P. Illes and H. Zimmermann (Eds.) Progress in Brain Research, Vol 120 0 1999 Elsevier Science BV. All rights reserved

CHAPTER 32

Diadenosine polyphosphates, extracellular function and catabolism

M. Teresa Miras-Portugal", Javier Gualix, Jesus Mateo, Miguel Diaz-Hemiindez, Rosa G6mez-Villafuertes, Enrique Castro and Jestis Pintor

Departamento de Bioquimica, Facultad de Veterinaria, UCM, 28040 Madrid, Spain

Introduction

Diadenosine polyphosphates (ApnA n = 2-6), are natural compounds that play an important role inside the cell in both the nucleus and at the cytosol (for review see McLennan, 1992.). Their levels are increased in cellular proliferation and stress situa- tions resulting in stimulation of DNA duplication and DNA repair (Rapaport and Zamecnick, 1976; Baker and Jacobson, 1986; Baxi and Vishwanatha, 1995). Besides, ApDA can be strong inhibitors of the enzymes that balance the energetic phosphate groups among the cytosolic nucleotides, such as adenylate lunase and adenosine kinase (Leinhardt and Secemsky, 1973; Rotllan and Miras-Portugal, 1985). Recent findings indicate that the cytosolic face of some plasma membrane proteins exhibits regulatory sites modulated by various types of ApnA. Among the most important of these proteins are the ATP regulated K' channels, and the nucleoside transporters (Delicado et al., 1994; Jovanovic and Terzic, 1996; Ripoll et al., 1996).

The transport of ApnA to cytoplasmatic storage granules provides both the end of their intracellular actions, and at the same time the way to be released by controlled exocytosis (Gualix et al., 1996, 1997). The storage organella so far found to contain

*Corresponding author. Tel.: + 34-9 1-394-3894; fax: + 34-9 1-394-3909; e-mail: mtmiras@eucmax.sim. ucm.es

ApnA, include the serotoninergic dense granules of platelets, the noradrenergic and adrenergic granules from adrenal medulla, and the acethylcholinergic vesicles from the Torpedo electric organ (Luthje and Ogilvie, 1983; Rodriguez del Castillo et al., 1988; Pintor et al., 1991a, 1992a, 1992b; Schliiter et al., 1994).

For further interpretations it is relevant to consider the concentration levels expected once released to the extracellular media. These are in the low pM range in the surrounding area of platelets aggregation and at the vicinity of the exocytotic event in the cultured neurochromaffin cell model. More diluted concentrations of ApnA could be expected after it has diffused or undergone local enzymatic hydrolysis. Values could then be expected in the low nM range (Pintor et al., 1991b). Values in the nM range have also been detected in brain perfusion after amphetamine stimulation (Pintor et al., 1995). Binding studies and autor- adiography indicate the existence of receptors with very high and high affinity (Kd values for Ap,A in rat brain synaptic terminals of 0.1 nM and 0.57 pM respectively), which is concordant with this con- centration range. The values in neurochromaffin cells from adrenal medulla and plasma membranes from the Torpedo are very similar (Pintor et al., 1991b, 1993; Walker et al., 1993; Rodriguez- Pascual et al., 1997).

From a functional point of view (and given to its chemical structure) ApnA could act through the

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ATP receptors, which are well characterised, and have been cloned and classified. Two main families of nucleotide receptors exist: P2Xl, and the P2Y both of them with a still increasing number of components (Abbracchio and Burnstock, 1994; Boarder et al., 1995; Fredholm et al., 1997). Dinucleoside polyphosphates have proved to be agonists for some of these receptors, but consider- ing the concentrations required and their physiological concentrations, these actions could perhaps be considered to be “pharmacological”. This is the case for the action on the P2Xl receptors of the vas deferens (Hoyle et al., 1995). Another possibility for the diadenosine polyphosphates is to behave as allosteric effectors. This is the case for the homomeric P2X2 expressed in oocytes, where the Ap,A activates the calcium entrance induced by ATP at low nM range (Pintor et al., 1996). The dinucleotide effect on P2Y metabotropic receptors has been documented in endothelial cells on a P2Y and cloned P2Y2 (Lazarowski et al., 1995; Mateo et al., 1996). The existence of dinucleotide specific metabotropic and ionotropic receptors can be deduced from the data reported below, although they have not yet been cloned. Recently, potentia- tion effects of Ap,A on the Ca2+ signals mediated by P2Y receptors, stimulated by 2-MeS-ATP or UTP in cerebellar astrocytes have been reported, and it is worth noting that the concentrations required were in the nM concentration range (1-10 nM), and that potentiation effect lasted for almost 6 h. In this case the structure and second messenger pathway of this receptor is still unknown (Jimenez et al., 1998).

The ionotropic presynaptic dinucleotide receptor from rat midbrain synaptic terminals, also called the P4 receptor, is the best characterised of the ionotropic dinucleotide receptors, and most of the data presented in this report refers to it (Pintor and Miras-Portugal, 1995). In well characterised neural preparations such as the neurochromaffin cell cultures, only the cells containing noradrenaline exhibit ionotropic receptors for ATP, these corre- spond to the P2X2 subtype, and the Ap,A has neither an agonistic, nor an antagonistic effect (Castro et al., 1995). A similar situation occurs in the cultured Purkinje cells from immature rat brains, where the ionotropic receptors for ATP at

the cell soma, that are mainly P2X,, do not respond to ApnA (Mateo et al., 1998).

Dinucleotide receptors from rat midbrain synaptic terminals

Brain synaptic terminals, known as synaptosomes, are among the most frequently employed neural preparations when studying the functioning of the nervous system. Nevertheless, they are extremely heterogeneous, both in their neurotransmitter molecular species and the presence of presynaptic modulatory receptors. However, in spite of these drawbacks, and in the absence of any specific model, this preparation provides a first approach to the study of a substance expected to be a neuro- transmitter or a neuromodulator.

In our studies, rat midbrain synaptic terminals were used because this area is very rich in aminergic and cholinergic terminals, and by anal- ogy with the peripheral models, ApnA should be co-stored with them and ATP. In these synaptic terminals, Ap,A, which is the best effector (Fig. lA), was able to induce Ca2+ transients, which were not cross desensitised by ATP or its non- hydrolysable analogues. On the other hand, they were not blocked by any of the toxins available for the voltage dependent calcium channels (VDCC) and only modulated after the first initial calcium spike by the w-conotoxin G-VI-A, which is an N- type VDCC inhibitor (Pintor and Miras-Portugal, 1995), similar ionotropic responses were found in guinea pig and deer mouse terminals (Pintor et al., 1995, 1997c; Pivorum and Nordone, 1996).

The diadenosine polyphosphates are not the only agonists for the presynaptic receptor; the guanine dinucleotides and the ethenoderivatives exhibit similar agonistic effects. Figure 2 shows the effect of various agonists, Ap5A being the best natural agonist with an EC,, value of 55 pM. It is relevant to point out that both parameters, the affinity and the maximal calcium response should be con- sidered. The dose response studies gave the following potency and effectiveness ranking: for the EC,, values Gp,G > Ap,A = Ap,A > Gp,G = E-

Ap4A = &-Ap,A > Gp,G > Ap,A = &-Ap,A and for the maximal values in calcium response Ap,A=Gp,G=&-Ap5A>Ap,A=Gp,G=&-Ap,A>

399

Fig. 1. Structure of the diadenosine pentaphosphate and main derivatives. (A) Diadenosine pentaphosphate. Natural agonist of dinucleotide-P4-receptors. Its presence has been described in the dense granules of platelets, chromaffin granules from adrenal medulla, and synaptic vesicles from rat brain and Torpedo electric organ. (El) Diinosine pentaphosphate. Antagonist of dinucleotide- P4-receptors. It is synthesised by the enzymatic action of adenylic acid deaminase from Aspergillus sp. (Pintor et al., 1997a). It has not yet been found as a natural compound, although it could be supposed. (C) Ethenoadenosine pentaphosphate. Fluorescent derivative employed in continuous fluorescent measurements of enzymatic hydrolysis. The etheno-derivatives are synthesised by condensation reaction with 2-CI-acetaldehide (Rot& et al.. 199 I).

400

+ 25 - - 20 -

O 15-

CI

G ii

8 10-

c, E 5

c,

E h m

E H

-6.0 -5.5 -5.0 -4.5 -4.0 -3.5 -3.0

Log [Dinucleotide] M

Fig. 2. Dose response curve of Ap,A, and structural analogues. Concentration-response relationship for Ap5A, GplG and E-A~,A on intracellular calcium increase in rat brain synaptosomes. Synaptosomes were prepared from midbrains of male Wistar rats that had been cervically dislocated and decapitated (Pintor and Miras-Portugal, 1995). Synaptosomal pellets containing 1 mg of protein were used. The cytosolic free calcium concentration was determined with fura-2 as described by Grynkiewicz et al. (1985).

Gp,G = Ap,A = E-A~,A. Three main families appear to exist when ranked for maximal effect: Np,N > Np,N > Np,N.

In this experimental model diinosine pentaphos- phate (Fig. lB), (which is enzymatically synthesised from Ap,A by the action of the enzyme adenylic acid deaminase from Aspergillus sp.) proved to be an excellent inhibitor of this receptor (ICso = 4.2 nM), having an affinity 6,000 times higher than that exhibited with the ionotropic presynaptic ATP receptor, also described in this preparation (ICs0 = 27.7 pM) (Pintor et al., 1997a). In the vas deferens, where the ATP receptors are supposed to be mostly homomeric P2X,, the Ip,I shows a PA, value of 6,4 (which is roughly 0.2 pM) with respect to ATP (Hoyle et al., 1997). This differential inhibitory effect clearly confirms the existence of separate receptors for ATP and ApnA on the presynaptic terminals.

how the dinucleotide ionotropic receptor (which is also presynaptic) could be influenced by this coexistence (Herrero et al., 1992, 1996). It should be borne in mind that ApnA are co-stored with most of the aminergic neurotransmitters, and also with acetylcholine, in all models studied so far (Richard- son and Brown, 1987). Moreover, these compounds have well characterised presynaptic receptors. As there is a wide range of possible membrane receptors, the first experimental approaches were undertaken by directly acting on protein kinases and protein phosphatases, or intracellular second messengers (Pintor et al., 1997b).

As is shown in Fig. 3A, direct activation of PKC by phorbol esters inhibits the maximal response of the Ap,A induced calcium signal. Conversely, inhibition of PKC by staurosporin or the inhibitory peptide results in an increase in the calcium signal, with respect to the control. The activation of PKA by dibutyril CAMP and activation of adenylylcy- clase by forskolin both result in a reduction of the calcium signal. The inhibition of the enzyme with

Regulation of dinucleotide receptors by protein kinases and phosphatases

A large number of metabotropic receptors have been found to be involved in the modulation of presynaptic terminals. This raises the question of

the PKA-inhibitory peptide results in- a clear increase in the effect through the dinucleotide receptor. Further studies have shown that PKA

40 1

activation causes a drastic decrease in the receptor affinity for Ap,A, and PKA inhibition produced receptor affinity states with EC,, values close to the nM range (Miras-Portugal et al., 1998).

Protein phosphatase inhibition by okadaic acid (which at the concentrations used is non-specific),

gj 6o A 45 6

* * * *

microcystin (a better inhibitor of protein phospha- tase 2A), and cyclosporin-A (a substance which is specific for protein phosphatase 2B, also known as calcineurin) all brought about a drastic reduction in the calcium transients elicited by Ap,A (Fig. 3B). Both protein phosphatases 2A and 2B appear to

* * I-

U Control €I PDBU PKC-IP

Forskolin PKA-IP 0 Stauros.

8 30 A

3 I. 20

m 6

a 1 m

a 8 10

Q o

Control a Microcystin

Cyclosporin Okadaic Acid

Fig. 3. Effect of protein kinase and protein phosphatase modulators on the calcium responses elicited by Ap,A. Rat midbrain synaptic terminals were pre-treated for 2 min with the different effectors and the calcium response to 50 pM Ap,A measured. When non- permeable substances were assayed, such as the inhibitory peptides IP-PKA, IP-PKC, or the protein phosphatase inhibitor microcystin, the brain homogeneization was carried out in the presence of these compounds to allow their entrance before the resealing of synaptic terminals. (A) Effect of Protein Kinases modulation. Forskolin, an adenylate cyclase activator, was used at 100 pM concentration. PDBu, the allosteric activator of PKC was used at a 1 pM concentration. The inhibitory peptides PKA-IP and PKC-IP were added at the first homogeneization step at concentrations of 25 p M and 5 pM, respectively. Staurosporine, the PKC inhibitor, was used at a concentration of 100 nM. (B) Effect of protein phosphatase inhibitors. Cyclosporin-A, the specific inhibitor of PPase 2B was used at 1 pg/ml. Microcystin an inhibitor of PPase 2A, was added to the first homogenization step at a concentration of 15 pM. Okadaic acid was added at a concentration of 100 nM. Under these conditions the both previously cited PPases were inhibited.

402

F

Fig. 4. [Ca*'], responses in isolated single nerve terminals. Panel A : viewfield image of isolated nerve terminals (synaptosomes) glued to a coverslip with poly-L-lysine and loaded with fura-2. Arrows indicate terminals analysed in other panels. Panel B: representative images of analysed terminals (1 and 2) before, during and after the stimulation. Pseudocolour scale represent F380 intensity (lower at higher [Ca2'],). The consecutive images correspond to time points (a)-(h) identified with lines in the time-course traces. Panel C: time course of fluorescence changes (as F/Fo after correcting photo-bleaching) recorded for terminals 1 and 2 in panel A. The stimulation periods are indicated by the solid bars and images in B by a-h lines. Methods: synaptosomes were diluted (0.5 mglml) and spreaded onto coverslips coated with poly-L-lysine for attachment and then loaded with fura-2/AM (1 h, 37°C). The coverslips were washed with saline solution and mounted in a small superfusion chamber in the stage of a Nikon TE-200 microscope. Fura-2 fluorescence was excited at 380 nm and collected at 510 nm (Omega Optical bandpass filters, 430 nm dichroic mirror) through a fluor x 100, 1.3 NA, objective (Nikon). The 12-bit images were collected every 1.023 s with a Hamamatsu C4880-80 CCD camera controlled by Kinetic (UK) software. Time course data represent the average light intensity in a small elliptical region inside each terminal. Continuous fading due to photo-bleaching was corrected by local fitting of an exponential function to the data (Raw(t) =F(t).exp(-k. t)). The data is represented as the normalized ratio F/F,,, that decreases with [Ca*'], increases (Lev-Ram et al., 1992).

play a significant role in the signal recovery after phosphorylation and the inhibition of both is required for the maximal inhibitory effect.

The logical question that arises from these results is how to know which are the presynaptic receptors that (by means of protein kinases and phosphatases) are relevant in the synaptic terminal containing the dinucleotide receptor. As has been mentioned, it is a complex picture, and one that needs to be studied one step at a time. As ATP is always co-stored with dinucleotides, and could also be at the extracellular level after controlled release or due to the fragility of synaptosomal prepara- tions. Thus its effects were the first to be analysed. Activation of metabotropic P2Y receptors resulted in a decrease in the calcium signal induced by Ap,A. Furthermore, as ATP breaks down to adeno- sine at the extracellular level via the ecto-nucleotidases cascade, this nucleoside was studied and found to exert a powerful modulatory effect through the A1 presynaptic receptor. The main effect of this modulatory action is to increase the affinity of the dinucleotide receptors to Ap,A. Changes in the ECSo values from 55 FM to 10 nM, for Ap,A, in the absence or presence respectively of an A1 receptor agonist is currently obtained (Miras- Portugal et al., 1998). A scheme on the complexity of presynaptic dinucleotide receptors regulation is represented in Fig. 5.

Thus, in spite of the complexity that may be envisaged at the presynaptic terminals, the regula- tion of dinucleotide receptors by the action of protein kinases and phosphatases could be a subtle way of adapting the receptors to the physiological levels of their natural agonists, or to prepare the

terminal to be more effective in the exocytotic event that is a Ca2' dependent process.

Studies of calcium responses in isolated single synaptic terminals by microfluorimetry

Until now the Ca2' responses induced by Ap,A have been studied in synaptosomal preparations using aproximately 1 mg of protein to obtain an optimal fluorimetric response. However, in our laboratory single cell microfluorimetry techniques have now being used for some time to study the presence of receptors, and their pharmacology, in cultured or recently isolated single cells (Castro et al., 1996; Mateo et al., 1998). Today, this technique has been improved, and it is possible to study the calcium responses induced by ApnA in perfused single synaptic terminals by using the calcium dye Fura-2. Figure 4 shows the response elicited by Ap,A in a synaptic terminal compared with the Ca2' transients induced by K' depolarisation. Most of the synaptic terminals exhibit a clear response to K' depolarisation, but only 20% of them showed a clear dinucleotide response in midbrain synaptoso- ma1 preparations.

This technique opens new possible experimental approaches to the study of ionotropic presynaptic receptors, not only for Ap,A, but also for more classical transmitters such as acetylcholine (nico- tinic) receptors, glutamate ionotropic receptors, and ATP receptors (Wonnacott, 1997; Clarke et al., 1997; Gu and Macdermott, 1997; Kaiser et al., 1998). The coexistence of these ionotropic pre- synaptic receptors and their mutual interactions through ionotropic or metabotropic receptors is

403

A

B

C

404

Fig. 5 . Regulation of presynaptic dinucleotide -P4- receptors by metabotropic A1 and P2Y receptors and their coupled second messengers. Activation of protein kinases A and/or C results in a clear inhibition in the calcium response elicited by Ap5A. Inhibition of protein phosphatases 2A and/or 2B maintains the phosphorylated form of this receptor and a reduced calcium response. When the ATP metabotropic receptors are stimulated, the PLC is activated producing diacylglycerol (DG) and IP3, resulting in PKC activation. This enzyme is able to phosphorylate many proteins from the presynaptic terminals, and phosphorylation of the P4 receptor by PKC results in a considerable reduction of calcium influx, without a significant change on the P4-receptor affinity for diadenosine polyphosphates. Adenosine receptors appear to play a leading role in P4-receptor modulation, through the A1 receptor, whose signalling pathway via Gi protein avoids adenylate cyclase activation, further increase in CAMP, and the subsequent activation of PKA. It should be noted that if the PKA phosphorylation pathway was blocked at whatever level, the affinity of the P4-receptor was able to reach EC5,, values in the low pM range. Inhibition of protein-phosphatases results in a significant decrease of the Ca2+ influx to synaptic terminals induced by diadenosine polyphosphates.

expected to be one of the most productive and to lead the resultant mononucleotides to further challenging areas on the road to understanding degradation via the ecto-nucleotidases cascade presynaptic terminal functioning (Fig. 5 ) . Thus (Zimmermann, 1996). The presence of ApnA in the perhaps the question about the physiological rea- dense granules of platelets, make the vascular sons for the existence of a presynaptic ionotropic endothelial cells a suitable place to search for ecto- receptor, could have a suitable answer. diadenosine polyphosphate hydrolase activity, and

it was the first tissue in which this it was reported (Goldman et al., 1986; Ogilvie et al., 1989). Besides, the presence of these comuounds in neuro-

Ecto-diadenosine polyphosphate hydrolase from neural origin

Ecto-enzymes able to hydrolyse diadenosine poly- secretory granules has provided a stimulus to the phosphates are required to terminate the search for ecto-diadenosine polyphosphate hydro- extracellular activity of ApllA, and at the same time lase activities in neural tissues (Rodriguez-Pascual

Fig. 6. Hydrolysis of ethenodiadenosine polyphosphates. The E-A~,A structure and the hydrolysis products, E - A ~ , and E-AMP, after the ecto-diadenosine polyphosphate hydrolase action is represented. The enzyme always acts in an asymmetrical way, AMP being always one of the reaction products. The HPLC profiles illustrating the time dependent hydrolysis of E - A ~ , A by Torpedo plasma membranes (100 p,g/ml), incubated at 37°C in the presence of 1 pM &-Ap,A and represented as consecutive chromatograms.

405

Diadenosine Polyphosphate

450

3 C 300 W

0 0 10 20 30 40

Elution time (min)

406

et al., 1992a, 1992b). From an experimental point of view, the first enzymatic assays required the processing of samples by HPLC, to make an accurate quantification of substrate hydrolysis, or to employ radiometric techniques. Thus, the con- tinuous fluorimetric technique developed by Rotllh et al. (1991), using the etheno-Ap,A derivatives as substrate analogues, allowed an accurate and rapid approach to the enzymes present in both the isolated cells and the plasma membrane preparations.

In Fig. 6 the etheno-Ap,A is represented together with the hydrolysis products, &-adenosine tetra- phosphate (e-Ap,) and &-AMP. Independent of their origin, the ecto-enzymes studied so far always cut the phosphate bridge asymmetrically, i.e. pro- ducing AMP and the mononucleotide with n - 1 phosphates (Fig. 6). This is the case for the endothelial enzymes from bovine adrenal capillar- ies and aortic vessels (Goldman et al., 1986; Ogilvie et al., 1989; Mateo et al., 1997b) and all types of neural enzymes studied so far (Ramos et al., 1995; Mateo et al., 1997a).

Comparative studies of enzymes from different sources have shown there to be variation in ionic requirements, and at least two groups can be distinguished. First, the enzymes of vascular endo- thelium origin, which are inhibited by Ca”, and second, neural enzymes in which Ca2+ is an activator (Mateo et al., 1997a, 1997b). Studies carried out with isolated synaptic terminals showed a hydrolysis rate 20-50 times slower for these compounds than that of ATP. It is also noteworthy that in cultured chromaffin cells the V,,, for the ecto-ATPase activity is between 500-1,000 times higher than that for the ecto-diadenosine poly- phosphate hydrolase (Rodn’guez-Pascual et al., 1992a). This finding explains the longer half-life of Ap,A in biological samples. In the case of perfuss ed brain from conscious rats, after amphetamine stimulation, ATP and ADP are present in such small quantities that they are almost undetectable, but Ap4A and Ap,A are present at nM concentration levels (Pintor et al., 1995).

The presynaptic neural enzyme was approached in the colinergic model of Torpedo. This enzyme exhibited the same ionic requirements as the neurochromaffin one, and the affinities for the

etheno-derivatives showed K,,, values of 0.39 pM, 0.42 pM and 0.37 pM for &-Ap3A, &-Ap4A and &-Ap,A respectively. This submicromolar affinity is perhaps related to the levels reached by the natural compounds after exocytosis and strengths their role in cellular and neural signalling.

Figure 6 shows the hydrolysis of &-Ap,A by the Torpedo electric organ membranes as a function of the time. It is worthy to notice that the nucleotide adenosine tetraphosphate -Ap,- accumulates with the experimental time, exhibiting a very low rate of hydrolysis. The enzymatic production of Ap,, at the synaptic terminal, made it worth studying the effect of this compound on the nucleotide receptors. Ap, behaved as an agonist on the ATP ionotropic presynaptic receptor, but not on the dinucleotide receptor, and its affinity was similar to that of ATP, but with much better stability, and it should be considered a natural agonist of ATP receptors (Gbmez-Villafuertes, 1998).

Conclusions

Diadenosine polyphosphates Ap,A are co-stored and co-released from a large variety of synaptic terminals. Their physiological extracellular concen- tration range can be expected to be between the nM and low pM. Binding studies have detected the presence of high affinity sites, correlating with the physiological concentrations reported. From a functional point of view, specific receptors, called dinucleotide receptors, or P4 receptors, have been described in presynaptic terminals, and elicit cal- cium entrance through ionotropic receptors, which are not susceptible to inhibition by voltage depend- ent calcium channel toxins. Diinosine polyphosphates are good inhibitors of the P4 receptor, Ip,I exhibiting an IC5,, value of 4 nM.

The presynaptic dinucleotide receptors are mod- ulated by the action of protein kinases and protein phosphatases. An increase in the levels of phos- phorylation results in a decrease in both receptor affinity to its agonists and maximal calcium entrance through the receptor. Adenosine, via its A1 receptor, coupled to a Gi protein and inhibition of the adenylyl cyclase, therefore prevents phosphor- ylation through PKA, and is able to increase the affinity of this receptor to the pM range of Ap,A. The effects of adenosine are mimicked by CHA

407

(cyclohexyladenosine) and reversed by adenosine deaminase, or the addition of the antagonist DPCPX (8-cyclopenty l- 1,3-dipropylxanthine). Activation of the P2Y receptors by ATP, or its non- hydrolyzable analogues, results in PKC activation and a significant reduction on the maximal calcium response, with minor effects on receptor affinity. The effect of ATP can be reversed by hydrolysis of the extracellular nucleotides by alkaline phospha- tase, or by using the antagonist PPADS.

The extracellular actions of diadenosine poly- phosphates are terminated by the enzymatic hydrolysis by an ecto-diadenosine polyphosphate hydrolase. The neural type, from Torpedo synaptic terminals, exhibit K,,, values close to 0.4 p,M, for all the dinucleotides tested and present similar ionic requirements than other neural enzymes.

Acknowledgements

This work was supported by grants from DGICYT (PM 95-0072), EU. Biomed-2 (PL 95-0676), Multidisciplinar U.C.M. (1995) and the Areces Foundation Neurosciences programme. J. G. is a fellowship from the Spanish Ministry of Education and Culture, M. D-H from the U.C.M. and R. G-V from the C.A.M. We thank Duncan Gilson for his help in preparing the manuscript.

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