Purinergic Signaling Controls Spontaneous Activity in the Auditory System throughout Early Development.

Spontaneous bursts of electrical activity in the developing auditory system arise within the cochlea before hearing onset and propagate through future sound-processing circuits of the brain to promote maturation of auditory neurons. Studies in isolated cochleae revealed that this intrinsically generated activity is initiated by ATP release from inner supporting cells (ISCs), resulting in activation of purinergic autoreceptors, K+ efflux, and subsequent depolarization of inner hair cells. However, it is unknown when this activity emerges or whether different mechanisms induce activity during distinct stages of development. Here we show that spontaneous electrical activity in mouse cochlea from both sexes emerges within ISCs during the late embryonic period, preceding the onset of spontaneous correlated activity in inner hair cells and spiral ganglion neurons, which begins at birth and follows a base to apex developmental gradient. At all developmental ages, pharmacological inhibition of P2Y1 purinergic receptors dramatically reduced spontaneous activity in these three cell types. Moreover, in vivo imaging within the inferior colliculus revealed that auditory neurons within future isofrequency zones exhibit coordinated neural activity at birth. The frequency of these discrete bursts increased progressively during the postnatal prehearing period yet remained dependent on P2RY1. Analysis of mice with disrupted cholinergic signaling in the cochlea indicate that this efferent input modulates, rather than initiates, spontaneous activity before hearing onset. Thus, the auditory system uses a consistent mechanism involving ATP release from ISCs and activation of P2RY1 autoreceptors to elicit coordinated excitation of neurons that will process similar frequencies of sound.SIGNIFICANCE STATEMENT In developing sensory systems, groups of neurons that will process information from similar sensory space exhibit highly correlated electrical activity that is critical for proper maturation and circuit refinement. Defining the period when this activity is present, the mechanisms responsible and the features of this activity are crucial for understanding how spontaneous activity influences circuit development. We show that, from birth to hearing onset, the auditory system relies on a consistent mechanism to elicit correlate firing of neurons that will process similar frequencies of sound. Targeted disruption of this activity will increase our understanding of how these early circuits mature and may provide insight into processes responsible for developmental disorders of the auditory system.

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.

Preserved Calretinin Interneurons in an App Model of Alzheimer's Disease Disrupt Hippocampal Inhibition via Upregulated P2Y1 Purinoreceptors.

To understand the pathogenesis of specific neuronal circuit dysfunction in Alzheimer's disease (AD), we investigated the fate of three subclasses of "modulatory interneurons" in hippocampal CA1 using the AppNL-F/NL-F knock-in mouse model of AD. Cholecystokinin- and somatostatin-expressing interneurons were aberrantly hyperactive preceding the presence of the typical AD hallmarks: neuroinflammation and amyloid-ß (Aß) accumulation. These interneurons showed an age-dependent vulnerability to Aß penetration and a reduction in density and coexpression of the inhibitory neurotransmitter GABA synthesis enzyme, glutamic acid decarboxylase 67 (GAD67), suggesting a loss in their inhibitory function. However, calretinin (CR) interneurons-specialized to govern only inhibition, showed resilience to Aß accumulation, preservation of structure, and displayed synaptic hyperinhibition, despite the lack of inhibitory control of CA1 excitatory pyramidal cells from midstages of the disease. This aberrant inhibitory homeostasis observed in CA1 CR cells and pyramidal cells was "normalized" by blocking P2Y1 purinoreceptors, which were "upregulated" and strongly expressed in CR cells and astrocytes in AppNL-F/NL-F mice in the later stages of AD. In summary, AD-associated cell-type selective destruction of inhibitory interneurons and disrupted inhibitory homeostasis rectified by modulation of the upregulated purinoreceptor system may serve as a novel therapeutic strategy to normalize selective dysfunctional synaptic homeostasis during pathogenesis of AD.

Context-Specific Switch from Anti- to Pro-epileptogenic Function of the P2Y1 Receptor in Experimental Epilepsy.

Extracellular ATP activates inflammatory responses to tissue injury. It is also implicated in establishing lasting network hyperexcitability in the brain by acting upon independent receptor systems. Whereas the fast-acting P2X channels have well-established roles driving neuroinflammation and increasing hyperexcitability, the slower-acting metabotropic P2Y receptors have received much less attention. Recent studies of P2Y1 receptor function in seizures and epilepsy have produced contradictory results, suggesting that the role of this receptor during seizure pathology may be highly sensitive to context. Here, by using male mice, we demonstrate that the metabotropic P2Y1 receptor mediates either proconvulsive or anticonvulsive responses, dependent on the time point of activation in relation to the induction of status epilepticus. P2Y1 deficiency or a P2Y1 antagonist (MRS2500) administered before a chemoconvulsant, exacerbates epileptiform activity, whereas a P2Y1 agonist (MRS2365) administered at this time point is anticonvulsant. When these drugs are administered after the onset of status epilepticus, however, their effect on seizure severity is reversed, with the antagonist now anticonvulsant and the agonist proconvulsant. This result was consistent across two different mouse models of status epilepticus (intra-amygdala kainic acid and intraperitoneal pilocarpine). Pharmacologic P2Y1 blockade during status epilepticus reduces also associated brain damage, delays the development of epilepsy and, when applied during epilepsy, suppresses spontaneous seizures, in mice. Our data show a context-specific role for P2Y1 during seizure pathology and demonstrate that blocking P2Y1 after status epilepticus and during epilepsy has potent anticonvulsive effects, suggesting that P2Y1 may be a novel candidate for the treatment of drug-refractory status epilepticus and epilepsy.SIGNIFICANCE STATEMENT This is the first study to fully characterize the contribution of a metabotropic purinergic P2Y receptor during acute seizures and epilepsy. The findings suggest that targeting P2Y1 may offer a potential novel treatment strategy for drug-refractory status epilepticus and epilepsy. Our data demonstrate a context-specific role of P2Y1 activation during seizures, switching from a proconvulsive to an anticonvulsive role depending on physiopathological context. Thus, our study provides a possible explanation for seemingly conflicting results obtained between studies of different brain diseases where P2Y1 targeting has been proposed as a potential treatment strategy and highlights that the timing of pharmacological interventions is of critical importance to the understanding of how receptors contribute to the generation of seizures and the development of epilepsy.

Role of Purinergic Receptor P2Y1 in Spatiotemporal Ca2+ Dynamics in Astrocytes.

Fine processes of astrocytes enwrap synapses and are well positioned to sense neuronal information via synaptic transmission. In rodents, astrocyte processes sense synaptic transmission via Gq-protein coupled receptors (GqPCR), including the P2Y1 receptor (P2Y1R), to generate Ca2+ signals. Astrocytes display numerous spontaneous microdomain Ca2+ signals; however, it is not clear whether such signals are due to local synaptic transmission and/or in what timeframe astrocytes sense local synaptic transmission. To ask whether GqPCRs mediate microdomain Ca2+ signals, we engineered mice (both sexes) to specifically overexpress P2Y1Rs in astrocytes, and we visualized Ca2+ signals via a genetically encoded Ca2+ indicator, GCaMP6f, in astrocytes from adult mice. Astrocytes overexpressing P2Y1Rs showed significantly larger Ca2+ signals in response to exogenously applied ligand and to repetitive electrical stimulation of axons compared with controls. However, we found no evidence of increased microdomain Ca2+ signals. Instead, Ca2+ waves appeared and propagated to occupy areas that were up to 80-fold larger than microdomain Ca2+ signals. These Ca2+ waves accounted for only 2% of total Ca2+ events, but they were 1.9-fold larger and 2.9-fold longer in duration than microdomain Ca2+ signals at processes. Ca2+ waves did not require action potentials for their generation and occurred in a probenecid-sensitive manner, indicating that the endogenous ligand for P2Y1R is elevated independently of synaptic transmission. Our data suggest that spontaneous microdomain Ca2+ signals occur independently of P2Y1R activation and that astrocytes may not encode neuronal information in response to synaptic transmission at a point source of neurotransmitter release.SIGNIFICANCE STATEMENT Astrocytes are thought to enwrap synapses with their processes to receive neuronal information via Gq-protein coupled receptors (GqPCRs). Astrocyte processes display numerous microdomain Ca2+ signals that occur spontaneously. To determine whether GqPCRs play a role in microdomain Ca2+ signals and the timeframe in which astrocytes sense neuronal information, we engineered mice whose astrocytes specifically overexpress the P2Y1 receptor, a major GqPCR in astrocytes. We found that overexpression of P2Y1 receptors in astrocytes did not increase microdomain Ca2+ signals in astrocyte processes but caused Ca2+ wavelike signals. Our data indicate that spontaneous microdomain Ca2+ signals do not require activation of P2Y1 receptors.

Release of ATP by pre-Bötzinger complex astrocytes contributes to the hypoxic ventilatory response via a Ca2+ -dependent P2Y1 receptor mechanism.

KEY POINTS: The ventilatory response to reduced oxygen (hypoxia) is biphasic, comprising an initial increase in ventilation followed by a secondary depression. Our findings indicate that, during hypoxia, astrocytes in the pre-Bötzinger complex (preBötC), a critical site of inspiratory rhythm generation, release a gliotransmitter that acts via P2Y1 receptors to stimulate ventilation and reduce the secondary depression. In vitro analyses reveal that ATP excitation of the preBötC involves P2Y1 receptor-mediated release of Ca2+ from intracellular stores. By identifying a role for gliotransmission and the sites, P2 receptor subtype, and signalling mechanisms via which ATP modulates breathing during hypoxia, these data advance our understanding of the mechanisms underlying the hypoxic ventilatory response and highlight the significance of purinergic signalling and gliotransmission in homeostatic control. Clinically, these findings are relevant to conditions in which hypoxia and respiratory depression are implicated, including apnoea of prematurity, sleep disordered breathing and congestive heart failure. ABSTRACT: The hypoxic ventilatory response (HVR) is biphasic, consisting of a phase I increase in ventilation followed by a secondary depression (to a steady-state phase II) that can be life-threatening in premature infants who suffer from frequent apnoeas and respiratory depression. ATP released in the ventrolateral medulla oblongata during hypoxia attenuates the secondary depression. We explored a working hypothesis that vesicular release of ATP by astrocytes in the pre-Bötzinger Complex (preBötC) inspiratory rhythm-generating network acts via P2Y1 receptors to mediate this effect. Blockade of vesicular exocytosis in preBötC astrocytes bilaterally (using an adenoviral vector to specifically express tetanus toxin light chain in astrocytes) reduced the HVR in anaesthetized rats, indicating that exocytotic release of a gliotransmitter within the preBötC contributes to the hypoxia-induced increases in ventilation. Unilateral blockade of P2Y1 receptors in the preBötC via local antagonist injection enhanced the secondary respiratory depression, suggesting that a significant component of the phase II increase in ventilation is mediated by ATP acting at P2Y1 receptors. In vitro responses of the preBötC inspiratory network, preBötC inspiratory neurons and cultured preBötC glia to purinergic agents demonstrated that the P2Y1 receptor-mediated increase in fictive inspiratory frequency involves Ca2+ recruitment from intracellular stores leading to increases in intracellular Ca2+ ([Ca2+ ]i ) in inspiratory neurons and glia. These data suggest that ATP is released by preBötC astrocytes during hypoxia and acts via P2Y1 receptors on inspiratory neurons (and/or glia) to evoke Ca2+ release from intracellular stores and an increase in ventilation that counteracts the hypoxic respiratory depression.

KV 7/M channels as targets for lipopolysaccharide-induced inflammatory neuronal hyperexcitability.

KEY POINTS: Neuroinflammation associated with CNS insults leads to neuronal hyperexcitability, which may culminate in epileptiform discharges. Application of the endotoxin lipopolysaccharide (LPS) to brain tissue initiates a neuroinflammatory cascade, providing an experimental model to study the mechanisms of neuroinflammatory neuronal hyperexcitability. Here we show that LPS application to hippocampal slices markedly enhances the excitability of CA1 pyramidal cells by inhibiting a specific potassium current, the M-current, generated by KV 7/M channels, which controls the excitability of almost every neuron in the CNS. The LPS-induced M-current inhibition is triggered by sequential activation of microglia, astrocytes and pyramidal cells, mediated by metabotropic purinergic and glutamatergic transmission, leading to blockade of KV 7/M channels by calcium released from intracellular stores. The identification of the downstream molecular target of neuroinflammation, namely the KV 7/M channel, potentially has far reaching implications for the understanding and treatment of many acute and chronic brain disorders. ABSTRACT: Acute brain insults and many chronic brain diseases manifest an innate inflammatory response. The hallmark of this response is glia activation, which promotes repair of damaged tissue, but also induces structural and functional changes that may lead to an increase in neuronal excitability. We have investigated the mechanisms involved in the modulation of neuronal activity by acute inflammation. Initiating inflammatory responses in hippocampal tissue rapidly led to neuronal depolarization and repetitive firing even in the absence of active synaptic transmission. This action was mediated by a complex metabotropic purinergic and glutamatergic glia-to-neuron signalling cascade, leading to the blockade of neuronal KV 7/M channels by Ca2+ released from internal stores. These channels generate the low voltage-activating, non-inactivating M-type K+ current (M-current) that controls intrinsic neuronal excitability, and its inhibition was the predominant cause of the inflammation-induced hyperexcitability. Our discovery that the ubiquitous KV 7/M channels are the downstream target of the inflammation-induced cascade, has far reaching implications for the understanding and treatment of many acute and chronic brain disorders.

Impaired Purinergic Regulation of the Glial (Müller) Cell Volume in the Retina of Transgenic Rats Expressing Defective Polycystin-2.

Retinal glial (Müller) cells possess an endogenous purinergic signal transduction cascade which normally prevents cellular swelling in osmotic stress. The cascade can be activated by osmotic or glutamate receptor-dependent ATP release. We determined whether activation of this cascade is altered in Müller cells of transgenic rats that suffer from a slow photoreceptor degeneration due to the expression of a truncated human cilia gene polycystin-2 (CMV-PKD21/703 HA). Age-matched Sprague-Dawley rats served as control. Retinal slices were superfused with a hypoosmotic solution (60 % osmolarity). Müller cells in retinas of PKD21/703 rats swelled immediately in hypoosmotic stress; this was not observed in control retinas. Pharmacological blockade of P2Y1 or adenosine A1 receptors induced osmotic swelling of Müller cells from control rats. The swelling induced by the P2Y1 receptor antagonist was mediated by induction of oxidative-nitrosative stress, mitochondrial dysfunction, production of inflammatory lipid mediators, and a sodium influx from the extracellular space. Exogenous VEGF or glutamate prevented the hypoosmotic swelling of Müller cells from PKD21/703 rats; this effect was mediated by activation of the purinergic signaling cascade. In neuroretinas of PKD21/703 rats, the gene expression levels of P2Y1 and A1 receptors, pannexin-1, connexin 45, NTPDases 1 and 2, and various subtypes of nucleoside transporters are elevated compared to control. The data may suggest that the osmotic swelling of Müller cells from PKD21/703 rats is caused by an abrogation of the osmotic ATP release while the glutamate-induced ATP release is functional. In the normal retina, ATP release and autocrine P2Y1 receptor activation serve to inhibit the induction of oxidative-nitrosative stress, mitochondrial dysfunction, and production of inflammatory lipid mediators, which otherwise will induce a sodium influx and cytotoxic Müller cell swelling under anisoosmotic conditions. Purinergic receptors may represent a target for the protection of retinal glial cells from mitochondrial oxidative stress.

A mechanistic model of a PDGFRα(+) cell.

A novel platelet-derived growth factor receptor alpha-positive cell (PDGFRα(+)) has recently been identified as part of the purinergic inhibitory neural control mechanism in the gastrointestinal (GI) tract. The mechanism through which PDGFRα(+) cells mediate GI muscle relaxation has been found to be associated with the purine receptors P2Y1 and apamin-sensitive SK3 channels that are highly expressed in these cells. This study aims to develop a mechanistic model elucidating a proposed mechanism through which PDGFRα(+) cells contribute to purinergic inhibitory neuromuscular transmission. In accordance with recent experimental findings, the model describes how the binding of neurotransmitters, released from enteric neurons, triggers the release of Ca(2+) from the endoplasmic reticulum in the PDGFRα(+) cells, and how this subsequently leads to large amplitude transient outward currents, which in turn hyperpolarize the cell. The model has been validated against experimental recordings and good agreement was found under normal and pharmacologically-altered conditions. This model demonstrates the feasibility of the proposed mechanism and provides a basis for understanding the mechanism underlying purinergic control of colonic motility.

Endogenous electric currents might guide rostral migration of neuroblasts.

Mechanisms that guide directional migration of neuroblasts from the subventricular zone (SVZ) are not well understood. We report here that endogenous electric currents serve as a guidance cue for neuroblast migration. We identify the existence of naturally occurring electric currents (1.5±0.6 µA/cm(2), average field strength of ∼3 mV/mm) along the rostral migration path in adult mouse brain. Electric fields of similar strength direct migration of neuroblasts from the SVZ in culture and in brain slices. The purinergic receptor P2Y1 mediates this migration. The results indicate that naturally occurring electric currents serve as a new guidance mechanism for rostral neuronal migration.

Purinergic signaling in human pluripotent stem cells is regulated by the housekeeping gene encoding hypoxanthine guanine phosphoribosyltransferase.

Lesch-Nyhan disease (LND) is an X-linked genetic disorder caused by mutations of the hypoxanthine guanine phosphoribosyltransferase (HPRT) purine biosynthesis gene and characterized by aberrant purine metabolism, deficient basal ganglia dopamine levels, dystonia, and severe neurobehavioral manifestations, including compulsive self-injurious behavior. Although available evidence has identified important roles for purinergic signaling in brain development, the mechanisms linking HPRT deficiency, purinergic pathways, and neural dysfunction of LND are poorly understood. In these studies aimed at characterizing purinergic signaling in HPRT deficiency, we used a lentivirus vector stably expressing an shRNA targeted to the HPRT gene to produce HPRT-deficient human CVB induced pluripotent stem cells and human HUES11 embryonic stem cells. Both CVB and HUES11 cells show >99% HPRT knockdown and demonstrate markedly decreased expression of the purinergic P2Y1 receptor mRNA. In CVB cells, P2Y1 mRNA and protein down-regulation by HPRT knockdown is refractory to activation by the P2Y1 receptor agonist ATP and shows aberrant purinergic signaling, as reflected by marked deficiency of the transcription factor pCREB and constitutive activation of the MAP kinases phospho-ERK1/2. Moreover, HPRT-knockdown CVB cells also demonstrate marked reduction of phosphorylated ß-catenin. These results indicate that the housekeeping gene HPRT regulates purinergic signaling in pluripotent human stem cells, and that this regulation occurs at least partly through aberrant P2Y1-mediated expression and signaling. We propose that such mechanisms may play a role in the neuropathology of HPRT-deficiency LND and may point to potential molecular targets for modulation of this intractable neurological phenotype.

P2Y1 receptor-mediated potentiation of inspiratory motor output in neonatal rat in vitro.

PreBötzinger complex inspiratory rhythm-generating networks are excited by metabotropic purinergic receptor subtype 1 (P2Y1R) activation. Despite this, and the fact that inspiratory MNs express P2Y1Rs, the role of P2Y1Rs in modulating motor output is not known for any MN pool. We used rhythmically active brainstem-spinal cord and medullary slice preparations from neonatal rats to investigate the effects of P2Y1R signalling on inspiratory output of phrenic and XII MNs that innervate diaphragm and airway muscles, respectively. MRS2365 (P2Y1R agonist, 0.1 mm) potentiated XII inspiratory burst amplitude by 60 ± 9%; 10-fold higher concentrations potentiated C4 burst amplitude by 25 ± 7%. In whole-cell voltage-clamped XII MNs, MRS2365 evoked small inward currents and potentiated spontaneous EPSCs and inspiratory synaptic currents, but these effects were absent in TTX at resting membrane potential. Voltage ramps revealed a persistent inward current (PIC) that was attenuated by: flufenamic acid (FFA), a blocker of the Ca(2+)-dependent non-selective cation current ICAN; high intracellular concentrations of BAPTA, which buffers Ca(2+) increases necessary for activation of ICAN; and 9-phenanthrol, a selective blocker of TRPM4 channels (candidate for ICAN). Real-time PCR analysis of mRNA extracted from XII punches and laser-microdissected XII MNs revealed the transcript for TRPM4. MRS2365 potentiated the PIC and this potentiation was blocked by FFA, which also blocked the MRS2365 potentiation of glutamate currents. These data suggest that XII MNs are more sensitive to P2Y1R modulation than phrenic MNs and that the P2Y1R potentiation of inspiratory output occurs in part via potentiation of TRPM4-mediated ICAN, which amplifies inspiratory inputs.

Purinergic inhibitory regulation of murine detrusor muscles mediated by PDGFRα+ interstitial cells.

Purines induce transient contraction and prolonged relaxation of detrusor muscles. Transient contraction could be due to activation of inward currents in smooth muscle cells, but the mechanism of purinergic relaxation has not been determined. We recently reported a new class of interstitial cells in detrusor muscles and showed that these cells could be identified with antibodies against platelet-derived growth factor receptor-α (PDGFRα(+) cells). The current density of small conductance Ca(2+)-activated K(+) (SK) channels in these cells is far higher (∼100 times) than in smooth muscle cells. Thus, we examined purinergic receptor (P2Y) mediated SK channel activation as a mechanism for purinergic relaxation. P2Y receptors (mainly P2ry1 gene) were highly expressed in PDGFRα(+) cells. Under voltage clamp conditions, ATP activated large outward currents in PDGFRα(+) cells that were inhibited by blockers of SK channels. ATP also induced significant hyperpolarization under current clamp conditions. A P2Y1 agonist, MRS2365, mimicked the effects of ATP, and a P2Y1 antagonist, MRS2500, inhibited ATP-activated SK currents. Responses to ATP were largely abolished in PDGFRα(+) cells of P2ry1(-/-) mice, and no response was elicited by MRS2365 in these cells. A P2X receptor agonist had no effect on PDGFRα(+) cells but, like ATP, activated transient inward currents in smooth muscle cells (SMCs). A P2Y1 antagonist decreased nerve-evoked relaxation. These data suggest that purines activate SK currents via mainly P2Y1 receptors in PDGFRα(+) cells. Our findings provide an explanation for purinergic relaxation in detrusor muscles and show that there are no discrete inhibitory nerve fibres. A dual receptive field for purines provides the basis for inhibitory neural regulation of excitability.

[Purine receptors and associated signaling systems in regulation of platelet function].

Purine receptors of platelets play key role in hemostasis and thrombogenesis and they are used as targets for antiplatelet therapy. Platelets express three subtypes of purine receptors--P2X1, P2Y1 and P2Y12. The expression, ligands and sensitivity of purine receptors of platelets and their role in thrombogenesis are discussed in this article. Authors debate information about basic molecular mechanisms, links with intracellular signaling and biological effects of purine receptors activation. In conclusion, understanding of signaling mechanisms of purine receptors is a basis for further pharmacological strategy and effective antiplatelet therapy development.

Purinergic signaling promotes proliferation of adult mouse subventricular zone cells.

In adult mammalian brains, neural stem cells (NSCs) exist in the subventricular zone (SVZ), where persistent neurogenesis continues throughout life. Those NSCs produce neuroblasts that migrate into the olfactory bulb via formation of transit-amplifying cells, which are committed precursor cells of the neuronal lineage. In this SVZ niche, cell-cell communications conducted by diffusible factors as well as physical cell-cell contacts are important for the regulation of the proliferation and fate determination of NSCs. Previous studies have suggested that extracellular purinergic signaling, which is mediated by purine compounds such as ATP, plays important roles in cell-cell communication in the CNS. Purinergic signaling also promotes the proliferation of adult NSCs in vitro. However, the in vivo roles of purinergic signaling in the neurogenic niche still remain unknown. In this study, ATP infusion into the lateral ventricle of the mouse brain resulted in an increase in the numbers of rapidly dividing cells and Mash1-positive transit-amplifying cells (Type C cells) in the SVZ. Mash1-positive cells express the P2Y1 purinergic signaling receptor and infusion of the P2Y1 receptor-specific antagonist MRS2179 decreased the number of rapidly dividing bromodeoxyuridine (BrdU)-positive cells and Type C cells. Moreover, a 17% reduction of rapidly dividing BrdU-positive cells and a 19% reduction of Mash1-positive cells were observed in P2Y1 knock-out mice. Together, these results suggest that purinergic signaling promotes the proliferation of rapidly dividing cells and transit-amplifying cells, in the SVZ niche through the P2Y1 receptor.

Involvement of glial P2Y1 receptors in cognitive deficit after focal cerebral stroke in a rodent model.

BACKGROUND: Neuroinflammation is associated with many conditions that lead to dementia, such as cerebrovascular disorders or Alzheimer's disease. However, the specific role of neuroinflammation in the progression of cognitive deficits remains unclear. To understand the molecular mechanisms underlying these events we used a rodent model of focal cerebral stroke, which causes deficits in hippocampus-dependent cognitive function. METHODS: Cerebral stroke was induced by middle cerebral artery occlusion (MCAO). Hippocampus-dependent cognitive function was evaluated by a contextual fear conditioning test. The glial neuroinflammatory responses were investigated by immunohistochemical evaluation and diffusion tensor MRI (DTI). We used knockout mice for P2Y1 (P2Y1KO), a glial ADP/ATP receptor that induces the release of proinflammatory cytokines, to examine the links among P2Y1-mediated signaling, the neuroinflammatory response, and cognitive function. RESULTS: Declines in cognitive function and glial neuroinflammatory response were observed after MCAO in both rats and mice. Changes in the hippocampal tissue were detected by DTI as the mean diffusivity (MD) value, which corresponded with the cognitive decline at 4 days, 1 week, 3 weeks, and 2 months after MCAO. Interestingly, the P2Y1KO mice with MCAO showed a decline in sensory-motor function, but not in cognition. Furthermore, the P2Y1KO mice showed neither a hippocampal glial neuroinflammatory response (as assessed by immunohistochemistry) nor a change in hippocampal MD value after MCAO. In addition, wild-type mice treated with a P2Y1-specific antagonist immediately after reperfusion did not show cognitive decline. CONCLUSION: Our findings indicate that glial P2Y1 receptors are involved in the hippocampal inflammatory response. The findings from this study may contribute to the development of a therapeutic strategy for brain infarction, targeting the P2Y1 receptor.

[Molecular mechanisms of platelet activation]. / Les mécanismes moléculaires de l'activation plaquettaire.

The main role of blood platelets is to ensure vascular integrity and hemostasis in case of vascular damage. The platelet functions involved in these physiological processes are also at work in arterial thrombosis, which is a dramatic complication of atherosclerosis that may lead to vascular occlusion. These functions of platelets include their ability to adhere to the injured vessel wall, to be activated by contact with various substrates and soluble activators, and to form aggregates stabilized by a fibrin network. Platelets are also involved in metastasis, various inflammatory processes, innate and adaptive immune defenses, and embryonic development. These roles are supported by multiple molecular mechanisms, some of which are common to several functions while others are distinct. Defects in one or other of these mechanisms do not necessarily disrupt all platelet functions.

Major contribution of the P2Y1receptor in purinergic regulation of TNFα-induced vascular inflammation.

BACKGROUND: Atherosclerosis is an inflammatory disease, and extracellular nucleotides are one of the factors possibly involved in vascular inflammation. The P2Y(1) receptor for adenosine 5'-diphosphate has been shown to be involved in the development of atherosclerosis in apolipoprotein E--deficient mice. Our aim is to determine whether the endothelial P2Y(1) receptor plays a role in leukocyte recruitment during vascular inflammation and characterize underlying mechanisms. METHODS AND RESULTS: We show here that the P2Y(1) receptor plays a role in leukocyte recruitment in inflamed mouse femoral arteries. Moreover, in wild-type bone marrow--transplanted chimeric P2Y(1)-deficient mice with an apolipoprotein E--deficient background, a strong reduction of adhesion molecule--dependent leukocyte recruitment was observed after local injection of tumor necrosis factor and interleukin 1ß, excluding a role for the platelet or other hematopoietic cell type P2Y(1) in these events. Similarly, the in vitro adhesion of isolated mouse monocytes to tumor necrosis factor α--stimulated murine endothelial cell monolayers and their migration across the cell layers were strongly reduced in P2Y(1)-deficient compared with wild-type endothelial cells, as was the expression of the adhesion molecules P-selectin, Vascular cell adhesion molecule 1, and intercellular adhesion molecule 1. Pharmacological inhibition using the selective antagonist MRS2500 also resulted in decreased expression of adhesion molecules. These events are related to the p38 mitogen-activated protein kinase and activating transcription factor 2 pathway. Finally, in vivo administration of MRS2500 resulted in strong reduction of leukocyte recruitment in inflamed femoral arteries of apolipoprotein E--deficient mice. CONCLUSIONS: The data highlight a key role of the endothelial P2Y(1) receptor in acute vascular inflammation. Pharmacological targeting the P2Y(1) receptor could represent a promising approach for the treatment of vascular inflammation.

P2Y1 receptor modulation of Ca2+-activated K+ currents in medium-sized neurons from neonatal rat striatal slices.

ATP signaling to neurons and glia in the nervous system occurs via activation of both P2Y and P2X receptors. Here, we investigated the effects of P2Y(1) receptor stimulation in developing striatal medium-sized neurons using patch-clamp recordings from acute brain slices of 7- and 28-day-old rats. Application of the selective P2Y(1) receptor agonist 2-(Methylthio) ADP trisodium salt (2-MeSADP; 250 nM) increased outward K(+) currents evoked by a ramp depolarization protocol in voltage-clamp recordings. This effect was observed in 59 out of 82 cells (72%) and was blocked completely by the P2Y(1) antagonist, 2'-deoxy-N(6)-methyl adenosine 3',5'-diphosphate. The averaged 2-MeSADP-sensitive conductance was fitted by the sum of a linear conductance and a Boltzmann relation, giving one-half activation voltage of -14.2 mV and an equivalent charge of 2.91. The 2MeSADP-mediated effect was sensitive to submillimolar concentrations of tetraethylammonium (TEA; 200 µM), to 200 nM iberiotoxin and to 100 nM apamin, suggesting the involvement of both big and small potassium (BK and SK, respectively) calcium-activated channels. In current-clamp experiments, 2-MeSADP decreased depolarization-evoked action potential (AP) firing in all 26 cells investigated, and this effect was reversed by TEA and by apamin but not by iberiotoxin. We conclude that the stimulation of P2Y(1) receptors in developing striatal neurons leads to activation of calcium-activated potassium channels [I(K(Ca))] of both BK and SK subtypes, the latter responsible for decreasing the frequency of AP firing in response to current injection. Therefore, P2Y(1) signaling leading to activation of I(K(Ca)) may be important in regulating the activity of medium-sized neurons in the striatum.

P2Y1 and P2Y13 purinergic receptors mediate Ca2+ signaling and proliferative responses in pulmonary artery vasa vasorum endothelial cells.

Extracellular ATP and ADP have been shown to exhibit potent angiogenic effects on pulmonary artery adventitial vasa vasorum endothelial cells (VVEC). However, the molecular signaling mechanisms of extracellular nucleotide-mediated angiogenesis remain not fully elucidated. Since elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) is required for cell proliferation and occurs in response to extracellular nucleotides, this study was undertaken to delineate the purinergic receptor subtypes involved in Ca(2+) signaling and extracellular nucleotide-mediated mitogenic responses in VVEC. Our data indicate that stimulation of VVEC with extracellular ATP resulted in the elevation of [Ca(2+)](i) via Ca(2+) influx through plasma membrane channels as well as Ca(2+) mobilization from intracellular stores. Moreover, extracellular ATP induced simultaneous Ca(2+) responses in both cytosolic and nuclear compartments. An increase in [Ca(2+)](i) was observed in response to a wide range of purinergic receptor agonists, including ATP, ADP, ATPγS, ADPßS, UTP, UDP, 2-methylthio-ATP (MeSATP), 2-methylthio-ADP (MeSADP), and BzATP, but not adenosine, AMP, diadenosine tetraphosphate, αßMeATP, and ßγMeATP. Using RT-PCR, we identified mRNA for the P2Y1, P2Y2, P2Y4, P2Y13, P2Y14, P2X2, P2X5, P2X7, A1, A2b, and A3 purinergic receptors in VVEC. Preincubation of VVEC with the P2Y1 selective antagonist MRS2179 and the P2Y13 selective antagonist MRS2211, as well as with pertussis toxin, attenuated at varying degrees agonist-induced intracellular Ca(2+) responses and activation of ERK1/2, Akt, and S6 ribosomal protein, indicating that P2Y1 and P2Y13 receptors play a major role in VVEC growth responses. Considering the broad physiological implications of purinergic signaling in the regulation of angiogenesis and vascular homeostasis, our findings suggest that P2Y1 and P2Y13 receptors may represent novel and specific targets for treatment of pathological vascular remodeling involving vasa vasorum expansion.