrophosphatase phosphodiesterase activity
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Fig. 5. Effects of Triton X-100 treatment on hydrolysis of e-(ApnA) by brain synaptosomes. Preparations of intact synaptosomes from forebrain were divided into two half moieties. One was subjected to lysis by 2% Triton X-100, centrifuged to obtain a clear supernatant and used for enzymatic assays; the untreated moiety served as control. Aliquots of control and detergent-solubilised synaptosomes were assayed for ApnA-cleaving activity by continuous fluorimetric assays using the substrates e-(Ap2A), e-(Ap3A) or e-(Ap4A) at 20 mM and in presence of several ions (Ca2+ 4 mM, Mg2+ 4 mM, Zn2+ 200 mM or F, 1 mM), as indicated. Assays were conducted in Tris–HCl 25 mM pH 7.5, CaCl2 4 mM or MgCl2 as indicated, glucose 5 mM and sucrose 0.25 M (250 ml final volume). Hydrolysis of e-(Ap2A) was not modified after synaptosome lysis and Zn2+ had no effect. Increased hydrolysis rates of e-(Ap3A) and e-(Ap4A) after Triton X-100 treatment and their inhibition by Zn2+ and F, respectively, indicate release of Ap3Aase and Ap4Aase.
(Fig. 7A). Further fractionation by gel filtration chromatography revealed three well-differentiated activity peaks (Fig. 7B). The first peak again grouped superimposed activity profiles on e-(ApnA), n = 2–5, e-NAD+ and TMPpnp, indicating strict co-elution of both nucleotide pyrophosphatase phosphodiesterase and dinucleotide-cleaving activities. This peak eluted close to elution volume, between blue dextran and b-amylase, indicating a molecular mass somewhat higher than 200 kDa for the enzyme involved. The second and third peaks, which co-eluted within the broad specificity activity peak in the previous ion-exchange chromatography step, were due to enzymes only active on e-(Ap3A) and e-(Ap4A), respectively. Such activities were very sensitive to inhibition by Zn2+ and coeluted in gel filtration with authentic Ap3Aase (32 kDa) and Table 1 Kinetic parameters for e-(ApnA) hydrolysis by plasma and synaptic membranes Substrate
Synaptic membranes
Plasma membranes
Km
Vmax
Km
Vmax
e-(Ap2A) e-(Ap3A) e-(Ap4A)
6.0 1.4 5.9 2.8 7.9 2.2
1.17 0.29 1.25 0.37 1.01 0.41
4.0 1.9 3.1 1.2 3.2 1.7
1.47 0.27 1.26 0.19 0.96 0.17
Km and Vmax values in mM and in nmol/min/mg protein (mU/mg protein), respectively. Results are means S.E.M. of three determinations, each performed in triplicate.
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phosphate were released from all the substrates (Fig. 8). The dinucleotides ApnA n = 2–5 were substrates as demonstrated by HPLC with UV detection and originated the expected AMP and adenosine 50 (n 1) phosphate moieties; all behaved as competitive inhibitors of e-(Ap2A) hydrolysis with similar Ki values around 5 mM. The mononucleotides AMP and ATP and the nucleotide analogues a,b-methyleneADP, ADPbS ATPgS, b,g-methyleneATP also behaved as inhibitors (Fig. 9). Heparin was a competitive inhibitor of e-(Ap2A) hydrolysis; considering that heparin in our preparation had an average molecular mass of 3 kDa (supplier data), a Ki value of 25 mM was calculated (Fig. 9). DEPC produced dose-dependent inhibition which was almost complete at 5 mM inhibitor (Fig. 9). Inhibition was additionally confirmed by HPLC analysis by assessing the decrease in e-AMP production from the substrate e-(Ap2A). Fig. 6. Kinetics of hydrolysis of e-(Ap2A) by synaptic membranes and synaptosomes. Preparations of synaptic membranes and intact synaptosomes from forebrain were incubated at 37 8C in the presence of e-(Ap2A) in the concentration range 0–20 mM and hydrolysis rates measured by continuous fluorimetric assays and expressed as percentage of Vmax values. Synaptic membranes were incubated in Tris–HCl 50 mM pH 7.5, CaCl2 4 mM, and synaptosomes in Tris–HCl 25 mM pH 7.5, CaCl2 4 mM, glucose 5 mM and sucrose 0.25 M in 250 ml final volume. Graphs represent the means of two experiments, each performed in triplicate.
Ap4Aase (20 kDa) previously purified from rat brain cytosolic fraction (Asensio et al., 2006), data not shown. The broad specificity peaks were concentrated and subjected to further biochemical characterization. HPLC analysis of purified ApnA-cleaving activity from plasma and synaptic membranes derived from forebrain and cerebellum demonstrated a unique hydrolytic cleavage pattern for e-(ApnA) n = 2–5; only peaks identified as e-AMP and e-Ado 50 (n 1)
2.3. Distribution of ectoenzymatic ApnA-cleaving activity in brain and ageing-related changes This activity in plasma and synaptic membranes derived from several brain regions of young adult and aged rats is presented in Fig. 10. The substrate e-(Ap2A), the best marker of ectoenzymatic ApnA-cleavage, was chosen. Among synaptic membranes, those from cerebellum, hypothalamus and hippocampus presented the highest activity, cortex and striatum presenting lower and similar values. No differences were found between young adult (0.6–2.60 mU/mg) and aged animals (0.6–2.8 mU/mg). Plasma membranes showed a more homogeneous distribution, with less variation between regions. However, in aged animals (2.6–5.5 mU/mg), a general increase of about two-fold was noted in ectoenzymatic ApnA-cleaving activity of plasma membranes relative to young adults (1.5– 2.5 mU/mg). In all brain regions analysed, ectoenzymatic activity was higher in the plasma membranes than in synaptic
Fig. 7. Chromatographic fractionation of ectoenzymatic ApnA-cleaving activity present in plasma and synaptic membranes from forebrain and cerebellum. (A) Anion-exchange chromatography. Triton X-100 solubilised protein extracts were applied onto a DEAE-Sephacel column equilibrated in 20 mM Tris–HCl pH 7.5, 0.2% Triton X-100 and eluted by a NaCl gradient 0–0.3 M. Fractions containing hydrolytic activity towards e-(ApnA) n = 2–4, e-NAD+ and TMPpnp were pooled and concentrated. (B) Gel filtration chromatography. Concentrated fractions obtained after ion-exchange chromatography were applied onto a Hi-prep column (Sephacryl S-200) equilibrated and eluted with 20 mM Tris–HCl pH 7.5, 0.1 M NaCl, 0.2% Triton X-100. Enzyme activities in chromatographic eluates were detected using the substrates e-(ApnA) n = 2–4 and e-NAD+ (non-continuous fluorimetric assays, FU) and TMPpnp (spectrophotometric assays, A400). Protein elution was assessed by measuring A295 in chromatographic eluates. Figure shows representative profiles obtained from forebrain plasma membranes; similar profiles were derived from synaptic membranes from forebrain and cerebellum.
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Fig. 8. Hydrolysis of e-(ApnA) by purified ectoenzymatic ApnA-cleaving activity. Aliquots of final preparations of purified ectoenzyme from forebrain and cerebellum plasma membranes and from forebrain and cerebellum synaptosomes were incubated at 37 8C in eppendorf tubes (Tris–HCl 50 mM pH 9.0, CaCl2 4 mM, 0.2% Triton X-100, final vol 100 ml) with e(ApnA), n = 2–5, and reaction mixtures (10 ml) at 10 min of incubation, subjected to HPLC analysis. Only e-AMP and the corresponding e-Ado (n 1) phosphate moieties appear as products of e-(ApnA) cleavage, indicating the absence of mononucleotide dephosphorylating activities; compare with Fig. 1. Figure shows representative HPLC profiles generated by ectoenzyme purified from forebrain plasma membranes; identical results were obtained for cerebellum ectoenzyme.
ones; however, cerebellum synaptic membranes from young adults displayed higher activity than corresponding plasma membranes. 3. Discussion Plasma and synaptic membranes and synaptosomes isolated from rat brain were used to gain insights into the ectonucleotidases involved in the hydrolysis of extracellular signalling nucleotides ApnA in nervous system. Earlier work in our laboratory showed that combined use of highly purified membrane preparations and the fluorogenic substrates e(ApnA) provides useful biochemical information on ApnAcleaving ecto-nucleotidases, avoiding complications derived from cell cultures (Ramos et al., 1995; Rotlla´n et al., 2002). In accordance with research on particulate fractions of rat brain and liver (Cameselle et al., 1984; Garcı´a-Agu´ndez et al., 1991), we found that membranes obtained by sucrose-gradient
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ultracentrifugation display typical Ap3Aase and Ap4Aase activities, in addition to the ectoenzymatic ApnA-cleaving activity reported. Since no evidence for the presence of Ap3Aase and Ap4Aase was found when using intact cultured cells (Ramos et al., 1995), any association to plasma membranes must be on the cytosolic side. An artifactual association produced during tissue homogenisation cannot be ruled out. In any case, interference by these specific hydrolases was overcome by using e-(Ap2A) as an ectoenzyme substrate, since dinucleotides with two phosphoryl groups are not substrates of Ap3Aase and Ap4Aase; or otherwise by including Zn2+ in assay mixtures to inhibit Ap3Aase and Ap4Aase when using e-(Ap3A) or e-(Ap4A) as substrates. Chromatographic analysis of e-(ApnA) degradation by membranes suggests that ApnA catabolism in the nervous system proceeds via a first hydrolytic cleavage ApnA ! AMP + adenosine 50 (n 1) phosphate, followed by dephosphorylation of released moieties down to adenosine. Dinucleotide cleavage was confirmed by observing that e-AMP and e-Ado (n 1) phosphate were the only products of e-(ApnA) hydrolysis by purified ectoenzyme. A short report indicated that cortical rat brain synaptosomes degraded Ap4A and Ap5A time and protein dependently, but no other biochemical data were provided (Emanuelli et al., 1998). General characteristics of ectoenzymatic ApnA-cleaving activity from brain and cerebellum, e.g. alkaline optimum pH, activation by Ca2+ and Mg2+ and inhibition by EDTA or Km values for dinucleotide hydrolysis in the low micromolar range, are similar to those reported in adrenochromaffin cells and Torpedo electric organ synaptosomes (Ramos et al., 1995; Mateo et al., 1997b), airway epithelial cells (Picher and Boucher, 2000) or Xenopus oocytes (Aguilar et al., 2001). These characteristics, along with others like a molecular mass somewhat higher than 200 kDa, ability to cleave TMPpnp and e-NAD+, and inhibition by DEPC, fit well with those of E-NPP family members (NPP1, NPP2 and NPP3) that hydrolyse TMPpnp, ATP and dinucleotides like ApnA or NAD+ (Goding et al., 1998; Bollen et al., 2000; Zimmermann, 2001; Vollmayer et al., 2003). This strongly suggests the involvement of NPP1–3 isoenzymes in the inactivation of extracellular ApnA in nervous system. It is difficult to discriminate between NPP isoenzymes present in a cell or tissue by biochemical criteria because of their similar catalytic properties; this question being further complicated by observing that several NPP ectoenzymes may be located in the same cell, e.g. in hepatocytes, and that their expression is developmentally regulated (Goding et al., 1998; Stefan et al., 1999). This activity clearly differs from the ApnAcleaving ectoenzymatic activity investigated in vascular endothelial cells, which is inhibited by Ca2+ (Mateo et al., 1997a). The ability of synaptosomes to cleave dinucleotides points to an immediate contribution of neurons to inactivating exocytotically released ApnA. Nevertheless, activity found in plasma membranes additionally suggests that dinucleotides diffused from synaptic clefts may also be cleaved far from release sites by the neurons themselves and by the more abundant glial cells. Previous investigations have provided
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Fig. 9. Inhibition of ectoenzymatic ApnA-cleaving activity purified from cerebellum plasma membranes by heparin, nucleotides and DEPC. Activities were measured by continuous fluorimetric assays using e-(Ap2A) as substrate. (A) Ectoenzyme samples were assayed at variable substrate concentrations (1–20 mM) in absence and presence of heparin (20 and 100 mM) and results presented as Lineweaver–Burk plots. (B and C) Ectoenzyme samples were assayed at 10 mM substrate in the absence (control) and in presence of 50 mM of indicated nucleotides (B) or at variable DEPC concentrations (0–5 mM). Activities expressed as percentage of fluorescence trace slopes in the presence of inhibitor relative to control slopes without inhibitor. Graphs represent the means of two experiments, each performed in triplicate.
information on the expression of NPP1–3 ectoenzymes in nervous system and in neural tumoural cells. NPP1 was not detected in brain neurons and glial cells but its expression has been reported in C6 and C6Bu-1 glioma cells (Goding et al., 2003; Grobben et al., 1999; Ohkubo et al., 2001). NPP2 was strongly expressed in glial cells from brain and cerebellum but not detected in neurons or adrenal medulla (Narita et al., 1994; Fuss et al., 1997). NPP3 was detected in some glial precursor cells in immature rat brain, in 9L glioma cells but not in the adult rat brain nor in C6 glioma cells (Deissler et al., 1995; Andoh et al., 1999). Accordingly, expression data could suggest participation of glial cells, mediated by NPP2, in the ectoenzymatic cleavage of ApnA in the adult central nervous system. However, the lack of inhibition of ApnA-cleaving activity in plasma and synaptic membranes observed here by the potent and selective NPP2 inhibitor LPA (Meeteren et al., 2005) and recent results showing that NPP2 is a secreted enzyme (Stefan et al., 2005; Koike et al., 2006) do not support such a role for NPP2. Our data on inhibition of ApnA cleavage in plasma and synaptic membranes by heparin and suramin, both reported as NPP1 inhibitors which do not affect NPP3 (Hosoda et al., 1999; Vollmayer et al., 2003), rather point to NPP1 as a main ectoenzyme involved in the cleavage of ApnA by glial cells and neurons. In this context it is worth noting that adrenal medulla, similarly to brain neurons and glial cells, was first reported as a negative tissue for NPP1 expression (Harahap and Goding, 1988) but later work demonstrated that adrenomedullary chromaffin cells, typical paraneurons, readily hydrolyse extracellular ApnA and express NPP1 isoenzyme (Rodrı´guez-Pascual et al., 1992; Ramos et al., 1995; Gasmi et al., 1998). Future efforts will be necessary to draw a detailed
distribution map of NPP isoenzymes in the different neural cells. Specific activities found in plasma and synaptic membranes are of the order of those determined in plasma and synaptic membranes from chromaffin cells and Torpedo electric organ (Ramos et al., 1995; Mateo et al., 1997b). However, distribution of ectoenzymatic activity in membranes from different regions was not homogeneous. For instance, in young adults, hypothalamus displayed high ectoenzymatic activity in both plasma and synaptic membranes, while cerebellum presented low activity in plasma membranes but the highest in synaptic ones. Other ecto-nucleotidases like 50 -nucleotidase and NTPDases 1 and 2, enzymes involved in the hydrolysis of mononucleotides generated from ApnA to adenosine, also display a markedly heterogeneous distribution (Kukulski et al., 2004). Most probably this reflects the high cellular and functional diversity of brain, suggesting that ApnA actions differ, and/or are differentially regulated, in different brain regions. Ectoenzymatic activity in synaptic membranes remained unchanged in young adults and aged animals, indicating that synaptic ectoenzyme function is preserved in ageing. This contrasted with general increases in activity observed in old animal plasma membranes suggesting that extracellular ApnA levels could be lowered through ageing. The biological significance of enhanced ectoenzymatic ApnAcleaving activity in plasma membranes is unclear but some considerations can be raised in the light of recent results. We reported that perfusion with e-(Ap4A) in rat striatum induced decreases in extracellular glutamate levels (Oaknin et al., 2001), while Wang et al. (2003), using rat-defined models of stroke and Parkinson’s disease, found neuroprotective actions
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oxidative damage and glutamate excitotoxicity in the aged brain. Acknowledgments This work was partially funded by grants PM-960081 from DGESIC, TR5-2002 and TR-2003/006 from Gobierno de Canarias (with the participation of Hospiten SA, NovoNordisk Ibe´rica SA and Aventis Behring SA) and C03/06 (Red CIEN) from ISCIII. References
Fig. 10. Distribution of ectoenzymatic ApnA-cleaving activity in plasma and synaptic membranes isolated from brain. Preparations of plasma and synaptic membranes from several brain regions (hypothalamus, HP; hippocampus, HC; temporal cortex, TC; frontal cortex, FC; striatum, ST) and cerebellum (CB) of young adults (3 months old) and aged (24 months old) male rats were analyzed for ectoenzymatic ApnA-cleaving activity using continuous fluorimetric assays. 250 ml reaction mixtures contained Tris–HCl 50 mM pH 9.0, CaCl2 4 mM, 100 mM ZnSO4, 20 mM e-(Ap2A) and 50–100 mg of membrane protein. Bars show means S.E.M. of three groups of five animals of young adult and aged rats. Activities in HP, HC, FC and ST in plasma membranes in aged rats were statistically different ( p < 0.004, p < 0.004, p < 0.001, p < 0.007, p < 0.002, respectively, *) from those in young adult rats (t-test). Analysis was carried out using the SPSS software.
of extracellular Ap4A, attenuating apoptotic cell death triggered by ischemia and 6-hydroxydopamine injuries in brain. Ischemia-induced increases in extracellular glutamate levels are responsible for excitotoxicity causing extensive neuronal and glial death (Schubert and Piasecki, 2001; Matute et al., 2002; Hurtado et al., 2003) and enhancement of oxidative stress through brain ageing is involved in the cellular damage observed in stroke and other ageing-related diseases like Parkinson’s and Alzheimer’s (Floyd and Hensley, 2002; Zuch et al., 2000). So, enhancement of ectoenzymatic ApnA-cleaving activity in plasma membranes in aged animals, due to its potential ability to decrease extracellular dinucleotide levels, could be envisaged as a deleterious ageingrelated factor limiting neuroprotective actions of extracellular Ap4A and leading to increased cellular vulnerability to
Aguilar, J.S., Reyes, R., Asensio, A.C., Oaknin, S., Rotlla´n, P., Miledi, R., 2001. Ectoenzymatic breakdown of diadenosine polyhosphates by Xenopus laevis oocytes. Eur. J. Biochem. 268, 1289–1297. Asensio, A.C., Rodrı´guez-Ferrer, C.R., Oaknin, S., Rotlla´n, P., 2006. Biochemical and immunochemical characterisation of human diadenosine triphosphatase provides evidence for its identification with the tumour suppressor Fhit protein. Biochimie 88, 461–471. Andoh, K., Piao, J.-H., Terashima, K., Nakamura, H., Sano, K., 1999. Genomic structure and promoter analysis of ecto-phosphodiestersae I gene (PDNP3) expressed in glial cells. Biochim. Biophys. Acta 1446, 213–224. Bollen, M., Gijsbers, R., Ceulemans, H., Stalmans, W., Stefan, C., 2000. Nucleotide pyrophosphatases/phosphodiesterases on the move. Crit. Rev. Biochem. Mol. Biol. 35, 393–432. Cameselle, J.C., Costas, M.J., Gu¨nther-Sillero, M.A., Sillero, A., 1984. Two low Km hydrolytic activities on dinucleoside 50 ,5000 -P1,P4-tetraphosphates in rat liver. Characterization as the specific dinucleoside tetraphosphatase and a phosphodiesterase I-like enzyme. J. Biol. Chem. 259, 2879–2885. Deissler, H., Lottspeich, F., Rajewsky, M.F., 1995. Affinity purification and cDNA cloning of rat neural differentiation and tumor cell surface antigen gp130 RB13-6 reveals relationship to human and murine PC-1. J. Biol. Chem. 270, 9849–9855. Dı´az-Herna´ndez, M., Pintor, J., Castro, E., Miras-Portugal, M.T., 2001. Independent receptors for diadenosine pentaphosphate and ATP in rat midbrain single synaptic terminals. Eur. J. Neurosci. 14, 918–926. Emanuelli, T., Bonan, C.D., Sarkis, J.J.F., Battastini, A.M.O., 1998. Catabolism of Ap4A and Ap5A by rat brain synaptosomes. Braz. J. Med. Biol. Res. 31, 1529–1532. Floyd, R.A., Hensley, K., 2002. Oxidative stress in brain aging. Implications for therapeutics of neurodegenerative diseases. Neurobiol. Aging 23, 795–807. Fuss, B., Baba, H., Phan, T., Tuhoy, V.K., Macklin, W.B., 1997. Phosphodiesterase I, a novel adhesion molecule and/or cytokine involved in oligodendrocyte function. J. Neurosci. 17, 9095–9103. Garcı´a-Agu´ndez, J.A., Cameselle, J.C., Costas, M.J., Gu¨nther-Sillero, M.A., Sillero, A., 1991. Particulate diadenosine 50 ,5000 -P1,P3-triphosphate hydrolases in rat brain: two specific dinucleoside triphosphatases and two phosphodiesterase I-like hydrolases. Biochim. Biophys. Acta 1073, 402– 409. Gasmi, L., Cartwright, J.L., McLennan, A.G., 1998. The hydrolytic activity of bovine adrenal medullary plasma membranes towards diadenosine polyphosphates is due to alkaline phosphodiesterase-I. Biochim. Biophys. Acta 1405, 121–127. Goding, J.W., Grobben, B., Slegers, H., 2003. Physiological and pathophysiological functions of the ecto-nucleotide pyrophosphatase/phosphodiesterase family. Biochim. Biophys. Acta 1638, 1–19. Goding, J.W., Terkeltaub, R., Maurice, M., Deterre, P., Sali, A., Belli, S.I., 1998. Ecto-phosphodiesterase/pyrophosphatase of lymphocytes and non-lymphoid cells: structure and function of the PC-1 family. Immunol. Rev. 161, 11–26. Grobben, B., Anciaux, K., Roymans, D., Stefan, C., Bollen, M., Esmans, E.L., Slegers, H., 1999. An ecto-nucleotide pyrophosphatase is one of the main enzymes in the extracellular metabolism of ATP in rat C6 glioma. J. Neurochem. 72, 826–834.
590
A.C. Asensio et al. / Neurochemistry International 50 (2007) 581–590
Guranowski, A., Sillero, A., 1992. Enzymes cleaving dinucleoside polyphosphates. In: McLennan, A.G. (Ed.), Ap4A and Other Dinucleoside Polyphosphates. CRC Press, Boca Rato´n, pp. 81–133. Harahap, A.R., Goding, J.W., 1988. Distribution of the murine plasma cell antigen PC-1 in non lymphoid tissues. J. Immunol. 141, 2317–2320. Hilderman, R.H., Pojoga, L.H., Haghiac, M., Cooper, H.M., Rosenberg, C.R., 2001. The role of extracellular adenosine dinucleotides as vasoregulators. In: Recent Research Developments in Biophysics and Biochemistry, vol. 1, Research Signpost, Trivandrum, pp 59–76. Hosoda, N., Hoshino, S.-I., Kanda, Y., Katada, T., 1999. Inhibition of phosphodiesterase/pyrophosphatase activity of PC-1 by its association with glycosaminoglycans. Eur. J. Biochem. 265, 763–770. Hoyle, C.H.V., Hilderman, R.H., Pintor, J., Schlu¨tter, H., King, B.F., 2001. Diadenosine polyphosphates as extracellular signal molecules. Drug Dev. Res. 52, 260–273. Hurtado, O., Cristo´bal, J., Sa´nchez, V., Lizasoain, I., Ca´rdenas, A., Pereira, M.P., Colado, M.I., Leza, J.C., Lorenzo, P., Moro, M.A., 2003. Inhibition of glutamate release by delaying ATP fall accounts fo neuroprotective effects of antioxidants in experimental stroke. FASEB J. 17, 2082–2084. Jankowski, J., Jankowski, V., Seibt, L., Henning, L., Zidek, W., Schlu¨tter, H., 2003. Identification of dinucleoside polyphosphates in adrenal glands. Biochim. Biophys. Res. Commun. 304, 365–370. Jankowski, V., Tolle, M., Vanholder, R., Schonfelder, G., van der Giet, M., Henning, L., Schlu¨tter, H., Paul, M., Zidek, W., Jankowski, J., 2005. Uridine adenosine tetraphosphate: a novel endothelium-derived vasoconstrictive factor. Nat. Med. 11, 223–227. Jime´nez, A.I., Castro, E., Delicado, E.G., Miras-Portugal, M.T., 1998. Potentiation of adenosine 50 -triphosphate calcium responses by diadenosine pentaphosphate in individual rat cerebellar astrocytes. Neurosci. Lett. 246, 109– 111. Koike, S., Keino-Masu, K., Ohto, T., Masu, M., 2006. The N-terminal hydrophobic sequence of autotoxin (ENPP2) functions as a signal peptide. Genes Cells 11, 133–142. Kukulski, F., Se´vigny, J., Komoszynski, M., 2004. Comparative hydrolysis of extracellular adenine nucleotides in synaptic membranes from porcine brain cortex, hippocampus, cerebellum and medulla oblongata. Brain Res. 1030, 49–56. Mateo, J., Miras-Portugal, M.T., Rotlla´n, P., 1997a. Ecto-enzymatic hydrolysis of diadenosine polyphosphates by cultured adrenomedullary vascular endothelial cells. Am. J. Physiol. 273, C918–C927. Mateo, J., Rotlla´n, P., Martı´, E., Go´mez de Aranda, I., Solsona, C., MirasPortugal, M.T., 1997b. Diadenosine polyphosphate hydrolase from presynaptic plasma membranes of Torpedo electric organ. Biochem. J. 323, 677– 684. Matute, C., Alberdi, E., Ibarretxe, G., Sa´nchez-Go´mez, M.V., 2002. Excitotoxicity in gial cells. Eur. J. Pharmacol. 447, 239–246. McLennan, A.G., 2000. Dinucleoside polyphosphates—friend or foe? Pharmacol. Ther. 87, 73–89. Meeteren, L.A., Ruurs, P., Christodoulos, E., Goding, J.W., Takakusa, H., Kikuchi, K., Perrakis, A., Nagano, T., Moolenaar, W.H., 2005. Inhibition of autotaxin by lysophosphatidic acid and sphingosine 1-phosphate. J. Biol. Chem. 280, 21155–21161. Narita, M., Goji, J., Nakamura, H., Sano, K., 1994. Molecular cloning, expression and localization of a brain-specific phosphodiesterase I/nucleotide pyrophosphatase (PD-Ia) from rat brain. J. Biol. Chem. 269, 28235– 28242. Oaknin, S., Rodrı´guez-Ferrer, C.R., Aguilar, J.S., Ramos, A., Rotlla´n, P., 2001. Receptor binding properties of di (1, N6 –ethenoadenosine) 50 ,5000 -P1,P4-
tetraphosphate and its modulatory effect on extracellular glutamate levels in rat striatum. Neurosci. Lett. 309, 177–180. Ohkubo, S., Kumazawa, K., Sagawa, K., Kimura, J., Matsuoka, I., 2001. b,gMethylene ATP-induced cAMP formation in C6Bu-1 cells: involvement of local metabolism and subsequent stimulation of an adenosine A2B receptor. J. Neurochem. 76, 872–880. Pereira, M.F., Diaz-Herna´ndez, M., Pintor, J., Miras-Portugal, M.T., Cunha, R.A., Ribeiro, J.A., 2000. Diadenosine polyphosphates facilitate the evoked release of acetylcholine from rat hippocampal nerve terminals. Brain Res. 879, 50–54. Picher, M., Boucher, R.C., 2000. Biochemical evidence for an ecto alkaline phosphodiesterase I in human airways. Am. J. Respir. Cell Mol. Biol. 23, 255–261. Pintor, J., Dı´az-Herna´ndez, M., Go´mez-Villafuertes, R., Gualix, J., Hernando, F., Miras-Portugal, M.T., 2000. Diadenosine polyphosphate receptors: from rat and guinea pig to human central nervous system. Pharmacol. Ther. 87, 103–115. Pintor, J., Miras-Portugal, M.T., 1995. A novel receptor for diadenosine polyphosphates coupled to the Ca2+ increase in rat brain synaptosomes. Br. J. Pharmacol. 115, 895–902. Ramos, A., Pintor, J., Miras-Portugal, M.T., Rotlla´n, P., 1995. Use of fluorogenic substrates for detection and investigation of ectoenzymatic hydrolysis of diadenosine polyphosphates: a fluorimetric study on chromaffin cells. Anal. Biochem. 228, 74–82. Ramos, A., Rotlla´n, P., 1995. Specific dinucleoside polyphosphate cleaving enzymes from chromaffin cells: A fluorimetric study. Biochim. Biophys. Acta 1253, 103–111. Rodrı´guez-Pascual, F., Torres, M., Rotlla´n, P., Miras-Portugal, M.T., 1992. Extracellular hydrolysis of diadenosine polyphosphates, ApnA, by bovine chromaffin cells in culture. Arch. Biochem. Biophys. 176–183. Rotlla´n, P., Asensio, A.C., Ramos, A., Rodrı´guez-Ferrer, C.R., Oaknin, S., 2002. Ectoenzymatic hydrolysis of the signalling nucleotides diadenosine polyphosphates. In: Recent Research Developments in Biochemistry, vol. 3, Research Signpost, Trivandrum, pp. 191–209. Stefan, C., Gijsbers, R., Stalmans, W., Bollen, M., 1999. Differential regulation of the expression of nucleotide pyrophosphatases/phosphodiesterases in rat liver. Biochim. Biophys. Acta 1450, 45–52. Stefan, C., Jansen, S., Bollen, M., 2005. NPP-type ectophosphodiesterases: unity in diversity. TIBS 30, 542–550. Scha¨fer, R., Reiser, G., 1997. Characterization of [35S]-ATPaS and [3H]-a,bMeATP binding sites in rat brain cortical synaptosomes: regulation of ligand binding by divalent cations. Br. J. Pharmacol. 121, 913–922. Schubert, D., Piasecki, D., 2001. Oxidative glutamate toxicity can be a component of the excitotoxicity cascade. J. Neurosci. 21, 7455–7462. Vollmayer, P., Clair, T., Goding, J.W., Sano, K., Servos, J., Zimmermann, H., 2003. Hydrolysis of diadenosine polyphosphates by nucleotide pyrophosphatases/phosphodiesterases. Eur. J. Biochem. 270, 2971–2978. Wang, Y., Chang, C.F., Morales, M., Chiang, Y.H., Harvey, B.K., Su, T.P., Tsao, L.I., Chen, S., Thiemermann, C., 2003. Diadenosine tetraphosphate protects against injuries induced by ischemia and 6-hydroxydopamine in rat brain. J. Neurosci. 23, 7958–7965. Zimmermann, H., 2001. Ecto-Nucleotidases. In: Abracchio, M.P., Williams, M. (Eds.), Handbook of Experimental Pharmacology. Purinergic and Pyrimidergic Signalling. Springer-Verlag, Heidelberg, pp. 209–250. Zuch, C.L., Nordstroem, V.K., Briedrick, L.A., Hoernig, G.R., Granholm, A.C., Bickford, P.C., 2000. Time course of degenerative alterations in nigral dopaminergic neurons following a 6-hidroxydopamine lesion. J. Comp. Neurol. 427, 440–454.
