Action Of Adenosine Research Articles

Adenosine: Direct and Indirect Actions on Gastric Acid Secretion.

Composed by a molecule of adenine and a molecule of ribose, adenosine is a paradigm of recyclable nucleoside with a multiplicity of functions that occupies a privileged position in the metabolic and regulatory contexts. Adenosine is formed continuously in intracellular and extracellular locations of all tissues. Extracellular adenosine is a signaling molecule, able to modulate a vast range of physiologic responses in many cells and organs, including digestive organs. The adenosine A1, A2A, A2B, and A3 receptors are P1 purinergic receptors, G protein-coupled proteins implicated in tissue protection. This review is focused on gastric acid secretion, a process centered on the parietal cell of the stomach, which contains large amounts of H+/K+-ATPase, the proton pump responsible for proton extrusion during acid secretion. Gastric acid secretion is regulated by an extensive collection of neural stimuli and endocrine and paracrine agents, which act either directly at membrane receptors of the parietal cell or indirectly through other regulatory cells of the gastric mucosa, as well as mechanic and chemic stimuli. In this review, after briefly introducing these points, we condense the current body of knowledge about the modulating action of adenosine on the pathophysiology of gastric acid secretion and update its significance based on recent findings in gastric mucosa and parietal cells in humans and animal models.

Heterozygous Gnal Mice Are a Novel Animal Model with Which to Study Dystonia Pathophysiology.

Dystonia is a movement disorder characterized by sustained or intermittent muscle contractions and its pathophysiological mechanisms are still poorly understood. Dominant mutations of the GNAL gene are a cause of isolated dystonia (DYT25) in patients. Some mutations result in a complete loss of function of the encoded protein, Gαolf, an adenylyl-cyclase-stimulatory G-protein highly enriched in striatal projection neurons, where it mediates the actions of dopamine and adenosine. We used male and female heterozygous Gnal knock-out mice (Gnal+/-) to study how GNAL haplodeficiency is implicated in dystonia. In basal conditions, no overt dystonic movements or postures or change in locomotor activity were observed. However, Gnal haploinsufficiency altered self-grooming, motor coordination, and apparent motivation in operant conditioning, as well as spine morphology and phospho-CaMKIIβ in the striatum. After systemic administration of oxotremorine, an unselective cholinergic agonist, Gnal+/- mice developed more abnormal postures and movements than WT mice. These effects were not caused by seizures as indicated by EEG recordings. They were prevented by the M1-preferring muscarinic antagonists, telenzepine, pirenzepine, and trihexyphenidyl, which alleviate dystonic symptoms in patients. The motor defects were worsened by mecamylamine, a selective nicotinic antagonist. These oxotremorine-induced abnormalities in Gnal+/- mice were replicated by oxotremorine infusion into the striatum, but not into the cerebellum, indicating that defects in striatal neurons favor the appearance of dystonia-like movement alterations after oxotremorine. Untreated and oxotremorine-treated Gnal+/- mice provide a model of presymptomic and symptomatic stages of DYT25-associated dystonia, respectively, and clues about the mechanisms underlying dystonia pathogenesis.SIGNIFICANCE STATEMENT Adult-onset dystonia DYT25 is caused by dominant loss-of-function mutations of GNAL, a gene encoding the stimulatory G-protein Gαolf, which is critical for activation of the cAMP pathway in the striatal projection neurons. Here, we demonstrate that Gnal-haplodeficient mice have a mild neurological phenotype and display vulnerability to developing dystonic movements after systemic or intrastriatal injection of the cholinergic agonist oxotremorine. Therefore, impairment of the cAMP pathway in association with an increased cholinergic tone creates alterations in striatal neuron functions that can promote the onset of dystonia. Our results also provide evidence that untreated and oxotremorine-treated Gnal-haplodeficient mice are powerful models with which to study presymptomic and symptomatic stages of DYT25-associated dystonia, respectively.

A semi-quantitative translational pharmacology analysis to understand the relationship between in vitro ENT1 inhibition and the clinical incidence of dyspnoea and bronchospasm

Adenosine contributes to the pathophysiology of respiratory disease, and adenosine challenge leads to bronchospasm and dyspnoea in patients. The equilibrative nucleoside transporter 1 (ENT1) terminates the action of adenosine by removal from the extracellular environment. Therefore, it is proposed that inhibition of ENT1 in respiratory disease patients leads to increased adenosine concentrations, triggering bronchospasm and dyspnoea. This study aims to assess the translation of in vitro ENT1 inhibition to the clinical incidence of bronchospasm and dyspnoea in respiratory disease, cardiovascular disease and healthy volunteer populations. Four marketed drugs with ENT1 activity were assessed; dipyridamole, ticagrelor, draflazine, cilostazol. For each patient population, the relationship between in vitro ENT1 [3H]-NBTI binding affinity (Ki) and [3H]-adenosine uptake (IC50) to the incidence of: (1) bronchospasm/severe dyspnoea; (2) tolerated dyspnoea and; (3) no adverse effects, was evaluated. A high degree of ENT1 inhibition (≥13.3x Ki, ≥4x IC50) associated with increased incidence of bronchospasm/severe dyspnoea for patients with respiratory disease only, whereas a lower degree of ENT1 inhibition (≥0.1x Ki, ≥0.05x IC50) associated with a tolerable level of dyspnoea in both respiratory and cardiovascular disease patients. ENT1 inhibition had no effect in healthy volunteers. Furthermore, physicochemical properties correlative with ENT1 binding were assessed using a set of 1625 diverse molecules. Binding to ENT1 was relatively promiscuous (22% compounds Ki<1μM) especially for neutral or basic molecules, and greater incidence tracked with higher lipophilicity (clogP >5). This study rationalises inclusion of an assessment of ENT1 activity during early safety profiling for programs targeting respiratory disorders.

Profound hypothermia after adenosine kinase inhibition in A1AR-deficient mice suggests a receptor-independent effect of intracellular adenosine.

Administration of the nucleoside adenosine has been shown to induce hypothermia in a number of species, an effect mediated predominantly by the adenosine 1 receptor (A1AR) subtype. The present experiments were performed to explore the possibility that the rise of intracellular adenosine levels expected to accompany adenosine administration may contribute to the hypothermic effect of adenosine independent of A1AR activation. Since phosphorylation of adenosine by adenosine kinase (ADK) is causal in the maintenance of low intracellular adenosine, we have examined the effect of ADK inhibition on core body temperature (CBT). Our data show that inhibition of ADK by A-134974 causes a long-lasting deep hypothermia in wild-type mice. Since there was an about 4-fold increase of adenosine plasma levels, experiments were repeated in A1AR-/- mice. ADK inhibition caused deep hypothermia despite the absence of A1AR, although the effect was significantly reduced compared to WT. Furthermore, the dose-dependent hypothermia caused by adenosine administration in WT mice was found to be reduced, but not abolished in A1AR-/- mice. To assess the possible role of A2AR and A3AR activation in our experimental setting, we compared the effects of the agonists CPA (A1AR), CGS21680 (A2AR), and IB-MECA (A3AR) on CBT. Hypothermia induced by CPA was much greater than that caused by CGS21680 or IB-MECA indicating that A1AR activation is the major receptor-dependent pathway for adenosine-induced hypothermia under our experimental conditions. Induction of deep hypothermia by inhibition of ADK, maintenance of this effect in A1AR-/- mice, and maintenance of adenosine-induced hypothermia in A1AR-deficient mice suggest that a receptor-independent action of adenosine requiring intact function of adenosine kinase contributes importantly to the hypothermia induced by adenosine.

A2A Adenosine Receptor Antagonism Reverts the Blood-Brain Barrier Dysfunction Induced by Sleep Restriction.

Chronic sleep restriction induces blood-brain barrier disruption and increases pro-inflammatory mediators in rodents. Those inflammatory mediators may modulate the blood-brain barrier and constitute a link between sleep loss and blood-brain barrier physiology. We propose that adenosine action on its A2A receptor may be modulating the blood-brain barrier dynamics in sleep-restricted rats. We administrated a selective A2A adenosine receptor antagonist (SCH58261) in sleep-restricted rats at the 10th day of sleep restriction and evaluated the blood-brain barrier permeability to dextrans coupled to fluorescein (FITC-dextrans) and Evans blue. In addition, we evaluated by western blot the expression of tight junction proteins (claudin-5, occludin, ZO-1), adherens junction protein (E-cadherin), A2A adenosine receptor, adenosine-synthesizing enzyme (CD73), and neuroinflammatory markers (Iba-1 and GFAP) in the cerebral cortex, hippocampus, basal nuclei and cerebellar vermis. Sleep restriction increased blood-brain barrier permeability to FITC-dextrans and Evans blue, and the effect was reverted by the administration of SCH58261 in almost all brain regions, excluding the cerebellum. Sleep restriction increased the expression of A2A adenosine receptor only in the hippocampus and basal nuclei without changing the expression of CD73 in all brain regions. Sleep restriction reduced the expression of tight junction proteins in all brain regions, except in the cerebellum; and SCH58261 restored the levels of tight junction proteins in the cortex, hippocampus and basal nuclei. Finally, sleep restriction induced GFAP and Iba-1 overexpression that was attenuated with the administration of SCH58261. These data suggest that the action of adenosine on its A2A receptor may have a crucial role in blood-brain barrier dysfunction during sleep loss probably by direct modulation of brain endothelial cell permeability or through a mechanism that involves gliosis with subsequent inflammation and increased blood-brain barrier permeability.

A1 Adenosine Receptor Activation Modulates Central Nervous System Development and Repair.

Adenosine is an endogenous, autacoid purine nucleoside which performs many important biological roles, particularly during stressful events. Adenosine can signal through four adenosine receptor (AR) subtypes: A1, A2A, A2B, and A3. Of these, adenosine A1 receptor (A1AR) has a broad, wide distribution throughout different vertebrate cell types and the highest affinity to adenosine. The A1AR-dependent action of adenosine is well documented in reports from numerous studies that have used different selective A1AR agonists and antagonists as well as in animals that have a genetically manipulated A1AR gene. Despite its wide distribution and function, A1AR homo/hetero-oligomerization with other adenosine and non-adenosine receptors extends its biological role during developmental, physiological, and pathological situations. In this review, we initially discuss the A1AR structure and most important signaling pathway triggered by its activation. Next, we summarize some of the most well-known biological effects of A1AR in the central nervous system (CNS) during development and adulthood, in addition to its role in nervous system regeneration and repair.

Effects of ATP and adenosine on contraction amplitude of rat soleus muscle at different temperatures.

The aim of this study was to evaluate the effects of adenosine 5'-triphosphate (ATP) and adenosine on the contractility of mammalian skeletal muscle under hypothermic conditions. Contractions of isolated rat soleus muscle were induced by either electrical stimulation (ES) or carbachol at physiological temperatures (37°C) and hypothermic conditions (30-14°C) and recorded in the presence of ATP, adenosine, suramin, and 8-(p-sulfophenyl)-theophylline (8-SPT). At 37°C, incubation of the muscles with ATP inhibited ES-induced contractions; the inhibitory effect of ATP disappeared at 14°C. Adenosine inhibited ES-induced contractions at all temperature levels; 8-SPT fully prevented the action of adenosine. ATP and adenosine did not significantly affect carbachol-induced contractions at 37°C, while at lower temperatures ATP potentiated them. Suramin fully prevented effects of ATP. ATP is involved in both pre- and postsynaptic regulation of rat soleus muscle contractility, and these processes are significantly more pronounced at low temperatures. Muscle Nerve 55: 417-423, 2017.

In Utero Caffeine Exposure Induces Transgenerational Effects on the Adult Heart.

Each year millions of pregnant woman are exposed to caffeine, which acts to antagonize adenosine action. The long-term consequences of this exposure on the developing fetus are largely unknown, although in animal models we have found adverse effects on cardiac function. To assess if these effects are transmitted transgenerationally, we exposed pregnant mice to caffeine equivalent to 2–4 cups of coffee at two embryonic stages. Embryos (F1 generation) exposed to caffeine early from embryonic (E) day 6.5–9.5 developed a phenotype similar to dilated cardiomyopathy by 1 year of age. Embryos exposed to caffeine later (E10.5–13.5) were not affected. We next examined the F2 generation and F3 generation of mice exposed to caffeine from E10.5–13.5, as this coincides with germ cell development. These F2 generation adult mice developed a cardiac phenotype similar to hypertrophic cardiomyopathy. The F3 generation exhibited morphological changes in adult hearts, including increased mass. This report shows that in utero caffeine exposure has long-term effects into adulthood and that prenatal caffeine exposure can exert adverse transgenerational effects on adult cardiac function.

Susceptibility to seizure-induced sudden death in DBA/2 mice is altered by adenosine

Sudden unexpected death in epilepsy (SUDEP) is rare but is an important public health burden due to the number of patient years lost. Respiratory dysfunction following generalized convulsive seizure is a common sequence of events in witnessed SUDEP cases. The DBA/2 mouse model of SUDEP exhibits generalized convulsive audiogenic seizures (AGSz), which result in seizure-induced respiratory arrest (S-IRA) in ∼75% of these animals, while the remaining DBA/2 mice exhibit AGSz without S-IRA. SUDEP induction may involve actions of adenosine, which is released during generalized seizures in animals and patients and is known to depress respiration. This study examined the effects of systemic administration of agents that alter the actions of adenosine on the incidence of S-IRA in DBA/2 mice. DBA/2 mice that consistently exhibited AGSz without S-IRA showed a significantly increased incidence of S-IRA following treatment with 5-iodotubercidin, which blocks adenosine metabolism. Treatment of DBA/2 mice that consistently exhibited AGSz followed by S-IRA with a non-selective adenosine antagonist, caffeine, or an A2A adenosine receptor subtype-selective antagonist (SCH 442416) significantly reduced S-IRA incidence. By contrast, an A1 adenosine receptor antagonist (DPCPX) was not effective in reducing S-IRA incidence. These findings suggest that preventative approaches for SUDEP should consider agents that reduce the actions of adenosine.

Contractile effects and receptor analysis of adenosine-receptors in human detrusor muscle from stable and neuropathic bladders.

To measure the relative transcription of adenosine receptor subtypes and the contractile effects of adenosine and selective receptor-subtype ligands on detrusor smooth muscle from patients with neuropathic overactive (NDO) and stable bladders and also from guinea-pigs. Contractile function was measured at 37°C in vitro from detrusor smooth muscle strips. Contractions were elicited by superfusate agonists or by electrical field stimulation. Adenosine-receptor (A1, A2A, A2B, A3) transcription was measured by RT-PCR. Adenosine attenuated nerve-mediated responses with equivalent efficacy in human and guinea-pig tissue (pIC50 3.65–3.86); the action was more effective at low (1–8 Hz) compared to high (20–40 Hz) stimulation frequencies in human NDO and guinea-pig tissue. With guinea-pig detrusor the action of adenosine was mirrored by the A1/A2-agonist N-ethylcarboxamidoadenosine (NECA), partly abolished in turn by the A2B-selectve antagonist alloxazine, as well as the A1-selective agonist N6- cyclopentyladenosine (CPA). With detrusor from stable human bladders the effects of NECA and CPA were much smaller than that of adenosine. Adenosine also attenuated carbachol contractures, but mirrored by NECA (in turn blocked by alloxazine) only in guinea-pig tissue. Adenosine receptor subtype transcription was measured in human detrusor and was similar in both groups, except reduced A2A levels in overactive bladder. Suppression of the carbachol contracture in human detrusor is independent of A-receptor activation, in contrast to an A2B-dependent action with guinea-pig tissue. Adenosine also reduced nerve-mediated contractions, by an A1- dependent action suppressing ATP neurotransmitter action.Electronic supplementary materialThe online version of this article (doi:10.1007/s00210-016-1255-1) contains supplementary material, which is available to authorized users.

The Effect of Endogenous Adenosine on Neuronal Activity in Rats: An FDG PET Study

ABSTRACT2–18F‐fluorodeoxy‐D‐glucose (FDG) is a glucose analog that is taken up by cells and phosphorylated. The amount of FDG accumulated by cells is a measure of the rate of glycolysis, which reflects cellular activity. As the levels and actions of the neuromodulator adenosine are dynamically regulated by neuronal activity, this study was designed to test whether endogenous adenosine affects tissue accumulation of FDG as assessed by positron emission tomography (PET) or by postmortem analysis of tissue radioactivity. Rats were given an intraperitoneal injection of the adenosine A1 receptor antagonist 8‐cyclopentyl‐1,3‐dipropyl‐xanthine (DPCPX, 3 mg/kg), the adenosine kinase inhibitor ABT‐702 (3 mg/kg), or vehicle 10 minutes prior to an intravenous injection of FDG (15.4 ± 0.7 MBq per rat). Rats were then subjected to a 15 minute static PET scan. Reconstructed images were normalized to FDG PET template for rats and standard uptake values (SUVs) were calculated. To examine the regional effect of active treatment compared to vehicle, statistical parametric mapping analysis was performed. Whole‐brain FDG uptake was not affected by drug treatment. Significant regional hypometabolism was detected, particularly in cerebellum, of DPCPX‐ and ABT‐702 treated rats, relative to vehicle‐treated rats. Thus, endogenous adenosine can affect FDG accumulation although this effect is modest in quiescent rats.

Adenosine can thwart antitumor immune responses elicited by radiotherapy : Therapeutic strategies alleviating protumor ADO activities.

By studying the bioenergetic status we could show that the development of tumor hypoxia is accompanied, apart from myriad other biologically relevant effects, by a substantial accumulation of adenosine (ADO). ADO has been shown to act as a strong immunosuppressive agent in tumors by modulating the innate and adaptive immune system. In contrast to ADO, standard radiotherapy (RT) can either stimulate or abrogate antitumor immune responses. Herein, we present ADO-mediated mechanisms that may thwart antitumor immune responses elicited by RT. An overview of the generation, accumulation, and ADO-related multifaceted inhibition of immune functions, contrasted with the antitumor immune effects of RT, is provided. Upon hypoxic stress, cancer cells release ATP into the extracellular space where nucleotides are converted into ADO by hypoxia-sensitive, membrane-bound ectoenzymes (CD39/CD73). ADO actions are mediated upon binding to surface receptors, mainly A2A receptors on tumor and immune cells. Receptor activation leads to a broad spectrum of strong immunosuppressive properties facilitating tumor escape from immune control. Mechanisms include (1) impaired activity of CD4 (+) T and CD8 (+) T, NK cells and dendritic cells (DC), decreased production of immuno-stimulatory lymphokines, and (2) activation of Treg cells, expansion of MDSCs, promotion of M2 macrophages, and increased activity of major immunosuppressive cytokines. In addition, ADO can directly stimulate tumor proliferation and angiogenesis. ADO mechanisms described can thwart antitumor immune responses elicited by RT. Therapeutic strategies alleviating tumor-promoting activities of ADO include respiratory hyperoxia or mild hyperthermia, inhibition of CD39/CD73 ectoenzymes or blockade of A2A receptors, and inhibition of ATP-release channels or ADO transporters.

Adenosine metabolism of human mesenchymal stromal cells isolated from patients with head and neck squamous cell carcinoma

BackgroundMesenchymal stromal cells (MSC) are a major component of the tumor microenvironment in patients with head and neck squamous cell carcinoma (HNSCC). MSC display innate and regulatory immunologic functions, very similar to many hematopoietic ‘classical’ immune cells. Conversion of ATP to immunosuppressive adenosine is an immunosuppressive mechanism utilized by other hematopoietic immune cells. The present study explores the adenosine metabolism of tumor derived MSC in comparison to autologous MSC from non-malignant tissue. MethodsFrom HNSCC patients (n=10), paired MSC were generated from tumor tissue (tMSC) and autologous healthy control tissue (cMSC). Differentiation properties and phenotype (CD105, CD73, CD39, CD90, CD26, CD29) were compared by flow cytometry. Production of immunosuppressive adenosine (ADO) by functionally active ectonucleotidases, CD39 and CD73, was determined by luminescence and mass spectrometry. Suppressive activity of ADO was tested in CFSE proliferation assays of isolated T-cells. Plasticity of cMSC was explored after incubation with tumor-cell conditioned media. ResultsDifferentiation into osteogenic, chondrogenic and adipogenic directions was comparable in tMSC and cMSC. Expression of ectonucleotidases, CD39 and CD73, was decreased in tMSC as compared to corresponding cMSC, which correlated with decreased ATP metabolism in mass spectrometry. Proliferation of CD4+ T-cells was significantly suppressed by exogenous ADO. Tumor-conditioned medium was unable to down-regulate ADO production in cMSC. ConclusionWe identified MSC of the oropharyngeal mucosa as an important producer of exogenous ADO. In patients with HNSCC, reduced expression of ADO may contribute to excessive inflammation and tumor growth.

Adenosine A2A receptors in neuronal outgrowth: a target for nerve regeneration?

Adenosine A2A receptors in neuronal outgrowth: a target for nerve regeneration?

ATP-induced changes in rat skeletal muscle contractility.

Extracellular purine compounds, adenosine triphosphate (ATP) and adenosine, are involved in regulation of many cell functions, engaging in rapid and long-term cellular processes. The nucleotides, including ATP, exert their extracellular effects by influencing membrane P2 receptors. ATP outside of the cell rapidly is metabolized by the ecto-enzyme system to produce adenosine, which acts on separate adenosine (P1) receptors. Since adenosine and ATP often are functional antagonists, ATP degradation not only limits its effect, but also brings new ligand with different, often opposing, properties. Great variety and widespread of P2 and adenosine receptors in the body emphasize the important physiological and pathophysiological significance of these receptors, and make them very attractive as targets for potential drug action.The existence of several subtypes of P2 and adenosine receptors has been shown in the skeletal muscles. ATP as a co-transmitter is densely packed together with classical neurotransmitters in the presynaptic vesicles of vertebral motor units but until recently ATP was refused to have its own functional role there and was recognized only as a source of adenosine. However, on the eve of the third millennium there appeared data that ATP, released from the nerve ending and acting on presynaptic P2 receptors, suppresses subsequent quantum release of acetylcholine. The final product of its degradation, adenosine, performs a similar inhibitory effect acting on presynaptic adenosine receptors.Despite the fact that the mechanisms of presynaptic inhibitory action of ATP and other purines were studied earlier, the object of those studies was usually neuromuscular synapse of cold-blooded animals. The few studies, in which experiments were carried out on preparations of warm-blooded animals, described the basic effects of purines. These often were guided by the convenience of preparation of the synapses of the diaphragm. We think that those results cannot be considered as typical effects of ATP and other purines on skeletal muscles and could not be extrapolated to all warm-blooded animals. Furthermore the role of ATP and its derivatives in the accumulation of vertebrate muscular effort has not been investigated.It is known that in physiological conditions vertebrates may mobilize only up to a third of the maximum muscle force. Why the two-thirds of muscular strength are not used normally but may be used at stress, remains unknown.It is known that the body's adaptive response to stress is a change in the activity of the endocrine system. The leading role in this is given to catechol amines and glucocorticoids, mobilized in significant quantities in blood under stress.We have found previously that incubation of frog sartorius muscle with hydrocortisone resulted in a decrease of contraction amplitude. However, when hydrocortisone was used in combination with ATP, its inhibitory effect on contractile responses disappeared. It is interesting that hydrocortisone had no effect on the inhibitory effect of adenosine. In the following experiments, assessing the effect of hydrocortisone on rat soleus muscle, it was established that hydrocortisone and purines had similar inhibitory effect. When ATP and hydrocortisone were given together the same oppression occurred.To study the effects of ATP and adenosine on contraction parameters of rat skeletal muscle and assess the impact of the catechol amines on these processes.Contractions of rat soleus muscles were recorded isometrically by mechanical sensor Linton FSG-01 (UK) according to standard procedures. The average of muscle parameters received within 30 seconds (30 responses) was treated as one result. Amplitude and time characteristics of the curve reductions were estimated. During all experiments standard Krebs solution flowed through the bath continuously to which agents were added at necessary concentrations. All experimental animals were maintained and prepared for dissection under the European Convention for the Protection of Vertebrate Animals used in scientific experiments. All agents used in the study were supplied by Sigma Chemical Company Ltd. (UK), Tocris Cookson and Research Biochemicals International (USA).The concentration of 100 μM for adenosine is close to saturation [1], and for its predecessor ATP this concentration is created after the passage of a pulse through the synapse [2]. We used this concentration of purines to study the mechanism of action of adenosine and ATP on neuromuscular synapse.The effect of adenosine was partially inhibited in the presence of 100 μM 8-SPT, an antagonist of adenosine receptors. The contraction force of "fast" and "slow" rat skeletal muscles was raised by half in the presence of norepinephrine. In the presence of norepinephrine adenosine exerted its effect fully, but ATP by half reduced its depressor effect on the contraction force of both muscles.1. Norepinephrine increases half times of the reduction of "fast" and "slow" skeletal muscle.2. In the presence of norepinephrine, inhibitory effect of adenosine on contraction force is maintained.3. Inhibitory effect of ATP on contraction force of studied skeletal muscles becomes twice less pronounced in the presence of norepinephrine.We think that reduction of ATP depressive effect on the skeletal muscle by norepinephrine may be an adaptive response to acute stress.

Basal adenosine modulates the functional properties of AMPA receptors in mouse hippocampal neurons through the activation of A1R A2AR and A3R.

Adenosine is a widespread neuromodulator within the CNS and its extracellular level is increased during hypoxia or intense synaptic activity, modulating pre- and postsynaptic sites. We studied the neuromodulatory action of adenosine on glutamatergic currents in the hippocampus, showing that activation of multiple adenosine receptors (ARs) by basal adenosine impacts postsynaptic site. Specifically, the stimulation of both A1R and A3R reduces AMPA currents, while A2AR has an opposite potentiating effect. The effect of ARs stimulation on glutamatergic currents in hippocampal cultures was investigated using pharmacological and genetic approaches. A3R inhibition by MRS1523 increased GluR1-Ser845 phosphorylation and potentiated AMPA current amplitude, increasing the apparent affinity for the agonist. A similar effect was observed blocking A1R with DPCPX or by genetic deletion of either A3R or A1R. Conversely, impairment of A2AR reduced AMPA currents, and decreased agonist sensitivity. Consistently, in hippocampal slices, ARs activation by AR agonist NECA modulated glutamatergic current amplitude evoked by AMPA application or afferent fiber stimulation. Opposite effects of AR subtypes stimulation are likely associated to changes in GluR1 phosphorylation and represent a novel mechanism of physiological modulation of glutamatergic transmission by adenosine, likely acting in normal conditions in the brain, depending on the level of extracellular adenosine and the distribution of AR subtypes.

Adenosine A2A receptor activation is determinant for BDNF actions upon GABA and glutamate release from rat hippocampal synaptosomes.

Adenosine, through A(2A) receptor (A(2A)R) activation, can act as a metamodulator, controlling the actions of other modulators, as brain-derived neurotrophic factor (BDNF). Most of the metamodulatory actions of adenosine in the hippocampus have been evaluated in excitatory synapses. However, adenosine and BDNF can also influence GABAergic transmission. We thus evaluated the role of A(2A)R on the modulatory effect of BDNF upon glutamate and GABA release from isolated hippocampal nerve terminals (synaptosomes). BDNF (30 ng/ml) enhanced K(+)-evoked [(3)H]glutamate release and inhibited the K(+)-evoked [(3)H]GABA release from synaptosomes. The effect of BDNF on both glutamate and GABA release requires tonic activation of adenosine A(2A)R since for both neurotransmitters, the BDNF action was blocked by the A(2A)R antagonist SCH 58261 (50 nM). In the presence of the A(2A)R agonist, CGS21680 (30 nM), the effect of BDNF on either glutamate or GABA release was, however, not potentiated. It is concluded that both the inhibitory actions of BDNF on GABA release as well as the facilitatory action of the neurotrophin on glutamate release are dependent on the activation of adenosine A(2A)R by endogenous adenosine. However, these actions could not be further enhanced by exogenous activation of A(2A)R.

Interactions between local dilator and sympathetic vasoconstrictor influences in skeletal muscle in acute and chronic hypoxia.

What is the topic of this review? This review considers how local dilator mechanisms and increased sympathetic nerve activity interact during acute systemic hypoxia and then reviews current understanding of some of the modifications induced by chronic hypoxia. What advances does it highlight? During acute hypoxia, local levels of hypoxia determine the release of vasodilators and magnitude of arteriolar dilatation, as well as the extent to which sympathetically evoked vasoconstriction is blunted, so maximizing distribution of O2 to muscle fibres. Chronic hypoxia in adult life and fetal programming induced by chronic hypoxia in utero lead to increased responsiveness to acute hypoxia and further blunting of sympathetic vasoconstriction, but are also associated with hypertension. In resting skeletal muscle, acute systemic hypoxia evokes vasodilatation, while vasoconstriction evoked by increased muscle sympathetic nerve activity is blunted, referred to herein as hypoxic sympatholysis. This review considers the contributions of adenosine, prostaglandin I2 , nitric oxide, ATP and endothelium-derived hyperpolarizing factors to the muscle vasodilatation, with particular attention being given to the release and actions of adenosine, which plays a dominant role. It is argued that the dilator substances are released in proportion to the local level of hypoxia, notably, allowing terminal arterioles to regulate O2 distribution through the capillaries. Correspondingly, hypoxic sympatholysis can be attributed to the ability of local hypoxia to blunt vasoconstriction evoked by noradrenaline acting on α1 - and α2 -adrenoceptors. The synergistic actions of ATP as cotransmitter may be depressed in parallel, but the actions of neuropeptide Y persist. Consideration is also given to the changes induced by chronic hypoxia in adult life and to the consequences in adult life of fetal programming induced by chronic hypoxia during pregnancy. In both conditions, dilator responsiveness to acute hypoxia is maintained, but the action or release of adenosine is altered in ways that are not yet understood. Both conditions are also accompanied by blunted sympathetically evoked vasoconstriction, tonically raised muscle sympathetic nerve activity, and increased muscular vascular tone and arterial blood pressure. With hypoxia-induced fetal programming, arterial pressure is increased in young adults and increases with age. The mechanisms underlying these changes are discussed, and it is argued that chronic hypoxia in adult life or in utero may facilitate development of hypertension.

Abstract 95: Pharmacological Characterization of Adenosine Effects on Spontaneously Beating Human-IPSC-Derived Cardiomyocytes

Purified human-Induced Pluripotent Stem Cell-derived cardiomyocytes (hIPSC-CMs: iCells®, Cellular Dynamics) form electrically coupled, spontaneously beating cell monolayers (syncytia) upon plating and continuous culture. In this study, we investigate the pharmacology of the hIPSC-CM adenosine (ADO) response via the use of selective A1 and A2A-receptor antagonists and equilibrative nucleoside transporter type-1 (ENT1) inhibitors. After a 14-day culture in 96-well impedance electrode-plates (ACEA Biosciences), the spontaneous iCell beating rates are highly stable from well to well, with a mean beat period of 1.7 sec at 37°C, and a coefficient of variation (CV) &lt; 1% . Extracellularly applied ADO inhibits the spontaneous beating rate of iCells with an IC 50 = 2.2 μM, and a saturation value of near 50% inhibition. ADO also destabilizes the beat period (CV EC 50 = 1.2 μM) with a maximum CV of 20% at saturating ADO concentrations. The ADO IC 50 is shifted to 248 μM upon pre-incubation of the iCells with 1 μM of an A1-selective antagonist DPCPX (A1 K i =3.9 nM), but not the A2A-selective antagonist ZM241385 (A2A K i =1nM). The duration of the depressive effect of ADO on beating rate is transient, even at super-saturating ADO concentrations, and depends on the well ADO concentration. The ENT1 inhibitor Draflazine (ENT1 pK i =9.5) pre-applied at 0.1 μM increases ADO sensitivity by 4-fold one hour after ADO application. Moreover, at any given ADO concentration, the duration of beating rate depression can be prolonged in a concentration-dependent manner by ENT1 inhibitors, with the prolongation-response curve nearly matching the ENT1 IC 50 s for tested ENT1 inhibitors (Draflazine, NBTI, Dilazep, and Dipyridamole). These findings suggest that the pool of ADO in the test well is cleared by uptake via endogenous ENT1 and support the role of equilibrative ADO uptake in limiting the duration of action of ADO on A1 receptors in cardiomyocytes. We are extending our investigation to the downstream target of A1 receptor activity, and the putative role of IK ACH/ADO as the underlying mechanism of beat rate slowing and irregularity. In conclusion, the hIPSC-CM model provides a useful model for investigation of ADO pharmacology of the heart.

Current Status of A 1 Adenosine Receptor Allosteric Enhancers

Adenosine is an ubiquitous nucleoside involved in various physiological and pathological functions by stimulating A1, A2A, A2B and A3 adenosine receptors (ARs). Allosteric enhancers to A1ARs may represent novel therapeutic agents because they increase the activity of these receptors by mediating a shift to their active form in the A1AR-G protein ternary complex. In this manner, they are able to amplify the action of endogenous adenosine, which is produced in high concentrations under conditions of metabolic stress. A1AR allosteric enhancers could be used as a justifiable alternative to the exogenous agonists that are characterized by receptor desensitization and downregulation. In this review, an analysis of some of the most interesting allosteric modulators of A1ARs has been reported.