The role of ATP and adenosine in the brain under normoxic and ischemic conditions By taking advantage of some recently synthesized compounds that are able to block ecto-ATPase activity , we demonstrated that adenosine triphosphate -LRB- ATP -RRB- in the hippocampus exerts an inhibitory action independent of its degradation to adenosine . In addition , tonic activation of P2 receptors contributes to the normally recorded excitatory neurotransmission . The role of P2 receptors becomes critical during ischemia when extracellular ATP concentrations increase . Under such conditions , P2 antagonism is protective . Although ATP exerts a detrimental role under ischemia , it also exerts a trophic role in terms of cell division and differentiation . We recently reported that ATP is spontaneously released from human mesenchymal stem cells -LRB- hMSCs -RRB- in culture . Moreover , it decreases hMSC proliferation rate at early stages of culture . Increased hMSC differentiation could account for an ATP-induced decrease in cell proliferation . ATP as a homeostatic regulator might exert a different effect on cell trophism according to the rate of its efflux and receptor expression during the cell life cycle . During ischemia , adenosine formed by intracellular ATP escapes from cells through the equilibrative transporter . The protective role of adenosine A1 receptors during ischemia is well accepted . However , the use of selective A1 agonists is hampered by unwanted peripheral effects , thus attention has been focused on A2A and A3 receptors . The protective effects of A2A antagonists in brain ischemia may be largely due to reduced glutamate outflow from neurones and glial cells . Reduced activation of p38 mitogen-activated protein kinases that are involved in neuronal death through transcriptional mechanisms may also contribute to protection by A2A antagonism . Evidence that A3 receptor antagonism may be protective after ischemia is also reported . Historical overview The physiological roles of adenosine triphosphate -LRB- ATP -RRB- and its metabolite adenosine have been historically linked to cell metabolism since ATP is an ubiquitous intracellular energy source in a number of enzymatic processes . However , over the years both metabolites have emerged as very versatile molecules of biological systems , being implicated in a variety of cell processes , from platelet aggregation to neurotransmission . The term ` purinergic signalling ' was first introduced in the scientific literature by Burnstock -LSB- 1 -RSB- . An implicit concept for sustaining the hypothesis of purinergic neurotransmission proposed in the 1970s was the existence of purinergic receptors . The first evidence in this direction suggested the existence of two different subfamilies of such ` purinoceptors ' , identified as P1 and P2 receptors , selective for adenosine and ATP respectively -LSB- 2 -RSB- . Four different subtypes of P1-G-protein-coupled receptors : A1 , A2A , A2B and A3 are known at present -LSB- 3 -RSB- , whereas P2 purinoceptors belong to two major families : P2X ligand-gated ion channel receptors and P2Y G-protein-coupled receptors -LSB- 4 -RSB- . Cloning experiments supported this classification and helped to subdivide P2 receptors into seven P2X and eight P2Y subtypes -LSB- 5 -RSB- -LRB- plus the recently deorphanized GPR17 receptor -LSB- 6 -RSB- -RRB- . Roles of ATP in neurotransmission under normoxic conditions The first studies on the role of extracellular ATP in hippocampal neurotransmission indicated prominent inhibitory action of this purinergic nucleotide on synaptic activity -LSB- 7 , 8 -RSB- . However , several lines of evidence led to the hypothesis that ATP-mediated inhibition of hippocampal neurotransmission was probably mediated by adenosine acting on A1 receptors . In fact , this response was theophylline-sensitive -LSB- 7 -- 9 -RSB- and absent in A1 knockout mice -LSB- 10 -RSB- . This assumption was supported by the fact that , in the hippocampus , extracellular ATP is rapidly converted into adenosine by ecto-ATPases and ecto-nucleotidases -LSB- 11 -RSB- and that adenosine acting on A1 receptors exerts a well-described inhibitory role on CA1 excitatory neurotransmission -LSB- 3 -RSB- . In recent years , a more detailed observation of ATP-evoked effects , supported by the synthesis of new pharmacological tools , has helped to clarify the effective role of purinergic nucleotides in the hippocampus . In particular , it emerged that ATP mediates inhibitory effects by P2 receptor activation -LSB- 12 -- 14 -RSB- . In a recent paper , we contributed to elucidating the role of ATP on CA1 hippocampal neurotransmission , and we reinforced the concept that ATP-mediated effects are not necessarily linked to adenosine formation in this brain region -LSB- 15 -RSB- . ATP is hydrolysed by ecto-NTPDase , enzymes located on the cell surface in the CNS that limit ATP , ADP and AMP spatio-temporal activity -LSB- 11 , 16 -RSB- . There are three different known NTPDases : NTPDase1 hydrolyzes ATP and ADP equally well , NTPDase2 has a high preference for ATP , NTPDase3 is a functional intermediate , preferably hydrolyzing ATP -LSB- 17 -RSB- . In our study , we took advantage of some recently synthesized compounds : ARL 67156 , which , at micromolar concentrations , inhibits rat NTPDase1 and 3 transiently transfected in Chinese hamster ovary cells , showing negligible activity on NTPDase2 -LSB- 18 -RSB- ; BGO 136 , a new inhibitor described as a selective NTPDase1 and 2 blocker with Ki values in the high micromolar range -LSB- 19 -RSB- ; and the recently synthesized PV4 , which strongly inhibits rat NTPDase1 , 2 and 3 with Ki values in the nanomolar range -LSB- 20 -RSB- . By using these inhibitors that are able to block NTPDase activity without interfering with P2 receptor activation , we demonstrated that ATP exerts an inhibitory action , independent of its degradation to adenosine . As shown in Fig. 1 , the application of ATP during a NTPDase activity blockade still elicits a decrease in evoked synaptic responses which is even more pronounced than that evoked by ATP alone . Fig. 1a , bThe inhibitory effect induced by ATP on fEPSP amplitude is potentiated in the presence of different NTPDase inhibitors . a Time-course of fEPSP amplitude before , during and after the application of ATP in the absence or in the presence of the NTPDase1 ,2,3 inhibitor PV4 . Each point in the graph represents the mean ± SE of fEPSP value measured as percent of baseline , pre-drug level . b Columns in the graph summarize the average amplitude -LRB- mean ± SE -RRB- of evoked fEPSP recorded from CA1 hippocampal region in control conditions , 5 min after superfusion of ATP alone and 5 min after ATP superfusion in the presence of different ecto-ATPases inhibitors . Note that the inhibitory effect of ATP on fEPSP amplitude is potentiated by BGO 136 , PV4 and ARL 67156 . * P < 0.05 one-way ANOVA , Newman-Keuls multiple comparison post-hoc test versus pre-drug value . § P < 0.05 , one-way ANOVA , Newman-Keuls multiple comparison post-hoc test versus 10 μM ATP treated slices . -LRB- Modified from -LSB- 15 -RSB- -RRB- An excitatory effect of exogenous ATP on hippocampal neurotransmission has also been reported by different authors . This effect , which was observed after drug removal , was described to persist in in vitro preparations for a relatively prolonged period , up to 1 h -LSB- 21 -- 25 -RSB- . This ` long-lasting ' potentiation of synaptic responses was compared to electrically evoked LTP , firstly described in the hippocampus by Abrams and Kandel -LSB- 26 -RSB- and called ` ATP-induced LTP ' . In agreement , we observed a potentiation of the synaptic responses after drug removal when slices were superfused in the presence of the metabolically stable ATP-analogue ATPγS -LRB- Fig. 2 -RRB- -LSB- 15 -RSB- . In addition to this ` long-lasting ' excitatory effect evoked by the exogenous application of P2 agonists , we also demonstrated an excitatory tone exerted by endogenous ATP . Slices superfused with P2 antagonists show a small but significant reduction in synaptic transmission -LRB- Fig. 3 -RRB- . These data demonstrate that tonic activation of P2 receptors contributes to glutamatergic excitatory neurotransmission in the hippocampus , an observation that is in line with previous work -LSB- 27 -RSB- . Fig. 2Inhibitory and excitatory effects of the stable ATP analogue ATPγS . Averaged time-course -LRB- n = 4 -RRB- of PS amplitude before , during and after the application of different concentrations of ATPγS . PS amplitude -LRB- mean ± SE -RRB- is measured as percent of baseline level . Upper panels represent single traces recorded in a typical experiment before , during and after ATPγS application at different concentrationsFig . 3Excitatory effects of endogenous ATP . Bars in the graphs represent the average of fEPSP amplitude in the presence of P2 antagonists : PPADS -LRB- 30 μM -RRB- and MRS 2179 -LRB- 10 μM -RRB- . * P < 0.05 , paired Student 's t-test . -LRB- Modified from -LSB- 15 -RSB- -RRB- Roles of ATP in ischemic conditions The role of ATP may become critical during pathological conditions such as ischemia , when extracellular ATP concentrations increase . An enhanced outflow of radioactive ATP from hippocampal slices during in vitro ischemic-like insults was first reported by Juranyi and co-workers -LSB- 28 -RSB- , and the first demonstration that ATP outflow increases in vivo during the induction of focal ischemia in the rat was reported by Melani and colleagues -LSB- 29 -RSB- . Thus , at the ischemic site , levels of extracellular nucleotides may remain elevated for long periods of time after injury . Evidence supports the idea that , under such pathological conditions , released ATP may exert an excitotoxic role by acting on its receptors , thus enhancing Ca2 + inward currents and altering synaptic activity and cellular plasticity . Non-selective antagonists of P2 receptors , suramin and PPADS , and the selective antagonists , BBG and MRS 2179 of P2X7 and P2Y1 receptors respectively , prevent the irreversible failure of neurotransmission induced by a prolonged period of OGD in hippocampal slices -LSB- 15 -RSB- . Moreover , they protect from development of anoxic depolarization -LRB- AD -RRB- , which is a rapid and regenerative wave of depolarization that propagates in tissue and represents an unequivocal sign of sufferance -LSB- 15 -RSB- . Antagonists of P2 receptors have been proved protective against cell death induced by either ATP itself , hypoglycemia or glutamate exposure in primary cultures of brain-derived neurones -LSB- 30 -- 33 -RSB- . Intrastriatal ATP injection in rats induced , 24 h later , a clearly lesioned area -LSB- 34 -RSB- . The ATP-induced damage was concentration-dependent , mimicked by ATPγS and α , β meATP -LRB- but not by ADP or adenosine -RRB- and blocked by RB2 , a non-specific P2 antagonist -LSB- 34 -RSB- . Suramin , another non-specific P2 receptor antagonist , administered 30 min before occlusion of the middle cerebral artery , resulted in a significant decrease in infarct and oedema volume 6 h after brain injury -LSB- 35 -RSB- . In agreement , it was demonstrated that RB2 -LSB- 36 -RSB- and PPADS -LSB- 37 -RSB- improve neurological deficit and reduce the damage induced in rats in a model of focal ischemia in vivo . Moreover , RB2 induces the expression of P2X7 receptors on reactive microglia in the remote ipsi and contralateral cingulate and medial frontal cortex and striatum . Although a pro-apoptotic role has been attributed to the P2X7 receptor , results suggest that microglial cells expressing the P2X7 receptor can be implicated in tissue damage as well as in the defence and repairing processes in the remote ipsi and contralateral undamaged areas -LSB- 36 -RSB- . In considering the effect of ATP during ischemia , it must be taken into account that ATP itself is involved in control of cerebrovascular regulation -LSB- 38 -RSB- and that the metabolite of ATP , ADP , that interacts with P2Y1/P2Y12 receptors , is a potent platelet aggregator . However , clinical studies that checked the antiplatelet therapy of the selective P2Y12 receptor antagonist clopidogrel reported a response variability -LSB- 39 , 40 -RSB- . Although evidence suggests that ATP exerts a detrimental role under ischemia , there are reports that it may exert a trophic role in terms of cell division and differentiation in both differentiated and undifferentiated cells -LSB- 41 -- 43 -RSB- , including adult neural stem cells -LSB- 44 -RSB- . Interesting data correlate these trophic effects with the occurrence of spontaneous Ca2 + waves that propagate among adjacent cells in a self-renewing manner . Kawano et al. -LSB- 45 -RSB- reported that ATP-induced initiation and propagation of intracellular Ca2 + waves in human mesenchymal stem cells -LRB- hMSCs -RRB- promote activation of transcription factors -LRB- e.g. NFAT -RRB- that are involved in cell differentiation . In the same study , they demonstrated that ATP-induced Ca2 + waves disappear in the fully differentiated adipogenic phenotype . We recently reported that ATP is spontaneously released from hMSCs during the early stages of culture -LRB- P0-P5 -RRB- . Moreover , we reported that ATP decreases proliferation rate -LRB- Fig. 4 -RRB- and modulates specific ionic current hMSCs -LSB- 46 -RSB- . A high extracellular ATP concentration at earlier cell culture passages suggests an important role of ATP in regulating cell differentiation . Increased hMSC differentiation may account for an ATP-induced decrease in cell proliferation . Fig. 4a , bHuman mesenchymal stem cells in culture spontaneously release ATP that modulates cell proliferation . a Extracellular concentrations of ATP were measured in the medium containing hMSCs and in control medium not containing cells . Data are expressed as mean ± SE , n = 11 , unpaired Student 's t-test : * P < 0.0001 vs medium alone . b Effect on hMSC proliferation after daily application of ATP -LRB- 10 μM -RRB- and P2 antagonists , PPADS -LRB- 30 μM -RRB- and MRS 2179 -LRB- 10 μM -RRB- . Data are expressed as percentage of proliferation . Proliferation of untreated cells was assumed as 100 % . The cell number was determined after 5 days of culture by a culture counter . Each column bar represents the mean ± SE of n = 4 for each experimental condition . Paired Student 's t-test : * P < 0.05 vs respective control ; one-way ANOVA , Newman-Keuls post-test : § P < 0.05 vs 10 μM ATP-treated cells . -LRB- Modified from -LSB- 46 -RSB- -RRB- Therefore , it can be envisaged that ATP , as an autocrine/paracrine homeostatic regulator , exerts different effects on cell trophism according to its extracellular concentrations , distinct cell populations involved , differential expression and recruitment of P2 receptors and of more or less sustained stimulation of the same receptor . Balancing these effects may be relevant in the post-ischemic brain , when a neuroregenerative process could promote tissue repair -LSB- 47 -RSB- . On this basis , determination of ATP extracellular concentrations at various times after ischemia induction may help to identify which types of receptors may be stimulated under ischemia and explain the role of detrimental versus trophic ATP during ischemia . ATP present in the extracellular space is rapidly metabolised by membrane-bound NTPDase . Extracellular ATP concentrations , evaluated in the brain to date , have been underestimated , since it has not been possible to selectively inhibit ecto-NTPDase . In fact , the selective inhibitor ARL 67156 , tentatively used thus far to inhibit ecto-NTPDase , interferes with the ATP assay method -LSB- 29 -RSB- . Role of adenosine in cerebral transmission under normoxic conditions Adenosine exerts an important tonic modulation of synaptic transmission in the brain . This tonic inhibition of synaptic transmission is evoked by stimulation of A1 receptors , as demonstrated in several brain regions , such as the hippocampus , striatum and olfactory cortex -LSB- 48 , 49 -RSB- . The inhibitory effect of adenosine A1 receptor stimulation has a pre - and postsynaptic component . Activation of the presynaptic A1 receptors reduces Ca2 + influx through the preferential inhibition of N-type and , probably , Q-type channels -LSB- 50 , 51 -RSB- . Inhibition of presynaptic calcium currents decreases transmitter release -LSB- 52 -RSB- , and adenosine , by stimulation of A1 receptors , has been found to inhibit the release of virtually all classical neurotransmitters : glutamate , acetylcholine , dopamine , noradrenaline and serotonin -LRB- see in -LSB- 53 -RSB- -RRB- . In particular , powerful suppression of glutamate release from presynaptic terminals has been described in the hippocampus -LSB- 54 , 55 -RSB- , where adenosine A1 receptor activation reduces the number of quanta released -LRB- but not the size of individual quanta nor postsynaptic glutamate receptor sensitivity -RRB- in the Schaffer collateral-commissural pathway -LSB- 56 -RSB- . The postsynaptic effect of A1 receptors consists of direct hyperpolarisation of neurones via activation of GIRK channels -LRB- Kir 3.2 and 3.4 channels : potassium inward rectifiers -RRB- -LSB- 57 , 58 -RSB- . Endogenous adenosine exerts tonic inhibition of excitatory neurotransmission . The selective A1 antagonist , DPCPX , causes a 15 % increase in synaptic potential amplitude in in vitro brain slices -LSB- 59 -RSB- . This is an expected result in a brain region where the adenosine concentration at a receptor level was calculated around 200 nM -LSB- 59 , 60 -RSB- and A1 receptors , whose affinity for adenosine is in the low nanomolar range , are highly expressed . These data are confirmed by the fact that , in slices taken from homozygous A1 receptor knockout mice , no evidence was found for an endogenous inhibitory action by adenosine in the Schaffer collateral pathway in the CA1 region of the hippocampus or at the mossy fibre synapses in the CA3 region -LSB- 61 -RSB- . Opposite effects from A1-mediated synaptic inhibition are elicited by A2A receptor activation , which has been shown to mediate excitatory actions in synaptic function -LSB- 62 -- 64 -RSB- . In the hippocampus in vitro , A2A receptor stimulation results in a Ca2 + - dependent release of acetylcholine -LSB- 65 , 66 -RSB- . Furthermore , the application of CGS 21680 , a selective A2A receptor agonist , decreases the ability of A1 receptor agonists to inhibit excitatory neurotransmission -LSB- 9 , 67 -RSB- . This effect suggests that A2A receptor stimulation increases synaptic transmission through A1 receptor desensitisation -LSB- 68 , 69 -RSB- . However , there is also evidence that A2A receptors increase excitatory amino acid release . In fact , the selective stimulation of adenosine A2A receptors augments the amount of glutamate released in the hippocampus and striatum of young rats -LSB- 70 -- 72 -RSB- . In spite of the excitatory role in neurotransmission brought about by A2A receptors , the net effect of adenosine is an inhibitory tonus on neurotransmission , in accordance with observations suggesting that activation of A2A receptors requires protracted stimulation to induce evident effects on synaptic transmission -LSB- 59 -RSB- . It is worth noticing that the role of A2A receptors in the striatum is recently gaining interest in light of their heterodimerisation with D2 dopamine receptors . The association between A2A and D2 receptors results in an antagonistic interaction that provides a rationale for evaluating A2A-selective antagonists in Parkinson 's disease , supported by epidemiological evidence indicating an inverse relationship between caffeine consumption and the risk of developing this pathology -LSB- 73 , 74 -RSB- . It was suggested that A2A antagonists not only provide symptomatic relief but also decelerate dopaminergic neurone degeneration in patients . Discrepancies about the role of adenosine A3 receptors in the brain are present in the literature . Activation of this receptor subtype has been associated with both excitatory and inhibitory effects , even in the same brain region . An excitatory role of A3 receptors has been supported by evidence indicating that , in the rat hippocampus , their activation attenuates LTD and allows induction of LTP elicited by a subliminal weak-burst protocol -LSB- 75 -RSB- . In addition , in the same brain area , A3 receptor activation through a selective adenosine A3 agonist has been shown to antagonize the adenosine A1 receptor-mediated inhibition of excitatory neurotransmission -LSB- 8 -RSB- . Moreover , A3 receptor stimulation always attenuates the inhibition of hippocampal slice neurotransmission , in a PKC-dependent manner , caused by presynaptic metabotropic glutamate receptors -LSB- 76 -RSB- . Whole-cell patch clamp recordings in CA3 hippocampal pyramidal neurones demonstrate that A3 receptor activation results in a significant potentiation of high threshold hippocampal Ca2 + currents by a PKA-dependent mechanism -LSB- 77 -RSB- . Finally , facilitation of the onset of epileptiform discharge has been observed in the presence of the selective A3 receptor agonist Cl-IB-MECA -LSB- 78 -RSB- , and a reduction in such epileptic activity was observed when A3 receptors , activated by endogenously released adenosine during seizures , were blocked by the selective antagonist MRS 1191 -LSB- 79 -RSB- . Contrary to previous results , an inhibitory action has been attributed to A3 receptors by Brand and colleagues -LSB- 80 -RSB- , who demonstrated that , in rat cortical neurones , the selective activation of this adenosine receptor subtype is involved in inhibition of excitatory neurotransmission , suggesting a synergistic action with the inhibitory effect of adenosine brought about by A1 receptor activation . Despite results obtained by A3 receptor stimulation , evidence that a selective block of A3 receptors does not affect neurotransmission in the CA1 region of the hippocampus under normoxic conditions indicates that endogenous adenosine at physiological concentrations does not exert tonic activation of A3 receptors -LSB- 8 , 81 -RSB- . This is in line with evidence that A3 receptor activation requires micromolar levels of extracellular adenosine , which can be reached only during pathological conditions of impairment in energy supply -LRB- for example during hypoxia or ischemia -LSB- 82 -- 85 -RSB- -RRB- . Role of adenosine in ischemic conditions Extracellular adenosine concentrations increase dramatically during ischemia -LSB- 82 , 86 , 87 -RSB- . During ischemia , following the imbalance between ATP degradation and resynthesis , the intracellular concentration of adenosine increases . Therefore , increased adenosine concentrations in the extracellular space during ischemia are likely due to the equilibrative transporter that carries adenosine out of cells . Adenosine formation can also take place at the extracellular level , through the hydrolysis of extracellular ATP operated by NTPDases and ecto-5 ′ - nucleotidase -LRB- e5 ′ - NTs -RRB- -LRB- see Fig. 5 -RRB- . Hence , these enzymes have a dual function in modulating purinergic neurotransmission : -LRB- 1 -RRB- they rapidly interrupt ATP-mediated signalling by degrading extracellular nucleotides and -LRB- 2 -RRB- they give rise to extracellular adenosine , which activates P1 receptors . However , recent evidence in vitro suggests that released ATP does not substantially contribute to the adenosine concentration in the extracellular milieu during ischemia -LSB- 88 -RSB- . Fig. 5Schematic drawing of intracellular and extracellular adenosine formation . In the extracellular space , adenosine and ATP act on own purinergic receptor subtypes : P1 and P2 receptors , respectively . ADP adenosine diphosphate , AMP adenosine monophosphate , ATP adenosine triphosphate , e5 ′ - NT ecto-5 ′ - nucleotidase , 5 ′ - NT 5 ′ - nucleotidase , NTDPase ecto-nucleoside triphosphate diphosphohydrolases , P1 adenosine receptor , P2 ATP receptor , SAH S-adenosylhomocysteine , T bidirectional nucleoside transporter . -LRB- Modified from -LSB- 142 -RSB- -RRB- Adenosine-potentiating agents , which elevate endogenous adenosine levels by either inhibiting its metabolism by adenosine deaminase or kinase -LSB- 89 , 90 -RSB- or preventing its transport -LSB- 91 , 92 -RSB- , offer protection against ischemic neuronal damage in different in vivo ischemia models . Moreover , adenosine infusion into the ischemic striatum during transient focal ischemia proves to significantly ameliorate the neurological outcome and reduce infarct volume -LSB- 93 -RSB- . A temporal correlation exists between adenosine outflow and synaptic potential inhibition in rat hippocampal slices during ischemia-like conditions -LSB- 84 , 88 -RSB- . Synaptic inhibition during ischemia is greatly dependent on adenosine , which , by stimulating A1 receptors , exerts a protective role by reducing the Ca2 + influx , thus counteracting the presynaptic release of excitatory neurotransmitters -LSB- 55 , 94 -RSB- and , in particular , glutamate , which exerts an excitotoxic role during ischemia mainly by overstimulation of NMDA receptors -LSB- 95 -RSB- . By directly increasing the K + and Cl − ion conductances , adenosine stabilises the neuronal membrane potentials , thus reducing neuronal excitability -LSB- 96 -RSB- . Consequent reductions in cellular metabolism and energy consumption -LSB- 97 -RSB- and moderate lowering of the body/brain temperature -LSB- 98 -RSB- are protective in ischemia . A1 receptor agonists are shown to attenuate ischemic or excitotoxic neuronal damage in both in vitro and in vivo models of cerebral ischemia -LRB- for review , see -LSB- 99 , 100 -RSB- -RRB- . In accordance , adenosine A1 antagonists given acutely exacerbate the damage induced by ischemia in different animal models of ischemia . An unselective A1 receptor antagonist , theophylline , increased mortality -LSB- 101 , 102 -RSB- . Unlike acute treatment , chronic administration of A1 agonists worsened survival and increased neuronal loss -LSB- 103 -RSB- , a phenomenon thought to depend on A1 receptor desensitization . Although data converge in demonstrating a neuroprotective effect of adenosine through A1 receptors during ischemia , the use of selective A1 agonists is hampered by unwanted peripheral effects , e.g. sedation , bradycardia , hypotension -LSB- 104 -RSB- . Von Lubitz and co-workers -LSB- 105 -RSB- have reported that post-ischemic administration of the A1 receptor agonist adenosine amine congener -LRB- ADAC -RRB- , which induces fewer undesirable effects , increases survival in gerbils . Moreover , we may consider that administration of agents that elevate the local concentration of adenosine at the injury site , by inhibiting its metabolism to inosine or rephosphorylation to AMP or reuptake , may have the advantage of restricting the effect of such inhibitors to areas of injury-induced adenosine release -LSB- 106 -RSB- . More recently , the role of A2A receptors in ischemic neuroprotection has been studied . Gao and Phillis -LSB- 107 -RSB- demonstrated for the first time that the non-selective A2A receptor antagonist CGS 15943 reduces cerebral ischemic injury in the gerbil following global forebrain ischemia . Subsequently , many reports have confirmed the neuroprotective role of A2A receptor antagonists in different models of ischemia . The selective A2A receptor antagonist SCH 58261 reduced ischemic brain damage in neonatal -LSB- 108 -RSB- and adult -LSB- 83 , 109 -RSB- rat models of focal cerebral ischaemia . The same antagonist , subchronically administered , was protective against both brain damage and neurological deficit in the adult rat model of focal cerebral ischemia -LSB- 110 , 111 -RSB- . Studies in A2A receptor knockout mice supported the neuroprotective role of A2A receptor antagonists on ischemic brain damage -LSB- 112 -RSB- . The beneficial effects of A2A antagonists in stroke were mainly attributed to reduced glutamate outflow -LSB- 83 , 110 , 113 -RSB- . Prolonged application of the A2A selective agonist CGS 21680 significantly reduced synaptic depression brought about by OGD in the hippocampus -LSB- 59 -RSB- and the same agonist promotes glutamate release under normoxic and ischemic conditions -LSB- 70 , 71 , 114 , 115 -RSB- . In addition to neurones , adenosine A2A receptors are located on microglia -LSB- 116 , 117 -RSB- and astrocytes -LSB- 118 -RSB- . On glial cells , A2A receptors mediate inhibition of the glutamate uptake transporter , GLT-1 , and stimulate glutamate outflow -LSB- 119 -RSB- . A2A antagonists prevent the increase in glutamate levels induced by glutamate uptake inhibitors -LSB- 120 -RSB- . Therefore , the protective effects of A2A antagonists in brain ischemia may be largely due to reduced glutamate outflow from neurones and glial cells . Further support of a major role for glia in the neuroprotective effect of A2A antagonism in ischemia comes from the observation that subchronic administration of the A2A receptor antagonist SCH 58261 reduces p38 mitogen-activated protein kinase -LRB- MAPK -RRB- activation in striatal and cortical microglia 24 h after permanent focal ischemia -LSB- 111 -RSB- . Evidence indicates that p38 MAPK is activated in glia up to 24 h after ischemia -LSB- 121 , 122 -RSB- and is involved , through transcriptional mechanisms , in neuronal death -LSB- 123 , 124 -RSB- . Since SCH 58261 reduces glutamate outflow in the first hours after ischemia -LSB- 83 -RSB- , reduced p38 MAPK activation may be due to a direct effect on glial A2A receptors or may be secondary to a reduction in the excitotoxic cascade that primes p38 activation -LSB- 125 -RSB- . The importance of A2A receptors under ischemia is highlighted by the observation that A2A receptor expression increases on neurones and microglia after ischemia -LSB- 126 -RSB- . Selective inactivation of A2A receptors on bone-marrow-derived cells -LRB- wild-type mice transplanted with A2A receptor knockout bone marrow cells -RRB- attenuates infarct volumes and ischemia-induced expression of several proinflammatory cytokines in the brain -LSB- 127 -RSB- . Therefore , protective effects of A2A antagonists may be attributed to inhibition of inflammation product production . In several studies , A2A receptor agonists have been found to be protective in the global ischemia model in the gerbil -LSB- 128 , 129 -RSB- . Jones and co-workers -LSB- 130 -RSB- show that peripheral administration of the A2A receptor agonist CGS 21680 protects the hippocampus against kainate-induced excitotoxicity . However , the direct injection of CGS 21680 into the hippocampus failed to afford protection , while the A2A antagonist ZM 241385 , when injected directly into the hippocampus , reduced kainate-induced neuronal damage -LSB- 131 -RSB- . These data suggest that the neuroprotective properties of A2A agonists are mainly due to peripherally mediated effects . Major mechanisms that may account for A2A-mediated protection include inhibition of platelet aggregation and vasodilation -LSB- 89 -RSB- and anti-inflammatory actions . A2A receptors on neutrophils may account for inhibition of adhesion to endothelial cells and ensuing production of free radicals -LSB- 132 , 133 -RSB- . On this basis , adenosine and its receptors are considered targets for therapeutic implementation in the treatment of stroke . At the moment , a possible adenosynergic therapeutic strategy after ischaemia that is worth consideration is that of increasing adenosine concentrations at the ischemic sites by inhibitors of adenosine metabolism or reuptake in association with adenosine A2A antagonists . When considering the possible use of adenosine kinase inhibitors , it should be taken into account that adenosine represents only a small percentage of nucleotide content -LSB- 134 -RSB- , therefore inhibition of its rephosphorylation to ATP by adenosine kinase inhibitors does not weigh upon the ATP content . The few studies present in the literature concerning the role of A3 receptors in the pathophysiology of cerebral ischemia are rather contradictory . We have demonstrated that selective antagonism of A3 receptors facilitates the recovery of synaptic activity induced by ischemic preconditioning in rat hippocampal slices -LSB- 81 -RSB- . A harmful role of A3 receptors during in vitro OGD was confirmed by our observation that blocking the A3 adenosine receptor consistently abolishes or delays the occurrence of anoxic depolarization -LRB- AD -RRB- and significantly protects from the irreversible disruption of excitatory neurotransmission caused by a severe ischemic episode -LSB- 135 -RSB- . These results are in agreement with the observation that acute administration of a selective adenosine A3 agonist exacerbates the damage elicited by global ischemia in the gerbil -LSB- 136 -RSB- . On the contrary , it was demonstrated that chronic pre-ischemic administration of an A3 agonist protects against ischemic neuronal damage -LSB- 136 -RSB- . This effect may be attributed to desensitisation of A3 receptors . In fact , both human and rat A3 receptors are desensitised within a few minutes after agonist exposure -LSB- 137 , 138 -RSB- . Contrary to the above information , Hentschel and colleagues -LSB- 139 -RSB- demonstrated that under hypoxic conditions , selective activation of A3 adenosine receptors brings about an inhibition of excitatory neurotransmission on cortical neurones , indicating that A3 receptors may sustain the neuroprotective action of adenosine induced by A1 receptors . Consistent with these reports , mice lacking A3 adenosine receptors show increased neurodegeneration in response to repeated episodes of moderate hypoxia -LSB- 140 -RSB- or an increase in cerebral infarction after transient ligation of the middle cerebral artery -LSB- 141 -RSB- . These opposite results regarding an excitatory or inhibitory role of A3 receptors on synaptic activity under hypoxia/ischemia may be reconciled by our recent data -LSB- 135 -RSB- , suggesting that in a first phase of ischemia , A3 receptors play a protective synergistic role with A1 receptors . Severe ischemia would transform the A3 receptor-mediated effects from protective to injurious . Taken together , these data suggest that the outcome of A3 receptor stimulation on synaptic transmission during hypoxic/ischemic phenomena depends on the intensity and duration of stimulation . Concluding remarks Purinergic signalling , e.g. adenosine and ATP , by activating specific membrane receptors -LRB- P1 and P2 respectively -RRB- , is strictly correlated in orchestrating brain cell functions either under physiological normoxic or ischemic circumstances . However , the respective contribution of each single element is hard to discern from the total outcome , due to the rapid and ubiquitous enzymatic interconversion of these molecules . In the brain , both compounds are likely to play a role in neuronal activity , and the inhibitory action of endogenous adenosine in excitatory neurotransmission -LRB- mainly due to A1 receptor activation -RRB- is well documented . Conversely our data , in line with previous observations in the literature , suggest that tonic activation of P2 receptors contributes to excitatory transmission in the hippocampus . Such an effect is modest , but it is unmasked by the application of P2 purinergic antagonists that reduce evoked synaptic responses . An inhibitory effect elicited by exogenous ATP application has been frequently described and is mostly due to enzymatic degradation to adenosine and the subsequent activation of A1 receptors . However , our evidence demonstrates that ATP itself may inhibit hippocampal synaptic transmission since an even more pronounced effect is observed when enzymatic ATP degradation is blocked . Moreover , in our studies in the hippocampus , a potentiation of neurotransmission by the ATP analogue , ATPγS , develops a few minutes after the inhibitory effect has been washed out , and it persists for 45 min to 1 h. Within the brain , ATP is involved in the synaptic plasticity phenomenon and may be an important mediator of long-lasting effects in synaptic plasticity . Either adenosine or ATP seems to become a particularly important signalling molecule under pathological conditions , such as ischemia , when the extracellular concentration of either compound drastically rises . It is well known that adenosine exerts important neuroprotective effects during brain ischemic insults by activating adenosine A1 receptors , which profoundly inhibit synaptic transmission and in particular the release of glutamate , known to contribute to ischemic damage . Under severe ischemia , a prolonged stimulation of both A2A and A3 receptors may be deleterious . The role of endogenously released ATP during cerebral ischemia is mainly deleterious , as found in our work and in the literature , since the application of P2 antagonists always results in a reduction in ischemic damage . The mechanisms underlying these effects are still unknown , but they can be correlated to the tonic excitatory action of endogenous ATP found in many brain regions -LRB- for example in the hippocampus and prefrontal cortex -RRB- . Purinergic signalling has an ancient phylogenetic origin . Expression of P2 receptors on cell membrane and responses to extracellular ATP are found in primitive prokaryotic species up to evolved animals and plants , suggesting an important and highly conserved role of extracellular purine nucleotides during evolution . Similarly , ontogenetic development in several species seems to involve purinergic signalling , especially during its first stages . Our observation that hMSCs , an undifferentiated line of cells able to originate a number of different cell lineages -LRB- adipocytes , chondrocytes , osteoblasts , neurones -RRB- , spontaneously release ATP into the extracellular space and express functional purinergic P2 receptors , which modulate different kinds of membrane currents , and that ATP , during the early stages of culture , inhibits proliferation indicates an autocrine/paracrine mechanism of action for extracellular ATP in modulating cell functions of undifferentiated stem cells at early developmental stages . Such effects may be important in brain neurogenesis during development and in responses to neurodegenerative stimuli .

O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O B O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O B O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O B O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O B O O O O O O O O B O B O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O B O O O O B O O O O O O O O O B O O O O O B O O O O O O O O B O O O O O B O O O O O O B O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O B O O O O O O B O O O O O O O O O O O B O O O O O O O B O B O O O O O O O O O O O O O O O B O O O B O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O B O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

adenosine a2a receptors
ecto-atpase inhibitors
p2 purinergic receptors


Monitoring

the

expression

of

purinoceptors

and

nucleotide-metabolizing

ecto-enzymes

with

antibodies

directed

against

proteins

in

native

conformation

Following

their

release

from

cells

ATP

and

NAD

the

universal

currencies

of

energy

metabolism

function

as

extracellular

signalling

molecules

Mammalian

cells

express

numerous

purinoceptors

i.e.

the

nucleotide-gated

ion

channels

and

the

G-protein-coupled

receptors

Signalling

through

purinoceptors

is

controlled

by

nucleotide-metabolizing

ecto-enzymes

which

regulate

the

availability

of

extracellular

nucleotides

These

enzymes

include

ecto-nucleoside

triphosphate

diphosphohydrolases

-LRB-

ENTPD

family

-RRB-

and

ecto-nucleotide

pyrophosphatase/phosphodiesterases

-LRB-

ENPP

family

-RRB-

Investigation

of

these

receptors

and

enzymes

has

been

hampered

by

the

lack

of

available

antibodies

especially

ones

that

recognize

these

proteins

in

their

native

conformation

This

study

reports

the

use

of

genetic

immunization

to

generate

such

antibodies

against

and

Genetic

immunization

ensures

expression

of

the

native

protein

by

the

cells

of

the

immunized

animal

and

yields

antibodies

directed

against

proteins

in

native

conformation

-LRB-

ADAPINCs

-RRB-

Such

antibodies

are

especially

useful

for

immunofluorescence

and

immunoprecipitation

analyses

whereas

antibodies

against

synthetic

peptides

usually

function

well

only

in

Western-blot

analyses

Here

we

illustrate

the

utility

of

the

new

antibodies

to

monitor

the

cell

surface

expression

of

and

to

purify

some

key

players

of

purinergic

signalling

Introduction

Following

their

release

from

cells

the

universal

currencies

of

energy

metabolism

ATP

and

NAD

function

as

extracellular

signalling

molecules

-LSB-

-RSB-

Molecular

cloning

has

identified

nucleotide-gated

ion

channels

-LRB-

purinoceptors

-RRB-

and

nucleotide-activated

G-protein-coupled

receptors

-LRB-

purinoceptors

-RRB-

-LSB-

-RSB-

Signaling

through

purinoceptors

is

effectively

controlled

by

nucleotide-metabolizing

ecto-enzymes

which

regulate

the

availability

of

extracellular

nucleotides

-LSB-

-RSB-

Prominent

roles

are

played

by

members

of

two

families

of

ecto-enzymes

the

ecto-nucleoside

triphosphate

diphosphohydrolases

-LRB-

ENTPD

family

-RRB-

-LSB-

-RSB-

and

the

ecto-nucleotide

pyrophosphatase/phosphodiesterases

-LRB-

ENPP

family

-RRB-

-LSB-

-RSB-

The

mammalian

family

encompasses

seven

members

All

purinoceptors

contain

cytosolic

N

and

C-terminal

tails

two

membrane

spanning

domains

and

an

extracellular

domain

of

approximately

amino

acids

containing

the

ligand-binding

site

and

conserved

cysteine

residues

that

likely

form

five

intrachain

disulfide

bonds

-LSB-

-RSB-

The

extracellular

domains

of

the

respective

mouse

and

human

orthologues

show

high

-LRB-

ca.

-RRB-

sequence

identity

the

paralogues

within

a

given

species

show

lower

-LRB-

-RRB-

identity

The

mammalian

ENTPD

family

encompasses

eight

members

-LSB-

-RSB-

and

have

a

structure

similar

to

that

of

purinoceptors

i.e.

cytosolic

N

and

C-terminal

tails

two

transmembrane

spanning

domains

and

an

extracellular

domain

of

approximately

amino

acids

encompassing

the

ligand-binding

site

and

four

conserved

cysteines

likely

engaged

in

intrachain

disulfide

bridges

-LSB-

-RSB-

and

are

expressed

as

cell

surface

enzymes

and

as

luminal

enzymes

of

intracellular

organelles

ENTPDases

share

structural

similarities

with

the

kinase

superfamily

-LSB-

-RSB-

and

lack

the

second

transmembrane

region

Moreover

in

the

region

corresponding

to

the

first

transmembrane

domain

of

the

other

family

members

is

N-terminal

and

functions

as

a

signal

peptide

resulting

in

the

secretion

of

into

the

lumen

of

the

endoplasmic

reticulum

is

a

type

II

transmembrane

protein

ENTPD

orthologues

show

high

-LRB-

ca.

-RRB-

sequence

identity

the

paralogues

within

a

given

species

show

lower

-LRB-

-RRB-

identity

The

ENPP

family

encompasses

seven

members

-LSB-

-RSB-

are

type

II

transmembrane

proteins

with

a

structure

analogous

to

that

of

i.e.

an

N-terminal

cytosolic

tail

a

single

transmembrane

domain

and

an

extracellular

catalytic

domain

of

approximately

amino

acids

in

contrast

are

type

I

membrane

proteins

A

hydrophobic

N-terminus

acts

as

a

signal

peptide

the

extracellular

catalytic

domain

is

followed

by

a

single

transmembrane

domain

and

a

short

C-terminal

cytosolic

tail

ENPPs

consist

of

catalytic

domains

only

the

catalytic

domains

of

ENPPs

are

flanked

by

genetically

fused

upstream

somatomedin

B

and

downstream

nuclease-like

domains

-LSB-

-RSB-

ENPPs

share

structural

similarities

with

the

alkaline-phosphatase

superfamily

-LSB-

-RSB-

The

catalytic

domain

of

ENPP

orthologues

shows

high

-LRB-

ca.

-RRB-

sequence

identity

the

paralogues

within

a

given

species

show

lower

-LRB-

-RRB-

identity

Despite

their

relevance

for

purinergic

signalling

investigation

of

these

receptors

and

enzymes

has

been

hampered

by

the

lack

of

available

antibodies

especially

ones

that

recognize

the

enzymes

in

their

native

conformation

Antibodies

raised

by

immunization

with

synthetic

peptides

derived

from

the

known

amino

acid

sequence

of

a

protein

of

interest

generally

work

well

in

Western-blot

analyses

but

often

fail

to

recognize

the

native

protein

on

the

cell

surface

-LRB-

Fig.

-RRB-

Such

antibodies

can

be

used

successfully

for

monitoring

the

overall

expression

level

of

the

protein

of

interest

in

a

population

of

cells

but

can

not

be

used

to

assess

cell

surface

expression

on

individual

cells

Genetic

immunization

in

contrast

ensures

that

the

protein

of

interest

is

expressed

in

native

conformation

by

the

cells

of

the

immunized

animal

and

yields

antibodies

directed

against

proteins

in

native

conformation

-LRB-

ADAPINCs

-RRB-

-LSB-

-RSB-

Such

antibodies

function

well

in

immunofluorescence

FACS

ELISA

and

immunoprecipitation

analyses

i.e.

assays

in

which

antipeptide

antibodies

often

fail

We

have

previously

employed

this

technique

to

generate

highly

specific

polyclonal

and

monoclonal

antibodies

against

ecto-ADP-ribsoyltransferases

and

against

murine

-LSB-

-RSB-

Here

we

report

the

generation

and

characterization

of

similar

antibodies

against

other

key

players

of

purinergic

signalling

and

illustrate

the

utility

of

these

antibodies

for

monitoring

the

cell

surface

expression

of

their

cognate

antigens

Fig.

immunization

vs.

peptide

immunization

Materials

and

methods

Cloning

of

expression

vectors

and

cell

transfections

Clones

encoding

full-length

open-reading

frames

representing

members

of

the

ENTPD

and

ENPP

families

were

purchased

from

the

mammalian

genome

collection

-LRB-

-RRB-

Clones

that

were

not

already

in

an

expression

vector

-LRB-

-RRB-

were

subcloned

into

the

expression

vector

Further

expression

constructs

were

kindly

provided

by

James

Wiley

Sydney

-LRB-

human

-RRB-

Richard

J.

Evans

Leicester

-LRB-

human

-RRB-

François

Rassendren

Montpellier

-LRB-

mouse

-RRB-

Ruth

Murrell-Lagnado

Cambridge

-LRB-

rat

-RRB-

and

Astrid

Kehlen

Halle

-LRB-

-RRB-

An

expression

construct

for

nuclear

green

fluorescent

protein

was

generated

by

cloning

the

DNA-binding

domain

of

the

LKLF

transcription

factor

-LSB-

-RSB-

as

a

C-terminal

fusion

protein

into

the

vector

-LRB-

Clontech

-RRB-

Expression

constructs

were

transfected

into

CHO

and

HEK

cells

-LRB-

μg

for

purinoceptors

and

ecto-enzymes

and/or

μg

eGFP-LKLF

per

flask

containing

cells

-RRB-

with

the

JetPei

transfection

reagent

-LRB-

Q-Biogen

-RRB-

For

stable

transfections

plasmids

were

linearized

with

or

before

transfection

and

stable

transfectants

were

selected

by

cultivating

cells

in

the

presence

of

μg/ml

blasticidin

-LRB-

Sigma

-RRB-

Genetic

immunization

and

purification

of

antibodies

Genetic

immunizations

were

performed

as

described

-LSB-

-RSB-

In

brief

rabbits

or

rats

were

immunized

four

times

by

ballistic

DNA

immunization

with

plasmid-conjugated

gold

particles

Serum

samples

were

obtained

days

post-immunization

For

monoclonal

antibody

production

animals

received

a

final

boost

with

transiently

transfected

HEK

cells

-LRB-

cells

in

μl

PBS

i.v.

-RRB-

days

prior

to

sacrifice

Splenocytes

were

fused

to

myeloma

cells

and

screened

for

production

of

specific

antibodies

by

immunofluorescence

analysis

of

CHO

cells

that

had

been

co-transfected

h

earlier

with

constructs

encoding

eGFP-LKLF

and

the

protein

of

interest

Antibodies

were

purified

by

affinity

chromatography

on

Protein

G

Sepharose

-LRB-

Pharmacia

-RRB-

and

conjugated

to

according

to

the

manufacturer

's

-LRB-

Molecular

Probes

-RRB-

instructions

Antibodies

directed

against

the

C-terminal

peptides

of

purinoceptors

were

purchased

from

Caltag

-LRB-

rat

Cal

-RRB-

ABCAM

-LRB-

rat

Abc

-RRB-

and

Alomone

-LRB-

mouse

Al

-RRB-

The

anti-human

mAb

was

kindly

provided

by

Dr.

Ian

Chessel

at

Glaxo

Wellcome

-LRB-

Middlesex

UK

-RRB-

Immunofluorescence

analyses

CHO

cells

were

co-transfected

with

expression

constructs

for

purinoceptors

or

ecto-enzymes

and

nuclear

GFP

-LRB-

and

cells

respectively

-RRB-

Cells

were

transferred

onto

plates

-LRB-

cells/well

-RRB-

h

post-transfection

Then

h

after

transfection

cells

were

fixed

for

min

in

paraformaldehyde

-LRB-

PFA

-RRB-

and

then

stained

with

serial

dilutions

of

immune

serum

for

min

at

room

temperature

To

detect

which

is

not

bound

to

the

cell

surface

cells

were

fixed

in

PFA

and

subsequently

permeabilized

with

IGEPAL

-LRB-

Sigma

-RRB-

before

staining

Bound

antibody

was

detected

with

PE-conjugated

antirat

or

antirabbit

IgG

-LRB-

-RRB-

-LRB-

Dianova

-RRB-

Cell

nuclei

were

counterstained

with

Hoechst

Cells

were

analyzed

with

a

Zeiss

Axiovert

microscope

equipped

with

the

Canon

PowerShot

digital

camera

and

appropriate

filters

for

visualizing

Hoechst

stain

-LRB-

Zeiss

filter

set

-RRB-

green

fluorescent

protein

-LRB-

Zeiss

filter

set

-RRB-

and

PE

fluorochrome

-LRB-

Zeiss

filter

set

-RRB-

Photographs

were

taken

at

magnification

with

fixed

camera

settings

-LRB-

digital

zoom

aperture

exposure

time

s

for

GFP

and

PE

s

for

Hoechst

-RRB-

GFP

and

PE

images

were

merged

using

Adobe

Photoshop

software

FACS

analyses

For

FACS

analyses

cells

were

harvested

by

brief

trypsinization

-LRB-

min

at

room

temperature

-RRB-

and

washed

in

serum-containing

medium

Cells

-LRB-

μl

-RRB-

were

stained

either

directly

with

antibodies

-LRB-

μg

-RRB-

or

with

serially

diluted

immune

serum

followed

by

PE-conjugated

antirat

or

antirabbit

IgG

-LRB-

Dianova

-RRB-

Gating

was

performed

on

living

cells

on

the

basis

of

propidium

iodide

exclusion

Immunoprecipitation

analyses

Antibodies

were

conjugated

to

Protein

G

Sepharose

beads

-LRB-

Pharmacia

-RRB-

-LRB-

μg

μL

beads

in

μL

PBS

containing

Triton

-RRB-

by

incubation

on

a

roller

for

min

at

room

temperature

Beads

were

washed

twice

in

Triton

before

use

for

immunoprecipitation

Cells

-LRB-

-RRB-

were

lysed

in

PBS

mM

AEBSF

-LRB-

Sigma

-RRB-

for

min

at

°C

Insoluble

material

was

pelleted

by

centrifugation

and

the

cleared

lysates

-LRB-

cells

in

μL

-RRB-

were

subjected

to

immunoprecipitation

using

μL

antibody-conjugated

beads

by

incubation

on

a

roller

for

min

at

room

temperature

Beads

were

washed

four

times

in

Triton

Proteins

were

eluted

from

the

beads

by

incubation

in

SDS-PAGE

sample

buffer

for

min

at

°C

Eluted

proteins

were

size-fractionated

on

precast

SDS-PAGE

gels

-LRB-

Invitrogen

-RRB-

and

blotted

onto

PVDF

membranes

Purinoceptors

and

ecto-enzymes

were

detected

with

serially

diluted

serum

followed

by

peroxidase

-LRB-

PO

-RRB-

conjugated

antirabbit

IgG

-LRB-

-RRB-

using

the

ECL

system

-LRB-

Amersham

-RRB-

Immunoblot

analyses

Cells

-LRB-

-RRB-

were

pelleted

by

centrifugation

and

lysed

by

resuspension

in

SDS-PAGE

sample

buffer

-LRB-

Invitrogen

-RRB-

DNA

was

sheared

by

brief

ultrasonication

on

ice

-LRB-

two

pulses

with

an

MSE

ultrasonicator

at

maximum

power

setting

-RRB-

Insoluble

material

was

pelleted

by

centrifugation

and

soluble

proteins

-LRB-

cell

equivalents/lane

-RRB-

were

size-fractionated

on

precast

SDS-PAGE

gels

-LRB-

Invitrogen

-RRB-

under

reducing

conditions

and

blotted

onto

PVDF

membranes

Purinoceptors

and

ecto-enzymes

were

detected

with

serially

diluted

serum

followed

by

PO-conjugated

antirabbit

IgG

-LRB-

-RRB-

using

the

ECL

system

-LRB-

Amersham

-RRB-

Results

and

discussion

Use

of

transiently

transfected

CHO

cells

to

assess

the

specificity

and

titres

of

antisera

against

purinoceptors

and

ecto-enzymes

Antibodies

raised

by

genetic

immunization

of

rabbits

and

rats

with

cDNA

expression

constructs

for

purinoceptors

ENTPDs

and

ENPPs

were

assayed

for

reactivity

and

specificity

by

indirect

immunofluorescence

staining

of

CHO

cells

h

after

cotransfection

with

a

construct

encoding

nuclear

GFP

and

the

cDNA

expression

construct

used

for

immunization

-LRB-

Fig.

and

Table

-RRB-

By

this

time

transfected

cells

were

clearly

distinguishable

from

untransfected

cells

by

virtue

of

their

green

fluorescent

nuclei

-LRB-

Fig.

-RRB-

Specific

antibodies

were

detected

on

the

basis

of

bright

cell

surface

staining

of

transfected

cells

and

lack

of

reactivity

with

untransfected

cells

Antipeptide

antisera

against

the

same

antigens

in

contrast

did

not

show

any

detectable

reactivity

in

this

assay

-LRB-

Table

-RRB-

Fig.

analyses

of

CHO

cells

following

transient

cotransfection

with

expression

constructs

for

nuclear

green

fluorescent

protein

-LRB-

GFP

-RRB-

and

purinoceptors

-LRB-

a

-RRB-

ENTPDs

-LRB-

b

-RRB-

and

ENPPs

-LRB-

c

-RRB-

CHO

cells

were

cotransfected

with

nuclear

GFP

and

expression

constructs

for

the

indicated

purinoceptors

or

ecto-enzymes

Twenty

hours

after

transfection

cells

were

stained

with

serial

dilutions

of

the

indicated

antisera

followed

by

PE-conjugated

antirabbit

IgG

To

detect

non-membrane-bound

intracellular

staining

was

performed

by

fixing

and

permeabilizing

cells

Cell

nuclei

were

counterstained

with

Hoechst

Cells

were

analyzed

with

a

Zeiss

Axiovert

microscope

equipped

with

filters

for

visualizing

Hoechst

dye

GFP

and

PE

fluorochrome

Panels

show

merged

images

for

Hoechst

dye

and

GFP

-LRB-

b

c

left

panels

-RRB-

or

for

GFP

and

PE

-LRB-

a

c

right

panels

-RRB-

Table

specificities

and

applications

of

antibodies

against

members

of

the

families

of

purinoceptors

ENTPDs

and

ENPPsAntibodyHostTitreSpecificityFACS/IFTWestern

-LRB-

-RRB-

ENTPD

ENPP

nd

Monitoring

cell

surface

expression

of

purinoceptors

and

ecto-enzymes

by

flow

cytometry

The

utility

of

the

ADAPINC

antisera

for

monitoring

cell

surface

expression

was

assessed

further

by

flow

cytometry

-LRB-

FACS

analyses

-RRB-

of

transiently

transfected

HEK

cells

-LRB-

Fig.

-RRB-

The

results

confirm

that

the

antisera

react

specifically

with

appropriately

transfected

cells

and

that

they

do

not

react

with

untransfected

HEK

cells

Importantly

the

antisera

recognize

the

respective

purinoceptor/ecto-enzyme

on

the

cell

surface

of

living

cells

In

contrast

antipeptide

antisera

against

the

same

antigens

did

not

show

any

detectable

reactivity

with

the

transfected

cells

in

these

assays

Further

the

use

of

the

ADAPINC

antisera

for

FACS

analyses

permits

an

assessment

of

the

relative

expression

level

of

the

cognate

protein

on

the

cell

surface

of

individual

cells

as

well

as

an

assessment

of

the

number

of

transfected

vs.

untransfected

cells

in

the

cell

population

These

parameters

can

not

be

quantified

appropriately

by

Western-blot

analyses

since

for

these

analyses

lysates

are

prepared

from

cell

populations

We

further

used

the

FACS-based

assay

system

to

test

whether

the

antisera

crossreact

with

other

members

-LRB-

paralogues

-RRB-

of

the

receptor/enzyme

family

and/or

the

same

receptor/enzyme

-LRB-

orthologue

-RRB-

in

other

species

-LRB-

human

rat

or

mouse

-RRB-

The

results

reveal

that

most

antisera

are

specific

for

the

receptor/enzyme

used

for

immunization

and

do

not

cross-react

with

other

members

of

the

same

protein

family

Some

antisera

however

did

react

with

the

orthologous

protein

in

particular

at

low

dilutions

e.g.

antiserum

raised

against

mouse

shows

some

reactivity

with

the

human

orthologue

-LRB-

Fig.

-RRB-

Fig.

analyses

of

purinoceptor

-LRB-

a

-RRB-

and

ecto-enzyme

-LRB-

b

-RRB-

expression

by

transfected

HEK

cells

HEK

cells

were

transfected

with

expression

constructs

for

the

indicated

purinoceptors

and

ecto-enzymes

Then

h

post-transfection

cells

were

harvested

by

brief

trypsinization

and

single

cell

suspensions

were

stained

with

serial

dilutions

of

the

indicated

antisera

followed

by

PE-conjugated

antirabbit

IgG

before

FACS

analyses

Control

stainings

were

performed

with

untransfected

cells

-LRB-

shaded

histograms

in

a

dotted

lines

in

b

-RRB-

and

with

cells

transfected

with

the

species

orthologue

-LRB-

red

lines

in

a

-RRB-

Some

of

the

antisera

recognize

the

denatured

cognate

antigen

in

Western-blot

analyses

We

next

tested

the

utility

of

the

ADAPINC

antisera

for

monitoring

overall

protein

expression

levels

in

immunoblot

analyses

-LRB-

Fig.

-RRB-

To

this

end

we

size-fractionated

the

proteins

from

lysates

of

untransfected

and

of

transiently

transfected

HEK

cells

by

SDS-PAGE

followed

by

immunoblot

analyses

The

results

show

that

only

some

of

the

ADAPINC

antisera

detected

bands

of

the

expected

size

For

comparison

we

again

performed

parallel

analyses

with

antisera

raised

against

the

same

proteins

by

peptide

immunization

The

results

show

that

the

antipeptide

sera

generally

detected

bands

of

the

expected

size

The

C-terminal

peptide

is

commonly

used

for

peptide

immunization

In

the

case

of

this

sequence

is

highly

conserved

between

rat

mouse

and

human

orthologues

and

the

corresponding

antipeptide

serum

recognized

the

respective

orthologues

-LRB-

not

shown

-RRB-

In

contrast

in

the

case

of

the

mouse

and

human

sequences

differ

in

two

and

five

positions

respectively

from

the

rat

peptide

used

for

immunization

and

this

antipeptide

serum

shows

only

weak

cross-reactivity

with

these

orthologues

-LRB-

Fig.

Abc

-RRB-

Fig.

analyses

of

purinoceptor

-LRB-

a

-RRB-

and

ecto-enzyme

-LRB-

b

-RRB-

expression

by

transfected

HEK

cells

Untransfected

-LRB-

u

-RRB-

or

HEK

cells

transfected

with

mouse

-LRB-

m

-RRB-

or

human

-LRB-

h

-RRB-

purinoceptors

or

ecto-enzymes

were

solubilized

h

post-transfection

with

SDS-PAGE

sample

buffer

Proteins

in

cell

lysates

were

size-fractionated

by

SDS-PAGE

and

subjected

to

immunoblot

analyses

using

the

indicated

antisera

Bound

antibodies

were

detected

with

peroxidase-conjugated

secondary

antibody

and

the

ECL

system

Affinity

purification

of

purinoceptors

and

ecto-enzymes

Finally

we

tested

the

utility

of

the

ADAPINC

antisera

for

immunoprecipitation

of

the

cognate

proteins

from

cell

lysates

-LRB-

Fig.

-RRB-

To

this

end

we

prepared

lysates

of

untransfected

and

of

transiently

transfected

HEK

using

the

nondenaturing

nonionic

detergent

Lysates

were

incubated

with

ADAPINC

antibodies

immobilized

on

protein-G

sepharaose

beads

and

proteins

bound

to

washed

beads

were

subsequently

analyzed

by

SDS-PAGE

followed

by

immunoblot

analyses

with

antipeptide

antibodies

The

results

show

that

all

of

the

ADAPINC

antisera

efficiently

precipitated

proteins

of

the

expected

size

from

cell

lysates

This

underscores

the

utility

of

these

antibodies

for

purifying

native

receptors

and

ecto-enzymes

from

lysed

cells

Fig.

of

purinoceptors

by

antibodies

raised

via

genetic

immunization

Untransfected

-LRB-

u

-RRB-

and

transfected

-LRB-

t

-RRB-

HEK

cells

were

solubilized

h

post-transfection

with

Triton

Purinoceptors

were

immunoprecipitated

from

cell

lysates

by

incubation

with

the

indicated

antisera

bound

to

Protein

G

Sepharose

Precipitates

were

subjected

to

immunoblot

analysis

as

in

Fig.

using

the

indicated

antipeptide

antisera

for

detection

Conclusion

Using

genetic

immunization

we

have

raised

highly

specific

polyclonal

and

monoclonal

antibodies

against

key

players

of

purinergic

signalling

i.e.

and

purinoceptors

and

and

enzymes

Our

findings

underscore

the

utility

of

genetic

immunization

for

generating

highly

specific

polyclonal

and

monoclonal

antibodies

directed

against

proteins

in

native

conformation

-LRB-

ADAPINCs

-RRB-

-LSB-

-RSB-

These

antibodies

are

valuable

tools

for

assessing

the

expression

levels

of

the

native

protein

by

immunofluorescence

analyses

and

flow

cytometry

i.e.

assays

in

which

antipeptide

antibodies

often

fail

The

antibodies

described

here

provide

useful

tools

for

further

characterization

of

the

structure

and

function

of

these

purinoceptors

and

ecto-enzymes

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

monoclonal antibodies
purinoceptor
genetic immunization