Medical Therapy Group, Defence Research Establishment Suffield, P.O. Box 4000, Medicine Hat, Alberta, Canada T1A 8K6. E-mail: Paul.Lundy@dres.dnd.ca
ATP receptors of the P2X class have previously been identified on autonomic nerve endings and on a limited population of CNS neurons.
In the present study P2X receptors on mammalian cortical synaptosomes have been identified by a variety of functional and biochemical studies. In choline buffer ATP analogues caused concentration/time dependent Ca2+ influx. Relative to the effects caused by ATP, benzoylbenzoyl ATP (BzATP) was about seven times more active than ATP while 2-me-S-ATP and ATPγS were much less active. α,β-me- ATP and β,γ-me-ATP were virtually inactive. In sucrose buffer, relative to choline buffer, the activity of BzATP was more than doubled while activity in sodium buffer was reduced. Moreover, the P2X antagonists PPADS or Brilliant Blue G both significantly attenuated influx. These observations suggest the presence of P2X receptors on synaptosomes which subserve Ca2+ influx. This activity profile of the ATP analogues and the response to blocking agents are characteristic of responses of P2X7 receptors.
Influx was unaffected by the VSCC inhibitors ω-CTx-MVIIC and (−) 202 – 791, indicating that ATP induced Ca2+ influx occurred primarily through P2X receptors.
P2X7 receptor protein was identified by Western blotting and immunohistochemical staining. Purified preparations were devoid of significant concentrations of GFAP or the microglial marker OX-42 but contained greatly enriched amounts of syntaxin and SNAP 25.
The various pharmacological and biochemical studies were all consistent with the presence of functional P2X7 receptors.
Ionotropic P2X purinergic receptors are located on a large number of different cell membranes and are believed to transduce the effects of extracellular ATP (adenosine triphosphate). There are currently seven identified, cloned subtypes of the P2X receptor. Some of these subtypes appear to mediate fast excitatory transmission in the peripheral autonomic nervous system between neurons and/or between neurons and smooth muscle cells (Evans et al., 1992; Silinsky et al., 1992; Galligan & Bertrand, 1994). ATP evokes the release of other autonomic neurotransmitters such as noradrenaline from peripheral neurons through activation of P2X receptor subtypes, which function as ATP gated ion channels or pores (Boehm et al., 1995; Gibb & Halliday, 1996; Boehm, 1999; von Külgelgen et al., 1993; 1999; Sperlágh et al., 2000). Therefore, ATP receptors operating as cation channels may serve as an important alternate mechanism to the voltage sensitive calcium channels (VSCCs), through which the organism initiates or controls the release of neurotransmitters from a variety of autonomic nerves.
In the present study, pharmacological and molecular biological evidence is presented for the existence of functional P2X receptors, specifically of the P2X7 subtype (Surprenant et al., 1996) on purified synaptosomal preparations of rat cortex. We have employed a number of pharmacological manipulations of responses proposed as reliable measures of P2X receptor function (Michel et al., 1996; 1998; Virginio et al., 1997; Jiang et al., 2000; Hibell et al., 2001). We have also employed Western blotting techniques and immunohistochemistry to confirm the presence of specific P2X subtypes in order to support the identification of receptors carried out by pharmacological methods. These findings constitute a variety of evidence for the existence and functional significance of P2X7 receptors on central presynaptic elements.
Preparation of synaptosomes
Rat (male, Sprague – Dawley, wt 250 – 300 g) crude synaptosomes were prepared by homogenization of brain cortex in ice-cold 0.32 M sucrose using six strokes of a teflon/glass homogenizer. The homogenate was centrifuged at 1000×g for 10 min at 4°C. The supernatant was decanted and centrifuged at 12,400×g for 25 min and the resulting pellet (P2) was resuspended in one of three different buffers of composition outlined below for the subsequent measurement of ATP induced calcium influx.
Purified synaptosomes were prepared using the Percoll® gradient method (Dunkley et al., 1988). Briefly, the S1 sucrose/EDTA supernatant was layered over a discontinuous Percoll® (Sigma, St Louis, MO, U.S.A.) gradient consisting of 2 ml each of 3, 10, 15 and 23% (v v−1) Percoll® dissolved in 0.32 M sucrose containing 100 mg l−1 EDTA. This was centrifuged at 20,000×g for 5 min at 4°C in a Beckman preparative centrifuge. The 10/15% and 15/23% interfaces were combined and diluted 4 fold with Hanks balanced salt solution (HBSS) at pH 7.3, and then centrifuged at 12,500×g for 25 min. The resulting pellet was resuspended in HBSS. Aliquots of the Percoll® purified synaptosomes were washed twice with 10 vol of HBSS (composition see below) to ensure the removal of all Percoll® and deposited on treated glass microscope slides (500 μl, Cytospin) and used for fluorescence histochemistry (outlined below). The composition of HBSS was as follows (in mM): KCl 5.4, KH2PO4 0.5, NaCl 136, NaHPO4 0·7H20 0.34, D-glucose 5.6 and CaCl2 1.
The first series of experiments were carried out in choline buffer (composition outlined below) and provided the results described in the first two figures, as well as parts of subsequent figures as described in the text where appropriate. Ca2+ influx was carried out according to the method of Blaustein (1975), with modifications (Lundy et al., 1991). In those cases where drug pretreatments were required, the synaptosomes were incubated at 30°C in the presence or absence of antagonist drugs for 15 min. Experiments were carried out using synaptosomes diluted to obtain a final concentration of about 1 mg protein ml−1. Following drug pre-treatment, a 100 μl aliquot of the synaptosomal suspension was quickly injected into an equal volume of resting buffer (5 mM K+), depolarizing buffer (25 mM K+, final concentration), or resting buffer to which ATP or its analogues had been added (various concentrations), all containing 0.5 μCi 45Ca2+ (New England Nuclear, Boston, MA, U.S.A.). Triplicate estimates of basal and of K+ stimulated Ca2+ influx (3 s or adenine nucleotide stimulated influx (60 s) were carried out and terminated by rapid dilution of the buffer with 4 ml ice-cold Ca2+-free buffer containing 4 mM EGTA. Each suspension was rapidly filtered under vacuum through 0.45 μM membrane filters (Gelman Science, Ann Arbor, MI, U.S.A.) using a filtration apparatus (Hoeffer Scientific, San Francisco, CA, U.S.A.). The membrane filters were washed twice with 5 ml normal resting buffer to which excess Ca2+ had been added. Membrane filters were allowed to dry, placed in scintillation cocktail, and counted on a Wallac 1500 scintillation counter. Basal influx was subtracted from K+ or nucleotide-stimulated influx and results were expressed as nmoles Ca2+ influx mg protein ml−1. Experimental protocols were designed so that the effects of ATP, as well as five nucleotide analogues could be examined on each synaptosomal preparation. K+-evoked Ca2+ influx was included to assess the viability of each preparation. Subsequent to the first series of experiments, either the sodium chloride or the sucrose-based systems were also used.
Three buffers were used in the study of calcium influx. The first buffer was choline based and was a buffer that had been utilized in this laboratory for studies of calcium influx for several years. The other two were used as the result of reports in the literature concerning ionic effects on P2X7 receptor activity:
Choline based buffer was of the same composition used previously in this laboratory and is one of three different buffers employed in the measurement of calcium influx. In the results section the various buffers used are identified where appropriate. Choline buffer was made with the following composition (in mM): Choline Cl 132, KCl 5, MgCl2 1.3, CaCl2 1.5, NaH2PO4 12, D-glucose 10, HEPES 20, brought to pH 7.4 with TRIS base. Choline chloride was originally substituted for NaCl to negate the effects of Na+/Ca2+ exchange (Blaustein, 1975) but also choline has been recently reported to interfere less with ATP induced effects on P2X7 receptors than sodium rich buffer systems (Michel et al., 1996).
Sodium based buffer was identical to the choline buffer but sodium chloride was substituted for choline chloride.
Sucrose buffer was a low ionic strength medium described by Michel et al. (1996) and Surprenant et al. (1996) in order to maintain lower concentrations of certain ions which interfered with agonist activity. It consisted of the following (in mM): sucrose 280, CaCl2 0.5, KCl 5, D-Glucose 10, HEPES 10, N-methyl-D-glucamine 5, with the pH adjusted to 7.4.
Immunohistochemical staining was conducted as follows: slides were rinsed in phosphate buffered saline (PBS) for 10 min and blocking/diluent buffer was applied (PBS, 1.5% normal goat serum (NGS) was applied for 20 min. Primary antibodies (anti-P2X7, anti-syntaxin, and anti-SNAP-25, all rabbit (Alomone Labs, Jerusalem, Israel and Chemicon International Inc., Temecula, CA, U.S.A.) and anti- OX-42 (mouse, ICN Biochemicals, Aurora, OH, U.S.A.) were applied at 1/300 dilution with diluent buffer and incubated for 4 h at room temperature. Slides were rinsed three times with PBS. Secondary antibodies (Oregon Green 488-labelled goat anti-rabbit IgG and goat anti-mouse IgG, Molecular Probes, Eugene, OR, U.S.A.) were applied to sections at 1/200 dilution with diluent buffer for 2 h under the same conditions. Following incubation, the slides were washed in three changes of PBS and coverslipped using Prolong anti-fade mounting media (Molecular Probes, Eugene, OR, U.S.A.).
A negative procedural control, in which the primary antibody was omitted and replaced with diluent buffer only, was used with each series. Slides were visualized using fluorescence microscopy and representative images taken with a Spot 2 digital camera (24 bit colour images, Diagnostic Instruments Inc., Sterling Heights, MI, U.S.A.) Protein concentration was determined using the BCA Protein Assay Reagent kit (Pierce Chemical Co., Rockford, IL, U.S.A.) by the method of Bradford (1976).
Western blot characterization of P2X receptors
Pheochromocytoma PC12 cells (CRL-1721, American Type Culture Collection, Manassas, VA, U.S.A.), vas deferentia, cortical synaptosomes (both crude P2 and purified homogenates), or whole brain homogenates from the rat were incubated on ice for 20 min in 300 – 500 μl 1× SDS gel loading buffer of the following composition: 50 mM Tris-HCl (pH 8.6), 100 mM DTT, 2% SDS, 0.1% bromophenol blue and 10% glycerol. Tissues were centrifuged at 12,000×g for 3 min at 4°C and the supernatant was transferred to a fresh tube. Protein was measured using Coomassie protein reagent (Pierce, Rockford, IL, U.S.A.) according to the method of Bradford (1976). The protein samples were immersed in boiling water for 5 min and equal amounts of protein (20 μg of each) were electrophoresed on 8% SDS polyacrylamide gels and transblotted onto a nitro-cellulose membrane. Prestained protein standards (Bio-Rad, Mississauga, Ontario, Canada) were used to visualize successful transfer and to measure molecular weight of the subsequent signal. The membrane was blocked with PBS containing 0.1% Tween (PBST) and 5% skimmed milk overnight at 4°C, then washed four times at 10 min intervals with PBST. Washed membranes were incubated with 1 : 300 dilutions of anti-P2X1, anti-P2X2 (Alomone Labs, Jerusalem, Israel), or anti-P2X7 antibody (Alomone Labs, or Chemicon International, Temecula, CA, U.S.A.) in PBST solutions for 90 min. They were then rewashed and incubated with a peroxidase labelled anti-rabbit antibody (1 : 3000 dilution, included in ECL-kit Amersham Pharmacia Biotech, Baie d'Urfe, Quebec, Canada). The membranes were washed again and protein visualized with the enhanced chemiluminescence reagents according to the instructions of the manufacturer.
In order to test the purity of synaptosomes obtained by the Percoll® gradient method, we detected the expression of the synaptosome specific proteins syntaxin, SNAP-25, and glial fibrillary acidic protein (GFAP) using a Western blot. Rabbit anti-syntaxin, anti -SNAP-25 (Alomone Labs. Jerusalem, Israel) and mouse anti-GFAP (Pharmingen Labs, Mississauga, Ontario, Canada), and the corresponding cognate antibodies (Amersham Pharmacia, Quebec, Canada) were used as primary and secondary antibodies in experiments according to the above-mentioned Western blot protocol. 12% SDS-Poly-acrylamide gels were used for syntaxin, SNAP-25 and GFAP.
The following drugs were used: Adenosine 5-triphosphate disodium (ATP), adenosine 5′-O-(3-thiotriphosphate) (ATPγS), 2-methylthio-ATP, 2′ & 3′-O-(4-benzoylbenzoyl)-ATP (BzATP), α,β-methylene ATP, β,γ-methylene-L-ATP; pyridoxal-phosphate-6-azophenyl-2-disulphonic acid tetra sodium (PPADS), ω-conotoxin-MVIIC (ω-CTx-MVIIC) and Brilliant Blue G (BBG) (all purchased from Sigma St Louis, MO, U.S.A). All drugs were dissolved in distilled H2O and added to the appropriate incubation buffer.
Comparisons among data were carried out using a two-way ANOVA. In certain cases a Student's t-test was performed where appropriate, as stated in the results. Probability values less than 0.05 were considered significant.
Initial experiments were performed in choline buffer in which cortical synaptosomes were exposed to increasing concentrations of ATP or BzATP (Figure 1A) for 60 s, which was determined as the optimal incubation period (Figure 1B). The maximally effective concentration required for ATP and BzATP stimulated influx (stimulated minus resting 45Ca2+ influx) was determined (nMoles Ca2+mg protein−1). Both nucleotides evoked a dose dependent influx of extracellular Ca2+. Of the agonists examined, only ATP and BzATP acted as full agonists for which an EC50 could be calculated. The maximal responses for the two full agonists was achieved at 1 mM ATP or 100 μM BzATP. Dose-effect curves to 2-methylthioadenosine triphosphate (2-me-S-ATP) and to adenosine 5′-O-(3- thiophosphate, (ATPγS) were also carried out however these analogues acted as partial agonists and produced shallow dose response curves which did not reach maximal values at the highest concentration examined (1 mM). Therefore, each analogue was tested at the concentration of ATP which produced maximal effects (ATP EC100 or 1 mM). The results in Figure 2 indicate that at 1 mM the order of agonist efficacy was BzATP=ATP>2-me-S-ATP>ATPγS. The nucleotides α, β-methylene adenosine triphosphate (α,β-me-ATP) and β, γ-methylene-L-adenosine triphosphate (β,γ-me-ATP) were inactive. EC50 values for ATP and BzATP were calculated to be 214 and 30 μM (about a 7 fold difference) respectively. Therefore for the two agonists for which an EC50 was available, BzATP was considerably more potent (about seven times) than ATP. In each experiment some synaptosomes were exposed to 25 mM K+ for 3 s to confirm synaptosomal viability and to demonstrate the effective block of neuronal VSCCs by ω- conotoxin-MVIIC (ω-CTx-MVIIC). ATP induced influx (Figure 2 inset) was unaffected by a concentration of the VSCC blocker ω-CTx MVIIC (1 μM) sufficient to completely inhibit influx through all of the recognized neuronal VSCCs (Hillyard et al., 1992; Lundy et al., 1994, see also Figure 2). ATP stimulated influx was also unaffected in synaptosomes treated with the L-type calcium channel blocker (−)202 – 791 (Lundy et al., 1991, present results not shown).
The P2X receptor antagonist pyridoxal-phosphate-6-azophenyl-2′-4′ disulphonic acid (PPADS) (Lambrecht, 1996) at a concentration of 30 μM markedly reduced ATP-evoked Ca2+ influx (Figure 2).
Since P2X7 agonist activity has been reported to be greater in physiological buffers containing low concentrations of cations, experiments were repeated in two additional cation modified buffers (see Methods). The dose-effect curve for BzATP-stimulated influx was shifted to the left in sucrose, as compared to both choline and Na+ based buffers; (difference P<0.001, 2-way ANOVA), confirming previous studies concerning P2X7 receptor sensitivity in ionic media (Michel et al., 1998; Virginio et al., 1997). Results are shown in Figure 3A.
In order to provide more definitive data concerning the receptor subtype involved in the observed influx, lysates and homogenates were prepared from both whole brain and purified synaptosomes and were probed with antibodies for P2X2 and P2X7 receptors (Figure 4). P2X2 receptors were detected in whole brain homogenates (Br) but not in purified synaptosomes (Syn) (Figure 4A). Lysates of whole PC-12 cells served as a positive control for the presence of P2X2 receptors (Brake et al., 1994). P2X7 receptor protein was strongly expressed in both whole brain (Br) homogenates and in synaptosomal preparations (Syn) (Figure 4B). Homogenates of vas deferens (Vas) were used as negative controls for the presence of P2X7 receptors.
The occurrence of P2X7 receptors in brain has been attributed to their presence on microglia (Collo et al., 1997). However, contamination by non-neuronal elements was not detected in purified synaptosomes prepared by similar methods as those described here (Daniels & Vickroy, 1998; Hoogland et al., 1999). However, to preclude this possibility, we examined the presence of contaminants using specific antibodies to P2X7 receptors and OX-42 (specific for microglia). Background immunofluorescence was examined in whole brain (Figure 5A) and synaptosomes (Figure 5B).
Immunofluorescence for P2X7 receptors was located in brain and synaptosomes (Figure 5C, D) while OX-42 fluorescence was evident in whole brain (Figure 5E) but was absent in synaptosomes. The fluorescence in individual synaptosomes appears not to be uniform, probably as the result of the fact that they were concentrated (cytospun) and therefore could be at slightly different planes in the field or in some cases overlap one another. In similar experiments we also detected GFAP immunofluorescence in cortical tissue but not in purified synaptosomes (results not shown).
Although the lack of activity of the P2X1 agonist α,β-me-ATP appears to preclude a role for P2X1 receptors in ATP-stimulated Ca2+ influx, P2X1 receptors were weakly detected by Western blot techniques in whole brain and in purified synaptosomal preparations. Rat vas deferens was used as a positive control for P2X1 receptors (results not shown).
Western blots obtained using purified synaptosomal homogenates probed with anti-SNAP 25, anti-syntaxin and anti-GFAP antibodies, provided no evidence for GFAP in the purified synaptosomal preparation, but both synaptic markers were clearly evident (Figure 6). The purity of synaptic elements was further confirmed using immunohistochemistry to visualize the neuronal markers using fluorescent antibodies against SNAP-25 and syntaxin. These findings, which are in accord with the Western blot data, are shown in Figure 7.
The present study offers several lines of evidence, which suggests the existence of P2X receptors on presynaptic elements of mammalian brain. In these studies we have examined the effects of ATP analogues on 45Ca2+ influx, which provides a reliable functional assay for characterizing P2X receptor activity (Michel et al., 1996; 1998). In previous studies, the potency order of ATP and related nucleotides has been used to define P2X receptor subtypes pharmacologically in a variety of tissues (Abbracchio & Burnstock, 1994; Fredholm et al., 1994; 1997; Burnstock, 1999). In the present study, the synaptosomal calcium influx initiated by ATP or ATP analogues suggested both the presence of P2X receptors and a role for a specific subtype.
The P2X2 receptor subtype has previously been identified on mammalian CNS neurons (see Kanjhan et al., 1999 and references therein; Collo et al., 1996). This receptor subtype has also been proposed to exist both on peripheral nerve endings and a percentage of neurohypophysial neurons (Troadec et al., 1998; Boehm, 1999). These previous studies raise the possibility that P2X2 receptors might be responsible for the calcium influx observed in this synaptosomal preparation. However several observations argue that P2X2 receptors do not exist on presynaptic areas in the cortex and are not responsible for the calcium influx observed in the present study. For example, there is ample evidence that BzATP is a much weaker agonist at P2X2 receptors than ATP, 2-me-S-ATP and ATPγS (King et al., 1996; Bianchi et al., 1999; Michel et al., 1996). In the present work, BzATP was found to be approximately seven times more effective as an agonist than ATP, the next most active nucleotide, which was in turn much more active than were any of the other analogues. Moreover western blots from whole brain showed the presence of P2X2 receptors while the synaptosomal preparation did not. The lack of evidence of P2X2 receptors in the synaptosomes corresponds with other important recent studies in which these receptors also could not be identified on midbrain synaptosomes (Gómez-Villafuertes et al., 2001; Diáz-Hernández et al., 2001). Differences between the existence of the same functional structures like P2X2 receptors on peripheral and central nerve endings may be analogous to the situation with calcium channels. In the periphery, the N type VSCC is found almost exclusively in nerve endings whereas the P/Q type appears to be much more prevalent centrally (Lundy & Frew, 1996).
Several other lines of functional evidence all point to the existence of P2X7 receptors on synaptosomes. Ionic composition of the buffer in which P2X receptor/agonist interactions are measured, has a major impact on the effectiveness of these interactions, particularly on the activity of the P2X7 receptor subtype (Michel et al., 1996; 1998; 1999; Surprenant et al., 1996; Virginio et al., 1997). Sodium has been suggested to cause an increase in the interference in P2X7 agonist/receptor interactions (see ref. above and Wiley et al., 1992; Hibell et al., 2001). In our studies, BzATP was much more active in the sucrose based buffer than in either sodium or choline based buffers. The response profile is in good agreement with the activity of BzATP at P2X7 receptors described in previous studies (Michel et al., 1996; 1999). BBG and PPADS also inhibited agonist induced calcium influx. BBG, a selective P2X7 inhibitor (Jiang et al., 2000) was found in this study, to be an effective inhibitor of calcium influx at concentrations similar to those reported previously to be effective (Hibell et al., 2001). The inhibitory effects of BBG at these relatively low concentrations, further mitigates against the presence of most of the other receptor subtypes with the possible exception of P2X2.
Voltage and ligand gated ion channels are the principal Ca2+ entry pathways in excitable cells in general and in synaptosomes in particular (Bean, 1992; Benham, 1992). Previous studies have indicated different interactions between P2X receptors and VSCCs. For example, ATP has been reported to activate Ca2+ influx by activating both P2X receptors and also VSCCs in some (Rogers et al., 1997; von Külgelgen et al., 1999; Boehm, 1999) but not all peripheral preparations (Sperlágh et al., 2000). In the CNS, the relative role played by calcium channels and P2X receptors in response to nucleotides depends on the neurons examined (Gu & MacDermott, 1997; Khakh and Henderson 1998; 2000; Hugel & Schlicter, 2000). Influx through VSCCs and subsequent neurotransmitter release in rat cortical synaptosomes occurs via activation of three major types of VSCCs (N, P and Q) which are inhibited by ω-CTx-MVIIC (Hillyard et al., 1992; Wheeler et al., 1994; Lundy et al., 1994). In the present study, in order to determine whether nucleotide induced influx was the result of the activation of VSCCs, experiments were carried out in the presence of ω-CTx-MVIIC and the L-type channel blocker (−)202-791. The lack of effect of ω-CTx-MVIIC and (−)202-791 on nucleotide-evoked Ca2+ influx would appear to preclude ATP induced activation of VSCCs and suggests that the Ca2+ entry observed in the current study occurred directly via P2X receptors. These results and those in the literature are consistent with the proposal that various central neuronal populations may utilize different mechanisms to control Ca2+ influx following stimulation by ATP and related nucleotides. Therefore, nucleotide induced influx may be the result of activation of VSCCs, activation of P2X receptors or a combination of these two mechanisms, depending on the neurons and the species examined (Khakh and Henderson, 1998; 2000; Gu & MacDermott, 1997; Pintor et al., 1999; Hugel & Schlicter, 2000; Nörenberg & Illes, 2000).
Western blots revealed weak staining for the P2X1 receptor (results not shown) indicating its presence in synaptosomes. P2X1 receptors predominate in smooth muscle, and their existence has also been reported in limited areas of the CNS, such as the cerebellum (Kidd et al., 1995; Burnstock, 1999). However P2X1 (or P2X3) receptor mediated Ca2+ influx in this preparation was also precluded as the result of the lack of activity of α, β-me-ATP, a potent agonist at these receptor subtypes (Valera et al., 1994; rev. see Abbracchio & Burnstock, 1994). Furthermore, α,β-me-ATP, which desensitises P2X1 receptors (Kasakov & Burnstock, 1983) also failed to affect subsequent BzATP-evoked influx (P.M. Lundy and R. Frew, unpublished results). In addition, calcium influx was significantly reduced by BBG at concentrations that would not be expected to be active at P2X1 receptors. Effective antagonism of ATP-evoked Ca2+ influx by PPADS and the lack of selective activity of certain agonists, appears to preclude a role for P2X4 and P2X6 receptor subtypes (Evans et al., 1998). However, convincing evidence for the existence of P2X3 receptors in synaptosomes from rat midbrain has been recently reported (Gómez-Villafuertes et al., 2001; Diáz-Hernández et al., 2001). Although we did not determine the proportion of P2X7 receptors in our preparation, the presence of a significant proportion of P2X3 receptors on cortical synaptosomes appears unlikely since a high concentration of α-β-me-ATP (1 mM) a potent P2X3 agonist was inactive in the mediation of calcium influx in our studies. However it appears clear that more than a single P2X receptor subtype likely co-exists at least in certain brain areas (Gómez-Villafuertes et al., 2001; Diáz-Hernández et al., 2001). Determination of the presence of P2X3 receptors in cortical synaptosomes, or conversely of P2X7 receptors in midbrain synaptosomes would be very interesting studies to carry out.
The results which demonstrated that the selective P2X7 receptor agonist BzATP was of the order of seven times more potent with respect to evoking Ca2+ influx than ATP, the next most effective agonist, is in good agreement with a number of previous studies in other preparations containing P2X7 receptors (for example, Nuttle & Dubyak, 1994; Wiley et al., 1996; 1998; Evans et al., 1998; Surprenant et al., 1996; Chessell et al., 1998; Chung et al., 2000). However, some doubt has been raised recently concerning the actual P2X7 selectivity of BzATP. For example in certain assays, BzATP has been reported to be more potent at P2X1 than at P2X7 receptors (Bianchi et al., 1999). Reasons for differences reported among a variety of studies concerning the selectivity of BzATP are unclear, but the apparent controversy may complicate use of BzATP as a tool to distinguish between P2X1 and P2X7 subtypes. Oxidized ATP (2,3′-dialdehyde ATP), a P2X7 receptor antagonist, could not be used to further confirm the presence of the P2X7 receptor subtype since its inhibitory activity has been reported to develop only after a 2 h pretreatment period (Murgia et al., 1993) which is inconsistent with synaptosomal viability. Studies using KN-62, which has been reported to be a potent antagonist of the P2X7 receptor of human lymphocytes (Gargett & Wiley, 1997) failed to inhibit ATP-evoked Ca2+ influx in synaptosomes (P.M. Lundy and R. Frew, unpublished results) likely due to differences in its inhibitory activity between rat and human receptors (Humphreys et al., 1998).
P2X7 receptor subtypes detected in whole brain homogenates have also been attributed to their presence on microglia (Ballerini et al., 1996; Collo et al., 1997; Ferrari et al., 1997). It was important therefore to ensure that P2X7 protein was indeed located on synaptosomal terminals and was not due to non-neuronal contaminating elements. Percoll® gradient isolation of synaptosomes has been reported to be sufficient to eliminate non-synaptosomal contaminants (Daniels & Vickroy, 1998; Dugar et al., 1998; Hoogland et al., 1999). The presence in the purified synaptosomes of the markedly enhanced concentrations of the synaptic markers syntaxin and SNAP-25, and the almost complete absence of GFAP or the microglial marker OX-42 (Brook et al., 2001) confirmed the lack of glial or microglial contamination in these synaptosomes.
In summary, our study clearly demonstrates that P2X receptors exist on central presynaptic nerve terminals and that the evidence presented here suggests that these receptors are of the P2X7 subtype. This novel finding of P2X7 receptors on cortical synaptosomes suggests that these receptor subtypes play a role in neurotransmission, and probably in other Ca2+ dependant events distinct from those mediated by VSCCs in presynaptic terminals in the CNS. We are currently examining the role of this receptor on BzATP induced glutamate release.
This work was supported in part by the U.S. Army Medical Research and Development Command, Award DAMD17-99-2-9010. In performing the research described in this paper, the authors adhered to the ‘Guide to the Care and Use of Laboratory Animals’ and ‘The Ethics of Animal Experimentation’ published by the Canadian Council on Animal Care, Ottawa, Ontario, Canada.