ABSTRACT
Early life stressors, such as social isolation (SI), can disrupt brain development contributing to behavioral and neurochemical alterations in adulthood. Purinergic receptors and ectonucleotidases are key regulators of brain development in embryonic and postnatal periods, and they are involved in several psychiatric disorders, including schizophrenia. The extracellular ATP drives purinergic signaling by activating P2X and P2Y receptors and it is hydrolyzed by ectonucleotidases in adenosine, which activates P1 receptors. The purpose of this study was to investigate if SI, a rodent model used to replicate abnormal behavior relevant to schizophrenia, impacts purinergic signaling. Male Wistar rats were reared from weaning in group-housed or SI conditions for 8 weeks. SI rats exhibited impairment in prepulse inhibition and social interaction. SI presented increased ADP levels in cerebrospinal fluid and ADP hydrolysis in the hippocampus and striatum synaptosomes. Purinergic receptor expressions were upregulated in the prefrontal cortex and downregulated in the hippocampus and striatum. A2A receptors were differentially expressed in SI prefrontal cortex and the striatum, suggesting distinct roles in these brain structures. SI also presented decreased ADP, adenosine, and guanosine levels in the cerebrospinal fluid in response to D-amphetamine. Like patients with schizophrenia, uric acid levels were prominently increased in SI rats after D-amphetamine challenge. We suggest that the SI-induced deficits in prepulse inhibition might be related to the SI-induced changes in purinergic signaling. We provide new evidence that purinergic signaling is markedly affected in a rat model relevant to schizophrenia, pointing out the importance of purinergic system in psychiatry conditions.
Subject(s)
Receptors, Purinergic , Signal Transduction , Social Isolation , Adenosine Diphosphate/cerebrospinal fluid , Animals , Behavior, Animal , Central Nervous System Stimulants/pharmacology , Dextroamphetamine/pharmacology , Male , Nucleotidases/metabolism , Rats , Rats, Wistar , Receptor, Adenosine A2A/metabolism , Receptors, Purinergic P2X/metabolism , Receptors, Purinergic P2Y/metabolism , Reflex, Startle , Schizophrenic Psychology , Social Behavior , Social Isolation/psychology , WeaningABSTRACT
The incidence of infectious diseases affecting the central nervous system (CNS) has been increasing over the last several years. Among the reasons for the expansion of these diseases and the appearance of new neuropathogens are globalization, global warming, and the increased proximity between humans and wild animals due to human activities such as deforestation. Neurotropism affecting normal brain function is shared by organisms such as viruses, bacteria, fungi, and parasites. Neuroinfections caused by these agents activate immune responses, inducing neuroinflammation, excitotoxicity, and neurodegeneration. Purinergic signaling is an evolutionarily conserved signaling pathway associated with these neuropathologies. During neuroinfections, host cells release ATP as an extracellular danger signal with pro-inflammatory activities. ATP is metabolized to its derivatives by ectonucleotidases such as CD39 and CD73; ATP and its metabolites modulate neuronal and immune mechanisms through P1 and P2 purinergic receptors that are involved in pathophysiological mechanisms of neuroinfections. In this review we discuss the beneficial or deleterious effects of various components of the purinergic signaling pathway in infectious diseases that affect the CNS, including human immunodeficiency virus (HIV-1) infection, herpes simplex virus type 1 (HSV-1) infection, bacterial meningitis, sepsis, cryptococcosis, toxoplasmosis, and malaria. We also provide a description of this signaling pathway in emerging viral infections with neurological implications such as Zika and SARS-CoV-2.
Subject(s)
Central Nervous System Infections/metabolism , Receptors, Purinergic P1/metabolism , Receptors, Purinergic P2X/metabolism , Receptors, Purinergic P2Y/metabolism , AIDS Dementia Complex/metabolism , Betacoronavirus , COVID-19 , Coronavirus Infections/metabolism , Encephalitis, Herpes Simplex/metabolism , Humans , Malaria/metabolism , Meningitis, Bacterial/metabolism , Meningitis, Cryptococcal/metabolism , Pandemics , Pneumonia, Viral/metabolism , SARS-CoV-2 , Sepsis/metabolism , Signal Transduction , Toxoplasmosis, Cerebral/metabolism , Zika Virus Infection/metabolismABSTRACT
The most common cause of dementia is Alzheimer's disease. The etiology of the disease is unknown, although considerable evidence suggests a critical role for the soluble oligomers of amyloid beta peptide (Aß). Because Aß increases the expression of purinergic receptors (P2XRs) in vitro and in vivo, we studied the functional correlation between long-term exposure to Aß and the ability of P2XRs to modulate network synaptic tone. We used electrophysiological recordings and Ca2+ microfluorimetry to assess the effects of chronic exposure (24 h) to Aß oligomers (0.5 µM) together with known inhibitors of P2XRs, such as PPADS and apyrase on synaptic function. Changes in the expression of P2XR were quantified using RT-qPCR. We observed changes in the expression of P2X1R, P2X7R and an increase in P2X2R; and also in protein levels in PC12 cells (143%) and hippocampal neurons (120%) with Aß. In parallel, the reduction on the frequency and amplitude of mEPSCs (72% and 35%, respectively) were prevented by P2XR inhibition using a low PPADS concentration. Additionally, the current amplitude and intracellular Ca2+ signals evoked by extracellular ATP were increased (70% and 75%, respectively), suggesting an over activation of purinergic neurotransmission in cells pre-treated with Aß. Taken together, our findings suggest that Aß disrupts the main components of synaptic transmission at both pre- and post-synaptic sites, and induces changes in the expression of key P2XRs, especially P2X2R; changing the neuromodulator function of the purinergic tone that could involve the P2X2R as a key factor for cytotoxic mechanisms. These results identify novel targets for the treatment of dementia and other diseases characterized by increased purinergic transmission.
Subject(s)
Amyloid beta-Peptides/pharmacology , Excitatory Postsynaptic Potentials/drug effects , Neurons/drug effects , Peptide Fragments/pharmacology , Receptors, Purinergic P2X/metabolism , Adenosine Triphosphate/pharmacology , Amyloid beta-Peptides/chemistry , Animals , Cell Survival/drug effects , Cells, Cultured , Cerebral Cortex/cytology , Disks Large Homolog 4 Protein/metabolism , Embryo, Mammalian , Female , Microtubule-Associated Proteins/metabolism , Neurotransmitter Agents/pharmacology , Patch-Clamp Techniques , Platelet Aggregation Inhibitors/pharmacology , Pregnancy , Pyridoxal Phosphate/analogs & derivatives , Pyridoxal Phosphate/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, Purinergic P2X/geneticsABSTRACT
ATP (adenosine 5'-triphosphate), one of the most ancient neurotransmitters, exerts essential functions in the brain, including neurotransmission and modulation of synaptic activity. Moreover, this nucleotide has been attributed with trophic properties and experimental evidence points to the participation of ATP-activated P2X and P2Y purinergic receptors in embryonic brain development as well as in adult neurogenesis for maintenance of normal brain functions and neuroregeneration upon brain injury. We discuss here the available data on purinergic P2 receptor expression and function during brain development and in the neurogenic zones of the adult brain, as well as the insights based on the use of in vitro stem cell cultures. While several P2 receptor subtypes were shown to be expressed during in vitro and in vivo neurogenesis, specific functions have been proposed for P2Y1, P2Y2 metabotropic as well as P2X2 ionotropic receptors to promote neurogenesis. Further, the P2X7 receptor is suggested to function in the maintenance of pools of neural stem and progenitor cells through induction of proliferation or cell death, depending on the microenvironment. Pathophysiological actions have been proposed for this receptor in worsening damage in brain disease. The P2X7 receptor and possibly additional P2 receptor subtypes have been implicated in pathophysiology of neurological diseases including Parkinson's disease, Alzheimer's disease and epilepsy. New strategies in cell therapy could involve modulation of purinergic signaling, either in the achievement of more effective protocols to obtain viable and homogeneous cell populations or in the process of functional engraftment of transplanted cells into the damaged brain. This article is part of the Special Issue entitled 'Purines in Neurodegeneration and Neuroregeneration'.
Subject(s)
Adenosine Triphosphate/metabolism , Brain/metabolism , Neurogenesis , Receptors, Purinergic P2X/metabolism , Receptors, Purinergic P2Y/metabolism , Animals , Brain/embryology , Cell Differentiation , Epilepsy/metabolism , Epilepsy/therapy , Humans , Neural Stem Cells/metabolism , Parkinson Disease/metabolism , Parkinson Disease/therapy , Receptors, Purinergic P2X7/metabolism , Receptors, Purinergic P2Y1/metabolism , Signal Transduction , Stem Cell TransplantationABSTRACT
Recent studies suggest that the toxic effects of Aß can be attributed to its capability to insert in membranes and form pore-like structures, which are permeable to cations and molecules such as ATP. Our working hypothesis is that Aß increases extracellular ATP causing activation of P2X receptors and potentiating excitatory synaptic activity. We found that soluble oligomers of ß-amyloid peptide increased cytosolic Ca(2+) 4-fold above control (415 ± 28% of control). Also, ATP leakage (157 ± 10% of control) was independent of extracellular Ca(2+), suggesting that ATP traveled from the cytosol through an Aß pore-mediated efflux and not from exocytotic mechanisms. The subsequent activation of P2XR by ATP can contribute to the cytosolic Ca(2+) increase observed with Aß. Additionally, we found that ß-amyloid oligomers bind preferentially to excitatory neurons inducing an increase in excitatory synaptic current frequency (248.1 ± 32.7%) that was blocked by the use of P2XR antagonists such as PPADS (Aß + PPADS: 110.9 ± 18.35%) or Apyrase plus DPCPX (Aß + inhibitors: 98.97 ± 17.4%). Taken together, we suggest that Aß induces excitotoxicity by binding preferentially to excitatory neuron membranes forming a non-selective pore and by increasing intracellular calcium by itself and through P2XR activation by extracellular ATP leading to an augmention in mEPSC activity. All these effects were blocked with a non-specific P2XR antagonist, indicating that part of the neurotoxicity of Aß is mediated by P2XR activation and facilitation of excitatory neurotransmitter release. These findings suggest that P2XR can be considered as a potential new target for the development of drugs or pharmacological tools to treat Alzheimer's disease. This article is part of the Special Issue entitled 'Synaptopathy--from Biology to Therapy'.
Subject(s)
Adenosine Triphosphate/metabolism , Alzheimer Disease/metabolism , Amyloid beta-Peptides/toxicity , Excitatory Postsynaptic Potentials/drug effects , Hippocampus/metabolism , Neurons/metabolism , Receptors, Purinergic P2X/metabolism , Amyloid beta-Peptides/metabolism , Animals , Calcium/metabolism , Cells, Cultured , Disease Models, Animal , Hippocampus/drug effects , Neurons/drug effects , Rats, Sprague-DawleyABSTRACT
Ligand-gated ion channels (LGICs) are cell surface integral proteins that mediate the fast neurotransmission in the nervous system. LGICs require auxiliary subunits for their trafficking, assembly and pharmacological modulation. Auxiliary subunits do not form functional homomeric receptors, but are reported to assemble with the principal subunits in order to modulate their pharmacological profiles. For example, nACh receptors are built at least by co-assemble of α and ß subunits, and the neuronal auxiliary subunits ß3 and α5 and muscle type ß, δ, γ, and ϵ determine the agonist affinity of these receptors. Serotonergic 5-HT3B, 5-HT3C, 5-HT3D and 5-HT3E are reported to assemble with the 5-HT3A subunit to modulate its pharmacological profile. Functional studies evaluating the role of γ2 and δ auxiliary subunits of GABAA receptors have made important advances in the understanding of the action of benzodiazepines, ethanol and neurosteroids. Glycine receptors are composed principally by α1-3 subunits and the auxiliary subunit ß determines their synaptic location and their pharmacological response to propofol and ethanol. NMDA receptors appear to be functional as heterotetrameric channels. So far, the existence of NMDA auxiliary subunits is controversial. On the other hand, Kainate receptors are modulated by NETO 1 and 2. AMPA receptors are modulated by TARPs, Shisa 9, CKAMP44, CNIH2-3 auxiliary proteins reported that controls their trafficking, conductance and gating of channels. P2X receptors are able to associate with auxiliary Pannexin-1 protein to modulate P2X7 receptors. Considering the pharmacological relevance of different LGICs auxiliary subunits in the present work we will highlight the therapeutic potential of these modulator proteins.
Subject(s)
Ligand-Gated Ion Channels/drug effects , Animals , Humans , Ion Channel Gating/drug effects , Ligand-Gated Ion Channels/chemistry , Ligand-Gated Ion Channels/metabolism , Models, Molecular , Protein Subunits , Receptors, AMPA/chemistry , Receptors, AMPA/drug effects , Receptors, AMPA/metabolism , Receptors, GABA-A/chemistry , Receptors, GABA-A/drug effects , Receptors, GABA-A/metabolism , Receptors, Glutamate/chemistry , Receptors, Glutamate/drug effects , Receptors, Glutamate/metabolism , Receptors, Glycine/chemistry , Receptors, Glycine/drug effects , Receptors, Glycine/metabolism , Receptors, Kainic Acid/chemistry , Receptors, Kainic Acid/drug effects , Receptors, Kainic Acid/metabolism , Receptors, N-Methyl-D-Aspartate/chemistry , Receptors, N-Methyl-D-Aspartate/drug effects , Receptors, N-Methyl-D-Aspartate/metabolism , Receptors, Nicotinic/chemistry , Receptors, Nicotinic/drug effects , Receptors, Nicotinic/metabolism , Receptors, Purinergic P2X/chemistry , Receptors, Purinergic P2X/drug effects , Receptors, Purinergic P2X/metabolism , Receptors, Serotonin, 5-HT3/chemistry , Receptors, Serotonin, 5-HT3/drug effects , Receptors, Serotonin, 5-HT3/metabolismABSTRACT
ATP is a key energetic molecule, fundamental to cell function, which also has an important role in the extracellular milieu as a signaling molecule, acting as a chemoattractant for immune cells and as a neuro- and gliotransmitter. The ionotropic P2X receptors are members of an ATP-gated ion channels family. These ionotropic receptors are widely expressed through the body, with 7 subunits described in mammals, which are arranged in a trimeric configuration with a central pore permeable mainly to Ca(2+) and Na(+). All 7 subunits are expressed in different brain areas, being present in neurons and glia. ATP, through these ionotropic receptors, can act as a neuromodulator, facilitating the Ca(2+)-dependent release of neurotransmitters, inducing the cross-inhibition between P2XR and GABA receptors, and exercising by this way a modulation of synaptic plasticity. Growing evidence shows that P2XR play an important role in neuronal disorders and neurodegenerative diseases, like Parkinson's and Alzheimer's disease; this role involves changes on P2XR expression levels, activation of key pathways like GSK3ß, APP processing, oxidative stress and inflammatory response. This review is focused on the neuromodulatory function of P2XR on pathophysiological conditions of the brain; the recent evidence could open a window to a new therapeutic target.
Subject(s)
Nerve Net/metabolism , Nervous System Diseases/metabolism , Receptors, Purinergic P2X/metabolism , Adenosine Triphosphate/metabolism , Alzheimer Disease/drug therapy , Alzheimer Disease/etiology , Alzheimer Disease/metabolism , Animals , Calcium Signaling , Humans , Nervous System Diseases/drug therapy , Nervous System Diseases/etiology , Neurodegenerative Diseases/drug therapy , Neurodegenerative Diseases/etiology , Neurodegenerative Diseases/metabolism , Neuronal Plasticity , Receptors, Purinergic P2X/chemistryABSTRACT
Seven P2X purinergic receptor subunits have been identified: P2X1-P2X7. The overlapping expression of P2X2, P2X4 and P2X6 subunits has been shown in different cell types, and functional analysis of P2X receptors in Leydig cells suggests that the three subunits might interact. Here, His6-tagged P2X2, HA-tagged P2X4 and FLAG-tagged P2X6 subunits were co-expressed in tsA 201 cells. After sequential co-immunoprecipitation using anti-HA and anti-FLAG beads, all three subunits were present, demonstrating their interaction. Atomic force microscopy (AFM) imaging revealed receptors that were specifically decorated by both an anti-His6 antibody and an anti-HA Fab fragment, indicating the presence of a P2X2/4/6 heterotrimer. To our knowledge, this is the first report of a P2X receptor containing three different subunits.
Subject(s)
Microscopy, Atomic Force , Protein Multimerization , Receptors, Purinergic P2X/chemistry , Animals , HEK293 Cells , Humans , Protein Structure, Quaternary , Rats , Receptors, Purinergic P2/chemistry , Receptors, Purinergic P2X2/chemistry , Receptors, Purinergic P2X4/chemistryABSTRACT
Currently, adenosine 5'-triphosphate (ATP) is recognized as the extracellular messenger that acts through P2 receptors. P2 receptors are divided into two subtypes: P2Y metabotropic receptors and P2X ionotropic receptors, both of which are found in virtually all mammalian cell types studied. Due to the difficulty in studying membrane protein structures by X-ray crystallography or NMR techniques, there is little information about these structures available in the literature. Two structures of the P2X4 receptor in truncated form have been solved by crystallography. Molecular modeling has proven to be an excellent tool for studying ionotropic receptors. Recently, modeling studies carried out on P2X receptors have advanced our knowledge of the P2X receptor structure-function relationships. This review presents a brief history of ion channel structural studies and shows how modeling approaches can be used to address relevant questions about P2X receptors.
Subject(s)
Adenosine Triphosphate/chemistry , Models, Molecular , Protein Structure, Tertiary , Receptors, Purinergic P2X/chemistry , Adenosine Triphosphate/metabolism , Animals , Crystallography, X-Ray , Humans , Magnetic Resonance Spectroscopy , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Protein Multimerization , Receptors, Purinergic P2X/metabolismABSTRACT
The purpose of the present study was to investigate the modulation of spontaneous afferent activity by ATP during embryonic development in a preparation isolated chicken inner ear. This work was performed using multiunit and single-unit extracellular recordings from the posterior semicircular canal nerve and the basilar papilla nerve. α,ß-meATP, a P2X receptor agonist, notably increased the discharge frequency of the vestibular afferents between E15 and E18, but not in the basilar papilla. In contrast, the P2Y receptor agonist UTP produced a slight increase in the discharge frequency of basilar papilla afferents, without apparent changes in the vestibular afferent activity. 2-MeSATP, a P2Y agonist, increased the basal discharge of the primary afferents in a dose-age dependent way, but when we applied the antagonist of P2Y receptor, Reactive Blue 2 (10(-4)M), the effect of 2-MeSATP decreased significantly. This was observed both in vestibule and basilar papilla. Using RT-PCR the presence of P2X3, P2Y1, P2Y2 and P2Y6 mRNA was documented in the vestibular system with more important presence during the early stage (E15) than the later stage (E21), however in the basilar papilla we found only the P2Y1, P2Y2 and P2Y6 mRNA with the same temporal course as in the vestibule. These results confirm our pharmacological findings. Together this data suggests a role for P2X receptors-mediated purinergic signaling in vestibular synaptic organization. Temporal changes in P2Y receptors during development might be involved in the establishment of the endolymphatic ion composition needed for normal vestibular and auditory transduction and/or specific cellular differentiation.
Subject(s)
Ear, Inner/metabolism , Receptors, Purinergic P2X/metabolism , Receptors, Purinergic P2Y/metabolism , Action Potentials , Afferent Pathways/physiology , Animals , Chick Embryo , Ear, Inner/innervation , Nerve Fibers/physiology , Organ of Corti/metabolism , Purinergic P2X Receptor Agonists/pharmacology , Purinergic P2Y Receptor Agonists/pharmacology , Vestibule, Labyrinth/innervation , Vestibule, Labyrinth/metabolismABSTRACT
The aim of the present study was to investigate if P2X4 receptors are expressed in murine myenteric neurons and if these receptors contribute to form functional channels in the neuronal membrane by using molecular and electrophysiological techniques. The whole-cell recording technique was used to measure membrane currents induced by ATP (I(ATP)) in myenteric neurons. Compared with recombinant P2X4 receptor-channels (reported by others in a previous study), native myenteric P2X receptors have a relative lower sensitivity for ATP (EC50=102 µM) and α,ß methylene ATP (not effect at 30 or 100 µM). BzATP was a weak agonist for native P2X receptors. KN-62 had no effect on myenteric P2X channels whereas PPADS (IC50=0.54 µM) or suramin (IC50=134 µM) were more potent antagonists than on P2X4 homomeric channels. I(ATP) were potentiated by ivermectin (effect that is specific on P2X4 receptors) and zinc. Western blotting shows the presence of P2X4 protein and RT-PCR the corresponding mRNA transcript in the small intestine. Immunoreactivity for P2X4 receptors was found in most myenteric neurons in culture. Single-cell RT-PCR shows the presence of P2X4 mRNA in 90% of myenteric neurons. Our results indicate that P2X4 receptors are expressed in the majority of myenteric neurons, contribute to the membrane currents activated by ATP, and because most properties of I(ATP) does not correspond to P2X4 homomeric channels it is proposed that P2X4 are forming heteromeric channels in these neurons. P2X4 subunits have a widespread distribution within the myenteric plexus and would be expected to play an important role in cell signaling.
Subject(s)
Myenteric Plexus/metabolism , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Protein Subunits/metabolism , Receptors, Purinergic P2X4/metabolism , Receptors, Purinergic P2X/metabolism , Adenosine Triphosphate/metabolism , Animals , Cells, Cultured , Female , Jejunum/cytology , Jejunum/innervation , Jejunum/metabolism , Male , Membrane Potentials/drug effects , Mice , Mice, Inbred C57BL , Myenteric Plexus/cytology , Myenteric Plexus/drug effects , Nerve Tissue Proteins/agonists , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/genetics , Neurons/cytology , Neurons/drug effects , Patch-Clamp Techniques , Protein Subunits/agonists , Protein Subunits/antagonists & inhibitors , Protein Subunits/genetics , Purinergic P2X Receptor Agonists/pharmacology , Purinergic P2X Receptor Antagonists/pharmacology , Receptors, Purinergic P2X/chemistry , Receptors, Purinergic P2X4/chemistry , Receptors, Purinergic P2X4/genetics , Second Messenger Systems/drug effects , Synaptic Transmission/drug effectsABSTRACT
Morbidity and mortality from diabetes mellitus (DM) are serious worldwide concerns. By the year 2030, the estimated number of diabetic patients will reach a staggering 439 million worldwide. Diabetes mellitus type 2 (DM2), which involves disturbances in both insulin secretion and resistance, is the most common form of diabetes and affects approximately 5 to 7% of the world's population. When a patient with DM2 cannot regulate his or her blood glucose levels through diet, weight loss, or exercise, oral medications, such as hypoglycemic agents (i.e., sulphonylureas, biguanides, alpha glucosidase inhibitors and thiazolidinediones), are crucial. Here, we discuss some physiological aspects of P2 receptors on pancreatic ß-cells, which express a variety of P2 receptor isoforms. These receptors enhance glucose-dependent insulin release. In addition, we speculate on the potential of purinergic compounds as novel or additional treatments for Type 2 Diabetes mellitus.
Subject(s)
Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Type 2/drug therapy , Hypoglycemic Agents/pharmacology , Insulin-Secreting Cells/drug effects , Receptors, Purinergic P2X/drug effects , Receptors, Purinergic P2Y/drug effects , Receptors, Purinergic P2/drug effects , Animals , Cell Line , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Type 2/metabolism , Humans , Immunohistochemistry , Insulin-Secreting Cells/metabolism , Mice , Phosphorylation , Purinergic P2 Receptor Agonists/pharmacology , Purinergic P2Y Receptor Antagonists/pharmacology , Rats , Receptors, Purinergic P2/metabolism , Receptors, Purinergic P2X/metabolism , Receptors, Purinergic P2Y/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Extracellular ATP promotes an indirect contraction of airway smooth muscle via the secondary release of thromboxane A2 (TXA2) from airway epithelium. Our aim was to evaluate if common contractile agonists modify this response to ATP. Tracheas from sensitized guinea pigs were used to evaluate ATP-induced contractions before and after a transient contraction produced by histamine, carbachol, or serotonin. Epithelial mRNA for COX-1 and COX-2 was measured by RT-PCR and their expression assessed by immunohistochemistry. Compared with the initial response, ATP-induced contraction was potentiated by pretreatment with histamine, carbachol, or serotonin. Either suramin (antagonist of P2X and P2Y receptors) plus RB2 (antagonist of P2Y receptors) or indomethacin (inhibitor of COX-1 and COX-2) annulled the ATP-induced contraction, suggesting that it was mediated by P2Y receptor stimulation and TXA2 production. When COX-2 was inhibited by SC-58125 or thromboxane receptors were antagonized by SQ-29548, just the potentiation was abolished, leaving the basal response intact. Airway epithelial cells showed increased COX-2 mRNA after stimulation with histamine or carbachol, but not serotonin, while COX-1 mRNA was unaffected. Immunochemistry corroborated this upregulation of COX-2. In conclusion, we showed for the first time that histamine and carbachol cause hyperresponsiveness to ATP by upregulating COX-2 in airway epithelium, which likely increases TXA2 production. Serotonin-mediated hyperresponsiveness seems to be independent of COX-2 upregulation, but nonetheless is TXA2 dependent. Because acetylcholine, histamine, and serotonin can be present during asthmatic exacerbations, their potential interactions with ATP might be relevant in its pathophysiology.
Subject(s)
Adenosine Triphosphate/metabolism , Carbachol/pharmacology , Cyclooxygenase 2/metabolism , Histamine/pharmacology , Serotonin/pharmacology , Trachea/drug effects , Animals , Cyclooxygenase 1/genetics , Cyclooxygenase 1/metabolism , Cyclooxygenase 2/genetics , Cyclooxygenase 2 Inhibitors/pharmacology , Cyclooxygenase Inhibitors/pharmacology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Guinea Pigs , Male , Muscle Contraction/drug effects , Muscle Contraction/genetics , Muscle, Smooth/drug effects , Muscle, Smooth/metabolism , Purinergic P2X Receptor Antagonists/pharmacology , Purinergic P2Y Receptor Antagonists/pharmacology , RNA, Messenger/genetics , Receptors, Purinergic P2X/genetics , Receptors, Purinergic P2X/metabolism , Receptors, Purinergic P2Y/genetics , Receptors, Purinergic P2Y/metabolism , Respiratory Mucosa/drug effects , Respiratory Mucosa/metabolism , Thromboxane A2/genetics , Thromboxane A2/metabolism , Trachea/metabolism , Up-Regulation/drug effects , Up-Regulation/geneticsABSTRACT
P2X receptors are expressed on ventrolateral medulla projecting paraventricular nucleus (PVN) neurons. Here, we investigate the role of adenosine 5'-triphosphate (ATP) in modulating sympathetic nerve activity (SNA) at the level of the PVN. We used an in situ arterially perfused rat preparation to determine the effect of P2 receptor activation and the putative interaction between purinergic and glutamatergic neurotransmitter systems within the PVN on lumbar SNA (LSNA). Unilateral microinjection of ATP into the PVN induced a dose-related increase in the LSNA (1 nmol: 38 ± 6 %, 2.5 nmol: 72 ± 7 %, 5 nmol: 96 ±13 %). This increase was significantly attenuated by blockade of P2 receptors (pyridoxalphosphate-6-azophenyl-20,40-disulphonic acid, PPADS) and glutamate receptors (kynurenic acid, KYN) or a combination of both. The increase in LSNA elicited by L-glutamate microinjection into the PVN was not affected by a previous injection of PPADS. Selective blockade of non-N-methyl-D-aspartate receptors (6-cyano-7-nitroquinoxaline-2,3-dione disodium salt, CNQX), but not N-methyl-D-aspartate receptors (NMDA) receptors (DL-2-amino-5-phosphonopentanoic acid, AP5), attenuated the ATP-induced sympathoexcitatory effects at the PVN level. Taken together, our data show that purinergic neurotransmission within the PVN is involved in the control of SNA via P2 receptor activation. Moreover, we show an interaction between P2 receptors and non-NMDA glutamate receptors in the PVN suggesting that these functional interactions might be important in the regulation of sympathetic outflow.
Subject(s)
Adenosine Triphosphate/metabolism , Paraventricular Hypothalamic Nucleus/metabolism , Sympathetic Nervous System/physiology , Adenosine Triphosphate/pharmacology , Animals , Electrophysiology , Male , Paraventricular Hypothalamic Nucleus/drug effects , Phrenic Nerve/drug effects , Phrenic Nerve/physiology , Rats , Rats, Wistar , Receptors, Purinergic P2X/metabolism , Sympathetic Nervous System/drug effectsABSTRACT
The medullary raphe (MR) is a putative central chemoreceptor site, contributing to hypercapnic respiratory responses elicited by changes in brain PCO2/pH. Purinergic mechanisms in the central nervous system appear to contribute to central chemosensitivity. To further explore the role of P2 receptors within the rostral and caudal MR in relation to respiratory control in room air and hypercapnic conditions, we performed microinjections of PPADS, a non-selective P2X antagonist, in conscious rats. Microinjections of PPADS into the rostral or caudal MR produced no changes in the respiratory frequency, tidal volume and ventilation in room air condition. The ventilatory response to hypercapnia was attenuated after microinjection of PPADS into the rostral but not in the caudal MR when compared to the control group (vehicle microinjection). These data suggest that P2X receptors in the rostral MR contribute to the ventilatory response to CO2, but do not participate in the tonic maintenance of ventilation under room air condition in conscious rats.
Subject(s)
Hypercapnia/physiopathology , Pulmonary Ventilation/physiology , Raphe Nuclei/physiopathology , Receptors, Purinergic P2X/metabolism , Synaptic Transmission/physiology , Animals , Consciousness , Hypercapnia/metabolism , Male , Microinjections , Platelet Aggregation Inhibitors/administration & dosage , Purines/metabolism , Pyridoxal Phosphate/administration & dosage , Pyridoxal Phosphate/analogs & derivatives , Raphe Nuclei/drug effects , Raphe Nuclei/metabolism , Rats , Rats, Wistar , Synaptic Transmission/drug effectsABSTRACT
The Na(+)/Ca(2+)exchanger (NCX) principal function is taking 1 Ca(2+) out of the cytoplasm and introducing 3 Na(+). The increase of cytoplasmic Na(+) concentration induces the NCX reverse mode (NCX(REV)), favoring Ca(2+) influx. NCX(REV) can be inhibited by: KB-R7943 a non-specific compound that blocks voltage-dependent and store-operated Ca(2+) channels; SEA0400 that appears to be selective for NCX(REV), but difficult to obtain and SN-6, which efficacy has been shown only in cardiomyocytes. We found that PPADS, a P2X receptor antagonist, acts as a NCX(REV) inhibitor in guinea pig tracheal myocytes. In these cells, we characterized the NCX(REV) by substituting NaCl and NaHCO(3) with LiCl, resulting in the increase of the intracellular Ca(2+) concentration ([Ca(2+)]i) using fura 2-AM. We analyzed 5 consecutive responses of the NCX(REV) every 10 min, finding no differences among them. To evaluate the effect of different NCX(REV) blockers, concentration response curves to KB-R7943 (1, 3.2 and 10 µM), and SN-6 (3.2, 10 and 30 µM) were constructed, whereas PPADS effect was characterized as time- and concentration-dependent (1, 3.2, 10 and 30 µM). PPADS had similar potency and efficacy as KB-R7943, whereas SN-6 was the least effective. Furthermore, KCl-induced contraction, sensitive to D600 and nifedipine, was blocked by KB-R7943, but not by PPADS. KCl-induced [Ca(2+)]i increment in myocytes was also significantly decreased by KBR-7943 (10 µM). Our results demonstrate that PPADS can be used as a reliable pharmacological tool to inhibit NCX(REV), with the advantage that it is more specific than KB-R7943 because it does not affect L-type Ca(2+) channels.
Subject(s)
Muscle, Smooth/drug effects , Muscle, Smooth/metabolism , Purinergic P2X Receptor Antagonists/pharmacology , Pyridoxal Phosphate/analogs & derivatives , Receptors, Purinergic P2X/metabolism , Sodium-Calcium Exchanger/antagonists & inhibitors , Trachea/cytology , Animals , Calcium Channel Blockers/pharmacology , Calcium Channels, L-Type/metabolism , Guinea Pigs , Male , Muscle Cells/drug effects , Muscle Cells/metabolism , Muscle, Smooth/cytology , Pyridoxal Phosphate/pharmacology , Reproducibility of Results , Thiourea/analogs & derivatives , Thiourea/pharmacologyABSTRACT
Presympathetic neurons in the different anteroposterior aspects of rostral ventrolateral medulla (RVLM) are colocalized with expiratory [Bötzinger complex (BötC)] and inspiratory [pre-Bötzinger complex (pre-BötC)] neurons of ventral respiratory column (VRC), suggesting that this region integrates the cardiovascular and respiratory chemoreflex responses. In the present study, we evaluated in different anteroposterior aspects of RVLM of awake rats the role of ionotropic glutamate and purinergic receptors on cardiorespiratory responses to chemoreflex activation. The bilateral ionotropic glutamate receptors antagonism with kynurenic acid (KYN) (8 nmol/50 nl) in the rostral aspect of RVLM (RVLM/BötC) enhanced the tachypneic (120 ± 9 vs. 180 ± 9 cpm; P < 0.01) and attenuated the pressor response (55 ± 2 vs. 15 ± 1 mmHg; P < 0.001) to chemoreflex activation (n = 7). On the other hand, bilateral microinjection of KYN into the caudal aspect of RVLM (RVLM/pre-BötC) caused a respiratory arrest in four awake rats used in the present study. Bilateral P2X receptors antagonism with PPADS (0.25 nmol/50 nl) in the RVLM/BötC reduced chemoreflex tachypneic response (127 ± 6 vs. 70 ± 5 cpm; P < 0.001; n = 6), but did not change the chemoreflex pressor response. In addition, PPADS into the RVLM/BötC attenuated the enhancement of the tachypneic response to chemoreflex activation elicited by previous microinjections of KYN into the same subregion (188 ± 2 vs. 157 ± 3 cpm; P < 0.05; n = 5). Our findings indicate that: 1) L-glutamate, but not ATP, in the RVLM/BötC is required for pressor response to peripheral chemoreflex and 2) both transmitters in the RVLM/BötC are required for the processing of the ventilatory response to peripheral chemoreflex activation in awake rats.
Subject(s)
Adenosine Triphosphate/pharmacology , Chemoreceptor Cells/physiology , Consciousness/physiology , Glutamic Acid/pharmacology , Medulla Oblongata/physiology , Oxygen Consumption/drug effects , Oxygen Consumption/physiology , Adenosine Triphosphate/administration & dosage , Animals , Dose-Response Relationship, Drug , Glutamic Acid/administration & dosage , Kynurenic Acid/pharmacology , Male , Medulla Oblongata/drug effects , Microinjections , Models, Animal , Pyridoxal Phosphate/analogs & derivatives , Pyridoxal Phosphate/pharmacology , Rats , Rats, Wistar , Receptors, Glutamate/drug effects , Receptors, Glutamate/physiology , Receptors, Purinergic P2X/drug effects , Receptors, Purinergic P2X/physiology , Sympathetic Nervous System/physiologyABSTRACT
Zn²(+) is an essential ion that is stored in and co-released from glutamatergic synapses and it modulates neurotransmitter receptors involved in long-term potentiation (LTP). However, the mechanism(s) underlying Zn²(+) -induced modulation of LTP remain(s) unclear. As the purinergic P2X receptors are relevant targets for Zn²(+) action, we have studied their role in LTP modulation by Zn²(+) in the CA1 region of rat hippocampal slices. Induction of LTP in the presence of Zn²(+) revealed a biphasic effect - 5-50 µm enhanced LTP induction, whereas 100-300 µm Zn²(+) inhibited LTP. The involvement of a purinergic mechanism is supported by the fact that application of the P2X receptor antagonists 2',3'-O-(2,4,6-trinitrophenyl) ATP (TNP-ATP) and periodate-oxidized ATP fully abolished the facilitatory effect of Zn²(+) . Notably, application of the P2X7 receptor-specific antagonist Brilliant Blue G did not modify the Zn²(+) -dependent facilitation of LTP. Exogenous ATP also produced a biphasic effect - 0.1-1 µm ATP facilitated LTP, whereas 5-10 µm inhibited LTP. The facilitatory effect of ATP was abolished by the application of TNP-ATP and was modified in the presence of 5 µm Zn²(+) , suggesting that P2X receptors are involved in LTP induction and that Zn²(+) leads to an increase in the affinity of P2X receptors for ATP. The latter confirms our previous results from heterologous expression systems. Collectively, our results indicate that Zn²(+) at low concentrations enhances LTP by modulating P2X receptors. Although it is not yet clear which purinergic receptor subtype(s) is responsible for these effects on LTP, the data presented here suggest that P2X4 but not P2X7 is involved.
Subject(s)
CA1 Region, Hippocampal/drug effects , CA1 Region, Hippocampal/physiology , Long-Term Potentiation/drug effects , Receptors, Purinergic P2X/metabolism , Zinc/pharmacology , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/pharmacology , Animals , Electrophysiology , Long-Term Potentiation/physiology , Male , Purinergic P2X Receptor Agonists/pharmacology , Purinergic P2X Receptor Antagonists/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, GABA-A/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Receptors, Purinergic P2X4/metabolism , Receptors, Purinergic P2X7/metabolismABSTRACT
Phagocytosis plays an important role in controlling inflammation and antigen cross-presentation through the uptake of apoptotic bodies from dying cells. As dying cells are known to release nucleotides and other "danger signals", we investigated whether extracellular nucleotides may affect phagocytosis through binding to P2 purinergic receptors on phagocytic cells. We here show that the purinergic receptor agonists, ATP, ADP, α,ß-methylene ATP (α,ß-meATP), 3'-O-(4-benzoyl)benzoyl ATP, UTP and UDP, increased phagocytosis of latex beads, and some of them increased endocytosis and/or macropinocytosis of dextran by macrophages. The enhanced phagocytosis could be inhibited by pre-treatment with the P2X and P2Y antagonists, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid and suramin, and the P2Y1-selective antagonist, MRS2179. The nucleotides induced upregulation in macrophages of the ß2 integrin CD11b/CD18 (Mac-1) and the vitronectin receptor (α(v)ß3, CD51/CD61), both of which are involved in recognition and internalization of apoptotic cells. In addition, ATP and α,ß-meATP increased adhesion of apoptotic cells to macrophages, both in vitro and in vivo, and α,ß-meATP had a small effect on adhesion of necrotic cells. The nucleotides had no effect on adhesion of viable cells. We propose that engagement of the P2 receptors (P2X1, or P2X3) by extracellular nucleotides released from dying cells increases the ability of macrophages to bind apoptotic bodies, thus enhancing their ability to internalize and present antigens from the dying cells.