The role of ATP in sleep-wake regulation: In adenosine-dependent and -independent manner.

ATP plays a crucial role as an energy currency in the body's various physiological functions, including the regulation of the sleep-wake cycle. Evidence from genetics and pharmacology demonstrates a strong association between ATP metabolism and sleep. With the advent of new technologies such as optogenetics, genetically encoded biosensors, and novel ATP detection methods, the dynamic changes in ATP levels between different sleep states have been further uncovered. The classic mechanism for regulating sleep by ATP involves its conversion to adenosine, which increases sleep pressure when accumulated extracellularly. However, emerging evidence suggests that ATP can directly bind to P2 receptors and influence sleep-wake regulation through both adenosine-dependent and independent pathways. The outcome depends on the brain region where ATP acts and the expression type of P2 receptors. This review summarizes the experimental evidence on the relationship between ATP levels and changes in sleep states and outlines the mechanisms by which ATP is involved in regulating the sleep-wake cycle through both adenosine-dependent and independent pathways. Hopefully, this review will provide a comprehensive understanding of the current research basis and progress in this field and promote further investigations into the specific mechanisms of ATP in regulating sleep.

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.

Central role of purinergic receptors with inflammation in neuropathic pain-related macrophage-SGC-neuron triad.

Adenosine triphosphate (ATP) acts on P2 purinergic receptors as an extracellular signaling molecule. P2 purinergic receptors include P2X ionotropic receptors and P2Y metabotropic receptors. Satellite glial cells (SGCs) and macrophages express P2X and P2Y receptors. Inflammatory cytokines and pro-nociceptive mediators are released by activated macrophages and SGCs, which can act on neurons to promote excitability and firing. In the primary sensory ganglia, in response to signals of injury, SGCs and macrophages accumulate around primary sensory neurons, forming a macrophage-SGC-neuron triad. In addition to affecting the pathological alterations of inflammation-related neuropathic pain, inflammatory cytokines and pro-nociceptive mediators are released by the action of ATP on P2X and P2Y receptors in macrophages and SGCs. Macrophages and SGCs work together to enhance and prolong neuropathic pain. The macrophage-SGC-neuron triad communicates with each other through ATP and other inflammatory mediators and maintains and promotes the initiation and development of inflammation related-neuropathic pain. This article is part of the Special Issue on "Purinergic Signaling: 50 years".

ATP-mediated signalling in the central synapses.

ATP released from the synaptic terminals and astrocytes can activate neuronal P2 receptors at a variety of locations across the CNS. Although the postsynaptic ATP-mediated signalling does not bring a major contribution into the excitatory transmission, it is instrumental for slow and diffuse modulation of synaptic dynamics and neuronal firing in many CNS areas. Neuronal P2X and P2Y receptors can be activated by ATP released from the synaptic terminals, astrocytes and microglia and thereby can participate in the regulation of synaptic homeostasis and plasticity. There is growing evidence of importance of purinergic regulation of synaptic transmission in different physiological and pathological contexts. Here, we review the main mechanisms underlying the complexity and diversity of purinergic signalling and purinergic modulation in central neurons.

The microglial P2Y6 receptor mediates neuronal loss and memory deficits in neurodegeneration.

Microglia are implicated in neurodegeneration, potentially by phagocytosing neurons, but it is unclear how to block the detrimental effects of microglia while preserving their beneficial roles. The microglial P2Y6 receptor (P2Y6R) - activated by extracellular UDP released by stressed neurons - is required for microglial phagocytosis of neurons. We show here that injection of amyloid beta (Aß) into mouse brain induces microglial phagocytosis of neurons, followed by neuronal and memory loss, and this is all prevented by knockout of P2Y6R. In a chronic tau model of neurodegeneration (P301S TAU mice), P2Y6R knockout prevented TAU-induced neuronal and memory loss. In vitro, P2Y6R knockout blocked microglial phagocytosis of live but not dead targets and reduced tau-, Aß-, and UDP-induced neuronal loss in glial-neuronal cultures. Thus, the P2Y6 receptor appears to mediate Aß- and tau-induced neuronal and memory loss via microglial phagocytosis of neurons, suggesting that blocking this receptor may be beneficial in the treatment of neurodegenerative diseases.

P2Y4, P2Y6 and P2Y11 receptors: From the early days of cloning to their function.

The family of P2Y nucleotide receptors is composed of eight members differentiated by their pharmacology and their coupling to specific G-proteins and transduction mechanisms. The laboratory studying these nucleotide receptors at IRIBHM institute (Free University of Brussels) has participated actively in their cloning. We used classical cloning by homology strategies relying on polymerase chain reactions with degenerate primers or on DNA libraries screening with P2Y receptors-related primers or probes, respectively. We identified and characterised four of the eight human P2Y receptors cloned so far: P2Y4, P2Y6, P2Y11 and P2Y13 receptors. These human receptors displayed specific features in terms of pharmacology such as affinity for pyrimidine nucleotides for P2Y4 and P2Y6 receptors and differential G-protein coupling. Their specific and restricted tissue distribution compared to ubiquitous P2Y1 and P2Y2 receptors led us to study their physiological role in chosen cell systems or using mice deficient for these P2Y subtypes. These studies revealed over the years that the P2Y11 receptor was able to confer tolerogenic and tumorigenic properties to human dendritic cells and that P2Y4 and P2Y6 receptors were involved in mouse heart post-natal development and cardioprotection. P2Y receptors and their identified target genes could constitute therapeutic targets to regulate cardiac hypertrophy and regeneration. The multiple roles of P2Y receptors identified in the ischemic heart and cardiac adipose tissue could have multiple innovative clinical applications and present a major interest in the field of cardiovascular diseases. P2Y receptors can induce cardioprotection by the regulation of cardiac inflammation and the modulation of the volume and composition of cardiac adipose tissue. These findings might lead to the pre-clinical validation of P2Y receptors as new targets for the treatment of myocardial ischemia.

The purinergic receptor P2Y11 choreographs the polarization, mitochondrial metabolism, and migration of T lymphocytes.

T cells must migrate to encounter antigen-presenting cells and perform their roles in host defense. Here, we found that autocrine stimulation of the purinergic receptor P2Y11 regulates the migration of human CD4 T cells. P2Y11 receptors redistributed from the front to the back of polarized cells where they triggered intracellular cAMP/PKA signals that attenuated mitochondrial metabolism at the back. The absence of P2Y11 receptors at the front of cells resulted in hotspots of mitochondrial metabolism and localized ATP production that stimulated P2X4 receptors, Ca2+ influx, and pseudopod protrusion at the front. This regulatory function of P2Y11 receptors depended on their subcellular redistribution and autocrine stimulation by cellular ATP release and was perturbed by indiscriminate global stimulation. We conclude that excessive extracellular ATP-such as in response to inflammation, sepsis, and cancer-disrupts this autocrine feedback mechanism, which results in defective T cell migration, impaired T cell function, and loss of host immune defense.

Distinct P2Y Receptors Mediate Extension and Retraction of Microglial Processes in Epileptic and Peritumoral Human Tissue.

Microglia exhibit multiple, phenotype-dependent motility patterns often triggered by purinergic stimuli. However, little data exist on motility of human microglia in pathological situations. Here we examine motility of microglia stained with a fluorescent lectin in tissue slices from female and male epileptic patients diagnosed with mesial temporal lobe epilepsy or cortical glioma (peritumoral cortex). Microglial shape varied from ramified to amoeboid cells predominantly in regions of high neuronal loss or closer to a tumor. Live imaging revealed unstimulated or purine-induced microglial motilities, including surveillance movements, membrane ruffling, and process extension or retraction. At different concentrations, ADP triggered opposing motilities. Low doses triggered process extension. It was suppressed by P2Y12 receptor antagonists, which also reduced process length and surveillance movements. Higher purine doses caused process retraction and membrane ruffling, which were blocked by joint application of P2Y1 and P2Y13 receptor antagonists. Purinergic effects on motility were similar for all microglia tested. Both amoeboid and ramified cells from mesial temporal lobe epilepsy or peritumoral cortex tissue expressed P2Y12 receptors. A minority of microglia expressed the adenosine A2A receptor, which has been linked with process withdrawal of rodent cells. Laser-mediated tissue damage let us test the functional significance of these effects. Moderate damage induced microglial process extension, which was blocked by P2Y12 receptor antagonists. Overall, the purine-induced motility of human microglia in epileptic tissue is similar to that of rodent microglia in that the P2Y12 receptor initiates process extension. It differs in that retraction is triggered by joint activation of P2Y1/P2Y13 receptors.SIGNIFICANCE STATEMENT Microglial cells are brain-resident immune cells with multiple functions in healthy or diseased brains. These diverse functions are associated with distinct phenotypes, including different microglial shapes. In the rodent, purinergic signaling is associated with changes in cell shape, such as process extension toward tissue damage. However, there are little data on living human microglia, especially in diseased states. We developed a reliable technique to stain microglia from epileptic and glioma patients to examine responses to purines. Low-intensity purinergic stimuli induced process extension, as in rodents. In contrast, high-intensity stimuli triggered a process withdrawal mediated by both P2Y1 and P2Y13 receptors. P2Y1/P2Y13 receptor activation has not previously been linked to microglial morphological changes.

Extracellular Nucleotides and P2 Receptors in Renal Function.

The understanding of the nucleotide/P2 receptor system in the regulation of renal hemodynamics and transport function has grown exponentially over the last 20 yr. This review attempts to integrate the available data while also identifying areas of missing information. First, the determinants of nucleotide concentrations in the interstitial and tubular fluids of the kidney are described, including mechanisms of cellular release of nucleotides and their extracellular breakdown. Then the renal cell membrane expression of P2X and P2Y receptors is discussed in the context of their effects on renal vascular and tubular functions. Attention is paid to effects on the cortical vasculature and intraglomerular structures, autoregulation of renal blood flow, tubuloglomerular feedback, and the control of medullary blood flow. The role of the nucleotide/P2 receptor system in the autocrine/paracrine regulation of sodium and fluid transport in the tubular and collecting duct system is outlined together with its role in integrative sodium and fluid homeostasis and blood pressure control. The final section summarizes the rapidly growing evidence indicating a prominent role of the extracellular nucleotide/P2 receptor system in the pathophysiology of the kidney and aims to identify potential therapeutic opportunities, including hypertension, lithium-induced nephropathy, polycystic kidney disease, and kidney inflammation. We are only beginning to unravel the distinct physiological and pathophysiological influences of the extracellular nucleotide/P2 receptor system and the associated therapeutic perspectives.

Ligand binding and activation of UTP-activated G protein-coupled P2Y2 and P2Y4 receptors elucidated by mutagenesis, pharmacological and computational studies.

The nucleotide receptors P2Y2 and P2Y4 are the most closely related G protein-coupled receptors (GPCRs) of the P2Y receptor (P2YR) family. Both subtypes couple to Gq proteins and are activated by the pyrimidine nucleotide UTP, but only P2Y2R is also activated by the purine nucleotide ATP. Agonists and antagonists of both receptor subtypes have potential as drugs e.g. for neurodegenerative and inflammatory diseases. So far, potent and selective, "drug-like" ligands for both receptors are scarce, but would be required for target validation and as lead structures for drug development. Structural information on the receptors is lacking since no X-ray structures or cryo-electron microscopy images are available. Thus, we performed receptor homology modeling and docking studies combined with mutagenesis experiments on both receptors to address the question how ligand binding selectivity for these closely related P2YR subtypes can be achieved. The orthosteric binding site of P2Y2R appeared to be more spacious than that of P2Y4R. Mutation of Y197 to alanine in P2Y4R resulted in a gain of ATP sensitivity. Anthraquinone-derived antagonists are likely to bind to the orthosteric or an allosteric site depending on their substitution pattern and the nature of the orthosteric binding site of the respective P2YR subtype. These insights into the architecture of P2Y2- and P2Y4Rs and their interactions with structurally diverse agonists and antagonist provide a solid basis for the future design of potent and selective ligands.

Hypertension and sympathetic nervous system overactivity rely on the vascular tone of pial vessels of the rostral ventrolateral medulla in spontaneously hypertensive rats.

NEW FINDINGS: What is the central question of this study? Is purinergic signalling in the pial vessels involved in the control of vascular tone in the ventral surface of the brainstem, affecting high blood pressure and sympathetic overactivity in spontaneously hypertensive rats? What is the main finding and its importance? The regulation of vascular tone in the ventral surface of the brainstem is tailored to support neuronal functions, arterial pressure and sympathetic activity. This adds one more piece in the complex puzzle to understand the central mechanisms underlying the genesis of hypertension. ABSTRACT: Evidence suggests the rostral ventrolateral medulla (RVLM) region is chronically hypoperfused and hypoxic in spontaneously hypertensive rats (SHR), which can facilitate ATP release throughout the brainstem. Thus, we hypothesized that purinergic signalling plays a key role in the increased vascular tone in the RVLM region, which in turn could be responsible for the high sympathetic tone and blood pressure in the SHR. The application of an antagonist of P2 receptors, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (10 µm), or of P2Y1a receptors, MRS2179 (100 µm), on the surface of RVLM pial vessels of SHR produced an increase in the diameter of blood vessels (PPADS: 31 ± 1.4 µm or MRS2179: 32 ± 0.78 µm vs. saline: 27 ± 1.2 µm), an effect not observed in normotensive Wistar rats. In addition, the antagonism of P2 receptors was able to evoke a significant decrease in the arterial pressure, heart rate and splanchnic nerve activity in SHR, but not in Wistar rats. Our data show that SHR have higher vascular tone of pial vessels in the RVLM region when compared to the normotensive Wistar rats, a mechanism that relies on purinergic signalling through P2 receptors, suggesting a possible association with higher activity of sympathoexcitatory neurones, and sustained increases in blood pressure.

P2Y receptors in neuropathic pain.

This review summarizes and evaluates the relationship between neuropathic pain and P2Y receptors from inception to 2019. Purinergic receptors have been well studied in recent years using various molecular biological methods. The main research objective of this review is to determine the association of P2Y1, P2Y2, P2Y6, P2Y12 and P2Y13 receptors with neuropathic pain. This review includes the most comprehensive subtypes of P2Y that related to neuropathic pain and the current therapeutic method of neuropathic pain. G protein-coupled P2Y receptors are located on neurons, astrocytes, oligodendrocytes and microglial cells and regulate neurotransmission. Nerve injury is the prime reason for abnormal regulation of P2Y receptor mRNA expression, subsequently, inducing neuropathic pain. Neuropathic pain is a type of chronic pain that is divided into peripheral, central and mixed. Numerous studies demonstrated a positive correlation between the expression level of P2Y receptors and neuropathic pain generation. Also, several reports showed that P2Y short hairpin RNA (shRNA) and P2Y antagonist can be used as an analgesic to relieve neuropathic pain via decreasing P2Y receptor expression level and neural cell activation. However, the transformation process from basic experiments to clinical applications is a long process. Current deficiencies and future research directions are discussed at the end of this review.

P2Y4/TSP-1/TGF-ß1/pSmad2/3 pathway contributes to acute generalized seizures induced by kainic acid.

Epilepsy is accompanied by angiogenesis and blood-brain barrier (BBB) disruption. The transforming growth factor-ß1 (TGF-ß1)/phosphorylated small mothers against decapentaplegic 2 and 3 (pSmad2/3)/vascular endothelial growth factor (VEGF) pathway, activated by thrombospondin-1 (TSP-1), which is further regulated by Y type P2 purinergic receptor activity, may participate in angiogenesis. We sought to investigate the relationship between the P2R/TSP-1/TGF-ß1/pSmad2/3/VEGF pathway, angiogenesis, and BBB damage in a kainic acid (KA) model of acute generalized seizure. Our results demonstrated that KA-induced seizures were accompanied by angiogenesis and BBB damage. In addition, expression of TSP-1, TGF-ß1, and pSmad2/3 was increased. Rats treated with pyridoxal phosphate-6-azophenyl-2', 4'-disulfonic acid, a broad P2 receptor antagonist, or Reactive Blue 2, a potent P2Y4 receptor antagonist, showed significant attenuation of TSP-1 expression and Smad2/3 phosphorylation levels. Furthermore, angiogenesis, BBB damage, and acute seizure severity were also reduced. The inhibition of TSP-1 expression by siRNA or TGF-ß1 activation by Leu-Ser-Lys-Leu (LSKL) treatment prevented KA-induced phosphorylation of Smad2/3, angiogenesis, BBB damage, and acute seizures. Our results strongly indicate that the P2Y4/TSP-1/TGF-ß1/pSmad2/3/VEGF pathway plays an essential role in seizure pathophysiology and angiogenesis. Therapeutic interventions targeting this pathway may offer new treatment options for acute seizures.

Purinergic signaling in the organ of Corti: Potential therapeutic targets of sensorineural hearing losses.

Purinergic signaling is deeply involved in the development, functions and protective mechanisms of the cochlea. Release of ATP and activation of purinergic receptors on sensory and supporting/epithelial cells play a substantial role in cochlear (patho)physiology. Both the ionotropic P2X and the metabotropic P2Y receptors are widely distributed on the inner and outer hair cells as well as on the different supporting cells in the organ of Corti and on other epithelial cells in the scala media. Among others, they are implicated in the sensitivity adjustment of the receptor cells by a K+ shunt and can attenuate the cochlear amplification by modifying cochlear micromechanics acting on outer hair cells and supporting cells. Cochlear blood flow is also regulated by purines. Sensorineural hearing losses currently lack any specific or efficient pharmacotherapy. Decreasing hearing sensitivity and increasing cochlear blood supply by pharmacological targeting of purinergic signaling in the cochlea are potential new therapeutic approaches in these hearing disabilities, especially in the noise-induced ones.

Role of P2 receptors in normal brain development and in neurodevelopmental psychiatric disorders.

The purinergic signaling system, including P2 receptors, plays an important role in various central nervous system (CNS) disorders. Over the last few decades, a substantial amount of accumulated data suggest that most P2 receptor subtypes (P2X1, 2, 3, 4, 6, and 7, and P2Y1, 2, 6, 12, and 13) regulate neuronal/neuroglial developmental processes, such as proliferation, differentiation, migration of neuronal precursors, and neurite outgrowth. However, only a few of these subtypes (P2X2, P2X3, P2X4, P2X7, P2Y1, and P2Y2) have been investigated in the context of neurodevelopmental psychiatric disorders. The activation of these potential target receptors and their underlying mechanisms mainly influence the process of neuroinflammation. In particular, P2 receptor-mediated inflammatory cytokine release has been indicated to contribute to the complex mechanisms of a variety of CNS disorders. The released inflammatory cytokines could be utilized as biomarkers for neurodevelopmental and psychiatric disorders to improve the early diagnosis intervention, and prognosis. The population changes in gut microbiota after birth are closely linked to neurodevelopmental/neuropsychiatric disorders in later life; thus, the dynamic expression and function of P2 receptors on gut epithelial cells during disease processes indicate a novel avenue for the evaluation of disease progression and for the discovery of related therapeutic compounds.

P2Y12 regulates microglia activation and excitatory synaptic transmission in spinal lamina II neurons during neuropathic pain in rodents.

Peripheral nerve injury causes neuropathic pain and microglia activation. P2Y12 receptors on microglia are thought to be a key player in the surveillance of the local environment, but whether or how these receptors are engaged in the cross-talk between microglia and neurons of the dorsal horn remain ambiguous. Using a rodent model of nerve injury-induced pain, we investigated the roles of P2Y12 in microglia activation, excitatory synaptic transmission, and nociceptive allodynia. We found that spinal nerve ligation (SNL) significantly increased the level of P2Y12 receptors specifically in the microglia of the ipsilateral dorsal horn. Injections of P2Y12 antagonists (MRS2395 or clopidogrel) attenuated microglia activation and increased the paw withdrawal latency in response to thermal stimuli on the ipsilateral side without affecting the basal threshold on the contralateral side. These effects on pain behaviors were replicated in P2Y12 knockout mice. Patch-clamp recordings further revealed that partial sciatic nerve ligation (PSNL)-induced excessive miniature excitatory postsynaptic currents (mEPSCs) were significantly attenuated in P2Y12 knockout mice. Moreover, we found that SNL activates the GTP-RhoA/ROCK2 signaling pathway and elevates the level of phosphorylated p38 mitogen-activated protein kinase (MAPK), which was inhibited by the P2Y12 antagonist. The phosphorylation of p38 MAPK was inhibited by a ROCK inhibitor, but not vice versa, suggesting that p38 MAPK is downstream of ROCK activation. Our findings suggest that nerve injury engages the P2Y12 receptor-dependent GTP-RhoA/ROCK2 signaling pathway to upregulate excitatory synaptic transmission in the dorsal horn. This cross-talk ultimately participates in the manifestation of nociceptive allodynia, implicating P2Y12 receptor as a potential target for alleviating neuropathic pain.

Microvesicles generated following traumatic brain injury induce platelet dysfunction via adenosine diphosphate receptor.

BACKGROUND: Traumatic brain injury (TBI) can result in an acute coagulopathy including platelet dysfunction that can contribute to ongoing intracranial hemorrhage. Previous studies have shown adenosine diphosphate (ADP)-induced platelet aggregation to be reduced after TBI. In addition, circulating microvesicles (MVs) are increased following TBI and have been shown to play a role in post-TBI coagulopathy and platelet function. We hypothesized that post-TBI MVs would affect platelet aggregation in a murine head injury model. METHODS: Moderate TBI was performed using a weight-drop method in male C57BL6 mice. Whole blood, plasma, MVs, and MV-poor plasma were isolated from blood collected 10 minutes following TBI and were mixed separately with whole blood from uninjured mice. Platelet aggregation was measured with Multiplate impedance platelet aggregometry in response to ADP. The ADP P2Y12 receptor inhibitor, R-138727, was incubated with plasma and MVs from TBI mice, and platelet inhibition was again measured. RESULTS: Whole blood taken from 10-minute post-TBI mice demonstrated diminished ADP-induced platelet aggregation compared with sham mice. When mixed with normal donor blood, post-TBI plasma and MVs induced diminished ADP-induced platelet aggregation compared with sham plasma and sham MVs. By contrast, the addition of post-TBI MV-poor plasma to normal blood did not change ADP-induced platelet aggregation. The observed dysfunction in post-TBI ADP platelet aggregation was prevented by the pretreatment of post-TBI plasma with R-138727. Treatment of post-TBI MVs with R-138727 resulted in similar findings of improved ADP-induced platelet aggregation compared with nontreated post-TBI MVs. CONCLUSION: Adenosine diphosphate-induced platelet aggregation is inhibited acutely following TBI in a murine model. This platelet inhibition is reproduced in normal blood by the introduction of post-TBI plasma and MVs. Furthermore, observed platelet dysfunction is prevented when post-TBI plasma and MVs are treated with an inhibitor of the P2Y12 ADP receptor. Clinically observed post-TBI platelet dysfunction may therefore be partially explained by the presence of the ADP P2Y12 receptor within post-TBI MVs. LEVEL OF EVIDENCE: Level III.

P2 receptor interaction and signalling cascades in neuroprotection.

Nucleotides can contribute to the survival of different glial and neuronal models at the nervous system via activation of purinergic P2X and P2Y receptors. Their activation counteracts different proapoptotic events, such as excitotoxicity, mitochondrial impairment, oxidative stress and DNA damage, which concur to elicit cell loss in different processes of neurodegeneration and brain injury. Thus, it is frequent to find that different neuroprotective mediators converge in the activation of the same intracellular survival pathways to protect cells from death. The present review focuses on the role of P2Y1 and P2Y13 metabotropic receptors, and P2X7 ionotropic receptors to regulate the balance between survival and apoptosis. In particular, we analyze the intracellular pathways involved in the signaling of these nucleotide receptors to elicit survival, including calcium/PLC, PI3K/Akt/GSK3, MAPK cascades, and the expression of antioxidant and antiapoptotic genes. This review emphasizes the novel contribution of nucleotide receptors to maintain cell homeostasis through the regulation of MAP kinases and phosphatases. Unraveling the different roles found for nucleotide receptors in different models and cellular contexts may be crucial to delineate future therapeutic applications based on targeting nucleotide receptors for neuroprotection.

Novel Proinflammatory Function of Renal Intercalated Cells.

BACKGROUND: Serious and often fatal acute kidney injury (AKI) is frequently seen after major surgery, local and remote organ damage, and sepsis. It is associated with uncontrolled inflammation, and is usually diagnosed only after the kidneys have gone through significant and often irreversible damage. SUMMARY: During our work involving another type of kidney disease that leads to acid-base disorders of the blood, we unexpectedly found high levels of a protein called the P2Y14 "purinergic" receptor, in specialized kidney epithelial cells called intercalated cells (ICs). These cells are responsible for maintaining whole body acid-base balance by regulating the secretion of excess protons into the urine, which normalizes blood pH. However, it turns out that the P2Y14 receptor in these cells responds to a molecule called uridine diphosphate (UDP)-glucose, which is a danger signal released by damaged cells anywhere in the body. When UDP-glucose reaches the kidney, it stimulates ICs to produce chemoattractant cytokines; this results in renal inflammation and contributes to the onset of AKI. Key Message: Thus, our work now points to ICs as key mediators of renal inflammation and AKI, following surgery and/or damage to remote organs, sepsis, and also local insults to the kidney itself. The link between the proton secreting ICs of the kidney and AKI is an example of how a fundamental research project with a defined aim, in this case understanding acid-base homeostasis, can lead to a novel observation that has unexpected but major implications in another area of human health.

Mechanism underlying the contractile activity of UTP in the mammalian heart.

We previously reported that uridine 5'-triphosphate (UTP), a pyrimidine nucleoside triphosphate produced a concentration- and time-dependent increase in the contraction force in isolated right atrial preparations from patients undergoing cardiac bypass surgery due to angina pectoris. The stimulation of the force of contraction was sustained rather than transient. In the present study, we tried to elucidate the underlying receptor and signal transduction for this effect of UTP. Therefore, we measured the effect of UTP on force of contraction, phosphorylation of p38 and ERK1/2, in human atrial preparations, atrial preparations from genetically modified mice, cardiomyocytes from adult mice and cardiomyocytes from neonatal rats. UTP exerted a positive inotropic effect in isolated electrically driven left atrial preparations from wild-type (WT) mice and P2Y2-, P2Y4- and P2Y6-receptor knockout mice. Therefore, we concluded that these P2Y receptors did not mediate the inotropic effects of UTP in atrial preparations from mice. However, UTP (like ATP) increased the phosphorylation states of p38 and ERK1/2 in neonatal rat cardiomyocytes, adult mouse cardiomyocytes and human atrial tissue in vitro. U0126, a MEK 1/2- signal cascade inhibitor, attenuated this phosphorylation and the positive inotropic effects of UTP in murine and human atrial preparations. We suggest that presently unknown receptors mediate the positive inotropic effect of UTP in murine and human atria. We hypothesize that UTP stimulates inotropy via p38 or ERK1/2 phosphorylation. We speculate that UTP may be a valuable target in the development of new drugs aimed at treating human systolic heart failure.