Activity-dependent adenosine release may be linked to activation of Na+-K+ ATPase : an in vitro rat study

In the brain, extracellular adenosine increases as a result of neuronal activity. The mechanisms by which this occurs are only incompletely understood. Here we investigate the hypothesis that the Na+ influxes associated with neuronal signalling activate the Na+-K+ ATPase which, by consuming ATP, generates intracellular adenosine that is then released via transporters. By measuring adenosine release directly with microelectrode biosensors, we have demonstrated that AMPA-receptor evoked adenosine release in basal forebrain and cortex depends on extracellular Na+. We have simultaneously imaged intracellular Na+ and measured adenosine release. The accumulation of intracellular Na+ during AMPA receptor activation preceded adenosine release by some 90 s. By removing extracellular Ca2+, and thus preventing indiscriminate neuronal activation, we used ouabain to test the role of the Na+-K+ ATPase in the release of adenosine. Under conditions which caused a Na+ influx, brief applications of ouabain increased the accumulation of intracellular Na+ but conversely rapidly reduced extracellular adenosine levels. In addition, ouabain greatly reduced the amount of adenosine released during application of AMPA. Our data therefore suggest that activity of the Na+-K+ ATPase is directly linked to the efflux of adenosine and could provide a universal mechanism that couples adenosine release to neuronal activity. The Na+-K+ ATPase-dependent adenosine efflux is likely to provide adenosine-mediated activity-dependent negative feedback that will be important in many diverse functional contexts including the regulation of sleep

Activity-dependent release of Adenosine: a critical re-evaluation of mechanism

Adenosine is perhaps the most important and universal modulator in the brain. The current consensus is that it is primarily produced in the extracellular space from the breakdown of previously released ATP. It is also accepted that it can be released directly, as adenosine, during pathological events primarily by equilibrative transport. Nevertheless, there is a growing realization that adenosine can be rapidly released from the nervous system in a manner that is dependent upon the activity of neurons. We consider three competing classes of mechanism that could explain neuronal activity dependent adenosine release (exocytosis of ATP followed by extracellular conversion to adenosine; exocytotic release of an unspecified transmitter followed by direct non-exocytotic adenosine release from an interposed cell; and direct exocytotic release of adenosine) and outline discriminatory experimental tests to decide between them. We review several examples of activity dependent adenosine release and explore their underlying mechanisms where these are known. We discuss the limits of current experimental techniques in definitively discriminating between the competing models of release, and identify key areas where technologies need to advance to enable definitive discriminatory tests. Nevertheless, within the current limits, we conclude that there is evidence for a mechanism that strongly resembles direct exocytosis of adenosine underlying at least some examples of neuronal activity dependent adenosine release

CO2-dependent opening of an inwardly rectifying K+ channel

CO2 chemosensing is a vital function for the maintenance of life that helps to control acid–base balance. Most studies have reported that CO2 is measured via its proxy, pH. Here we report an inwardly rectifying channel, in outside-out excised patches from HeLa cells that was sensitive to modest changes in PCO2 under conditions of constant extracellular pH. As PCO2 increased, the open probability of the channel increased. The single-channel currents had a conductance of 6.7 pS and a reversal potential of –70 mV, which lay between the K+ and Cl– equilibrium potentials. This reversal potential was shifted by +61 mV following a tenfold increase in extracellular [K+] but was insensitive to variations of extracellular [Cl–]. The single-channel conductance increased with extracellular [K+]. We propose that this channel is a member of the Kir family. In addition to this K+ channel, we found that many of the excised patches also contained a conductance carried via a Cl–-selective channel. This CO2-sensitive Kir channel may hyperpolarize excitable cells and provides a potential mechanism for CO2-dependent inhibition during hypercapnia

Biosensor measurement of purine release from cerebellar cultures and slices

We have previously described an action-potential and Ca2+-dependent form of adenosine release in the molecular layer of cerebellar slices. The most likely source of the adenosine is the parallel fibres, the axons of granule cells. Using microelectrode biosensors, we have therefore investigated whether cultured granule cells (from postnatal day 7–8 rats) can release adenosine. Although no purine release could be detected in response to focal electrical stimulation, purine (adenosine, inosine or hypoxanthine) release occurred in response to an increase in extracellular K+ concentration from 3 to 25 mM coupled with addition of 1 mM glutamate. The mechanism of purine release was transport from the cytoplasm via an ENT transporter. This process did not require action-potential firing but was Ca2+dependent. The major purine released was not adenosine, but was either inosine or hypoxanthine. In order for inosine/hypoxanthine release to occur, cultures had to contain both granule cells and glial cells; neither cellular component was sufficient alone. Using the same stimulus in cerebellar slices (postnatal day 7–25), it was possible to release purines. The release however was not blocked by ENT blockers and there was a shift in the Ca2+ dependence during development. This data from cultures and slices further illustrates the complexities of purine release, which is dependent on cellular composition and developmental stage

\u3cem\u3eRhizobium leguminosarum\u3c/em\u3e CFN42 Lipopolysaccharide Antigenic Changes Induced by Environmental Conditions

Four monoclonal antibodies were raised against the lipopolysaccharide of Rhizobium leguminosarum bv. phaseoli CFN42 grown in tryptone and yeast extract. Two of these antibodies reacted relatively weakly with the lipopolysaccharide of bacteroids of this strain isolated from bean nodules. Growth ex planta of strain CFN42 at low pH, high temperature, low phosphate, or low oxygen concentration also eliminated binding of one or both of these antibodies. Lipopolysaccharide mobility on gel electrophoresis and reaction with other monoclonal antibodies and polyclonal antiserum indicated that the antigenic changes detected by these two antibodies did not represent major changes in lipopolysaccharide structure. The antigenic changes at low pH were dependent on growth of the bacteria but were independent of nitrogen and carbon sources and the rich or minimal quality of the medium. The Sym plasmid of this strain was not required for the changes induced ex planta. Analysis of bacterial mutants inferred to have truncated O-polysaccharides indicated that part, but not all, of the lipopolysaccharide O-polysaccharide portion was required for binding of these two antibodies. In addition, this analysis suggested that O-polysaccharide structures more distal to lipid A than the epitopes themselves were required for the modifications at low pH that prevented antibody binding. Two mutants were antigenically abnormal, even though they had abundant lipopolysaccharides of apparently normal size. One of these two mutants was constitutively unreactive toward three of the antibodies but indistinguishable from the wild type in symbiotic behavior. The other, whose bacteroids retained an epitope normally greatly diminished in bacteroids, was somewhat impaired in nodulation frequency and nodule development

Real-time measurement of adenosine and ATP release in the central nervous system

This brief review recounts how, stimulated by the work of Geoff Burnstock, I developed biosensors that allowed direct real-time measurement of ATP and adenosine during neural function. The initial impetus to create an adenosine biosensor came from trying to understand how ATP and adenosine-modulated motor pattern generation in the frog embryo spinal cord. Early biosensor measurements demonstrated slow accumulation of adenosine during motor activity. Subsequent application of these biosensors characterized real-time release of adenosine in in vitro models of brain ischaemia, and this line of work has recently led to clinical measurements of whole blood purine levels in patients undergoing carotid artery surgery or stroke. In parallel, the wish to understand the role of ATP signalling in the chemosensory regulation of breathing stimulated the development of ATP biosensors. This revealed that release of ATP from the chemosensory areas of the medulla oblongata preceded adaptive changes in breathing, triggered adaptive changes in breathing via activation of P2 receptors, and ultimately led to the discovery of connexin26 as a channel that mediates CO2-gated release of ATP from cells

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 (~3 µM). 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 µM) 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

A depletable pool of adenosine in area CA1 of the rat hippocampus

Adenosine plays a major modulatory and neuroprotective role in the mammalian CNS. During cerebral metabolic stress, such as hypoxia or ischemia, the increase in extracellular adenosine inhibits excitatory synaptic transmission onto vulnerable neurons via presynaptic adenosine A1 receptors, thereby reducing the activation of postsynaptic glutamate receptors. Using a combination of extracellular and whole-cell recordings in the CA1 region of hippocampal slices from 12- to 24-d-old rats, we have found that this protective depression of synaptic transmission weakens with repeated exposure to hypoxia, thereby allowing potentially damaging excitation to both persist for longer during oxygen deprivation and recover more rapidly on reoxygenation. This phenomenon is unlikely to involve A1 receptor desensitization or impaired nucleoside transport. Instead, by using the selective A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine and a novel adenosine sensor, we demonstrate that adenosine production is reduced with repeated episodes of hypoxia. Furthermore, this adenosine depletion can be reversed at least partially either by the application of exogenous adenosine, but not by a stable A1 agonist, N6-cyclopentyladenosine, or by endogenous means by prolonged (2 hr) recovery between hypoxic episodes. Given the vital neuroprotective role of adenosine, these findings suggest that depletion of adenosine may underlie the increased neuronal vulnerability to repetitive or secondary hypoxia/ischemia in cerebrovascular disease and head injury