Application Of ATP Research Articles

The Polarized Effect of Intracellular Calcium on the Renal Epithelial Sodium Channel Occurs as a Result of Subcellular Calcium Signaling Domains Maintained by Mitochondria

The renal epithelial sodium channel (ENaC) provides regulated sodium transport in the distal nephron. The effects of intracellular calcium ([Ca(2+)]i) on this channel are only beginning to be elucidated. It appears from previous studies that the [Ca(2+)]i increases downstream of ATP administration may have a polarized effect on ENaC, where apical application of ATP and the subsequent [Ca(2+)]i increase have an inhibitory effect on the channel, whereas basolateral ATP and [Ca(2+)]i have a stimulatory effect. We asked whether this polarized effect of ATP is, in fact, reflective of a polarized effect of increased [Ca(2+)]i on ENaC and what underlying mechanism is responsible. We began by performing patch clamp experiments in which ENaC activity was measured during apical or basolateral application of ionomycin to increase [Ca(2+)]i near the apical or basolateral membrane, respectively. We found that ENaC does indeed respond to increased [Ca(2+)]i in a polarized fashion, with apical increases being inhibitory and basolateral increases stimulating channel activity. In other epithelial cell types, mitochondria sequester [Ca(2+)]i, creating [Ca(2+)]i signaling microdomains within the cell that are dependent on mitochondrial localization. We found that mitochondria localize in bands just beneath the apical and basolateral membranes in two different cortical collecting duct principal cell lines and in cortical collecting duct principal cells in mouse kidney tissue. We found that inhibiting mitochondrial [Ca(2+)]i uptake destroyed the polarized response of ENaC to [Ca(2+)]i. Overall, our data suggest that ENaC is regulated by [Ca(2+)]i in a polarized fashion and that this polarization is maintained by mitochondrial [Ca(2+)]i sequestration.

Extracellular ATP functions in alleviating the decrease of PSII photochemistry caused by the infection of Xanthomonas campestris pv. phaseoli

Extracellular ATP (eATP) can function as a signalling molecule to regulate a wide range of cellular processes. This present work investigated the role of eATP in mediating the change of PSII (photosystem II) photochemistry of the tissues of Phaseolus vulgaris leaves infected with Xanthomonas campestris pv. phaseoli (Xcp). Infection of the leaves with Xcp caused a significant decrease in the the PSII maximal photochemical efficiency (Fv/Fm), the maximum quantum efficiency of PSII photochemistry at illumination (Fv′/Fm′), the PSII operating efficiency (ΦPSII), the rate of non‐cyclic electron transport through PSII (ETR), photochemical quenching (qP), and eATP level in the tissues of the infected leaves. At the same time, the levels of non‐photochemical quenching (qN) and the quantum yield of regulated energy dissipation of PSII (Y(NPQ)) were significantly increased. Application of exogenous ATP at 0·2 mm to uninfected leaves had no significant effect on any of the chlorophyll fluorescence parameters being measured. However, in the tissues of infected leaves, the application of exogenous ATP alleviated the decreases of the Fv/Fm, Fv′/Fm′, ΦPSII, ETR, qP and eATP level, and also abolished the increases of qN and Y(NPQ). These results suggest that the change of PSII photochemistry by pathogen infection could be mediated by eATP.

Electron microscopic recording of myosin head power stroke in hydrated myosin filaments.

Muscle contraction results from cyclic attachment and detachment between myosin heads and actin filaments, coupled with ATP hydrolysis. Despite extensive studies, however, the amplitude of myosin head power stroke still remains to be a mystery. Using the gas environmental chamber, we have succeeded in recording the power stroke of position-marked myosin heads in hydrated mixture of actin and myosin filaments in a nearly isometric condition, in which myosin heads do not produce gross myofilament sliding, but only stretch adjacent elastic structures. On application of ATP, individual myosin heads move by ~3.3 nm at the distal region, and by ~2.5 nm at the proximal region of myosin head catalytic domain. After exhaustion of applied ATP, individual myosin heads return towards their initial position. At low ionic strength, the amplitude of myosin head power stroke increases to >4 nm at both distal and proximal regions of myosin heads catalytic domain, being consistent with the report that the force generated by individual myosin heads in muscle fibers is enhanced at low ionic strength. The advantages of the present study over other in vitro motility assay systems, using myosin heads detached from myosin filaments, are discussed.

Expression and testing in plants of ArcLight, a genetically-encoded voltage indicator used in neuroscience research.

BackgroundIt is increasingly appreciated that electrical controls acting at the cellular and supra-cellular levels influence development and initiate rapid responses to environmental cues. An emerging method for non-invasive optical imaging of electrical activity at cell membranes uses genetically-encoded voltage indicators (GEVIs). Developed by neuroscientists to chart neuronal circuits in animals, GEVIs comprise a fluorescent protein that is fused to a voltage-sensing domain. One well-known GEVI, ArcLight, undergoes strong shifts in fluorescence intensity in response to voltage changes in mammalian cells. ArcLight consists of super-ecliptic (SE) pHluorin (pH-sensitive fluorescent protein) with an A227D substitution, which confers voltage sensitivity in neurons, fused to the voltage-sensing domain of the voltage-sensing phosphatase of Cionaintestinalis (Ci-VSD). In an ongoing effort to adapt tools of optical electrophysiology for plants, we describe here the expression and testing of ArcLight and various derivatives in different membranes of root cells in Arabidopsis thaliana.ResultsTransgenic constructs were designed to express ArcLight and various derivatives targeted to the plasma membrane and nuclear membranes of Arabidopsis root cells. In transgenic seedlings, changes in fluorescence intensity of these reporter proteins following extracellular ATP (eATP) application were monitored using a fluorescence microscope equipped with a high speed camera. Coordinate reductions in fluorescence intensity of ArcLight and Ci-VSD-containing derivatives were observed at both the plasma membrane and nuclear membranes following eATP treatments. However, similar responses were observed for derivatives lacking the Ci-VSD. The dispensability of the Ci-VSD suggests that in plants, where H+ ions contribute substantially to electrical activities, the voltage-sensing ability of ArcLight is subordinate to the pH sensitivity of its SEpHluorin base. The transient reduction of ArcLight fluorescence triggered by eATP most likely reflects changes in pH and not membrane voltage.ConclusionsThe pH sensitivity of ArcLight precludes its use as a direct sensor of membrane voltage in plants. Nevertheless, ArcLight and derivatives situated in the plasma membrane and nuclear membranes may offer robust, fluorescence intensity-based pH indicators for monitoring concurrent changes in pH at these discrete membrane systems. Such tools will assist analyses of pH as a signal and/or messenger at the cell surface and the nuclear periphery in living plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0633-z) contains supplementary material, which is available to authorized users.

Modulation of Root Skewing in Arabidopsis by Apyrases and Extracellular ATP.

When plant primary roots grow along a tilted surface that is impenetrable, they can undergo a slanted deviation from the direction of gravity called skewing. Skewing is induced by touch stimuli which the roots experience as they grow along the surface. Touch stimuli also induce the release of extracellular ATP (eATP) into the plant's extracellular matrix, and two apyrases (NTPDases) in Arabidopsis, APY1 and APY2, can help regulate the concentration of eATP. The primary roots of seedlings overexpressing APY1 show less skewing than wild-type plants. Plants suppressed in their expression of APY1 show more skewing than wild-type plants. Correspondingly, chemical inhibition of apyrase activity increased skewing in mutants and wild-type roots. Exogenous application of ATP or ATPγS also increased skewing in wild-type roots, which could be blocked by co-incubation with a purinergic receptor antagonist. These results suggest a model in which gradients of eATP set up by differential touch stimuli along roots help direct skewing in roots growing along an impenetrable surface.

Rhein antagonizes P2X7 receptor in rat peritoneal macrophages.

P2X7 receptor plays important roles in inflammation and immunity, and thereby it serves as a potential therapeutic target for inflammatory diseases. Rhein, an anthraquinone derivative, exhibits significant anti-inflammatory and immunosuppressive activities in therapy. However, the underlying mechanisms are largely unclear. Here, we aimed to investigate the effects of rhein on P2X7 receptor-mediated responses in vitro. In HEK293 cells expressing rat P2X7 receptor, we first found that rhein concentration-dependently blocked ATP-induced cytosolic calcium concentration ([Ca2+]c) elevation and pore formation of the plasma membrane, two hallmarks of the P2X7 receptor activation. These two inhibitory effects of rhein were also observed in rat peritoneal macrophages. Furthermore, rhein counteracted macrophage phagocytosis attenuation and suppressed reactive oxygen species (ROS) production triggered by ATP/BzATP. Meanwhile, rhein reduced ATP/BzATP-induced IL-1β release in lipopolysaccharide-activated macrophages. Prolonged application of ATP caused macrophage apoptosis, while the presence of rhein suppressed this cell cytotoxicity. Such ATP/BzATP-induced cellular reactions were also inhibited by a well-known rat P2X7 receptor antagonist, brilliant blue G, in a similar way to rhein. Together, our results demonstrate that rhein inhibit ATP/BzATP-induced [Ca2+]c increase, pore formation, ROS production, phagocytosis attenuation, IL-1β release and cell apoptosis by antagonizing the P2X7 receptor in rat peritoneal macrophages.

Dynein arms are strain-dependent direction-switching force generators.

Dynein is a minus-end-directed motor that can generate (forward) force to move along the microtubule toward its minus end. In addition, axonemal dyneins were reported to oscillate in the generation of forward force, and cytoplasmic dynein is observed to generate bidirectional forces in response to defined chemical states. Both dyneins can also respond to mechanically applied force. To test whether axonemal dynein can switch direction of force generation, we measured force using an optical trap and UV-photolysis of caged ATP. We observed that isolated dynein could repeatedly generate force in both directions along the microtubule. Bidirectional force was also observed for dynein arms that are still attached on the doublet microtubules. Axonemal dynein generated force to move backward (∼ 4 pN) as well as forward (5-6 pN) along microtubules. Furthermore, backward force could be stimulated by plus-end directed external force applied to axonemal dynein before ATP application. The results show that axonemal dynein is unique exhibiting multiple modes of force generation including backward and forward force, oscillatory force and slow, repetitive bidirectional force. The results also demonstrate that mechanical strain is important for switching the directionality of force generation in axonemal dyneins.

Contribution of the Juxtatransmembrane Intracellular Regions to the Time Course and Permeation of ATP-gated P2X7 Receptor Ion Channels

P2X7 receptors are ATP-gated ion channels that contribute to inflammation and cell death. They have the novel property of showing marked facilitation to repeated applications of agonist, and the intrinsic channel pore dilates to allow the passage of fluorescent dyes. A 60-s application of ATP to hP2X7 receptors expressed in Xenopus oocytes gave rise to a current that had a biphasic time course with initial and secondary slowly developing components. A second application of ATP evoked a response with a more rapid time to peak. This facilitation was reversed to initial levels following a 10-min agonist-free interval. A chimeric approach showed that replacement of the pre-TM1 amino-terminal region with the corresponding P2X2 receptor section (P2X7-2Nβ) gave responses that quickly reached a steady state and did not show facilitation. Subsequent point mutations of variant residues identified Asn-16 and Ser-23 as important contributors to the time course/facilitation. The P2X7 receptor is unique in having an intracellular carboxyl-terminal cysteine-rich region (Ccys). Deletion of this region removed the secondary slowly developing current, and, when expressed in HEK293 cells, ethidium bromide uptake was only ∼5% that of WT levels, indicating reduced large pore formation. Dye uptake was also reduced for the P2X7-2Nβ chimera. Surprisingly, combination of the chimera and the Ccys deletion (P2X7-2NβdelCcys) restored the current rise time and ethidium uptake to WT levels. These findings suggest that there is a coevolved interaction between the juxtatransmembrane amino and carboxyl termini in the regulation of P2X7 receptor gating.

Role of domain calcium in purinergic P2X2 receptor channel desensitization.

Activation of P2X2 receptor channels (P2X2Rs) is characterized by a rapid current growth accompanied by a decay of current during sustained ATP application, a phenomenon known as receptor desensitization. Using rat, mouse, and human receptors, we show here that two processes contribute to receptor desensitization: bath calcium-independent desensitization and calcium-dependent desensitization. Calcium-independent desensitization is minor and comparable during repetitive agonist application in cells expressing the full size of the receptor but is pronounced in cells expressing shorter versions of receptors, indicating a role of the COOH terminus in control of receptor desensitization. Calcium-dependent desensitization is substantial during initial agonist application and progressively increases during repetitive agonist application in bath ATP and calcium concentration-dependent manners. Experiments with substitution of bath Na(+) with N-methyl-d-glucamine (NMDG(+)), a large organic cation, indicate that receptor pore dilation is a calcium-independent process in contrast to receptor desensitization. A decrease in the driving force for calcium by changing the holding potential from -60 to +120 mV further indicates that calcium influx through the channel pores at least partially accounts for receptor desensitization. Experiments with various receptor chimeras also indicate that the transmembrane and/or intracellular domains of P2X2R are required for development of calcium-dependent desensitization and that a decrease in the amplitude of current slows receptor desensitization. Simultaneous calcium and current recording shows development of calcium-dependent desensitization without an increase in global intracellular calcium concentrations. Combined with experiments with clamping intrapipette concentrations of calcium at various levels, these experiments indicate that domain calcium is sufficient to establish calcium-dependent receptor desensitization in experiments with whole-cell recordings.

Extracellular ATP modulates synaptic plasticity induced by activation of metabotropic glutamate receptors in the hippocampus.

Synaptic plasticity is believed to be a cellular mechanism for memory formation in the brain. It has been known that the metabotropic glutamate receptor (mGluR) is required for persistent forms of memory and induction of synaptic plasticity. Application of mGluR agonists induces synaptic plasticity in the absence of electrical conditioning stimulation, such as high or low frequency stimulation. The direction of the mGluR-induced synaptic plasticity, i.e., either long-term potentiation (LTP) or long-term-depression (LTD), is dependent on whether N-methyl-D-aspartate receptors (NMDARs) are co-activated with mGluRs. ATP has modulatory effects on neuronal functions and, in particular, there is increasing evidence that it plays a crucial role in synaptic plasticity. LTP can be induced by application of ATP, and this effect is inhibited by NMDAR antagonist. Although cooperative effects of NMDARs and mGluRs and of NMDARs and extracellular ATP in synaptic plasticity have been revealed, the effect of extracellular ATP on mGluR-induced synaptic plasticity is unknown. In this article, we summarize published data on mGluR- and ATP-induced synaptic plasticity, and present new data showing that extracellular ATP facilitates both the LTP and LTD induced by mGluR activation.

Rapid Activation of Distinct Conducting States in P2X Receptor Channels

P2X receptors are trimeric cation channels that are activated by extracellular ATP. These purinergic receptors have been reported to undergo pore dilation following activation by high concentrations of ATP, a phenomenon thought to mediate apoptosis in the immune system. Here we demonstrate that the widely reported slow time-dependent increase in the relative permeability of NMDG to Na, thought to reflect pore dilation, results from a gradual inhibition of small-cation-selective channels following depletion of intracellular alkali ions. Moreover, we find that P2X receptors enter both small-cation-selective open states and large-cation-permeable open states within milliseconds of ATP application. Taken together, our results demonstrate that P2X receptors can rapidly enter a large-cation-permeable open state without requiring either high ATP concentrations or high channel density, indicating that the ability of ATP to permeabilize cells can occur under physiologically realistic conditions.

Extracellular ATP activates MAPK and ROS signaling during injury response in the fungus Trichoderma atroviride.

The response to mechanical damage is crucial for the survival of multicellular organisms, enabling their adaptation to hostile environments. Trichoderma atroviride, a filamentous fungus of great importance in the biological control of plant diseases, responds to mechanical damage by activating regenerative processes and asexual reproduction (conidiation). During this response, reactive oxygen species (ROS) are produced by the NADPH oxidase complex. To understand the underlying early signaling events, we evaluated molecules such as extracellular ATP (eATP) and Ca2+ that are known to trigger wound-induced responses in plants and animals. Concretely, we investigated the activation of mitogen-activated protein kinase (MAPK) pathways by eATP, Ca2+, and ROS. Indeed, application of exogenous ATP and Ca2+ triggered conidiation. Furthermore, eATP promoted the Nox1-dependent production of ROS and activated a MAPK pathway. Mutants in the MAPK-encoding genes tmk1 and tmk3 were affected in wound-induced conidiation, and phosphorylation of both Tmk1 and Tmk3 was triggered by eATP. We conclude that in this fungus, eATP acts as a damage-associated molecular pattern (DAMP). Our data indicate the existence of an eATP receptor and suggest that in fungi, eATP triggers pathways that converge to regulate asexual reproduction genes that are required for injury-induced conidiation. By contrast, Ca2+ is more likely to act as a downstream second messenger. The early steps of mechanical damage response in T. atroviride share conserved elements with those known from plants and animals.

P.2.a.019 Lipopolysaccharide administration alters brain neurotransmission in prenatally stressed female rats

Extracellular ATP can influence cells via activation of P2X purinoceptors, the distribution of which can be altered in the central and peripheral nervous systems following injury or tissue damage. Here we have investigated the effect of a unilateral section of the cervical vagus nerve on the distribution of P2X1, P2X2, P2X3, P2X4 and P2X7 receptor subunit immunoreactivity (R-IR) in the dorsal vagal motor nucleus (DVN) and the nucleus ambiguus (NA) in the medulla oblongata. As early as 2 days, and followed up to 14 days, there was a dramatic ipsilateral increase in P2X1, P2X2 and P2X4R-IR in the cell soma of vagal efferent neurones in the DVN following the nerve section, but not the NA. There were no changes in P2X3 and P2X7R-IR in either nuclei. To test for possible functional consequences of increased P2X receptor levels, whole-cell patch-clamp recordings were made from DVN cells in brainstem slices 4 days following unilateral vagotomy. Application of ATP revealed large cell-to-cell variance in the current amplitude in neurones from both sectioned and control DVN. However, when ATP responses were compared to those elicited by the nicotinic acetylcholine receptor agonist carbachol, the mean ratio of the peak ATP-evoked current to the peak carbachol-evoked current was significantly larger in DVN neurones ipsilateral to the section. Thus the increase in P2XR levels in DVN cells ipsilateral to a nerve section are likely to reflect an increase in expression of functional P2XRs on the cell surface.

P.2.a.020 Hippocampal P2X7 receptor expression is modulated by stress and antidepressant treatment in the learned helplessness model

Extracellular ATP can influence cells via activation of P2X purinoceptors, the distribution of which can be altered in the central and peripheral nervous systems following injury or tissue damage. Here we have investigated the effect of a unilateral section of the cervical vagus nerve on the distribution of P2X1, P2X2, P2X3, P2X4 and P2X7 receptor subunit immunoreactivity (R-IR) in the dorsal vagal motor nucleus (DVN) and the nucleus ambiguus (NA) in the medulla oblongata. As early as 2 days, and followed up to 14 days, there was a dramatic ipsilateral increase in P2X1, P2X2 and P2X4R-IR in the cell soma of vagal efferent neurones in the DVN following the nerve section, but not the NA. There were no changes in P2X3 and P2X7R-IR in either nuclei. To test for possible functional consequences of increased P2X receptor levels, whole-cell patch-clamp recordings were made from DVN cells in brainstem slices 4 days following unilateral vagotomy. Application of ATP revealed large cell-to-cell variance in the current amplitude in neurones from both sectioned and control DVN. However, when ATP responses were compared to those elicited by the nicotinic acetylcholine receptor agonist carbachol, the mean ratio of the peak ATP-evoked current to the peak carbachol-evoked current was significantly larger in DVN neurones ipsilateral to the section. Thus the increase in P2XR levels in DVN cells ipsilateral to a nerve section are likely to reflect an increase in expression of functional P2XRs on the cell surface.

Autocrine activation of P2Y1 receptors couples Ca2+ influx to Ca2+ release in human pancreatic beta cells

There is evidence that ATP acts as an autocrine signal in beta cells but the receptors and pathways involved are incompletely understood. Here we investigate the receptor subtype(s) and mechanism(s) mediating the effects of ATP on human beta cells. We examined the effects of purinergic agonists and antagonists on membrane potential, membrane currents, intracellular Ca(2+) ([Ca(2+)]i) and insulin secretion in human beta cells. Extracellular application of ATP evoked small inward currents (3.4 ± 0.7 pA) accompanied by depolarisation of the membrane potential (by 14.4 ± 2.4 mV) and stimulation of electrical activity at 6 mmol/l glucose. ATP increased [Ca(2+)]i by stimulating Ca(2+) influx and evoking Ca(2+) release via InsP3-receptors in the endoplasmic reticulum (ER). ATP-evoked Ca(2+) release was sufficient to trigger exocytosis in cells voltage-clamped at -70 mV. All effects of ATP were mimicked by the P2Y(1/12/13) agonist ADP and the P2Y1 agonist MRS-2365, whereas the P2X(1/3) agonist α,β-methyleneadenosine-5-triphosphate only had a small effect. The P2Y1 antagonists MRS-2279 and MRS-2500 hyperpolarised glucose-stimulated beta cells and lowered [Ca(2+)]i in the absence of exogenously added ATP and inhibited glucose-induced insulin secretion by 35%. In voltage-clamped cells subjected to action potential-like stimulation, MRS-2279 decreased [Ca(2+)]i and exocytosis without affecting Ca(2+) influx. These data demonstrate that ATP acts as a positive autocrine signal in human beta cells by activating P2Y1 receptors, stimulating electrical activity and coupling Ca(2+) influx to Ca(2+) release from ER stores.

Activation of neuronal NMDA receptors triggers transient ATP-mediated microglial process outgrowth.

Microglia are morphologically dynamic cells that rapidly extend their processes in response to various stimuli including extracellular ATP. In this study, we tested the hypothesis that stimulation of neuronal NMDARs trigger ATP release leading to communication with microglia. We used acute mouse hippocampal brain slices and two-photon laser scanning microscopy to study microglial dynamics and developed a novel protocol for fixation and immunolabeling of microglia processes. Similar to direct topical ATP application in vivo, short multiple applications of NMDA triggered transient microglia process outgrowth that was reversible and repeatable indicating that this was not due to excitotoxic damage. Stimulation of NMDAR was required as NMDAR antagonists, but not blockers of AMPA/kainate receptors or voltage-gated sodium channels, prevented microglial outgrowth. We report that ATP release, secondary to NMDAR activation, was the key mediator of this neuron-microglia communication as both blocking purinergic receptors and inhibiting hydrolysis of ATP to prevent locally generated gradients abolished outgrowth. Pharmacological and genetic analyses showed that the NMDA-triggered microglia process extension was independent of Pannexin 1, the ATP releasing channels, ATP release from astrocytes via connexins, and nitric oxide generation. Finally, using whole-cell patch clamping we demonstrate that activation of dendritic NMDAR on single neurons is sufficient to trigger microglia process outgrowth. Our results suggest that dendritic neuronal NMDAR activation triggers ATP release via a Pannexin 1-independent manner that induces outgrowth of microglia processes. This represents a novel uncharacterized form of neuron-microglial communication mediated by ATP.

Involvement of Ectodomain Leu 214 in ATP Binding and Channel Desensitization of the P2X4 Receptor

P2X receptors are trimeric ATP-gated cation permeable ion channels. When ATP binds, the extracellular head and dorsal fin domains are predicted to move closer to each other. However, there are scant functional data corroborating the role of the dorsal fin in ligand binding. Here using site-directed mutagenesis and electrophysiology, we show that a dorsal fin leucine, L214, contributes to ATP binding. Mutant receptors containing a single substitution of alanine, serine, glutamic acid, or phenylalanine at L214 of the rat P2X4 receptor exhibited markedly reduced sensitivities to ATP. Mutation of other dorsal fin side chains, S216, T223, and D224, did not significantly alter ATP sensitivity. Exposure of L214C to sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) or (2-aminoethyl) methanethiosulfonate hydrobromide in the absence of ATP blocked responses evoked by subsequent ATP application. In contrast, when MTSES(-) was applied in the presence of ATP, no current inhibition was observed. Furthermore, L214A also slightly reduced the inhibitory effect of the antagonist 2',3'-O-(2,4,6-trinitrophenyl)-ATP, and the blockade was more rapidly reversible after washout. Certain L214 mutants also showed effects on current desensitization in the continued presence of ATP. L214I exhibited an accelerated current decline, whereas L214M exhibited a slower rate. Taken together, these data reveal that position L214 participates in both ATP binding and conformational changes accompanying channel opening and desensitization, providing compelling evidence that the dorsal fin domain indeed has functional properties that are similar to those previously reported for the body domains.

Purinergic signaling contributes to CO2‐sensitivity of neurons in the retrotrapezoid nucleus but not nucleus of the solitary tract or medullary raphe (872.6)

Central chemoreception is the mechanism by which the brain controls breathing in response to changes in tissue CO2/H+. Several brainstem regions are thought to contribute to chemoreception including the caudal nucleus of the solitary tract (cNTS), medullary raphe and retrotrapezoid nucleus (RTN). Although the molecular basis of chemoreception remains unclear, evidence suggests that RTN chemoreception involves intrinsic H+ sensing by an unidentified K+ channel and purinergic signaling possibly from CO2‐sensitive astrocytes. However, it is not clear whether purinergic signaling influences CO2‐responsiveness of other putative chemoreceptors. The goal of this study is to determine whether purinergic signaling modulates basal activity or CO2‐responsivness of neurons in the cNTS or medullary raphe. We use cell‐attached current clamp techniques to characterize firing rate responses to 15% CO2 and focal application of ATP (1mM) under control conditions and in the presence of a P2‐receptor blocker (PPADS, 100 µM). Chemosensitive neurons in the cNTS or medullary raphe were identified in brain slices (300 µm thick) from neonatal rat pups (P7‐12) bases on a 蠅 0.5 Hz firing rate response to 15% CO2. We found that CO2‐sensitive neurons in the cNTS, but not the medullary raphe, respond to focal ATP application with ~3 fold increase in firing rate. However, bath application of PPADS had no effect on baseline activity or CO2‐responsivness of neurons in either region. Immunohistochemical analysis using an astrocyte‐specific marker (aldehyde dehydrogenase 1L1) suggests that the RTN has a higher density of astrocytes compared to the cNTS or medullary raphe. Together, these results suggest that purinergic signaling is a unique feature of RTN chemoreception.Grant Funding Source: Supported by NLBI (HL 104101) and AHA (11PRE7580037)

Purinergic signalling contributes to chemoreception in the retrotrapezoid nucleus but not the nucleus of the solitary tract or medullary raphe

Several brain regions are thought to function as important sites of chemoreception including the nucleus of the solitary tract (NTS), medullary raphe and retrotrapezoid nucleus (RTN). In the RTN, mechanisms of chemoreception involve direct H(+)-mediated activation of chemosensitive neurons and indirect modulation of chemosensitive neurons by purinergic signalling. Evidence suggests that RTN astrocytes are the source of CO2-evoked ATP release. However, it is not clear whether purinergic signalling also influences CO2/H(+) responsiveness of other putative chemoreceptors. The goals of this study are to determine if CO2/H(+)-sensitive neurons in the NTS and medullary raphe respond to ATP, and whether purinergic signalling in these regions influences CO2 responsiveness in vitro and in vivo. In brain slices, cell-attached recordings of membrane potential show that CO2/H(+)-sensitive NTS neurons are activated by focal ATP application; however, purinergic P2-receptor blockade did not affect their CO2/H(+) responsiveness. CO2/H(+)-sensitive raphe neurons were unaffected by ATP or P2-receptor blockade. In vivo, ATP injection into the NTS increased cardiorespiratory activity; however, injection of a P2-receptor blocker into this region had no effect on baseline breathing or CO2/H(+) responsiveness. Injections of ATP or a P2-receptor blocker into the medullary raphe had no effect on cardiorespiratory activity or the chemoreflex. As a positive control we confirmed that ATP injection into the RTN increased breathing and blood pressure by a P2-receptor-dependent mechanism. These results suggest that purinergic signalling is a unique feature of RTN chemoreception.

Corticosterone treatment results in enhanced release of peptidergic vesicles in astrocytes via cytoskeletal rearrangements

While the effect of stress on neuronal physiology is widely studied, its effect on the functionality of astrocytes is not well understood. We studied the effect of high doses of stress hormone corticosterone, on two physiological properties of astrocytes, i.e., gliotransmission and interastrocytic calcium waves. To study the release of peptidergic vesicles from astrocytes, hippocampal astrocyte cultures were transfected with a plasmid to express pro-atrial natriuretic peptide (ANP) fused with the emerald green fluorescent protein (ANP.emd). The rate of decrease in fluorescence of ANP.emd on application of ionomycin, a calcium ionophore was monitored. Significant increase in the rate of calcium-dependent exocytosis of ANP.emd was observed with the 100 nM and 1 μM corticosterone treatments for 3 h, which depended on the activation of the glucocorticoid receptor. ANP.emd tagged vesicles exhibited increased mobility in astrocyte culture upon corticosterone treatment. Increasing corticosterone concentrations also resulted in concomitant increase in the calcium wave propagation velocity, initiated by focal ATP application. Corticosterone treatment also resulted in increased GFAP expression and F-actin rearrangements. FITC-Phalloidin immunostaining revealed increased formation of cross linked F-actin networks with the 100 nM and 1 μM corticosterone treatment. Alternatively, blockade of actin polymerization and disruption of microtubules prevented the corticosterone-mediated increase in ANP.emd release kinetics. This study reports for the first time the effect of corticosterone on gliotransmission via modulation of cytoskeletal elements. As ANP acts on both neurons and blood vessels, modulation of its release could have functional implications in neurovascular coupling under pathophysiological conditions of stress.