Recommended tool compounds and drugs for blocking P2X and P2Y receptors

This review article presents a collection of tool compounds that selectively block and are recommended for studying P2Y and P2X receptor subtypes, investigating their roles in physiology and validating them as future drug targets. Moreover, drug candidates and approved drugs for P2 receptors will be discussed.     

Characterization of P2X3, P2Y1 and P2Y4 receptors in cultured HEK293-hP2X3 cells and their inhibition by ethanol and trichloroethanol

Membrane currents and changes in the intracellular Ca2+ concentration ([Ca2+]i) were measured in HEK293 cells transfected with the human P2X3 receptor (HEK293-hP2X3). RT-PCR and immunocytochemistry indicated the additional presence of endogenous P2Y1 and to some extent P2Y4 receptors. P2 receptor agonists induced inward currents in HEK293-hP2X3 cells with the rank order of potency alpha,beta-meATP approximately ATP > ADP-beta-S > UTP. A comparable rise in [Ca2+]i was observed after the slow superfusion of ATP, ADP-beta-S and UTP; alpha,beta-meATP was ineffective. These data, in conjunction with results obtained by using the P2 receptor antagonists TNP-ATP, PPADS and MRS2179 indicate that the current response to alpha,beta-meATP is due to P2X3 receptor activation, while the ATP-induced rise in [Ca2+]i is evoked by P2Y1 and P2Y4 receptor activation. TCE depressed the alpha,beta-meATP current in a manner compatible with a non-competitive antagonism. The ATP-induced increase of [Ca2+]i was much less sensitive to the inhibitory effect of TCE than the current response to alpha,beta-meATP. The present study indicates that in HEK293-hP2X3 cells, TCE, but not ethanol, potently inhibits ligand-gated P2X3 receptors and, in addition, moderately interferes with G protein-coupled P2Y1 and P2Y4 receptors. Such an effect may be relevant for the interruption of pain transmission in dorsal root ganglion neurons following ingestion of chloral hydrate or trichloroethylene.     

Neuroprotective roles of the P2Y2 receptor

Purinergic signaling plays a unique role in the brain by integrating neuronal and glial cellular circuits. The metabotropic P1 adenosine receptors and P2Y nucleotide receptors and ionotropic P2X receptors control numerous physiological functions of neuronal and glial cells and have been implicated in a wide variety of neuropathologies. Emerging research suggests that purinergic receptor interactions between cells of the central nervous system (CNS) have relevance in the prevention and attenuation of neurodegenerative diseases resulting from chronic inflammation. CNS responses to chronic inflammation are largely dependent on interactions between different cell types (i.e., neurons and glia) and activation of signaling molecules including P2X and P2Y receptors. Whereas numerous P2 receptors contribute to functions of the CNS, the P2Y(2) receptor is believed to play an important role in neuroprotection under inflammatory conditions. While acute inflammation is necessary for tissue repair due to injury, chronic inflammation contributes to neurodegeneration in Alzheimer's disease and occurs when glial cells undergo prolonged activation resulting in extended release of proinflammatory cytokines and nucleotides. This review describes cell-specific and tissue-integrated functions of P2 receptors in the CNS with an emphasis on P2Y(2) receptor signaling pathways in neurons, glia, and endothelium and their role in neuroprotection.     

P2Y12 receptor gene polymorphisms are associated with epilepsy

The basic research indicated that microglial P2Y12 receptors (P2Y12Rs) are involved in the pathophysiology of epilepsy through regulated microglial-neuronal interactions, aberrant neurogenesis, or immature neuronal projections. However, whether the clinic case of epilepsy would be associated with P2Y12 receptor gene polymorphisms is presented with few data. In our study, a total of 176 patients with epilepsy and 50 healthy controls were enrolled. Two single-nucleotide polymorphisms, namely rs1491974 and rs6798347, were selected for analysis. The results revealed that carriers of the G allele of rs1491974 G>A or rs6798347 G>A may be associated with an increased risk of epilepsy (OR = 0.576, 95% CI = 0.368–0.901, p = 0.015; OR = 0.603, 95% CI = 0.367–0.988, p = 0.043). Interestingly, we found that the rs1491974 G>A genotype and allele frequencies have only a significant difference in female instead of male case (p = 0.004 for genotype; p = 0.001 for allele). The subgroup analysis demonstrated that individuals with the rs1491974 G>A genotype might have more frequent seizure (OR = 0.476, 95% CI = 0.255–0.890; p = 0.019). These data implied that both rs1491974 and rs6798347 polymorphisms of P2Y12R would be able to play import roles in epilepsy susceptibility, whereas the rs1491974 polymorphism may be specifically related to seizure frequency.     

P2Y receptor mediated inhibitory modulation of noradrenaline release in response to electrical field stimulation and ischemic conditions in superfused rat hippocampus slices

In this study, the inhibitory regulation of the release of noradrenaline (NA) by P2 receptors was investigated in hippocampus slices pre-incubated with [³H]NA. Electrical field stimulation (EFS; 2 Hz, 240 shocks, and 1 ms) released NA in an outside [Ca²⁺]-dependent manner, and agonists of P2Y receptors inhibited the EFS-evoked [³H]NA release with pharmacological profile similar to that of the P2Y₁ and P2Y₁₃ receptor subtypes. This inhibitory modulation was counteracted by bicuculline and 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline + 2-amino-5-phosphonovalerate and 2-amino-4-phosphonobutyrate. In contrast, the excess release in response to 30 min combined oxygen and glucose deprivation was outside [Ca²⁺] independent, but still sensitive to the inhibition of both facilitatory P2X₁ and inhibitory P2Y₁ receptors. Whereas mRNA encoding P2Y₁₂ and P2Y₁₃ receptor subunits were expressed in the brainstem, P2Y₁ receptor immunoreactivity was localized to neuronal somata and dendrites innervated by the mossy fiber terminals in the CA3 region of the hippocampus, as well as somata of granule cells and interneurons in the dentate gyrus. In summary, in addition to the known facilitatory modulation via P2X receptors, EFS-evoked [³H]NA outflow in the hippocampus is subject to inhibitory modulation by P2Y₁/P2Y₁₃ receptors. Furthermore, endogenous activation of both facilitatory and inhibitory P2 receptors may participate in the modulation of pathological NA release under ischemic-like conditions.     

Selective induction of P2Y14 receptor by RANKL promotes osteoclast formation

The purinergic receptor P2Y, G protein coupled, 14 (P2Y14) receptor for UDP-glucose and other UDP-sugars has been implicated in the regulation of the stem cell compartment as well as neuroimmune function. However, the role of P2Y14 in osteoclast formation is completely unknown. We found that RANKL selectively induced P2Y14 among seven mammalian P2Y receptors when analysed at both the mRNA and protein level, but inhibitors of the mitogenactivated protein (MAP) kinase pathway suppressed induction of P2Y14 proteins. Extracellular addition of UDP-sugars such as UDP-glucose, UDP-galactose, UDP-glucuronic acid, and UDP-N-acetyl glucosamine promoted RANKL-induced osteoclastogenesis, while P2Y14 downregulation by RNA interference inhibited osteoclast formation. Taken together, these results suggest that P2Y14 may act as the receptor for UDP-sugars in osteoclast precusors and may regulate RANKL-induced osteoclastogenesis.     

P2Y receptors activate neuroprotective mechanisms in astrocytic cells

Mechanical or ischemic trauma to the CNS causes the release of nucleotides and other neurotransmitters into the extracellular space. Nucleotides can activate nucleotide receptors that modulate the expression of genes implicated in cellular adaptive responses. In this investigation, we used human 1321N1 astrocytoma cells expressing a recombinant P2Y2 receptor to assess the role of this receptor in the regulation of anti-apoptotic (bcl-2 and bcl-xl) and pro-apoptotic (bax) gene expression. Acute treatment with the P2Y2 receptor agonist UTP up-regulated bcl-2 and bcl-xl, and down-regulated bax, gene expression. Activation of P2Y2 receptors was also coupled to the phosphorylation of cyclic AMP responsive element binding protein that positively regulates bcl-2 and bcl-xl gene expression. Cyclic AMP responsive element decoy oligonucleotides markedly attenuated the UTP-induced increase in bcl-2 and bcl-xl mRNA levels. Activation of P2Y2 receptors induced the phosphorylation of the pro-apoptotic factor Bad and caused a reduction in bax/bcl-2 mRNA expression ratio. All these signaling pathways are known to be involved in cell survival mechanisms. Using cDNA microarray analysis and RT-PCR, P2Y2 receptors were found to up-regulate the expression of genes for neurotrophins, neuropeptides and growth factors including nerve growth factor 2; neurotrophin 3; glia-derived neurite-promoting factor, as well as extracellular matrix proteins CD44 and fibronectin precursor--genes known to regulate neuroprotection. Consistent with this observation, conditioned media from UTP-treated 1321N1 cells expressing P2Y2 receptors stimulated the outgrowth of neurites in PC-12 cells. Taken together, our results suggest an important novel role for the P2Y2 receptor in survival and neuroprotective mechanisms under pathological conditions.     

P2Y1嘌呤受体在心血管系统的研究进展

P2Y1嘌呤受体是最早被发现并克隆的P2Y受体家族成员,属G蛋白偶联受体,其内源性激动剂主要是ADP及ATP。随着研究深入,人们发现该受体在多个方面有着重要作用,参与机体多种生理病理过程。本文对该受体近年来在心血管系统方面的研究进行综述,提示P2Y1受体有可能成为疾病治疗的潜在靶点。     

Alanine‐(87)‐threonine polymorphism impairs signaling and internalization of the human P2Y11 receptor,when co‐expressed with the P2Y1 receptor

The P2Y₁₁ nucleotide receptor detects high extracellular ATP concentrations. Mutations of the human P2RY₁₁ gene can play a role in brain autoimmune responses, and the P2Y₁₁ receptor alanine‐87‐threonine (A87T) polymorphism has been suggested to affect immune‐system functions. We investigated receptor functionality of the P2Y₁₁A87T mutant using HEK293 and 1321N1 astrocytoma cells. In HEK293 cells, the P2Y₁₁ receptor agonist 3′‐O‐(4‐benzoylbenzoyl)adenosine 5′‐triphosphate (BzATP) was completely inactive in evoking intracellular calcium release while the potency of ATP was reduced. ATP was also less potent in triggering cAMP generation. However, 1321N1 astrocytoma cells, which lack any endogenous P2Y₁ receptors, did not display a reduction. Only when 1321N1 cells were co‐transfected with P2Y₁₁A87T and P2Y₁ receptors, the calcium responses to the P2Y₁₁ receptor‐specific agonist BzATP were reduced. It is already known that P2Y₁ and P2Y₁₁ receptors interact. We thus conclude that the physiological impact of A87T mutation of the P2Y₁₁ receptor derives from detrimental effects on P2Y₁–P2Y₁₁ receptor interaction. We additionally investigated alanine‐87‐serine and alanine‐87‐tyrosine P2Y₁₁ receptor mutants. Both mutations rescue the response to BzATP in HEK293 cells, thus ruling out polarity of amino acid‐87 to be the molecular basis for altered receptor characteristics. We further found that the P2Y₁₁A87T receptor shows complete loss of nucleotide‐induced internalization in HEK293 cells. Thus, we demonstrate impaired signaling of the P2Y₁₁ A87T‐mutated receptors when co‐operating with P2Y₁ receptors.

     

Current knowledge on the nucleotide agonists for the P2Y2 receptor

P2Y receptors are G-protein-coupled receptors (GPCRs) for extracellular nucleotides. There are eight mammalian P2Y receptor subtypes (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14). P2Y2 receptors are widely expressed and play important roles in multiple functionalities. Diquafosol tetrasodium, known as INS365, which was the first P2Y2 receptor agonists that had been approved in April 2010 and launched in Japan by Santen Pharmaceuticals. Besides, a series of similar agonists for the P2Y2 receptor are undergoing development to cure different diseases related to the P2Y2 receptor. This article illustrated the structure and functions of the P2Y2 receptor and focused on several kinds of agonists about their molecular structures, research progress and chemical synthesis methods. Last but not the least, we summarized the structures-activity relationship (SAR) of agonists for the P2Y2 receptor and expected more efficient agonists for the P2Y2 receptor.     

Involvement of purinergic P2Y1R in antidepressant-like effects of electroacupuncture treatment on social isolation stress mice

Depression is a common neuropsychiatric disorder with high incidence and disability. Electroacupuncture (EA) is effective in the treatment of depression. However, the underlying mechanisms are not fully understood. Social isolation stress during post-weaning period can impair purinergic signaling in the brain of rodents and has emerged as a major risk factor for depression. The purpose of this study was to investigate the involvement of P2Y1 receptor (P2Y1R) in the antidepressant-like effects of EA. In this study, C57BL/6 mice were randomly assigned to group-housed (GH) or social isolated (SI) groups at post-natal day 21. After 6 weeks of social isolation, EA was performed on acupoints “Bai-hui” (GV20) and “Yin-tang” (GV29), or non-acupoints for 4 weeks. The SI mice received either intracerebroventricular injection of a selective P2Y1R agonist, MRS2365 (1 nmol); or a selective P2Y1R antagonist, MRS2179 (2 μmol), before and after EA. We found that SI mice exhibited depression-like behaviors accompanied with anxiety-like behaviors. The expressions of P2Y1R were well co-localized with GFAP-positive astrocytes and increased in the prefrontal cortex and hippocampus of SI mice. After treated with MRS2179, the depression-like behaviors of SI mice were attenuated, but not with MRS2365. Meanwhile, we found that EA could attenuate social isolation caused depression- and anxiety-like behaviors, and inhibited the up-regulation of P2Y1R in the prefrontal cortex and hippocampus of SI mice. Notably, the positive effects of EA on depression-like behaviors of SI mice could be reversed by MRS2365, while MRS2365 had no effect on the anxiolytic-like effects of EA. Therefore, we provide new evidence that EA could ameliorate depression- and anxiety-like behaviors in social isolation stress mice, and P2Y1R was involved in the antidepressant-like effects of EA.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11302-021-09827-1.     

Effects of the allosteric modulator SCH-202676 on adenosine and P2Y receptors

The G protein-coupled receptor allosteric modulator SCH-202676 (N-(2,3-diphenyl-1,2,4-thiadiazol-5-(2H)-ylidene)methanamine), which affects a wide range of structurally unrelated G protein-coupled receptors, has highly divergent effects on purine receptors. SCH-202676 inhibited radioligand binding to human adenosine A(1), A(2A), and A(3) receptors (IC(50) = 0.5-0.8 microM) and affected dissociation kinetics, but at the human P2Y(1) nucleotide receptor it had no effect. SCH-202676 (10 microM) selectively accelerated agonist dissociation at adenosine A(3) receptors and either slowed (adenosine A(1) receptors) or accelerated (adenosine A(2A) receptors) antagonist dissociation. Thus, SCH-202676 differentially modulated A(1), A(2A), and A(3) receptors as well as agonist- and antagonist-occupied receptors.     

神经肽Y及其受体Y1、Y2对痛觉调制的研究进展

神经肽Y（neuropeptide Y,NPY）是一种由36个氨基酸残基组成的肽类激素,属胰多肽家族,广泛分布于中枢及外周神经组织的神经元中。NPY主要参与摄食行为、心血管活动、垂体分泌等生理功能的调节。NPY还参与了痛觉调制。NPY受体有Y1、Y2、Y3、Y4、Y5和Y6六种亚型。目前对Y1受体和Y2受体的研究较多,显示Y1受体和Y2受体参与痛觉调制。但现在对NPY在痛觉中的具体作用机制还不清楚。该文对NPY及其Y1受体、Y2受体在痛觉调制中的作用作一概述。     

P2Y receptors and pain transmission

It is widely accepted that the most important ATP receptors involved in pain transmission belong to the P2X₃ and P2X_2/3 subtypes, selectively expressed in small diameter dorsal root ganglion (DRG) neurons. However, several types of the metabotropic ATP (P2Y) receptors have also been found in primary afferent neurons; P2Y₁ and P2Y₂ receptors are typically expressed in small, nociceptive cells. Here we review the results available on the involvement of P2Y receptors in the modulation of pain transmission.     

The P2Y6 receptor signals through Gαq/Ca2+/PKCα and Gα13/ROCK pathways to drive the formation of membrane protrusions and dictate cell migration

Cell migration is a ubiquitous process necessary to maintain and restore tissue functions. However, in cancer, cell migration leads to metastasis development and thus worsens the prognosis. Although the mechanism of cell migration is well understood, the identification of new targets modulating cell migration and deciphering their signaling events could lead to new therapies to restore tissue functions in diseases, such as inflammatory bowel disease, or to block metastatic development in different forms of cancer. Previous research has identified the G-protein-coupled P2Y₆ receptor as an innovative target that could dictate cell migration under normal and pathological conditions. Surprisingly, there is little information on the cellular events triggered by activated P2Y₆ during cell migration. Here, we demonstrated that P2Y₆ activation stimulated A549 human lung cancer cells and Caco-2 colorectal cancer cell migration. Activated P2Y₆ increased the number of filopodia and focal adhesions; two migratory structures required for cell migration. The generation of these structures involved Gα_q/calcium/protein kinases C (PKC) and Gα₁₃/RHO-associated protein kinase-dependent pathways that dictate the formation of the migratory structures. These pathways led to the stabilization of the actin cytoskeleton through a PKC-dependent phosphorylation of cofilin. These results support the idea that the P2Y₆ receptor represents a target of interest to modulate cell migration and revealed an intricate dialogue between two Gα-protein signaling pathways.     

Burnstock and the legacy of the inhibitory junction potential and P2Y1 receptors

The synaptic event called the inhibitory junction potential (IJP) was arguably one of the more important discoveries made by Burnstock and arguably one of his finer legacies. The discovery of the IJP fundamentally changed how electromechanical coupling was visualised in gastrointestinal smooth muscle. Its discovery also set in motion the search for novel inhibitory neurotransmitters in the enteric nervous system, eventually leading to proposal that ATP or a related nucleotide was a major inhibitory transmitter. The subsequent development of purinergic signalling gave impetus to expanding the classification of surface receptors for extracellular ATP, not only in the GI tract but beyond, and then led to successive phases of medicinal chemistry as the P2 receptor field developed. Ultimately, the discovery of the IJP led to the successful cloning of the first P2Y receptor (chick P2Y1) and expansion of mammalian ATP receptors into two classes: metabotropic P2Y receptors (encompassing P2Y1, P2Y2, P2Y4, P2Y6, P2Y11–14 receptors) and ionotropic P2X receptors (encompassing homomeric P2X1–P2X7 receptors). Here, the causal relationship between the IJP and P2Y1 is explored, setting out the milestones reached and achievements made by Burnstock and his colleagues.

     

P2Y1R and P2Y2R: potential molecular triggers in muscle regeneration

Muscle regeneration is indispensable for skeletal muscle health and daily life when injury, muscular disease, and aging occur. Among the muscle regeneration, muscle stem cells’ (MuSCs) activation, proliferation, and differentiation play a key role in muscle regeneration. Purines bind to its specific receptors during muscle development, which transmit environmental stimuli and play a crucial role of modulator of muscle regeneration. Evidences proved P2R expression during development and regeneration of skeletal muscle, both in human and mouse. In contrast to P2XR, which have been extensively investigated in skeletal muscles, the knowledge of P2YR in this tissue is less comprehensive. This review summarized muscle regeneration via P2Y1R and P2Y2R and speculated that P2Y1R and P2Y2R might be potential molecular triggers for MuSCs’ activation and proliferation via the p-ERK1/2 and PLC pathways, explored their cascade effects on skeletal muscle, and proposed P2Y1/2 receptors as potential pharmacological targets in muscle regeneration, to advance the purinergic signaling within muscle and provide promising strategies for alleviating muscular disease.     

P2Y6 receptor-dependent microglial phagocytosis of synapses mediates synaptic and memory loss in aging

Aging causes loss of brain synapses and memory, and microglial phagocytosis of synapses may contribute to this loss. Stressed neurons can release the nucleotide UTP, which is rapidly converted into UDP, that in turn activates the P2Y₆ receptor (P2Y₆R) on the surface of microglia, inducing microglial phagocytosis of neurons. However, whether the activation of P2Y₆R affects microglial phagocytosis of synapses is unknown. We show here that inactivation of P2Y₆R decreases microglial phagocytosis of isolated synapses (synaptosomes) and synaptic loss in neuronal–glial co-cultures. In vivo, wild-type mice aged from 4 to 17 months exhibited reduced synaptic density in cortical and hippocampal regions, which correlated with increased internalization of synaptic material within microglia. However, this aging-induced synaptic loss and internalization were absent in P2Y₆R knockout mice, and these mice also lacked any aging-induced memory loss. Thus, P2Y₆R appears to mediate aging-induced loss of synapses and memory by increasing microglial phagocytosis of synapses. Consequently, blocking P2Y₆R has the potential to prevent age-associated memory impairment.     

Purinergic signaling: a potential therapeutic target for depression and chronic pain

The comorbid mechanism of depression and chronic pain has been a research hotspot in recent years. Until now, the role of purinergic signals in the comorbid mechanism of depression and chronic pain has not been fully understood. This review mainly summarizes the research results published in PubMed during the past 5 years and concludes that purinergic signaling is a potential therapeutic target for comorbid depression and chronic pain, and the purinergic receptors A1, A2A, P2X3, P2X4, and P2X7and P2Y₆, P2Y₁, and P2Y₁₂ may be important factors. The main potential pathways are as follows: A1 receptor-related G protein-dependent activation of introverted K⁺ channels (GIRKs), A2A receptor-related effects on the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) and MAPK/nuclear factor-κB (NF-κB) pathways, P2X3 receptor-related effects on dorsal root ganglia (DRG) excitability, P2X4 receptor-related effects on proinflammatory cytokines and inflammasome activation, P2X7 receptor-related effects on ion channels, the NLRP3 inflammasome and brain-derived neurotrophic factor (BDNF), and P2Y receptor-related effects on the phospholipase C (PLC)/inositol triphosphate (IP3)/Ca²⁺ signaling pathway. We hope that the conclusions of this review will provide key ideas for future research on the role of purinergic signaling in the comorbid mechanism of depression and chronic pain.