The involvement of P2Y12 receptors, NADPH oxidase, and lipid rafts in the action of extracellular ATP on synaptic transmission at the frog neuromuscular junction

© 2014 IBRO. Adenosine 5'-triphosphate (ATP) is the main co-transmitter accompanying the release of acetylcholine from motor nerve terminals. Previously, we revealed the direct inhibitory action of extracellular ATP on transmitter release via redox-dependent mechanism. However, the receptor mechanism of ATP action and ATP-induced sources of reactive oxygen sources (ROS) remained not fully understood. In the current study, using microelectrode recordings of synaptic currents from the frog neuromuscular junction, we analyzed the receptor subtype involved in synaptic action of ATP, receptor coupling to NADPH oxidase and potential location of ATP receptors within the lipid rafts. Using subtype-specific antagonists, we found that the P2Y13 blocker 2-[(2-chloro-5-nitrophenyl)azo]-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-4-pyridinecarboxaldehyde did not prevent the depressant action of ATP. In contrast, the P2Y12 antagonist 2-methylthioadenosine 5'-monophosphate abolished the inhibitory action of ATP, suggesting the key role of P2Y12 receptors in ATP action. As the action of ATP is redox-dependent, we also tested potential involvement of the NADPH oxidase, known as a common inducer of ROS. The depressant action of extracellular ATP was significantly reduced by diphenyleneiodonium chloride and 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, two structurally different inhibitors of NADPH oxidase, indicating that this enzyme indeed mediates the action of ATP. Since the location and activity of various receptors are often associated with lipid rafts, we next tested whether ATP-driven inhibition depends on lipid rafts. We found that the disruption of lipid rafts with methyl-beta-cyclodextrin reduced and largely delayed the action of ATP. Taken together, these data revealed key steps in the purinergic control of synaptic transmission via P2Y12 receptors associated with lipid rafts, and identified NADPH oxidase as the main source of ATP-induced inhibitory ROS at the neuromuscular junction. Our data suggest that the location of P2Y receptors in lipid rafts speeds up the modulatory effect of ATP. Uncovered mechanisms may contribute to motor dysfunctions and neuromuscular diseases associated with oxidative stress

Modeling of the two wave response of ATP receptors to jumps of the agonist concentration in pheochromocytoma cells | Modelirovanie dvukh voln otveta ATP-retseptorov kletok feokhromotsitomy na skachok kontsentratsii agonista.

A model for the kinetics of conformational transitions of ionotropic ATP receptors in pheochromocytoma cells was elaborated. The contribution of the states of ionotropic receptors (upon the blockage of the "open" channel state) to the kinetics of postsynaptic currents was estimated at mediator concentrations studied. The model enables one to determine the contribution of various conformational states of the receptor, in particular in the "closed" state, to the dynamics of ionic current that is registered upon stimulation of ATP receptors. It is shown that after the cessation of the agonist application, a secondary current wave can arise. The rate constants for conformational transitions of ATP receptors were determined

Hydrogen Sulfide Ameliorates Developmental Impairments of Rat Offspring with Prenatal Hyperhomocysteinemia

Maternal high levels of the redox active amino acid homocysteine-called hyperhomocysteinemia (hHCY)-can affect the health state of the progeny. The effects of hydrogen sulfide (H2S) treatment on rats with maternal hHCY remain unknown. In the present study, we characterized the physical development, reflex ontogeny, locomotion and exploratory activity, muscle strength, motor coordination, and brain redox state of pups with maternal hHCY and tested potential beneficial action of the H2S donor-sodium hydrosulfide (NaHS)-on these parameters. Our results indicate a significant decrease in litter size and body weight of pups from dams fed with methionine-rich diet. In hHCY pups, a delay in the formation of sensory-motor reflexes was observed. Locomotor activity tested in the open field by head rearings, crossed squares, and rearings of hHCY pups at all studied ages (P8, P16, and P26) was diminished. Exploratory activity was decreased, and emotionality was higher in rats with hHCY. Prenatal hHCY resulted in reduced muscle strength and motor coordination assessed by the paw grip endurance test and rotarod test. Remarkably, administration of NaHS to pregnant rats with hHCY prevented the observed deleterious effects of high homocysteine on fetus development. In rats with prenatal hHCY, the endogenous generation of H2S brain tissues was lower compared to control and NaHS administration restored the H2S level to control values. Moreover, using redox signaling assays, we found an increased level of malondialdehyde (MDA), the end product of lipid peroxidation, and decreased activity of antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GPx) in the brain tissues of rats of the hHCY group. Notably, NaHS treatment restored the level of MDA and the activity of SOD and GPx. Our data suggest that H2S has neuroprotective/antioxidant effects against homocysteine-induced neurotoxicity providing a potential strategy for the prevention of developmental impairments in newborns

Membrane current series monitoring: Essential reduction of data points to finite number of stable parameters

© 2014 Nigmatullin, Giniatullin and Skorinkin. In traditional studies of changes in cell membrane potential or trans-membrane currents a large part of the recorded data presents "a pure noise." This noise results mainly from the random openings of membrane ionic channels. Different types of stationary or non-stationary noise analysis have been used in electrophysiological experiments for identification of channels kinetic states. But these methods have a limited power and often cannot answer to the main question of the experimental study: do external factors induce a significant change of channels kinetics? A new method suggested in the current study is based on the scaling properties of the beta-distribution function that allows reducing the series containing 200,000 and more data points to analysis of only 10-20 stable parameters. The following clusterization using the generalized Pearson correlation function allows taking into account the influence of an external factor and combine/separate different parameters of interest into a statistical cluster considering the influential parameter. This method which we call BRC (Beta distribution-Reduction-Clusterization) opens new possibilities in creation of a largely reduced database while extracting specific fingerprints of the long-term series. The BRC method was validated using patch clamp current recordings containing 250,000 data points obtained from the living cells and from open tip electrode. The numerical distinction between these two series in terms of the reduced parameters was obtained

Bimodal action of protons on ATP currents of rat PC12 cells

The mode of action of extracellular protons on ATP-gated P2X2 receptors remains controversial as either enhancement or depression of ATP-mediated currents has been reported. By investigating, at different pH, the electrophysiological effect of ATP on P2X2 receptors and complementing it with receptor modelling, the present study suggests a unified mechanism for both potentiation and inactivation of ATP receptors by protons. Our experiments on patch-clamped PC12 cells showed that, on the same cell, mild acidification potentiated currents induced by low ATP concentrations (1 mM) with emergence of current fading and rebound. To clarify the nature of the ATP/H+ interaction, we used the Ding and Sachs's "loop" receptor model which best describes the behavior of such receptors with two open states linked via one inactivated state. No effects by protons could be ascribed to H+-mediated open channel block. However, by assuming that protons facilitated binding of ATP to resting as well as open receptors, the model could closely replicate H+-induced potentiation of currents evoked by low ATP doses plus fading and rebound induced by high ATP doses. The latter phenomenon was due to receptor transition to the inactive state. The present data suggest that the high concentration of protons released with ATP (and catecholamines) from secretory vesicles may allow a dual action of H+ on P2X2 receptors. This condition might also occur on P2X2 receptors of central neurons exposed to low pH during ischemia

Adenosine Promotes Endplate nAChR Channel Activity in Adult Mouse Skeletal Muscle Fibers via Low Affinity P1 Receptors

© 2018 IBRO Adenosine is a powerful modulator of skeletal neuromuscular transmission, operating via inhibitory or facilitatory purinergic-type P1 receptors. To date, studies have been focused mainly on the effect of adenosine on presynaptic P1 receptors controlling transmitter release. In this study, using two-microelectrode voltage-clamp and single-channel patch-clamp recording techniques, we have explored potential postsynaptic targets of adenosine and their modulatory effect on nicotinic acetylcholine receptor (nAChR)-mediated synaptic responses in adult mouse skeletal muscle fibers in vitro. In the whole-mount neuromuscular junction (NMJ) preparation, adenosine (100 μM) significantly reduced the frequency of the miniature endplate currents (MEPCs) and slowed their rising and decay time. Consistent with a postsynaptic site of action, adenosine and the potent P1 receptor agonist NECA significantly increased the open probability, the frequency and the open time of single nAChR channels, recorded at the endplate region. Using specific ligands for the P1 receptor subtypes, we found that the low-affinity P1 receptor subtype A2B was responsible for mediating the effects of adenosine on the nAChR channel openings. Our data suggest that at the adult mammalian NMJ, adenosine acts not only presynaptically to modulate acetylcholine transmitter release, but also at the postsynaptic level, to enhance the activity of nAChRs. Our findings open a new scenario in understanding of purinergic regulation of nAChR activity at the mammalian endplate region

Emerging role of (endo)cannabinoids in migraine

© 2018 Leimuranta, Khiroug and Giniatullin. In this mini-review, we summarize recent discoveries and present new hypotheses on the role of cannabinoids in controlling trigeminal nociceptive system underlying migraine pain. Individual sections of this review cover key aspects of this topic, such as: (i) the current knowledge on the endocannabinoid system (ECS) with emphasis on expression of its components in migraine related structures; (ii) distinguishing peripheral from central site of action of cannabinoids, (iii) proposed mechanisms of migraine pain and control of nociceptive traffic by cannabinoids at the level of meninges and in brainstem, (iv) therapeutic targeting in migraine of monoacylglycerol lipase and fatty acid amide hydrolase, enzymes which control the level of endocannabinoids; (v) dual (possibly opposing) actions of cannabinoids via anti-nociceptive CB1 and CB2 and pro-nociceptive TRPV1 receptors. We explore the cannabinoid-mediated mechanisms in the frame of the Clinical Endocannabinoid Deficiency (CECD) hypothesis, which implies reduced tone of endocannabinoids in migraine patients. We further discuss the control of cortical excitability by cannabinoids via inhibition of cortical spreading depression (CSD) underlying the migraine aura. Finally, we present our view on perspectives of Cannabis-derived (extracted or synthetized marijuana components) or novel endocannabinoid therapeutics in migraine treatment

Clustering Analysis for Sorting ATP-Induced Nociceptive Firing in rat Meninges

© 2016, Springer Science+Business Media New York.We previously reported the pronociceptive effect of extracellular ATP on meningeal branches of the trigeminal nerve. Based on this, we proposed ATP-driven purinergic mechanisms as important contributors to migraine pain. However, the neurochemical profile of the single nociceptive fibers composing the trigeminal nerve remained unsolved. Here, we present a clustering approach to separate and characterize ATP-induced nociceptive spikes obtained from the trigeminal nerve using a suction electrode. By combining a number of procedures to ensure adequate separation of spikes into clearly distinguishable clusters, we were able to reconstruct single fiber activity, which was characterized by a uniformity of spike shapes and specific signatures of the autocorrelation function. This allows us to reveal, at single fiber level, the sustained time-course of responses to ATP and their respective firing frequencies. These firing frequencies predicted the temporal summation of pain signals in the brainstem and spinal cord. Our findings contribute to a better understanding of the peripheral purinergic mechanisms of trigeminal pain, including migraine pain, and suggest a novel reliable approach for testing other pronociceptive agents potently implicated in migraine

High sensitivity of cerebellar neurons to homocysteine is determined by expression of GluN2C and GluN2D subunits of NMDA receptors

© 2018 Elsevier Inc. Homocysteine (HCY) induced neurotoxicity largely depends on interaction of this endogenous amino acid with glutamate NMDA receptors (NMDARs). This receptor type is composed by GluN1 and different GluN2 (A, B, C or D) subunits. However, the receptor activity of HCY in brain regions which differ in relative contribution of GluN2 subunits was not tested so far. In the current study, we explored the action of HCY on cerebellar neurons which natively express GluN2C and GluN2D subunits of NMDARs and compared this with the action of HCY on cortical neurons which are mainly composed by GluN2A and GluN2B subunits. To validate obtained results, we also studied the responses to HCY in recombinant GluN1/2C and GluN1/2D NMDARs expressed in HEK293T cells. Responses to HCY were compared to membrane currents evoked by glutamate or by the specific agonist NMDA. First, we found that on HEK cells expressing GluN1/2C or GluN1/2D NMDARs, HCY was full agonist producing membrane currents similar in amplitude to currents induced by glutamate. The EC50 values for these particular receptor subtype activation were 80 μM and 31 μM, respectively. Then, we found that HCY similarly to NMDA, evoked large slightly desensitizing membrane currents in native NMDARs of cerebellar and cortical neurons. In cortical neurons, the ratio of the respective currents (IHCY/INMDA) was 0.16 and did not significantly change during in vitro maturation. In sharp contrast, in cerebellar neurons, the ratio of currents evoked by HCY and NMDA was dramatically increased from 0.31 to 0.72 from 7 to 21 day in culture. We show that least 75% of HCY-induced currents in cerebellum were mediated by GluN2C- or GluN2D-containing NMDARs. Thus, our data revealed a large population of cerebellar NMDA receptors highly sensitive to HCY which suggest potential vulnerability of this brain region to pathological conditions associated with enhanced levels of this neurotoxic amino acid