Maria P. Abbracchio

Purinoceptors: Are there families of P2X and P2Y purinoceptors?

There has been an exponential growth in interest in purinoceptors since the potent effects of purines were first reported in 1929 and purinoceptors defined in 1978. A distinction between P1 (adenosine) and P2 (ATP/ADP) purinoceptors was recognized at that time and later, A1 and A2, as well as P2x and P2y subclasses of P1 and P2 purinoceptors were also defined. However, in recent years, many new subclasses have been claimed, particularly for the receptors to nucleotides, including P2t, P2z, P2u(n) and P2D, and there is some confusion now about how to incorporate additional discoveries concerning the responses of different tissues to purines. The studies beginning to appear defining the molecular structure of P2-purinoceptor subtypes are clearly going to be important in resolving this problem, as well as the introduction of new compounds that can discriminate pharmacologically between subtypes. Thus, in this review, on the basis of this new data and after a detailed analysis of the literature, we propose that: (1) P2X(ligand-gated) and P2Y(G-protein-coupled) purinoceptor families are established; (2) four subclasses of P2X-purinoceptor can be identified (P2X1-P2X4) to date; (3) the variously named P2-purinoceptors that are G-protein-coupled should be incorporated into numbered subclasses of the P2Y family. Thus: P2Y1 represents the recently cloned P2Y receptor (clone 803) from chick brain; P2Y2 represents the recently cloned P2u (or P2n) receptor from neuroblastoma, human epithelial and rat heart cells; P2Y3 represents the recently cloned P2Y receptor (clone 103) from chick brain that resembles the former P2t receptor; P2Y4-P2Y6 represent subclasses based on agonist potencies of newly synthesised analogues; P2Y7 represents the former P2D receptor for dinucleotides. This new framework for P2 purinoceptors would be fully consistent with what is emerging for the receptors to other major transmitters, such as acetylcholine, gamma-aminobutyric acid, glutamate and serotonin, where two main receptor families have been recognised, one mediating fast receptor responses directly linked to an ion channel, the other mediating slower responses through G-proteins. We fully expect discussion on the numbering of the different receptor subtypes within the P2X and P2Y families, but believe that this new way of defining receptors for nucleotides, based on agonist potency order, transduction mechanisms and molecular structure, will give a more ordered and logical approach to accommodating new findings. Moreover, based on the extensive literature analysis that led to this proposal, we suggest that the development of selective antagonists for the different P2-purinoceptor subtypes is now highly desirable, particularly for therapeutic purposes.

Purinergic signalling in the nervous system: an overview

Purinergic receptors, represented by several families, are arguably the most abundant receptors in living organisms and appeared early in evolution. After slow acceptance, purinergic signalling in both peripheral and central nervous systems is a rapidly expanding field. Here, we emphasize purinergic co-transmission, mechanisms of release and breakdown of ATP, ion channel and G-protein-coupled-receptor subtypes for purines and pyrimidines, the role of purines and pyrimidines in neuron-glial communication and interactions of this system with other transmitter systems. We also highlight recent data involving purinergic signalling in pathological conditions, including pain, trauma, ischaemia, epilepsy, migraine, psychiatric disorders and drug addiction, which we expect will lead to the development of therapeutic strategies for these disorders with novel mechanisms of action.

Trophic actions of extracellular nucleotides and nucleosides on glial and neuronal cells

In addition to their well-established roles as neurotransmitters and neuromodulators, growing evidence suggests that nucleotides and nucleosides might also act as trophic factors in both the central and peripheral nervous systems. Specific extracellular receptor subtypes for these compounds are expressed on neurons, glial and endothelial cells, where they mediate strikingly different effects. These range from induction of cell differentiation and apoptosis, mitogenesis and morphogenetic changes, to stimulation of synthesis or release, or both, of cytokines and neurotrophic factors, both under physiological and pathological conditions. Nucleotides and nucleosides might be involved in the regulation of development and plasticity of the nervous system, and in the pathophysiology of neurodegenerative disorders. Receptors for nucleotides and nucleosides could represent a novel target for the development of therapeutic strategies to treat incurable diseases of the nervous system, including trauma- and ischemia-associated neurodegeneration, demyelinating and aging-associated cognitive disorders.

Characterization of the UDP-glucose receptor (re-named here the P2Y14 receptor) adds diversity to the P2Y receptor family

The cloning of a human G-protein-coupled receptor (GPCR) that specifically responds to UDP-glucose and related sugar-nucleotides has been reported recently. This receptor has important structural similarities to known members of the P2Y receptor family but also shows a distinctly different pharmacological response profile. Here, the IUPHAR Subcommittee for P2Y receptor nomenclature and classification review the current knowledge of this receptor and present their reasons for including this receptor in the P2Y receptor family as the P2Y(14) receptor.

Towards a revised nomenclature for P1 and P2 receptors

The classification of receptors for adenosine, ATP and ADP (collectively called purinoceptors) has seen a number of developments in the past three years. The important division of receptors into two major classes1 (1) adenosine (P1) receptors and (2) P2 purinoceptors, first suggested by Burnstock in 1978 (Ref.2), has been an abiding one that has set the stage for further subdivision of P2 purinoceptors into P2X and P2Y subtypes on the basis of pharmacological properties3. Later, Dubyak4 summarized the evidence that ATP worked through two different transduction mechanisms: intrinsic ion channels and G protein-coupled receptors. This information, coupled with the cloning of purinoceptors in 1993/94, led Abbracchio and Burnstock5 to propose that purinoceptors should be classified in two families: G protein-coupled receptors termed P2Y purinoceptors, and intrinsic ion channels termed P2X purinoceptors. Developments in recent years have borne out these expectations and a revised nomenclature, essentially adopting the Abbracchio and Burnstock proposal, can now be proposed.

The orphan receptor GPR17 identified as a new dual uracil nucleotides/cysteinyl-leukotrienes receptor

Nucleotides and cysteinyl‐leukotrienes (CysLTs) are unrelated signaling molecules inducing multiple effects through separate G‐protein‐coupled receptors: the P2Y and the CysLT receptors. Here we show that GPR17, a Gi‐coupled orphan receptor at intermediate phylogenetic position between P2Y and CysLT receptors, is specifically activated by both families of endogenous ligands, leading to both adenylyl cyclase inhibition and intracellular calcium increases. Agonist‐response profile, as determined by [35S]GTPγS binding, was different from that of already known CysLT and P2Y receptors, with EC50 values in the nanomolar and micromolar range, for CysLTs and uracil nucleotides, respectively. Both rat and human receptors are highly expressed in the organs typically undergoing ischemic damage, that is, brain, heart and kidney. In vivo inhibition of GPR17 by either CysLT/P2Y receptor antagonists or antisense technology dramatically reduced ischemic damage in a rat focal ischemia model, suggesting GPR17 as the common molecular target mediating brain damage by nucleotides and CysLTs. In conclusion, the deorphanization of GPR17 revealed a dualistic receptor for two endogenous unrelated ligand families. These findings may lead to dualistic drugs of previously unexplored therapeutic potential.

Purinergic signalling: From normal behaviour to pathological brain function

Purinergic neurotransmission, involving release of ATP as an efferent neurotransmitter was first proposed in 1972. Later, ATP was recognised as a cotransmitter in peripheral nerves and more recently as a cotransmitter with glutamate, noradrenaline, GABA, acetylcholine and dopamine in the CNS. Both ATP, together with some of its enzymatic breakdown products (ADP and adenosine) and uracil nucleotides are now recognised to act via P2X ion channels and P1 and P2Y G protein-coupled receptors, which are widely expressed in the brain. They mediate both fast signalling in neurotransmission and neuromodulation and long-term (trophic) signalling in cell proliferation, differentiation and death. Purinergic signalling is prominent in neurone-glial cell interactions. In this review we discuss first the evidence implicating purinergic signalling in normal behaviour, including learning and memory, sleep and arousal, locomotor activity and exploration, feeding behaviour and mood and motivation. Then we turn to the involvement of P1 and P2 receptors in pathological brain function; firstly in trauma, ischemia and stroke, then in neurodegenerative diseases, including Alzheimers, Parkinsons and Huntingtons, as well as multiple sclerosis and amyotrophic lateral sclerosis. Finally, the role of purinergic signalling in neuropsychiatric diseases (including schizophrenia), epilepsy, migraine, cognitive impairment and neuropathic pain will be considered.

Nucleotide-mediated calcium signaling in rat cortical astrocytes: Role of P2X and P2Y receptors

ATP is the dominant messenger for astrocyte‐to‐astrocyte calcium‐mediated communication. Definition of the exact ATP/P2 receptors in astrocytes and of their coupling to intracellular calcium ([Ca2+]i) has important implications for brain physiology and pathology. We show that, with the only exception of the P2X6 receptor, primary rat cortical astrocytes express all cloned ligand‐gated P2X (i.e., P2X1–5 and P2X7) and G‐protein‐coupled P2Y receptors (i.e., P2Y1, P2Y2, P2Y4, P2Y6, and P2Y12). These cells also express the P2Y‐like UDP‐glucose receptor, which has been recently recognized as the P2Y14 receptor. Single‐cell image analysis showed that only some of these receptors are coupled to [Ca2+]i. While ATP induced rapid and transient [Ca2+]i increases (counteracted by the P2 antagonists suramin, pyridoxal‐phosphate‐6‐azophenyl‐2′‐4′‐disulfonic acid and oxidized ATP), the P2X1/P2X3 agonist αβmeATP produced no changes. Conversely, the P2X7 agonist BzATP markedly increased [Ca2+]i; the presence and function of the P2X7 receptor was also confirmed by the formation of the P2X7 pore. ADP and 2meSADP also produced [Ca2+]i increases antagonized by the P2Y1 antagonist MRS2179. Some cells also responded to UTP but not to UDP. Significant responses to sugar‐nucleotides were also detected, which represents the first functional response reported for the putative P2Y14 receptor in a native system. Based on agonist preference of known P2 receptors, we conclude that, in rat astrocytes, ATP‐induced calcium rises are at least mediated by P2X7 and P2Y1 receptors; additional receptors (i.e., P2X2, P2X4, P2X5, P2Y2, P2Y4, and P2Y14) may also contribute.

Modulation of astroglial cell proliferation by analogues of adenosine and ATP in primary cultures of rat striatum.

We have studied the possible purinoceptor-mediated modulation of astroglial cell proliferation in neuron-glia primary cultures obtained from rat corpus striatum. Cultures were grown for three days in the presence of either 2-chloro-adenosine or alpha beta-methylene-ATP (which behave as agonists of adenosine/P1 and ATP/P2 purinoceptors, respectively), and then immunostained with an antibody to glial fibrillary acidic protein. 2-Chloro-adenosine decreased and alpha beta-methylene-ATP increased the number of astroglial cells in culture. For both derivatives, the effect was dose-dependent. The effect of alpha beta-methylene-ATP was antagonized by the trypanoside suramin, suggesting the involvement of a suramin-sensitive P2 purinoceptor, whereas the effect of 2-chloro-adenosine was not reversed by the P1 purinoceptor antagonist p-sulphonyl-phenyl-theophylline, implying the activation of a xanthine-insensitive adenosine purinoceptor subtype. In order to evaluate the extent of astrocyte proliferation in the presence of these two analogues, some cultures were incubated with bromodeoxyuridine for 24 h before fixing, and then double-immunostained for glial fibrillary acidic protein and bromodeoxyuridine. The percentage of bromodeoxyuridine positive astrocytes was significantly increased after exposure to both agents. It is therefore concluded that purines can modulate astroglial cells in opposite ways, inducing decreases or increases of cell number by activation of P1 and P2 purinoceptors, respectively. For the P2 purinoceptor-mediated effect, there was a quantitative correlation between the percentage of bromodeoxyuridine positive astrocytes and the cell number. For the P1 purinoceptor-mediated effect, no apparent correlation between these two parameters was found. This suggests the activation of independent effects, which involve other mechanisms besides the stimulation of DNA synthesis, and which eventually result in a reduction of cell number. The possible relevance of these findings to in vivo regulation of astrocyte cell function as well as in trauma- and ischaemia-associated hypergliosis is discussed.

The Recently Identified P2Y-Like Receptor GPR17 Is a Sensor of Brain Damage and a New Target for Brain Repair

Deciphering the mechanisms regulating the generation of new neurons and new oligodendrocytes, the myelinating cells of the central nervous system, is of paramount importance to address new strategies to replace endogenous damaged cells in the adult brain and foster repair in neurodegenerative diseases. Upon brain injury, the extracellular concentrations of nucleotides and cysteinyl-leukotrienes (cysLTs), two families of endogenous signaling molecules, are markedly increased at the site of damage, suggesting that they may act as “danger signals” to alert responses to tissue damage and start repair. Here we show that, in brain telencephalon, GPR17, a recently deorphanized receptor for both uracil nucleotides and cysLTs (e.g., UDP-glucose and LTD4), is normally present on neurons and on a subset of parenchymal quiescent oligodendrocyte precursor cells. We also show that induction of brain injury using an established focal ischemia model in the rodent induces profound spatiotemporal-dependent changes of GPR17. In the lesioned area, we observed an early and transient up-regulation of GPR17 in neurons expressing the cellular stress marker heat shock protein 70. Magnetic Resonance Imaging in living mice showed that the in vivo pharmacological or biotechnological knock down of GPR17 markedly prevents brain infarct evolution, suggesting GPR17 as a mediator of neuronal death at this early ischemic stage. At later times after ischemia, GPR17 immuno-labeling appeared on microglia/macrophages infiltrating the lesioned area to indicate that GPR17 may also acts as a player in the remodeling of brain circuitries by microglia. At this later stage, parenchymal GPR17+ oligodendrocyte progenitors started proliferating in the peri-injured area, suggesting initiation of remyelination. To confirm a specific role for GPR17 in oligodendrocyte differentiation, the in vitro exposure of cortical pre-oligodendrocytes to the GPR17 endogenous ligands UDP-glucose and LTD4 promoted the expression of myelin basic protein, confirming progression toward mature oligodendrocytes. Thus, GPR17 may act as a “sensor” that is activated upon brain injury on several embryonically distinct cell types, and may play a key role in both inducing neuronal death inside the ischemic core and in orchestrating the local remodeling/repair response. Specifically, we suggest GPR17 as a novel target for therapeutic manipulation to foster repair of demyelinating wounds, the types of lesions that also occur in patients with multiple sclerosis.