The acid nature of CO2-evoked adenosine release in the CNS.
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Action Potentials↗
Biomedical subjects
Nicholas Dale
Publications and source records attributed to Nicholas Dale.
Comparative genomic and expression analysis of the conserved NTPDase gene family in Xenopus.
The purines, ATP and adenosine, are important signaling molecules in the nervous system. ATP is sequentially degraded to adenosine by the ectonucleotidase proteins. The NTPDase (or CD39) family is a subfamily of these enzymes, which consists of nine members in mammals. In Xenopus embryos, we have shown that ATP, and its antagonist adenosine, regulate the rundown of swimming and we therefore proposed that ectonucleotidase proteins are key regulators of locomotor activity. Here, we report the cloning of all nine members of the NTPDase family in Xenopus laevis and Xenopus tropicalis. Our phylogenetic analysis shows that this family is highly conserved between the frog species and also during vertebrate evolution. In the adult frog, NTPDase genes are broadly expressed. During development, all NTPDase genes, except for NTPDase8, are expressed and display a distinct specific expression pattern, suggesting potentially different functions of these proteins during embryogenesis of X. laevis.
Amino Acid Sequence↗
ATP is a mediator of chemosensory transduction in the central nervous system.
Extracellular signalling by the purine nucleotide ATP has long been associated with sensory function. In the periphery, ATP mediates nociception, mechanosensitivity, thermal sensitivity and O2 chemosensitivity. These processes share a common mechanism that involves the release of ATP to excite afferent fibres via activation of ionotropic P2X and/or metabotropic P2Y receptors. Chemosensors located in the brainstem are crucial for the maintenance of physiological levels of blood gases through the regulation of breathing. Here we show that an increase in pCO2 in the arterial blood triggers the immediate release of ATP from three chemosensitive regions located on the ventral surface of the medulla oblongata. Blockade of ATP receptors at these sites diminishes the chemosensory control of breathing, suggesting that ATP release constitutes a key step in central chemosensory transduction. These new data suggest that ATP, a phylogenetically ancient, unique and simple molecule, has been widely used in the evolution of afferent systems to mediate distinct forms of sensory transduction not only in the periphery but also within the central nervous system.
Adenosine Triphosphate↗
ATP released via gap junction hemichannels from the pigment epithelium regulates neural retinal progenitor proliferation.
The retinal pigment epithelium (RPE) plays an essential role in the normal development of the underlying neural retina, but the mechanisms by which this regulation occurs are largely unknown. Ca2+ transients, induced by the neurotransmitter ATP acting on purinergic receptors, both increase proliferation and stimulate DNA synthesis in neural retinal progenitor cells. Here, we show that the RPE regulates proliferation in the underlying neural retina by the release of a soluble factor and identify that factor as ATP. Further, we show that this ATP is released by efflux through gap junction connexin 43 hemichannels, the opening of which is evoked by spontaneous elevations of Ca2+ in trigger cells in the RPE. This release mechanism is localized within the RPE cells to the membranes facing the neural retina, a location ideally positioned to influence neural retinal development. ATP released from RPE hemichannels speeds both cell division and proliferation in the neural retina.
Adenosine Triphosphate↗
Microelectrode biosensor for real-time measurement of ATP in biological tissue.
The purines ATP, ADP, and adenosine are important extracellular signaling agents. Analysis of purinergic signaling has been slowed by lack of direct methods for measurement of purine release in real-time during physiological activity. We have previously reported microelectrode biosensors for adenosine, but similar sensors for ATP have remained elusive. We now describe an ATP biosensor formed by coating a Pt microelectrode with an ultrathin biolayer containing glycerol kinase and glycerol-3-phosphate oxidase. It responds rapidly (10-90% rise time <10 s) and exhibits a linear response to ATP over the physiologically relevant concentrations of 200 nM-50 microM and is very sensitive approximately 250 mA.M(-1).cm(-2). By including phosphocreatine kinase in the biolayer, we can optionally amplify the ATP signal and also make the sensor sensitive to external ADP. We have used our sensors to make the first demonstration that ATP is released from spinal networks in vivo during locomotor activity.
Adenosine Diphosphate↗
Release of ATP in the ventral medulla during hypoxia in rats: role in hypoxic ventilatory response.
P2X2 receptor subunits of the ATP-gated ion channels are expressed by physiologically identified respiratory neurons in the ventral respiratory column, implicating ATP in the control of respiratory activity. We now show that, during hypoxia, release of ATP in the ventrolateral medulla (VLM) plays an important role in the hypoxic ventilatory response in rats. By measuring ATP release in real time at the ventral surface of the medulla with novel amperometric biosensors, we found that hypoxia (10% O2; 5 min) induced a marked increase in the concentration of ATP (approximately 3 microm). This ATP release occurred after the initiation of enhanced respiratory activity but coincided with the later hypoxia-induced slowing of the respiratory rhythm. ATP was also released at the ventral surface of the medulla during hypoxia in peripherally chemodenervated animals (vagi, aortic, and carotid sinus nerve sectioned). By using horizontal slices of the rat medulla, we found that, during hypoxia, ATP is produced throughout the VLM in the locations corresponding to the ventral respiratory column. Blockade of ATP receptors in the VLM (microinjection of P2 receptor antagonist pyridoxal-5'-phosphate-6-azophenyl-2',4'-disulphonic acid; 100 mum) augmented the hypoxia-induced secondary slowing of the respiratory rhythm. Our findings suggest that ATP released within the ventral respiratory column is involved in maintenance of the respiratory activity in conditions when hypoxia-induced slowing of respiration occurs. These data illustrate a new functional role for ATP-mediated purinergic signaling in the medullary mechanisms controlling respiratory activity.
Adaptation, Physiological↗
Listening to the brain: microelectrode biosensors for neurochemicals.
Chemical signalling underlies every function of the nervous system, from those of which we are unaware, for example, control of the heart, to higher cognitive functions, such as emotions, learning and memory. Neurotransmitters and neuromodulators mediate communication between neurons and between neurons and non-neural cells such as glia and muscle. In the past, the means for studying the production and release of these signalling agents directly has been limited in its temporal and spatial resolution relative to the dynamics of chemical signalling and the structures of interest in the brain. Now microelectrode biosensors are becoming available that give unprecedented spatial and temporal resolution, enabling, for the first time, direct measurement in real time of the chemical conversations between cells in the nervous system.
Animals↗
Fever in systemic inflammation: roles of purines.
Extracellular purine nucleotide and nucleoside signalling molecules, such as ATP and adenosine, acting through specific receptors (P2 and P1, respectively) play significant roles in the mechanisms underlying the febrile response. A variety of P2 and P1 receptor subunits have been identified in the hypothalamus, the area of the brain that orchestrates the febrile response. Importantly, both ATP and adenosine have been shown to modulate release and/or action of cytokines that are implicated in fever, as well as to be involved in the central mechanisms of cardiovascular and respiratory control. Our data indicate that at the level of the anterior hypothalamus extracellular ATP is involved in the control of the development of fever. A population of warm-sensitive neurones in the anterior hypothalamus is likely to be the site of action of ATP on body temperature. ATP-induced cytokine release does not appear to play a significant role in the hypothalamic mechanisms leading to the development of the febrile response. However, the blockade of fever by P2 receptor antagonists given systemically suggests that ATP-mediated signalling may play a role in the release of pyrogenic cytokines in the periphery. At the level of the anterior hypothalamus adenosine appears to be released tonically, and acts to maintain body temperature under afebrile conditions. There is also evidence that adenosine-mediated signalling may play a role in the hypothalamic mechanisms controlling the degree of body temperature increase during fever. Our investigations have identified possible mechanisms by which purines modulate the febrile response. The actions of purines on body temperature during fever are most likely "site specific" (brain vs. periphery), may or may not involve their effect on cytokine release and/or action, and are likely to involve P2 and P1 receptors of different subtypes. Further extensive studies are needed to elucidate these mechanisms in greater detail and may lead to the development of new approaches for modifying febrile, cytokine and acute-phase responses to infection.
Adenosine↗
AICA riboside both activates AMP-activated protein kinase and competes with adenosine for the nucleoside transporter in the CA1 region of the rat hippocampus.
5-Aminoimidazole-4-carboxamide riboside (AICA riboside; Acadesine) activates AMP-activated protein kinase (AMPK) in intact cells, and is reported to exert protective effects in the mammalian CNS. In rat cerebrocortical brain slices, AMPK was activated by metabolic stress (ischaemia > hypoxia > aglycaemia) and AICA riboside (0.1-10 mm). Activation of AMPK by AICA riboside was greatly attenuated by inhibitors of equilibrative nucleoside transport. AICA riboside also depressed excitatory synaptic transmission in area CA1 of the rat hippocampus, which was prevented by an adenosine A1 receptor antagonist and reversed by application of adenosine deaminase. However, AICA riboside was neither a substrate for adenosine deaminase nor an agonist at adenosine receptors. We conclude that metabolic stress and AICA riboside both stimulate AMPK activity in mammalian brain, but that AICA riboside has an additional effect, i.e. competition with adenosine for uptake by the nucleoside transporter. This results in an increase in extracellular adenosine and subsequent activation of adenosine receptors. Neuroprotection by AICA riboside could be mediated by this mechanism as well as, or instead of, by AMPK activation. Caution should therefore be exercised in ascribing an effect of AICA riboside to AMPK activation, especially in systems where inhibition of adenosine re-uptake has physiological consequences.
AMP-Activated Protein Kinases↗
ATP is a key mediator of central and peripheral chemosensory transduction.
Recent evidence suggests that ATP is a mediator of central (within the ventral surface of the medulla) and peripheral (within the carotid body) chemosensory transduction. This short review discusses the data obtained in experiments in vivo and in vitro supporting this hypothesis. P2 receptors for ATP are expressed within the ventrolateral medulla as well as by the peripheral chemosensory afferent neurones. Blockade of P2 receptors in the ventrolateral medulla attenuates the CO2-induced increase in respiration while blockade of purinergic signalling impairs carotid body function and diminishes the ventilatory response to hypoxia. Furthermore, ATP is released from the ventral surface of the medulla during hypercapnia and from the carotid body during hypoxia. Finally, exogenous ATP applied on the ventral surface of the medulla evokes rapid increase in phrenic nerve activity, while ATP applied to the carotid body evokes marked excitation of the carotid sinus nerve afferents. We suggest that in the ventrolateral medulla ATP is produced following CO2/H(+)-induced activation of central chemosensory elements (neuronal and/or glial) and acts within the respiratory network to produce physiologically relevant changes in ventilation. In the carotid body, ATP contributes in a significant manner to the transmission of the sensitivity of the carotid body to changes in arterial PO2 and may be considered as a key transmitter released by chemoreceptor cells to activate endings of the sinus nerve afferent fibres.
Adenosine Triphosphate↗
Chemosensory control of the respiratory function. Working towards understanding the role of ATP-mediated purinergic signalling.
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Adenosine Triphosphate↗
A three-enzyme microelectrode sensor for detecting purine release from central nervous system.
As the purines, in particular adenosine, are important signaling agents in the nervous system we have devised a new biosensor for directly measuring their production in real time during physiological activity. Our amperometric adenosine biosensor is made by entrapping 3 enzymes (xanthine oxidase, purine nucleoside phosphorylase and adenosine deaminase) in a composite lactobionamide and amphiphillic polypyrrole matrix around a Pt microelectrode. The resulting sensors are small (25-100 microm diameter), fast responding (10-90% rise time, 2+/-0.23 s), sensitive (100-222 mA M(-1) cm(-2)) and stable (100% activity after 5 days). The sensor was used in vivo to demonstrate the spatial localization of release of adenosine from Xenopus embryo spinal cord during fictive swimming.
Animals↗
Xenopus embryonic spinal neurons recorded in situ with patch-clamp electrodes--conditional oscillators after all?
The central pattern generator for swimming Xenopus embryo is organized as two half-centres linked by reciprocal inhibition. Microelectrode recordings suggest that Xenopus neurons are poorly excitable, necessitating a key role for postinhibitory rebound in the operation of the central pattern generator. However the Xenopus central pattern generator seems unusual in that the component neurons apparently have no intrinsic or conditional rhythmogenic properties. We have re-examined the firing properties of Xenopus embryo spinal neurons by making patch-clamp recordings in situ from intact spinal cord. Recordings made from 99 neurons were divided into three groups. Central pattern generator neurons overwhelmingly (44/51) fired trains of action potentials in response to current injection. Just over half of the sensory interneurons (13/22) also fired trains of action potentials. Neurons that received no synaptic inputs during swimming mostly fired just one or two action potentials (22/26). Thirty-four neurons were identified morphologically. Commissural (8/12) and descending (6/6) interneurons, key components of the spinal central pattern generator, fired repetitive trains of action potentials during current injection. Neurons that were not part of the central pattern generator did not demonstrate this preponderance for repetitive firing. Analysis of the interspike intervals during current injection revealed that the majority of central pattern generators, descending and commissural interneurons, could readily fire at frequencies up to twice that of swimming. We suggest that Xenopus neurons can be considered as conditional oscillators: in the presence of unpatterned excitation they exhibit an ability to fire rhythmically. This property makes the Xenopus embryonic central pattern generator more similar to other model central pattern generators than has hitherto been appreciated.
Action Potentials↗
Development of Ca2+-channel and BK-channel expression in embryos and larvae of Xenopus laevis.
The patterns and density of channels expressed in neurons critically determine their electrical properties. We have examined developmental regulation of Ca2+-channel expression during the maturation of the spinal motor circuits in Xenopus as it develops from an embryo to a larva. In embryonic neurons approximately 60% of the current is carried by N-type channels, 8% by l-type channels and the remainder by an unidentified channel. As the embryo matures, omega-agatoxin-sensitive P/Q channels are gradually expressed and replace the unidentified HVA channel such that at stage 42 approximately 25% of the current is carried by P/Q channels. We have used fluorescent labelling of selective channel toxins to directly observe the distribution of P/Q, N and BK channels. The P/Q channel distribution was most prevalent on the cell surface proximal to the areas of the soma where processes emerge. Both N and BK channels were distributed throughout the soma but still exhibited concentration around the areas adjacent to the emergence of processes from the soma. The patterns of fluorescence labelling during development mirrored the development of the respective ionic currents. Both N and P/Q channels contribute roughly equally to activation of the BK current, suggesting that overlap in the distribution of the N, P/Q and BK channels is important in their functional interdependence. The newly expressed P/Q channels play a role in spike initiation and repetitive firing in larval spinal neurons and contribute to burst generation during swimming in the larva.
Aging↗
Resetting intrinsic purinergic modulation of neural activity: an associative mechanism?
The purines, ATP and adenosine, control the rundown and termination of swimming in the Xenopus embryo. This intrinsic purinergic modulation, unavoidably present during every swimming episode, could lead to stereotyped inflexible behavior and consequently could jeopardize the survival of the embryo. To explore whether this control system can exhibit adaptability, I have used a minimal simulation in which a model neuron released ATP that (1) inhibited K+ currents and (2) was converted by ectonucleotidases to adenosine, which then inhibited Ca2+ currents. The model neuron exhibited an accommodating spike train controlled by the actions of ATP and adenosine. Feedforward inhibition by the upstream metabolite ADP of the ecto-5'-nucleotidase that converts AMP to adenosine introduced adaptability and allowed the resetting of spike accommodation. The strength of feedforward inhibition determined the extent to which resetting could occur. I have tested these predictions by examining swimming in the real embryo. The rundown of swimming was reset in a manner similar to that predicted by the single-neuron model. By blocking the purinoceptors, I have demonstrated that resetting in the embryo is attributable to the actions of the purines and results from feedforward inhibition of adenosine production. The resetting of rundown in the motor systems can be reformulated as an associative mechanism in which the temporal coincidence of two stimuli can prolong network activity if they fall within a particular time window. The length of the time window and the magnitude of the prolongation of neural activity both depend on the strength of the feedforward ADP-mediated inhibition of the ecto-5'-nucleotidase.
5'-Nucleotidase↗
Rapid adenosine release in the nucleus tractus solitarii during defence response in rats: real-time measurement in vivo.
We have measured the release of adenosine and inosine from the dorsal surface of the brainstem and from within the nucleus tractus solitarii (NTS) during the defence response evoked by hypothalamic stimulation in the anaesthetised rat. At the surface of the brainstem, only release of inosine was detected on hypothalamic defence area stimulation. This inosine signal was greatly reduced by addition of the ecto-5'-nucleotidase inhibitor alpha,beta-methylene ADP (200 microM), suggesting that the inosine arose from adenosine that was produced in the extracellular space by the prior release of ATP. By placing a microelectrode biosensor into the NTS under stereotaxic control we have recorded release of adenosine within this nucleus. By contrast to the brainstem surface, a fast increase in adenosine, accompanied only by a much smaller change in inosine levels, was seen following stimulation of the hypothalamic defence area. The release of adenosine following hypothalamic stimulation was mainly confined to a narrow region of the NTS some 500 microm in length around the level of the obex. Interestingly the release of adenosine was depletable: when the defence reaction was evoked at short time intervals, much less adenosine was released on the second stimulus. Our novel techniques have given unprecedented real-time measurement and localisation of adenosine release in vivo and demonstrate that adenosine is released at the right time and in sufficient quantities to contribute to the cardiovascular components of the defence reaction.
Adenosine↗
Adenosine release in nucleus tractus solitarii does not appear to mediate hypoxia-induced respiratory depression in rats.
The time course of adenosine release in the nucleus tractus solitarii (NTS) and ventrolateral medulla (VLM) during acute systemic hypoxia was investigated in the anaesthetised rat by means of amperometric enzymatic sensors. It was found that acute hypoxia induced a significant delayed increase in adenosine level (reaching levels as high as 5 microM) in the NTS and that hypoxia-induced release of adenosine was similar at various regions of the NTS along its rostro-caudal axis. Significantly smaller or no increases in adenosine levels at all in response to hypoxia were observed in the VLM. The increase in adenosine level in the NTS occurred during reoxygenation after the termination of the hypoxic challenge and was accompanied by a smaller increase in inosine concentration. At the dorsal surface of the brainstem, only release of inosine was detected following acute hypoxia. Addition of the ecto-5'-nucleotidase inhibitor alpha,beta-methylene ADP (200 microM) to the dorsal surface of the brainstem completely abolished the signal evoked by hypoxia, suggesting that the inosine arose from adenosine that was produced in the extracellular space by the prior release of ATP. This study indicates that following systemic hypoxia, adenosine levels in the NTS increase to a significantly greater extent than in the VLM. However, the increase in adenosine concentration in the NTS occurs too late to be responsible for the hypoxia-induced depression of the respiratory activity.
Adenosine↗
Modulation of K(+) currents in Xenopus spinal neurons by p2y receptors: a role for ATP and ADP in motor pattern generation.
We have investigated the pharmacological properties and targets of p2y purinoceptors in Xenopus embryo spinal neurons. ATP reversibly inhibited the voltage-gated K(+) currents by 10 +/- 3 %. UTP and the analogues alpha,beta-methylene-ATP and 2-methylthio-ATP also inhibited K(+) currents. This agonist profile is similar to that reported for a p2y receptor cloned from Xenopus embryos. Voltage-gated K(+) currents could be inhibited by ADP (9 +/- 0.8 %) suggesting that a further p2y1-like receptor is also present in the embryo spinal cord. Unexpectedly we found that alpha,beta-methylene-ADP, often used to block the ecto-5'-nucleotidase, also inhibited voltage-gated K(+) currents (7 +/- 2.3 %). This inhibition was occluded by ADP, suggesting that alpha,beta-methylene-ADP is an agonist at p2y1 receptors. We have directly studied the properties of the ecto-5'-nucleotidase in Xenopus embryo spinal cord. Although ADP inhibited this enzyme, alpha,beta-methylene-ADP had no action. Caution therefore needs to be used when interpreting the actions of alpha,beta-methylene-ADP as it has previously unreported agonist activity at P2 receptors. Xenopus spinal neurons possess fast and slow voltage-gated K(+) currents. By using catechol to selectively block the fast current, we completely occluded the actions of ATP and ADP. Furthermore, the purines appeared to block only the fast relaxation component of the tail currents. We therefore conclude that the p2y receptors target only the fast component of the delayed rectifier. As ATP breakdown to ADP is rapid and ADP may accumulate at higher levels than ATP, the contribution of ADP acting through p2y1-like receptors may be an important additional mechanism for the control of spinal motor pattern generation.
Adenosine Diphosphate↗