P2Y13 receptor deficiency favors adipose tissue lipolysis and worsens insulin resistance and fatty liver disease.

Excessive lipolysis in white adipose tissue (WAT) leads to insulin resistance (IR) and ectopic fat accumulation in insulin-sensitive tissues. However, the impact of Gi-coupled receptors in restraining adipocyte lipolysis through inhibition of cAMP production remained poorly elucidated. Given that the Gi-coupled P2Y13 receptor (P2Y13-R) is a purinergic receptor expressed in WAT, we investigated its role in adipocyte lipolysis and its effect on IR and metabolic dysfunction-associated steatotic liver disease (MASLD). In humans, mRNA expression of P2Y13-R in WAT was negatively correlated to adipocyte lipolysis. In mice, adipocytes lacking P2Y13-R displayed higher intracellular cAMP levels, indicating impaired Gi signaling. Consistently, the absence of P2Y13-R was linked to increased lipolysis in adipocytes and WAT explants via hormone-sensitive lipase activation. Metabolic studies indicated that mice lacking P2Y13-R showed a greater susceptibility to diet-induced IR, systemic inflammation, and MASLD compared with their wild-type counterparts. Assays conducted on precision-cut liver slices exposed to WAT conditioned medium and on liver-specific P2Y13-R-knockdown mice suggested that P2Y13-R activity in WAT protects from hepatic steatosis, independently of liver P2Y13-R expression. In conclusion, our findings support the idea that targeting adipose P2Y13-R activity may represent a pharmacological strategy to prevent obesity-associated disorders, including type 2 diabetes and MASLD.

The role of P2Y6 receptor in the pathogenesis of cardiovascular and inflammatory diseases.

The purinergic receptor P2Y6 receptor (P2Y6R) is a member of the G protein-coupled receptors (GPCR) family. P2Y6R is widely expressed in various cell types and plays a critical role in physiological processes, where it is activated by extracellular uridine diphosphate (UDP) and mobilizes Ca2+ via the Gαq/11 protein pathway. We have recently discovered the pathophysiological role of P2Y6R in cardiovascular and inflammatory diseases, including inflammatory bowel disease and non-alcoholic fatty liver disease. Furthermore, we uncovered the redox-dependent internalization of P2Y6R. In this review, we provide a comprehensive overview of the pathophysiological activity of P2Y6R in cardiovascular and inflammatory diseases. Additionally, we discuss the concept of atypical internalization control of GPCRs, which may be applied in the prevention and treatment of intestinal inflammation and cardiovascular remodeling.

Macrophage P2Y6 receptor deletion attenuates atherosclerosis by limiting foam cell formation through phospholipase Cß/store-operated calcium entry/calreticulin/scavenger receptor A pathways.

BACKGROUND AND AIMS: Macrophage-derived foam cells play a causal role during the pathogenesis of atherosclerosis. P2Y6 receptor (P2Y6R) highly expressed has been considered as a disease-causing factor in atherogenesis, but the detailed mechanism remains unknown. This study aims to explore P2Y6R in regulation of macrophage foaming, atherogenesis, and its downstream pathways. Furthermore, the present study sought to find a potent P2Y6R antagonist and investigate the feasibility of P2Y6R-targeting therapy for atherosclerosis. METHODS: The P2Y6R expression was examined in human atherosclerotic plaques and mouse artery. Atherosclerosis animal models were established in whole-body P2Y6R or macrophage-specific P2Y6R knockout mice to evaluate the role of P2Y6R. RNA sequencing, DNA pull-down experiments, and proteomic approaches were performed to investigate the downstream mechanisms. High-throughput Glide docking pipeline from repurposing drug library was performed to find potent P2Y6R antagonists. RESULTS: The P2Y6R deficiency alleviated atherogenesis characterized by decreasing plaque formation and lipid deposition of the aorta. Mechanically, deletion of macrophage P2Y6R significantly inhibited uptake of oxidized low-density lipoprotein through decreasing scavenger receptor A expression mediated by phospholipase Cß/store-operated calcium entry pathways. More importantly, P2Y6R deficiency reduced the binding of scavenger receptor A to CALR, accompanied by dissociation of calreticulin and STIM1. Interestingly, thiamine pyrophosphate was found as a potent P2Y6R antagonist with excellent P2Y6R antagonistic activity and binding affinity, of which the pharmacodynamic effect and mechanism on atherosclerosis were verified. CONCLUSIONS: Macrophage P2Y6R regulates phospholipase Cß/store-operated calcium entry/calreticulin signalling pathway to increase scavenger receptor A protein level, thereby improving foam cell formation and atherosclerosis, indicating that the P2Y6R may be a potential therapeutic target for intervention of atherosclerotic diseases using P2Y6R antagonists including thiamine pyrophosphate.

P2Y13 receptor involved in HIV-1 gp120 induced neuropathy in superior cervical ganglia through NLRP3 inflammasome activation.

Cardiac autonomic neuropathy resulting from human immunodeficiency virus (HIV) infection is common; however, its mechanism remains unknown. The current work attempted to explore the function and mechanism of the P2Y13 receptor in HIV-glycoprotein 120 (gp120)-induced neuropathy in cervical sympathetic ganglion. The superior cervical ganglion (SCG) of the male SD rat was coated with HIV-gp120 to establish a model of autonomic neuropathy. In each group, we measured heart rate, blood pressure, heart rate variability, sympathetic nerve discharge and cardiac function. The expression of P2Y13 mRNA and protein in the SCG was tested by real-time polymerase chain reaction and western blotting. Additionally, this study focused on identifying the protein levels of NOD-like receptor family pyrin domain-containing 3 (NLRP3), Caspase-1, Gasdermin D (GSDMD), interleukin (IL)-1ß and IL-18 in the SCG using western blotting and immunofluorescence. In gp120 rats, increased blood pressure, heart rate, cardiac sympathetic nerve activity, P2Y13 receptor levels and decreased cardiac function could be found. P2Y13 shRNA or MRS2211 inhibited the above mentioned changes induced by gp120, suggesting that the P2Y13 receptor may be engaged in gp120-induced sympathetic nerve injury. Moreover, the levels of NLRP3, Caspase-1, GSDMD, IL-1ß and IL-18 in the gp120 group were increased, while significantly decreased by P2Y13 shRNA or MRS2211. Therefore, the P2Y13 receptor is involved in gp120-induced sympathetic neuropathy, and its molecular mechanism shows an association with the activation of the NLRP3 inflammasome, followed by GSDMD formation along with the release of inflammatory factors including IL-1ß and IL-18. This article is part of the Special Issue on "Purinergic Signaling: 50 years".

Tollip deficiency exaggerates airway type 2 inflammation in mice exposed to allergen and influenza A virus: role of the ATP/IL-33 signaling axis.

Toll-interacting protein (Tollip) is a negative regulator of the pro-inflammatory response to viruses, including influenza A virus (IAV). Genetic variation of Tollip has been associated with reduced airway epithelial Tollip expression and poor lung function in patients with asthma. Whether Tollip deficiency exaggerates type 2 inflammation (e.g., eosinophils) and viral infection in asthma remains unclear. We sought to address this critical, but unanswered question by using a Tollip deficient mouse asthma model with IAV infection. Further, we determined the underlying mechanisms by focusing on the role of the ATP/IL-33 signaling axis. Wild-type and Tollip KO mice were intranasally exposed to house dust mite (HDM) and IAV with or without inhibitors for IL-33 (i.e., soluble ST2, an IL-33 decoy receptor) and ATP signaling (i.e., an antagonist of the ATP receptor P2Y13). Tollip deficiency amplified airway type 2 inflammation (eosinophils, IL-5, IL-13 and mucins), and the release of ATP and IL-33. Blocking ATP receptor P2Y13 decreased IL-33 release during IAV infection in HDM-challenged Tollip KO mice. Furthermore, soluble ST2 attenuated airway eosinophilic inflammation in Tollip KO mice treated with HDM and IAV. HDM challenges decreased lung viral load in wild-type mice, but Tollip deficiency reduced the protective effects of HDM challenges on viral load. Our data suggests that during IAV infection, Tollip deficiency amplified type 2 inflammation and delayed viral clearance, in part by promoting ATP signaling and subsequent IL-33 release. Our findings may provide several therapeutic targets, including ATP and IL-33 signaling inhibition for attenuating excessive airway type 2 inflammation in human subjects with Tollip deficiency and IAV infection.

Traditional Chinese medicine alleviating neuropathic pain targeting purinergic receptor P2 in purinergic signaling: A review.

Past studies have suggested that Chinese herbal may alleviate neuropathic pain, and the mechanism might target the inhibition of purinergic receptor P2. This review discusses whether traditional Chinese medicine target P2 receptors in neuropathic pain and its mechanism in order to provide references for future clinical drug development. The related literatures were searched from Pubmed, Embase, Sinomed, and CNKI databases before June 2023. The search terms included"neuropathic pain", "purinergic receptor P2", "P2", "traditional Chinese medicine", "Chinese herbal medicine", and "herb". We described the traditional Chinese medicine alleviating neuropathic pain via purinergic receptor P2 signaling pathway including P2X2/3 R, P2X3R, P2X4R, P2X7R, P2Y1R. Inhibition of activating glial cells, changing synaptic transmission, increasing painful postsynaptic potential, and activating inflammatory signaling pathways maybe the mechanism. Purine receptor P2 can mediate the occurrence of neuropathic pain. And many of traditional Chinese medicines can target P2 receptors to relieve neuropathic pain, which provides reasonable evidences for the future development of drugs. Also, the safety and efficacy and mechanism need more in-depth experimental research.

Degranulation of human mast cells: modulation by P2 receptors' agonists.

Since the late 1970s, there has been an alarming increase in the incidence of asthma and its morbidity and mortality. Acute obstruction and inflammation of allergic asthmatic airways are frequently caused by inhalation of exogenous substances such as allergens cross-linking IgE receptors expressed on the surface of the human lung mast cells (HLMC). The degree of constriction of human airways produced by identical amounts of inhaled allergens may vary from day to day and even hour to hour. Endogenous factors in the human mast cell (HMC)'s microenvironment during allergen exposure may markedly modulate the degranulation response. An increase in allergic responsiveness may significantly enhance bronchoconstriction and breathlessness. This review focuses on the role that the ubiquitous endogenous purine nucleotide, extracellular adenosine 5'-triphosphate (ATP), which is a component of the damage-associated molecular patterns, plays in mast cells' physiology. ATP activates P2 purinergic cell-surface receptors (P2R) to trigger signaling cascades resulting in heightened inflammatory responses. ATP is the most potent enhancer of IgE-mediated HLMC degranulation described to date. Current knowledge of ATP as it relates to targeted receptor(s) on HMC along with most recent studies exploring HMC post-receptor activation pathways are discussed. In addition, the reviewed studies may explain why brief, minimal exposures to allergens (e.g., dust, cat, mouse, and grass) can unpredictably lead to intense clinical reactions. Furthermore, potential therapeutic approaches targeting ATP-related enhancement of allergic reactions are presented.

Immunological role and clinical prognostic significance of P2RY6 in lung adenocarcinoma: a multi-omics studies and single-cell sequencing analysis.

BACKGROUND: There is increasing evidence that recombinant human P2Y purinoceptor 6 (P2RY6) may be involved in inflammatory responses. However, the role of P2RY6 in lung adenocarcinoma (LUAD) remains unknown. METHODS: We used transcriptomic, genomic, single-cell transcriptomic, and methylation sequencing data from The Cancer Genome Atlas database to analyze the aberrant status and prognostic value of P2RY6 in a variety of tumors. The LUAD single-cell sequencing dataset was used to explore the effect of P2RY6 on the tumor microenvironment. Cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT) was used to quantify immune cells in the tumor microenvironment. We also analyzed the correlation of P2RY6 with immune checkpoints and immune regulation-related genes. The correlation of between tumor mutation burden (TMB), microsatellite instability (MSI), and P2RY6 expression was also analyzed simultaneously. Tissue microarray and immunohistochemistry were employed to assess the expression of P2RY6 in internal tumor samples. RESULTS: Our findings indicate that P2RY6 exhibits significantly higher expression levels in various cancer tissues, particularly in LUAD. High expression of P2RY6 was closely associated with a poor prognosis for patients, and it plays a role in regulating immune-related pathways, such as cytokine-cytokine receptor interaction. Notably, P2RY6 expression is closely linked to the abundance of CD8 + T cells. Furthermore, we have developed a P2RY6-related inflammation prediction model that demonstrates promising results in predicting the prognosis of LUAD patients, with an AUC (area under the curve) value of 0.83. This performance is significantly better than the traditional TNM staging system. Through single-cell transcriptome sequencing analysis, we observed that high P2RY6 expression is associated with increased intercellular communication. Additionally, pathway enrichment analysis revealed that P2RY6 influences antigen presentation and processing pathways within the LUAD microenvironment. CONCLUSIONS: This study suggests that P2RY6 would be a new target for immunotherapy in LUAD.

Extracellular ATP Neurotransmission and Nicotine Sex-Specifically Modulate Habenular Neuronal Activity in Adolescence.

The recent increase in the use of nicotine products by teenagers has revealed an urgent need to better understand the impact of nicotine on the adolescent brain. Here, we sought to examine the actions of extracellular ATP as a neurotransmitter and to investigate whether ATP and nicotinic signaling interact during adolescence. With the GRABATP (G-protein-coupled receptor activation-based ATP sensor), we first demonstrated that nicotine induces extracellular ATP release in the medial habenula, a brain region involved in nicotine aversion and withdrawal. Using patch-clamp electrophysiology, we then demonstrated that activation of the ATP receptors P2X or P2Y1 increases the neuronal firing of cholinergic neurons. Surprisingly, contrasting interactive effects were observed with nicotine exposure. For the P2X receptor, activation had no observable effect on acute nicotine-mediated activity, but during abstinence after 10 d of nicotine exposure, coexposure to nicotine and the P2X agonist potentiated neuronal activity in female, but not male, neurons. For P2Y1 signaling, a potentiated effect of the agonist and nicotine was observed with acute exposure, but not following extended nicotine exposure. These data reveal a complex interactive effect between nicotinic and ATP signaling in the adolescent brain and provide mechanistic insights into extracellular ATP signaling with sex-specific alterations of neuronal responses based on prior drug exposure.SIGNIFICANCE STATEMENT In these studies, it was discovered that nicotine induces extracellular ATP release in the medial habenula and subsequent activation of the ATP purinergic receptors increases habenular cholinergic neuronal firing in the adolescent brain. Interestingly, following extended nicotine exposure, nicotine was found to alter the interplay between purinergic and nicotinic signaling in a sex-specific manner. Together, these studies provide a novel understanding for the role of extracellular ATP in mediating habenular activity and reveal how nicotine exposure during adolescence alters these signaling mechanisms, which has important implications given the high incidence of e-cigarette/vape use by youth.

UDP-glucose sensing P2Y14R: A novel target for inflammation.

Uridine 5'-diphosphoglucose (UDP-G) as a preferential agonist, but also other UDP-sugars, such as UDP galactose, function as extracellular signaling molecules under conditions of cell injury and apoptosis. Consequently, UDP-G is regarded to function as a damage-associated molecular pattern (DAMP), regulating immune responses. UDP-G promotes neutrophil recruitment, leading to the release of pro-inflammatory chemokines. As a potent endogenous agonist with the highest affinity for the P2Y14 receptor (R), it accomplishes an exclusive relationship between P2Y14Rs in regulating inflammation via cyclic adenosine monophosphate (cAMP), nod-like receptor protein 3 (NLRP3) inflammasome, mitogen-activated protein kinases (MAPKs), and signal transducer and activator of transcription 1 (STAT1) pathways. In this review, we initially present a brief introduction into the expression and function of P2Y14Rs in combination with UDP-G. Subsequently, we summarize emerging roles of UDP-G/P2Y14R signaling pathways that modulate inflammatory responses in diverse systems, and discuss the underlying mechanisms of P2Y14R activation in inflammation-related diseases. Moreover, we also refer to the applications as well as effects of novel agonists/antagonists of P2Y14Rs in inflammatory conditions. In conclusion, due to the role of the P2Y14R in the immune system and inflammatory pathways, it may represent a novel target for anti-inflammatory therapy.

Alicyclic Ring Size Variation of 4-Phenyl-2-naphthoic Acid Derivatives as P2Y14 Receptor Antagonists.

P2Y14 receptor (P2Y14R) is activated by extracellular UDP-glucose, a damage-associated molecular pattern that promotes inflammation in the kidney, lung, fat tissue, and elsewhere. Thus, selective P2Y14R antagonists are potentially useful for inflammatory and metabolic diseases. The piperidine ring size of potent, competitive P2Y14R antagonist (4-phenyl-2-naphthoic acid derivative) PPTN 1 was varied from 4- to 8-membered rings, with bridging/functional substitution. Conformationally and sterically modified isosteres included N-containing spirocyclic (6-9), fused (11-13), and bridged (14, 15) or large (16-20) ring systems, either saturated or containing alkene or hydroxy/methoxy groups. The alicyclic amines displayed structural preference. An α-hydroxyl group increased the affinity of 4-(4-((1R,5S,6r)-6-hydroxy-3-azabicyclo[3.1.1]heptan-6-yl)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoic acid 15 (MRS4833) compared to 14 by 89-fold. 15 but not its double prodrug 50 reduced airway eosinophilia in a protease-mediated asthma model, and orally administered 15 and prodrugs reversed chronic neuropathic pain (mouse CCI model). Thus, we identified novel drug leads having in vivo efficacy.

P2 receptor-mediated signaling in the physiological and pathological brain: From development to aging and disease.

The purinergic pathway mediates both pro-inflammatory and anti-inflammatory responses, whereas the breakdown of adenosine triphosphate (ATP) is in a critical equilibrium. Under physiological conditions, extracellular ATP is maintained at a nanomolar concentration. Whether released into the medium following tissue damage, inflammation, or hypoxia, ATP is considered a clear indicator of cell damage and a marker of pathological conditions. In this overview, we provide an update on the participation of P2 receptor-mediated purinergic signaling in normal and pathological brain development, with special emphasis on neurodevelopmental psychiatric disorders. Since purinergic signaling is ubiquitous, it is not surprising that it plays a prominent role in developmental processes and pathological alterations. The main aim of this review is to conceptualize the time-dependent dynamic changes in the participation of different players in the purinome in shaping the normal and aberrant developmental patterns and diseases of the central nervous system over one's lifespan. This article is part of the Special Issue on "Purinergic Signaling: 50 years".

The Signaling Pathway of the ADP Receptor P2Y12 in the Immune System: Recent Discoveries and New Challenges.

P2Y12 is a G-protein-coupled receptor that is activated upon ADP binding. Considering its well-established role in platelet activation, blocking P2Y12 has been used as a therapeutic strategy for antiplatelet aggregation in cardiovascular disease patients. However, receptor studies have shown that P2Y12 is functionally expressed not only in platelets and the microglia but also in other cells of the immune system, such as in monocytes, dendritic cells, and T lymphocytes. As a result, studies were carried out investigating whether therapies targeting P2Y12 could also ameliorate inflammatory conditions, such as sepsis, rheumatoid arthritis, neuroinflammation, cancer, COVID-19, atherosclerosis, and diabetes-associated inflammation in animal models and human subjects. This review reports what is known about the expression of P2Y12 in the cells of the immune system and the effect of P2Y12 activation and/or inhibition in inflammatory conditions. Lastly, we will discuss the major problems and challenges in studying this receptor and provide insights on how they can be overcome.

Purinergic P2 Receptors: Structure and Function 2.0.

This Special Issue of the International Journal of Molecular Sciences (IJMS) is a direct continuation of the previous Special Issue of this journal, entitled "Purinergic P2 Receptors: Structure and Function" https://www [...].

The adenosinergic machinery in cancer: In-tandem insights from basic mechanisms to therapy.

Extracellular adenosine (eADO) signaling has emerged as an increasingly important regulator of immune responses, including tumor immunity. eADO is mainly produced from extracellular ATP (eATP) hydrolysis. eATP is rapidly accumulated in the extracellular space following cell death or cellular stress triggered by hypoxia, nutrient starvation, or inflammation. eATP plays a pro-inflammatory role by binding and activating the P2 purinergic receptors (P2X and P2Y), while eADO has been reported in many studies to mediate immunosuppression by activating the P1 purinergic receptors (A1, A2A, A2B, and A3) in diverse immune cells. Consequently, the hydrolysis of eATP to eADO alters the immunosurveillance in the tumor microenvironment (TME) not only by reducing eATP levels but also by enhancing adenosine receptor signaling. The effects of both P1 and P2 purinergic receptors are not restricted to immune cells. Here we review the most up-to-date understanding of the tumor adenosinergic system in all cell types, including immune cells, tumor cells, and stromal cells in TME. The potential novel directions of future adenosinergic therapies in immuno-oncology will be discussed.

The Pleiotropic Role of Extracellular ATP in Myocardial Remodelling.

Myocardial remodelling is a molecular, cellular, and interstitial adaptation of the heart in response to altered environmental demands. The heart undergoes reversible physiological remodelling in response to changes in mechanical loading or irreversible pathological remodelling induced by neurohumoral factors and chronic stress, leading to heart failure. Adenosine triphosphate (ATP) is one of the potent mediators in cardiovascular signalling that act on the ligand-gated (P2X) and G-protein-coupled (P2Y) purinoceptors via the autocrine or paracrine manners. These activations mediate numerous intracellular communications by modulating the production of other messengers, including calcium, growth factors, cytokines, and nitric oxide. ATP is known to play a pleiotropic role in cardiovascular pathophysiology, making it a reliable biomarker for cardiac protection. This review outlines the sources of ATP released under physiological and pathological stress and its cell-specific mechanism of action. We further highlight a series of cardiovascular cell-to-cell communications of extracellular ATP signalling cascades in cardiac remodelling, which can be seen in hypertension, ischemia/reperfusion injury, fibrosis, hypertrophy, and atrophy. Finally, we summarize current pharmacological intervention using the ATP network as a target for cardiac protection. A better understanding of ATP communication in myocardial remodelling could be worthwhile for future drug development and repurposing and the management of cardiovascular diseases.

Computational prediction and in vitro analysis of the potential ligand binding site within the extracellular ATP receptor, P2K2.

The plant extracellular ATP (eATP) receptor, P2K2, binds eATP with strong ligand affinity through its extracellular lectin domain. Ligand binding activates the intracellular kinase domain of P2K2 resulting in a variety of intracellular responses and, ultimately, increased plant immunity to invading fungal and bacterial pathogens. Here, using a computational prediction approach, we developed a tertiary structure model of the P2K2 extracellular lectin domain. In silico target docking of ATP to the P2K2-binding site predicted interaction with several residues through hydrophobic interactions and hydrogen bonding. Our confirmation of the modeling was obtained by showing that H99, R144, and S256 are key residues essential for in vitro binding of ATP by P2K2.

Hindbrain Adenosine 5-Triphosphate (ATP)-Purinergic Signaling Triggers LH Surge and Ovulation via Activation of AVPV Kisspeptin Neurons in Rats.

Ovulation disorders are a serious problem for humans and livestock. In female rodents, kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) are responsible for generating a luteinizing hormone (LH) surge and consequent ovulation. Here, we report that adenosine 5-triphosphate (ATP), a purinergic receptor ligand, is a possible neurotransmitter that stimulates AVPV kisspeptin neurons to induce an LH surge and consequent ovulation in rodents. Administration of an ATP receptor antagonist (PPADS) into the AVPV blocked the LH surge in ovariectomized (OVX) rats treated with a proestrous level of estrogen (OVX + high E2) and significantly reduced the ovulation rate in proestrous ovary-intact rats. AVPV ATP administration induced a surge-like LH increase in OVX + high E2 rats in the morning. Importantly, AVPV ATP administration could not induce the LH increase in Kiss1 KO rats. Furthermore, ATP significantly increased intracellular Ca2+ levels in immortalized kisspeptin neuronal cell line, and coadministration of PPADS blocked the ATP-induced Ca2+ increase. Histologic analysis revealed that the proestrous level of estrogen significantly increased the number of P2X2 receptor (an ATP receptor)-immunopositive AVPV kisspeptin neurons visualized by tdTomato in Kiss1-tdTomato rats. The proestrous level of estrogen significantly increased varicosity-like vesicular nucleotide transporter (a purinergic marker)-immunopositive fibers projecting to the vicinity of AVPV kisspeptin neurons. Furthermore, we found that some hindbrain vesicular nucleotide transporter-positive neurons projected to the AVPV and expressed estrogen receptor α, and the neurons were activated by the high E2 treatment. These results suggest that hindbrain ATP-purinergic signaling triggers ovulation via activation of AVPV kisspeptin neurons.SIGNIFICANCE STATEMENT Ovulation disorders, which cause infertility and low pregnancy rates, are a serious problem for humans and livestock. The present study provides evidence that adenosine 5-triphosphate, acting as a neurotransmitter in the brain, stimulates kisspeptin neurons in the anteroventral periventricular nucleus, known as the gonadotropin-releasing hormone surge generator, via purinergic receptors to induce the gonadotropin-releasing hormone/luteinizing hormone surge and ovulation in rats. In addition, histologic analyses indicate that adenosine 5-triphosphate is likely to be originated from the purinergic neurons in the A1 and A2 of the hindbrain. These findings may contribute to new therapeutic controls for hypothalamic ovulation disorders in humans and livestock.