Spontaneous Adenosine Research Articles

Pannexin1 channels regulate mechanically stimulated but not spontaneous adenosine release.

Fast-scan cyclic voltammetry (FSCV)is a rapid technique to measure neuromodulators, and using FSCV, two modes of rapid adenosine have been discovered. Spontaneous transients occur randomly in the brain, while mechanical stimulation also causes a rapid adenosine event. Pannexin1 channels are membrane channels that transport ions, including ATP, out of the cell where it is rapidly broken down into adenosine. Pannexin 1 channels (Panx1) have a flickering mode of rapid opening and are also mechanically stimulated. Here, we test the extent to which pannexin channels, specifically pannexin1 (Panx1) channels, are responsible for rapid adenosine events. Spontaneous adenosine release or mechanosensitive adenosine release were measured using fast-scan cyclic voltammetry in hippocampal (CA1) brain slices. In global Panx1KO mice, there is no significant difference in the frequency or concentration of spontaneous adenosine release, indicating Panx1 is not a release mechanism for spontaneous adenosine. Spontaneous adenosine frequency decreased slightly after administration of a large (100µM) dose of carbenoxolone, a nonspecific inhibitor of many pannexin and connexin channels, suggesting other hemichannels only play a small role at most. For mechanically stimulated adenosine release, the concentration of each adenosine event significantly decreased 30% in Panx1KO mice and the frequency of stimulations that evoked adenosine also decreased. The response was similar in WT mice with carbenoxolone. Thus, Panx1 is a release mechanism for mechanically stimulated adenosine release, but not the only mechanism. These results demonstrate that pannexin channels differentially regulate rapid adenosine release and could be targeted to differentially affect mechanically stimulated adenosine due to brain damage.

Dual-Channel Electrochemical Measurements Reveal Rapid Adenosine is Localized in Brain Slices.

Rapid adenosine signaling has been detected spontaneously or after mechanical stimulation in the brain, providing rapid neuromodulation in a local area. To measure rapid adenosine signaling, a single carbon-fiber microelectrode has traditionally been used, which limits spatial resolution and an understanding of regional coordination. In this study, we utilized dual-channel fast-scan cyclic voltammetry to measure the spontaneous or mechanically stimulated adenosine release at two electrodes placed at different spacings in hippocampal CA1 mouse brain slices. For mechanically stimulated adenosine release, adenosine can be detected up to 150 μm away from where it was stimulated, although the signal is smaller and delayed. While spontaneous adenosine transients were detected at both electrodes, only 10 percent of the events were detected concurrently, and that number was similar at 50 and 200 μm electrode spacings. Thus, most adenosine transients were not caused by the widespread coordination of release. There was no evidence of diffusion of spontaneous transients to a second electrode 50-200 μm away. This study shows that spontaneous adenosine events are very localized and thus provide only local neuromodulation. Injury, such as mechanical stimulation, allows adenosine to diffuse farther, but the neuroprotective effects are still regional. These results provide a better understanding of the spatial and temporal profiles of adenosine available to act at receptors, which is crucial for future studies that design neuroprotective treatments based on rapid adenosine signaling.

Spontaneous Adenosine and Dopamine Cotransmission in the Caudate-Putamen Is Regulated by Adenosine Receptors.

Transient changes in adenosine and dopamine have been measured in vivo, but no studies have examined if these transient changes occur simultaneously. In this study, we characterized spontaneous adenosine and dopamine transients in anesthetized mice, examining coincident release in the caudate-putamen for the first time. We found that in C57B mice, most of the dopamine transients (77%) were coincident with adenosine, but fewer adenosine transients (12%) were coincident with a dopamine transient. On average, the dopamine transient started 200 ms before its coincident adenosine transient, so they occurred concurrently. There was a positive correlation (r = 0.7292) of adenosine and dopamine concentrations during coincident release. ATP is quickly broken down to adenosine in the extracellular space, and the coincident events may be due to corelease, where dopaminergic vesicles are packaged with ATP, or cotransmission, where ATP is packaged in different vesicles released simultaneously with dopamine. The high frequency of adenosine transients compared to that of dopamine transients suggests that adenosine is also released from nondopaminergic vesicles. We investigated how A1 and A2A adenosine receptors regulate adenosine and dopamine transients using A1 and A2AKO mice. In A1KO mice, the frequency of adenosine and dopamine transients increased, while in A2AKO mice, the frequency of adenosine alone increased. Adenosine receptors modulate coincident transients and could be drug targets to modulate both dopamine and adenosine release. Many spontaneous dopamine transients have coincident adenosine release, and regulating adenosine and dopamine cotransmission could be important for designing treatments for dopamine diseases, such as Parkinson's or addiction.

Spontaneous, transient adenosine release is not enhanced in the CA1 region of hippocampus during severe ischemia models.

Ischemic stroke causes damage in the brain, and a slow buildup of adenosine is neuroprotective during ischemic injury. Spontaneous, transient adenosine signaling, lasting only 3s per event, has been discovered that increases in frequency in the caudate-putamen during early stages of mild ischemia-reperfusion injury. However, spontaneous adenosine changes have not been studied in the hippocampus during ischemia, an area highly susceptible to stroke. Here, we investigated changes of spontaneous, transient adenosine in the CA1 region of rat hippocampus during three different models of the varied intensity of ischemia. During the early stages of the milder bilateral common carotid artery occlusion (BCCAO) model, there were fewer spontaneous, transient adenosine, but no change in the concentration of individual events. In contrast, during the moderate 2 vertebral artery occlusion (2VAO) and severe 4 vessel occlusion (4VO) models, both the frequency of spontaneous, transient adenosine and the average event adenosine concentration decreased. Blood flow measurements validate that the ischemia models decreased blood flow, and corresponding pathological changes were observed by transmission electron microscopy (TEM). 4VO occlusion showed the most severe damage in histology and BCCAO showed the least. Overall, our data suggest that there is no enhanced spontaneous adenosine release in the hippocampus during moderate and severe ischemia, which could be due to depletion of the rapidly releasable adenosine pool. Thus, during ischemic stroke, there are fewer spontaneous adenosine events that could inhibit neurotransmission, which might lead to more damage and less neuroprotection in the hippocampus CA1 region. Read the Editorial Highlight for this article on page 800.

A1 and A2A Receptors Modulate Spontaneous Adenosine but Not Mechanically Stimulated Adenosine in the Caudate.

Adenosine is a neuromodulator, and rapid increases in adenosine in the brain occur spontaneously or after mechanical stimulation. However, the regulation of rapid adenosine by adenosine receptors is unclear, and understanding it would allow better manipulation of neuromodulation. The two main adenosine receptors in the brain are A1 receptors, which are inhibitory, and A2A receptors, which are excitatory. Here, we investigated the regulation of spontaneous adenosine and mechanically stimulated adenosine by adenosine receptors, using global A1 or A2A knockout mice. Results were compared in vivo and in brain slices' models. A1 KO mice have increased frequency of spontaneous adenosine events, but no change in the average concentration of an event, while A2A KO mice had no change in frequency but increased average event concentration. Thus, both A1 and A2A self-regulate spontaneous adenosine release; however, A1 acts on the frequency of events, while A2A receptors regulate concentration. The trends are similar both in vivo and slices, so brain slices are a good model system to study spontaneous adenosine release. For mechanically stimulated adenosine, there was no effect of A1 or A2A KO in vivo, but in brain slices, there was a significant increase in concentration evoked in A1KO mice. Mechanically stimulated release was largely unregulated by A1 and A2A receptors, likely because of a different release mechanism than spontaneous adenosine. Thus, A1 receptors affect the frequency of spontaneous adenosine transients, and A2A receptors affect the concentration. Therefore, future studies could probe drug treatments targeting A1 and A2A receptors to increase rapid adenosine neuromodulation.

Structural Similarity Image Analysis for Detection of Adenosine and Dopamine in Fast-Scan Cyclic Voltammetry Color Plots.

Fast-scan cyclic voltammetry (FSCV) is widely used for in vivo detection of neurotransmitters, but identifying analytes, particularly mixtures, is difficult. Data analysis has focused on identifying dopamine from cyclic voltammograms, but it would be better to analyze all the data in the three-dimensional FSCV color plot. Here, the goal was to use image analysis-based analysis of FSCV color plots for the first time, specifically the structural similarity index (SSIM), to identify rapid neurochemical events. Initially, we focused on identifying spontaneous adenosine events, as adenosine cyclic voltammograms have a primary oxidation at 1.3 V and a secondary oxidation peak that grows in over time. Using SSIM, sample FSCV color plots were compared with reference color plots, and the SSIM cutoff score was optimized to distinguish adenosine. High-pass digital filtering was also applied to remove the background drift and lower the noise, which produced a better LOD. The SSIM algorithm detected more adenosine events than a previous algorithm based on current versus time traces, with 99.5 ± 0.6% precision, 95 ± 3% recall, and 97 ± 2% F1 score (n = 15 experiments from three researchers). For selectivity, it successfully rejected signals from pH changes, histamine, and H2O2. To prove it is a broad strategy useful beyond adenosine, SSIM analysis was optimized for dopamine detection and is able to detect simultaneous events with dopamine and adenosine. Thus, SSIM is a general strategy for FSCV data analysis that uses three-dimensional data to detect multiple analytes in an efficient, automated analysis.

Complex sex and estrous cycle differences in spontaneous transient adenosine

Adenosine is a ubiquitous neuromodulator that plays a role in sleep, vasodilation, and immune response and manipulating the adenosine system could be therapeutic for Parkinson's disease or ischemic stroke. Spontaneous transient adenosine release provides rapid neuromodulation; however, little is known about the effect of sex as a biological variable on adenosine signaling and this is vital information for designing therapeutics. Here, we investigate sex differences in spontaneous, transient adenosine release using fast-scan cyclic voltammetry to measure adenosine in vivo in the hippocampus CA1, basolateral amygdala, and prefrontal cortex. The frequency and concentration of transient adenosine release were compared by sex and brain region, and in females, the stage of estrous. Females had larger concentration transients in the hippocampus (0.161±0.003µM) and the amygdala (0.182±0.006µM) than males (hippocampus: 0.134±0.003, amygdala: 0.115±0.002µM), but the males had a higher frequency of events. In the prefrontal cortex, the trends were reversed. Males had higher concentrations (0.189±0.003µM) than females (0.170±0.002µM), but females had higher frequencies. Examining stages of the estrous cycle, in the hippocampus, adenosine transients are higher concentration during proestrus and diestrus. In the cortex, adenosine transients were higher in concentration during proestrus, but were lower during all other stages. Thus, sex and estrous cycle differences in spontaneous adenosine are complex, and not completely consistent from region to region. Understanding these complex differences in spontaneous adenosine between the sexes and during different stages of estrous is important for designing effective treatments manipulating adenosine as a neuromodulator.

CD73 or CD39 Deletion Reveals Different Mechanisms of Formation for Spontaneous and Mechanically Stimulated Adenosine and Sex Specific Compensations in ATP Degradation.

Adenosine is important for local neuromodulation, and rapid adenosine signaling can occur spontaneously or after mechanical stimulation, but little is known about how adenosine is formed in the extracellular space for those stimulations. Here, we studied mechanically stimulated and spontaneous adenosine to determine if rapid adenosine is formed by extracellular breakdown of adenosine triphosphate (ATP) using mice globally deficient in extracellular breakdown enzymes, either CD39 (nucleoside triphosphate diphosphohydrolase 1, NTPDase1) or CD73 (ecto-5'-nucleotidase). CD39 knockout (KO) mice have a lower frequency of spontaneous adenosine events than wild-type (WT, C57BL/6). Surprisingly, CD73KO mice demonstrate sex differences in spontaneous adenosine; males maintain similar event frequencies as WT, but females have significantly fewer events and lower concentrations. Examining the mRNA expression of other enzymes that metabolize ATP revealed tissue nonspecific alkaline phosphatase (TNAP) was upregulated in male CD73KO mice, but not secreted prostatic acid phosphatase (PAP) or transmembrane PAP. Thus, TNAP upregulation compensates for CD73 loss in males but not in females. These sex differences highlight that spontaneous adenosine is formed by metabolism of extracellular ATP by many enzymes. For mechanically stimulated adenosine, CD39KO or CD73KO did not change stimulation frequency, concentration, or t1/2. Thus, the mechanism of formation for mechanically stimulated adenosine is likely direct release of adenosine, different than spontaneous adenosine. Understanding these different mechanisms of rapid adenosine formation will help to develop pharmacological treatments that differentially target modes of rapid adenosine signaling, and all treatments should be studied in both sexes, given possible differences in extracellular ATP degradation.

Comparison of spontaneous and mechanically-stimulated adenosine release in mice

Rapid adenosine signaling, on the time frame of seconds, has been discovered in the brain that can modulate neurotransmission or blood flow. Rapid adenosine release can occur spontaneously or be evoked after a mechanical stimulation, but these two modes of adenosine have not been compared. Here, we compared spontaneous and mechanically-stimulated adenosine release in the prefrontal cortex, striatum, and hippocampus of anesthetized mice. For spontaneous adenosine, the number of adenosine events in the prefrontal cortex (40 ± 4 per hour) was significantly lower than in the striatum (54 ± 3) or hippocampus (56 ± 3). Similarly, the concentration per transient was lower in the prefrontal cortex but highest in the striatum. For mechanically-stimulated adenosine, the peak concentration in the prefrontal cortex (8 ± 2 μM) and striatum (8 ± 1 μM) were significantly lower than in the hippocampus (16 ± 2 μM). Comparing the two modes, the hippocampus had high mechanically-stimulated concentration and high spontaneous frequency, while the prefrontal cortex had lower spontaneous frequency and mechanically-stimulated release. However, there is no pattern with the striatum and thus no direct correlations between spontaneous and mechanically-stimulated adenosine. Thus, there may be different pools of adenosine or mechanisms of formation for these two modes. Because of the high frequency of spontaneous events and high concentration of mechanically-stimulated release in the hippocampus, there may be some areas that have stronger adenosine signaling and thus stronger neuromodulatory control by adenosine.

Caffeine Modulates Spontaneous Adenosine and Oxygen Changes during Ischemia and Reperfusion.

Adenosine is an endogenous neuroprotectant that modulates vasodilation in the central nervous system. Oxygen changes occur when there is an increase in local cerebral blood flow and thus are a measure of vasodilation. Transient oxygen events following rapid adenosine events have been recently discovered, but the relationship between adenosine and blood flow change during ischemia/reperfusion (I/R) has not been characterized. Caffeine is a nonselective adenosine receptor antagonist that can modulate the effects of adenosine in the brain, but how it affects adenosine and oxygen levels during I/R is also unknown. In this study, extracellular changes in adenosine and oxygen were simultaneously monitored using fast-scan cyclic voltammetry during bilateral common carotid artery occlusion (BCCAO) and the effects of a specific A2A antagonist, SCH 442416, or general antagonist, caffeine, were studied. Measurements were made in the caudate-putamen for 1 h of normoxia, followed by 30 min of BCCAO and 30 min of reperfusion. The frequency and number of both adenosine and oxygen transient events significantly increased during I/R. The specific A2A antagonist, SCH 442416 (3 mg/kg, i.p.), eliminated the increase in adenosine and oxygen events caused by I/R. The general adenosine receptor antagonist, caffeine (100 mg/kg, i.p.), decreased the frequency of adenosine and oxygen transient events during I/R. These results demonstrate that, during BCCAO, there are more rapid release events of the neuromodulator adenosine and correlated local oxygen changes, and these rapid, local effects are dampened by caffeine and other A2A antagonists.

Exosomes in HNSCC plasma as surrogate markers of tumour progression and immune competence.

Exosomes in plasma of head and neck squamous cell carcinoma (HNSCC) patients comprise subsets of vesicles derived from various cells. Recently, we separated CD3(+) from CD3(-) exosomes by immune capture. CD3(-) exosomes were largely tumour-derived (CD44v3+ ). Both subsets carried immunosuppressive proteins and inhibited functions of human immune cells. The role of these subsets in immune cell reprogramming by the tumour was investigated by focusing on the adenosine pathway components. Spontaneous adenosine production by CD3(+) or CD3(-) exosomes was measured by mass spectrometry, as was the production of adenosine by CD4+ CD39+ regulatory T cells (Treg ) co-incubated with these exosomes. The highest level of CD39/CD73 ectoenzymes and of adenosine production was found in CD3(-) exosomes in patients with the stages III/IV HNSCCs). Also, the production of 5'-AMP and purines was significantly higher in Treg co-incubated with CD3(-) than CD3(+) exosomes. Consistently, CD26 and adenosine deaminase (ADA) levels were higher in CD3(+) than CD3(-) exosomes. ADA and CD26 levels in CD3(+) exosomes were significantly higher in patients with early (stages I/II) than advanced (stages III/IV) disease. HNSCC patients receiving and responding to photodynamic therapy had increased ADA levels in CD3(+) exosomes with no increase in CD3(-) exosomes. The opposite roles of CD3(+) ADA+ CD26+ and CD3(-) CD44v3+ adenosine-producing exosomes in early versus advanced HNSCC suggest that, like their parent cells, these exosomes serve as surrogates of immune suppression in cancer.

Transient Adenosine Release Is Modulated by NMDA and GABAB Receptors.

Adenosine is a neuroprotective agent that modulates neurotransmission and is modulated by other neurotransmitters. Spontaneous, transient adenosine is a recently discovered mode of signaling where adenosine is released and cleared from the extracellular space quickly, in less than three seconds. Spontaneous adenosine release is regulated by adenosine A1 and A2a receptors, but regulation by other neurotransmitter receptors has not been studied. Here, we examined the effect of glutamate and GABA receptors on the concentration and frequency of spontaneous, transient adenosine release by measuring adenosine with fast-scan cyclic voltammetry in the rat caudate-putamen. The glutamate NMDA antagonist, 3-(R-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP, 6.25 mg/kg i.p.), increased the frequency of adenosine transients and the concentration of individual transients, but NMDA (agonist, 50 mg/kg, i.p.) did not change the frequency. In contrast, antagonists of other glutamate receptors had no effect on the frequency or concentration of transient adenosine release, including the AMPA antagonist NBQX (15 mg/kg i.p.) and the mGlu2/3 glutamate receptor antagonist LY 341495 (5 mg/kg i.p.). The GABAB antagonist CGP 52432 (30 mg/kg i.p.) significantly decreased the number of adenosine release events while the GABAB agonist baclofen (4 mg/kg i.p.) increased the frequency of adenosine release. The GABAA antagonist bicuculline (5 mg/kg i.p.) had no significant effects on adenosine. NMDA and GABAB likely act presynaptically, affecting the overall cell excitability for vesicular release. The ability to regulate adenosine with NMDA and GABAB receptors will help control the modulatory effects of transient adenosine release.

Automated Algorithm for Detection of Transient Adenosine Release.

Spontaneous adenosine release events have been discovered in the brain that last only a few seconds. The identification of these adenosine events from fast-scan cyclic voltammetry (FSCV) data is difficult due to the random nature of adenosine release. In this study, we develop an algorithm that automatically identifies and characterizes adenosine transient features, including event time, concentration, and duration. Automating the data analysis reduces analysis time from 10 to 18 h to about 40 min per experiment. The algorithm identifies adenosine based on its two oxidation peaks, the time delay between them, and their current vs time peak ratios. In order to validate the program, four data sets from three independent researchers were analyzed by the algorithm and then compared to manual identification by an analyst. The algorithm resulted in 10 ± 4% false negatives and 9 ± 3% false positives. The specificity of the algorithm was verified by comparing calibration data for adenosine triphosphate (ATP), histamine, hydrogen peroxide, and pH changes and these analytes were not identified as adenosine. Stimulated histamine release in vivo was also not identified as adenosine. The code is modular in design and could be easily adjusted to detect features of spontaneous dopamine or other neurochemical transients in FSCV data.

Clearance of rapid adenosine release is regulated by nucleoside transporters and metabolism.

Adenosine is a neuromodulator that regulates neurotransmission in the brain and central nervous system. Recently, spontaneous adenosine release that is cleared in 3–4 sec was discovered in mouse spinal cord slices and anesthetized rat brains. Here, we examined the clearance of spontaneous adenosine in the rat caudate‐putamen and exogenously applied adenosine in caudate brain slices. The V max for clearance of exogenously applied adenosine in brain slices was 1.4 ± 0.1 μmol/L/sec. In vivo, the equilibrative nucleoside transport 1 (ENT1) inhibitor, S‐(4‐nitrobenzyl)‐6‐thioinosine (NBTI) (1 mg/kg, i.p.) significantly increased the duration of adenosine, while the ENT1/2 inhibitor, dipyridamole (10 mg/kg, i.p.), did not affect duration. 5‐(3‐Bromophenyl)‐7‐[6‐(4‐morpholinyl)‐3‐pyrido[2,3‐d]byrimidin‐4‐amine dihydrochloride (ABT‐702), an adenosine kinase inhibitor (5 mg/kg, i.p.), increased the duration of spontaneous adenosine release. The adenosine deaminase inhibitor, erythro‐9‐(2‐hydroxy‐3‐nonyl)adenine (EHNA) (10 mg/kg, i.p.), also increased the duration in vivo. Similarly, NBTI (10 μmol/L), ABT‐702 (100 nmol/L), or EHNA (20 μmol/L) also decreased the clearance rate of exogenously applied adenosine in brain slices. The increases in duration for blocking ENT1, adenosine kinase, or adenosine deaminase individually were similar, about 0.4 sec in vivo; thus, the removal of adenosine on a rapid time scale occurs through three mechanisms that have comparable effects. A cocktail of ABT‐702, NBTI, and EHNA significantly increased the duration by 0.7 sec, so the mechanisms are not additive and there may be additional mechanisms clearing adenosine on a rapid time scale. The presence of multiple mechanisms for adenosine clearance on a time scale of seconds demonstrates that adenosine is tightly regulated in the extracellular space.

Real time adenosine fluctuations detected with fast-scan cyclic voltammetry in the rat striatum and motor cortex

BackgroundAdenosine serves many functions within the CNS, including inhibitory and excitatory control of neurotransmission. The understanding of adenosine dynamics in the brain is of fundamental importance. The goal of the present study was to explore subsecond adenosine fluctuations in the rat brain in vivo. MethodLong Evans rats were anesthetized and a carbon fiber electrode was positioned in the motor cortex or dorsal striatum. Real time electrochemical recordings were made at the carbon fiber electrodes every 100ms by applying a triangular waveform (−0.4 to +1.5V, 400V/s). Adenosine spikes were identified by the background-subtracted cyclic voltammogram. ResultsThe frequency of detected adenosine spikes was relatively stable in both tested regions, and the time intervals between spikes were regular and lasted from 1 to 5s within an animal. Spike frequency ranged from 0.5 to 1.5Hz in both the motor cortex and the dorsal striatum. Average spike amplitudes were 85±11 and 66±7nM for the motor cortex and the dorsal striatum, respectively. Comparison with existing methodsThe current study established that adenosine signaling can operate on a fast time scale (within seconds) to modulate brain functions. ConclusionsThis finding suggests that spontaneous adenosine release may play a fast, dynamic role in regulating an organism's response to external events. Therefore, adenosine transmission in the brain may have characteristics similar to those of classical neurotransmitters, such as dopamine and norepinephrine.

Characterization of Real Time Adenosine Effluxes in the Brain of Anesthetized Rats using Fast‐Scan Cyclic Voltammetry

The present study was designed to characterize adenosine effluxes in the brain using fast scan cyclic voltammetry and explore how spontaneous adenosine release may be affected by a tail pinch. Long Evans rats were anesthetized and a carbon fiber electrode was positioned in the motor cortex or dorsal striatum. Voltammetric recordings were made every 100 ms by applying a triangular waveform from ‐0.4 to +1.5 V and back versus an Ag/AgCl reference electrode at a rate of 400 V/s. Frequency of adenosine spikes detected was relatively stable in both tested regions and the time intervals between spikes were regular and lasted from 1 to 6 seconds. The number of spikes per 10 seconds ranged from 7.5 ± 0.56 to 9.5 ± 0.62 in the motor cortex and 8.0 ± 1.24 to 9.8 ± 1.54 in the dorsal striatum. The average amplitude fluctuated from 75.0 ± 10.18 to 94.3 ± 18.12 nM in the motor cortex and from 122.7 ± 29.17 to 150.3 ± 43.20 nM in the dorsal striatum. Statistical analysis did not reveal any significant difference in the frequency or the amplitude between the two regions (n=6 rats, p>0.05). The striatal and cortical clearance rate of adenosine was equivalent (n=7 rats, 137.4 ± 23.23 and 195.9 ± 44.14 nM/s for the cortex and striatum, respectively). Furthermore, a 3 s tail pinch transiently increased adenosine signaling in both brain areas. The increase was significantly higher in the dorsal striatum than the motor cortex (n=6, p<0.05). Our study confirmed that adenosine signaling can operate on a faster time scale to modulate brain functions. This study was supported by NIAAA R01AA022449.

Characterization of Spontaneous, Transient Adenosine Release in the Caudate-Putamen and Prefrontal Cortex

Adenosine is a neuroprotective agent that inhibits neuronal activity and modulates neurotransmission. Previous research has shown adenosine gradually accumulates during pathologies such as stroke and regulates neurotransmission on the minute-to-hour time scale. Our lab developed a method using carbon-fiber microelectrodes to directly measure adenosine changes on a sub-second time scale with fast-scan cyclic voltammetry (FSCV). Recently, adenosine release lasting a couple of seconds has been found in murine spinal cord slices. In this study, we characterized spontaneous, transient adenosine release in vivo, in the caudate-putamen and prefrontal cortex of anesthetized rats. The average concentration of adenosine release was 0.17±0.01 µM in the caudate and 0.19±0.01 µM in the prefrontal cortex, although the range was large, from 0.04 to 3.2 µM. The average duration of spontaneous adenosine release was 2.9±0.1 seconds and 2.8±0.1 seconds in the caudate and prefrontal cortex, respectively. The concentration and number of transients detected do not change over a four hour period, suggesting spontaneous events are not caused by electrode implantation. The frequency of adenosine transients was higher in the prefrontal cortex than the caudate-putamen and was modulated by A1 receptors. The A1 antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine, 6 mg/kg i.p.) increased the frequency of spontaneous adenosine release, while the A1 agonist CPA (N6-cyclopentyladenosine, 1 mg/kg i.p.) decreased the frequency. These findings are a paradigm shift for understanding the time course of adenosine signaling, demonstrating that there is a rapid mode of adenosine signaling that could cause transient, local neuromodulation.

Slow spontaneous [Ca2+]i oscillations reflect nucleotide release from renal epithelia

Renal epithelia can be provoked mechanically to release nucleotides, which subsequently increases the intracellular Ca(2+) concentration [Ca(2+)](i) through activation of purinergic (P2) receptors. Cultured cells often show spontaneous [Ca(2+)](i) oscillations, a feature suggested to involve nucleotide signalling. In this study, fluo-4 loaded Madin-Darby canine kidney (MDCK) cells are used as a model for quantification and characterisation of spontaneous [Ca(2+)](i) increases in renal epithelia. Spontaneous [Ca(2+)](i) increases occurred randomly as single cell events. During an observation period of 1 min, 10.9 +/- 6.7% (n = 23) of the cells showed spontaneous [Ca(2+)](i) increases. Spontaneous adenosine triphosphate (ATP) release from MDCK cells was detected directly by luciferin/luciferase. Scavenging of ATP by apyrase or hexokinase markedly reduced the [Ca(2+)](i) oscillatory activity, whereas inhibition of ecto-ATPases (ARL67156) enhanced the [Ca(2+)](i) oscillatory activity. The association between spontaneous [Ca(2+)](i) increases and nucleotide signalling was further tested in 132-1N1 cells lacking P2 receptors. These cells hardly showed any spontaneous [Ca(2+)](i) increases. Transfection with either hP2Y(6) or hP2Y(2) receptors revealed a striking degree of oscillations. Similar spontaneous [Ca(2+)](i) increases were observed in freshly isolated, perfused mouse medullary thick ascending limb (mTAL). The oscillatory activity was reduced by basolateral apyrase and substantially lower in mTAL from P2Y(2) knock out mice (0.050 +/- 0.020 events per second, n = 8) compared to the wild type (0.147 +/- 0.018 events per second, n = 9). These findings indicate that renal epithelia spontaneously release nucleotides leading to P2-receptor-dependent [Ca(2+)](i) oscillations. Thus, tonic nucleotide release is likely to modify steady state renal function.

Comparison of the effects of unfractionated heparin and the low molecular weight heparins, dalteparin and enoxaparin, on spontaneous platelet aggregation and adenosine diphosphate activity in platelets during the third trimester of pregnancy

Low-molecular-weight heparins (LMWHs) have demonstrable pharmacokinetic, pharmacodynamic and safety advantages over unfractionated heparin (UH) in routine clinical use and are now the preferred agents in routine anticoagulant therapy. However, the utility and impact of the LMWH compared with that of UH has not been studied extensively in human pregnancy, wherein the prophylaxis against venous thromboembolism is imperative. Human pregnancy is a hypercoagulable state with an increase in spontaneous platelet aggregation (SPA) in vivo. We evaluated and compared the effects of UH and the LMWHs dalteparin and enoxaparin (10 U/ml) on SPA in citrated whole blood with an ultraflow platelet counter in pregnancy and also investigated the role of adenosine diphosphate (ADP) in heparin-induced platelet aggregation in the third trimester of pregnant women (aged 28 +/- 3 years, gestational age 34 +/- 5 weeks) and in healthy, age-matched nonpregnant women. Pregnant women showed a significantly increased SPA of 37% 6 5% compared with 16% 6 3% in nonpregnant women (p < 0.01). UH exerted a significantly greater proaggregatory effect on SPA compared with that of LMWHs or saline (p < 0.0002; ANOVA). The maximum values of SPA were as follows: UH, 69% +/- 5%; dalteparin, 46% +/- 5%; and enoxaparin, 54% +/- 3%. There was no difference between SPA induced by LMWHs and saline or between enoxaparin and dalteparin. At 480 s, there was no difference in SPA induced by LMWH between pregnant and nonpregnant women, but UH substantially and specifically increased SPA in pregnant women compared with that in nonpregnant women (p < 0.01). This heparin-induced platelet activation and thrombocytopenic response was reversed by apyrase grade II (ADP scavenger) that also inhibited SPA in pregnancy to a level similar to that of nonpregnant women (p < 0.0002; ANOVA). These results indicate that the LMWHs dalteparin and enoxaparin cause significantly less platelet aggregation in whole blood in pregnancy and in the nonpregnant state when compared with UH. The proaggregatory platelet effects of UH is substantially enhanced in pregnancy, a property not shared by LMWHs. The reversal of the heparin-induced platelet activation by apyrase grade II suggests that the mechanism is, at least in part, mediated by copious ADP release from platelets or red cells by heparin but not LMWHs.