Stimulation Of Adenosine Research Articles

Direct or indirect stimulation of adenosine A2A receptors enhances bone regeneration as well as bone morphogenetic protein-2.

Promoting bone regeneration and repair of bone defects is a need that has not been well met to date. We have previously found that adenosine, acting via A2A receptors (A2AR) promotes wound healing and inhibits inflammatory osteolysis and hypothesized that A2AR might be a novel target to promote bone regeneration. Therefore, we determined whether direct A2AR stimulation or increasing endogenous adenosine concentrations via purine transport blockade with dipyridamole regulates bone formation. We determined whether coverage of a 3 mm trephine defect in a mouse skull with a collagen scaffold soaked in saline, bone morphogenetic protein-2 (BMP-2; 200 ng), 1 μM CGS21680 (A2AR agonist, EC50 = 160 nM), or 1 μM dipyridamole (EC50 = 32 nM) promoted bone regeneration. Microcomputed tomography examination demonstrated that CGS21680 and dipyridamole markedly enhanced bone regeneration as well as BMP-2 8 wk after surgery (60 ± 2%, 79 ± 2%, and 75 ± 1% bone regeneration, respectively, vs. 32 ± 2% in control, P < 0.001). Blockade by a selective A2AR antagonist (ZM241385, 1 μM) or deletion of A2AR abrogated the effect of CGS21680 and dipyridamole on bone regeneration. Both CGS21680 and dipyridamole treatment increased alkaline phosphatase-positive osteoblasts and diminished tartrate resistance acid phosphatase-positive osteoclasts in the defects. In vivo imaging with a fluorescent dye for new bone formation revealed a strong fluorescent signal in treated animals that was equivalent to BMP-2. In conclusion, stimulation of A2AR by specific agonists or by increasing endogenous adenosine levels stimulates new bone formation as well as BMP-2 and represents a novel approach to stimulating bone regeneration.

Abstract 9789: Transient Elevation of Highly Sensitive Troponin T Levels in Patients With Documented Coronary Artery Disease Following Stress Myocardial Perfusion Imaging

Introduction: Cardiac troponins are the preferred biomarkers of myocardial injury. More sensitive assays led to the detection of circulating troponins not only in myocardial infarction, but also in apparently healthy subjects after prolonged exercise. Hypothesis: In the era of highly sensitive troponin T (hs-TnT) tests we assesed the hypothesis that brief but intense exercise during diagnostic tests might cause elevated troponin levels in patients with stable coronary artery disease (CAD). Methods: Seventy-two patients (71% male, age 64±10 years) with documented CAD, LVEF &gt;45%, normal renal function and normal baseline hs-TnT levels (&lt;14 ng/l) underwent myocardial perfusion imaging (MPI, Tc-99m sestamibi) using bicycle stress test (n=19) or adenosine stimulation (n=55). Hs-TnT levels were detected immediately before and immediately after stress, and again after 4 hours and 24 hours. The extent of stress-induced myocardial ischemia was estimated from perfusion scores derived from single-photon emission computed tomography (SPECT) post stress and rest images. Significant reversible myocardial ischemia was prospectively defined as regional perfusion abnormality ≥10% of the left ventricular myocardium. Results: Transient elevations of hs-TnT levels above the normal limit occurred in 5/72 patients (7%) after diagnostic stress testing with a peak level at 4 hours after stress. Hs-TnT elevations were more common in patients with stress-induced myocardial ischemia (3/12, 25%) compared to patients without ischemia (2/60, 3%; p=0.038). However, no relation between peak hs-TnT levels and the extent of reversible myocardial ischemia was found (r=-0.16, n=72, p=NS). Furthermore, peak hs-TnT levels were similar with both types of stress (bicycle 6.6 [4.0 – 10.5] pg/l versus adenosine 5.1 [3.1 – 8.6] pg/l, median [IQR]). Conclusions: Transient hs-TnT elevations are quite common after diagnostic stress testing and occur more frequently in patients with documented reversible ischemia. No relation between peak hs-TnT levels and the extent of reversible myocardial ischemia at MPI was obtained. In conclusion this suggests that hs-TNT elevation during stress testing depends on other factors then those leading to SPECT-detected myocardial ischemia.

Adenosine as a probing tool for the mechanistic study of acupuncture treatment.

To date, acupuncture has been widely used despite a lack of solid clinical evidence in the East and West. However, there are few validated in vitro models for the mechanistic studies of acupuncture. We hypothesized that adenosine could be used as a probing tool in the mechanistic studies of acupuncture because of its critical role in the action of acupuncture. Subsequently, we tested this hypothesis using both in vitro and in vivo experiments. First, we found that adenosine stimulation mimicked the effect of acupuncture on microRNA profiling (including miR-339, miR-145 and miR-451) and protein level (including Sirt2) in nerve growth factor-induced differentiated PC12 cells. These miRNA and proteins have been found to be regulated by acupuncture treatment in the brain of spontaneously hypertensive rats. Next, we found that adenosine stimulation downregulated miR-339 expression through adenosine A1 receptor-mediated pathway. Finally, we showed that the concentration of adenosine was actually decreased in the brain of spontaneously hypertensive rats after acupuncture treatment at Taichong acupoint. Taken together, these findings suggest that adenosine could be used as a useful probing tool for acupuncture mechanistic studies, while more validation studies are certainly warranted.

Correction: The Stimulatory Adenosine Receptor ADORA2B Regulates Serotonin (5-HT) Synthesis and Release in Oxygen-Depleted EC Cells in Inflammatory Bowel Disease

Correction: The Stimulatory Adenosine Receptor ADORA2B Regulates Serotonin (5-HT) Synthesis and Release in Oxygen-Depleted EC Cells in Inflammatory Bowel Disease

The Stimulatory Adenosine Receptor ADORA2B Regulates Serotonin (5-HT) Synthesis and Release in Oxygen-Depleted EC Cells in Inflammatory Bowel Disease

ObjectiveWe recently demonstrated that hypoxia, a key feature of IBD, increases enterochromaffin (EC) cell 5-HT secretion, which is also physiologically regulated by the ADORA2B mechanoreceptor. Since hypoxia is associated with increased extracellular adenosine, we wanted to examine whether this nucleotide amplifies HIF-1α-mediated 5-HT secretion.DesignThe effects of hypoxia were studied on IBD mucosa, isolated IBD-EC cells, isolated normal EC cells and the EC cell tumor derived cell line KRJ-1. Hypoxia (0.5% O2) was compared to NECA (adenosine agonist), MRS1754 (ADORA2B receptor antagonist) and SCH442146 (ADORA2A antagonist) on HIF signaling and 5-HT secretion. Antisense approaches were used to mechanistically evaluate EC cells in vitro. PCR and western blot were used to analyze transcript and protein levels of HIF-1α signaling and neuroendocrine cell function. An animal model of colitis was evaluated to confirm hypoxia:adenosine signaling in vivo.ResultsHIF-1α is upregulated in IBD mucosa and IBD-EC cells, the majority (∼90%) of which express an activated phenotype in situ. Hypoxia stimulated 5-HT release maximally at 30 mins, an effect amplified by NECA and selectively inhibited by MRS1754, through phosphorylation of TPH-1 and activation of VMAT-1. Transient transfection with Renilla luciferase under hypoxia transcriptional response element (HRE) control identified that ADORA2B activated HIF-1α signaling under hypoxic conditions. Additional signaling pathways associated with hypoxia:adenosine included MAP kinase and CREB. Antisense approaches mechanistically confirmed that ADORA2B signaling was linked to these pathways and 5-HT release under hypoxic conditions. Hypoxia:adenosine activation which could be reversed by 5′-ASA treatment was confirmed in a TNBS-model.ConclusionHypoxia induced 5-HT synthesis and secretion is amplified by ADORA2B signaling via MAPK/CREB and TPH-1 activation. Targeting ADORA2s may decrease EC cell 5-HT production and secretion in IBD.

Pulmonary blood flow and its distribution in highly trained endurance athletes and healthy control subjects

Pulmonary blood flow (PBF) is an important determinant of endurance sports performance, yet studies investigating adaptations of the pulmonary circulation in athletes are scarce. In the present study, we investigated PBF, its distribution, and heterogeneity at baseline and during intravenous systemic adenosine infusion in 10 highly trained male endurance athletes and 10 untrained but fit healthy controls, using positron emission tomography and [(15)O]water at rest and during adenosine infusion at supine body posture. Our results indicate that PBF at rest and during adenosine stimulation was similar in both groups (213 ± 55 and 563 ± 138 ml·100 ml(-1)·min(-1) in athletes and 206 ± 83 and 473 ± 212 ml·100 ml(-1)·min(-1) in controls, respectively). Although the PBF response to adenosine was thus unchanged in athletes, overall PBF heterogeneity was reduced from rest to adenosine infusion (from 84 ± 18 to 70 ± 19%, P < 0.05), while remaining unchanged in healthy controls (77 ± 16 to 85 ± 33%, P = 0.4). Additionally, there was a marked gravitational influence on general PBF distribution so that clear dorsal dominance was observed both at rest and during adenosine infusion, but training status did not have an effect on this distribution. Regional blood flow heterogeneity was markedly lower in the high-perfusion dorsal areas, both at rest and during adenosine, in all subjects, but flow heterogeneity in dorsal area tended to further decrease in response to adenosine in athletes. In conclusion, reduced blood flow heterogeneity in response to adenosine in endurance athletes may be a reflection of capillary reserve, which is more extensively recruitable in athletes than in matched healthy control subjects.

Synthesis of N6-alkyl(aryl)-2-alkyl(aryl)thioadenosines as antiplatelet agents

A series of novel N6-alkyl(aryl)-2-alkyl(aryl)thioadenosines were synthesized, and their human antiplatelet aggregation activities were evaluated by the stimulation of adenosine 5′-diphosphate (ADP). Some of these compounds showed strong activity, among which compound 5b11 displayed the highest activity with an IC50 value of 29±3μM. Furthermore, five compounds were tested against arachidonic acid (AA)-induced human platelet aggregation. The results showed that compound 5b10 exhibited the highest activity with an IC50 value of 3±2μM. The adenosine derivatives substituted with a phenethyl group at the N6 position and a methylthio or ethylthio group at the C-2 position displayed high antiplatelet aggregation activity.

Suppression of Adenosine-Activated Chloride Transport by Ethanol in Airway Epithelia

Alcohol abuse is associated with increased lung infections. Molecular understanding of the underlying mechanisms is not complete. Airway epithelial ion transport regulates the homeostasis of airway surface liquid, essential for airway mucosal immunity and lung host defense. Here, air-liquid interface cultures of Calu-3 epithelial cells were basolaterally exposed to physiologically relevant concentrations of ethanol (0, 25, 50 and 100 mM) for 24 hours and adenosine-stimulated ion transport was measured by Ussing chamber. The ethanol exposure reduced the epithelial short-circuit currents (ISC) in a dose-dependent manner. The ion currents activated by adenosine were chloride conductance mediated by cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel. Alloxazine, a specific inhibitor for A2B adenosine receptor (A2BAR), largely abolished the adenosine-stimulated chloride transport, suggesting that A2BAR is a major receptor responsible for regulating the chloride transport of the cells. Ethanol significantly reduced intracellular cAMP production upon adenosine stimulation. Moreover, ethanol-suppression of the chloride secretion was able to be restored by cAMP analogs or by inhibitors to block cAMP degradation. These results imply that ethanol exposure dysregulates CFTR-mediated chloride transport in airways by suppression of adenosine-A2BAR-cAMP signaling pathway, which might contribute to alcohol-associated lung infections.

Activation of PDE10 and PDE11 Phosphodiesterases

The most recently identified cyclic nucleotide phosphodiesterases, PDE10 and PDE11, contain a tandem of so-called GAF domains in their N-terminal regulatory regions. In PDE2 and PDE5, the GAF domains mediate cGMP stimulation; however, their function in PDE10 and PDE11 remains controversial. Although the GAF domains of PDE10 mediate cAMP-induced stimulation of chimeric adenylyl cyclases, cAMP binding did not stimulate the PDE10 holoenzyme. Comparable data about cGMP and the PDE11 GAF domains exist. Here, we identified synthetic ligands for the GAF domains of PDE10 and PDE11 to reduce interference of the GAF ligand with the catalytic reaction of PDE. With these ligands, GAF-mediated stimulation of the PDE10 and PDE11 holoenzymes is demonstrated for the first time. Furthermore, PDE10 is shown to be activated by cAMP, which paradoxically results in potent competitive inhibition of cGMP turnover by cAMP. PDE11, albeit susceptible to GAF-dependent stimulation, is not activated by the native cyclic nucleotides cAMP and cGMP. In summary, PDE11 can be stimulated by GAF domain ligands, but its native ligand remains to be identified, and PDE10 is the only PDE activated by cAMP.

Polydeoxyribonucleotide reduces cytokine production and the severity of collagen‐induced arthritis by stimulation of adenosine A2A receptor

Broad antiinflammatory effects following adenosine A(₂A) receptor stimulation have been demonstrated in acute inflammatory diseases, including arthritis. Polydeoxyribonucleotide (PDRN) activates the adenosine A(₂A) receptor. This study was undertaken to investigate the effects of PDRN in collagen-induced arthritis (CIA) in mice. Arthritis was induced in DBA/1 mice by an intradermal injection of 100 μl of bovine type II collagen in Freund's complete adjuvant. Mice were immunized a second time 21 days later. Control animals received 100 μl of a saline solution. Animals with CIA were randomized to receive one of the following: vehicle (1 ml/kg); PDRN (8 mg/kg intraperitoneally daily); 3,7-dimethyl-propargylxanthine (DMPX), a specific adenosine A(₂A) receptor antagonist (0.1 mg/kg intraperitoneally daily); or PDRN plus DMPX. The treatment was initiated immediately after the second immunization and continued to day 45. Clinical evaluation of arthritis was performed throughout the study. On day 45, the animals were killed and the severity of arthritis was evaluated histologically. Cartilage expression and circulating levels of high mobility group box chromosomal protein 1 (HMGB-1), tumor necrosis factor α (TNFα), interleukin-6 (IL-6), and IL-10 were investigated. Inflammatory cytokine production was also evaluated in stimulated human chondrocytes treated with PDRN. PDRN treatment significantly ameliorated clinical signs of arthritis, improved histologic damage, reduced the cartilage expression and circulating levels of HMGB-1, TNFα, and IL-6, and enhanced IL-10 expression. The concomitant administration of DMPX and PDRN ablated the PDRN-induced protective effect in experimental arthritis. PDRN also reduced cytokine production from stimulated human chondrocytes. Our findings indicate that PDRN may represent a new alternative for the treatment of arthritis.

R3F, a novel membrane‐associated glycogen targeting subunit of protein phosphatase 1 regulates glycogen synthase in astrocytoma cells in response to glucose and extracellular signals

Abnormal regulation of brain glycogen metabolism is believed to underlie insulin-induced hypoglycaemia, which may be serious or fatal in diabetic patients on insulin therapy. A key regulator of glycogen levels is glycogen targeted protein phosphatase 1 (PP1), which dephosphorylates and activates glycogen synthase (GS) leading to an increase in glycogen synthesis. In this study, we show that the gene PPP1R3F expresses a glycogen-binding protein (R3F) of 82.8 kDa, present at the high levels in rodent brain. R3F binds to PP1 through a classical 'RVxF' binding motif and substitution of Phe39 for Ala in this motif abrogates PP1 binding. A hydrophobic domain at the carboxy-terminus of R3F has similarities to the putative membrane binding domain near the carboxy-terminus of striated muscle glycogen targeting subunit G(M)/R(GL), and R3F is shown to bind not only to glycogen but also to membranes. GS interacts with PP1-R3F and is hyperphosphorylated at glycogen synthase kinase-3 sites (Ser640 and Ser644) when bound to R3F(Phe39Ala). Deprivation of glucose or stimulation with adenosine or noradrenaline leads to an increased phosphorylation of PP1-R3F bound GS at Ser640 and Ser644 curtailing glycogen synthesis and facilitating glycogen degradation to provide glucose in astrocytoma cells. Adenosine stimulation also modulates phosphorylation of R3F at Ser14/Ser18.

Extracellular signal‐regulated protein kinases mediate protein kinase C‐ and reactive oxygen species‐dependent stimulation of intracellular cAMP in human eosinophils

The role of extracellular signal‐regulated protein kinases 1/2 (ERK1/2) in the signaling pathway of the novel protein kinase C (PKC)‐ and reactive oxygen species (ROS)‐dependent stimulation of intracellular adenosine 3′,5′‐cyclic monophosphate (cAMP) in human eosinophils was studied. Highly purified human eosinophils were stimulated with the PKC activator, phorbol myristate acetate (PMA) and the cAMP response was determined by ELISA. The role of ERK1/2 was studied using specific inhibitors and Western blot analysis. PMA‐stimulated increase in cAMP was confirmed to be dependent on PKC and ROS by using specific inhibitors: Ro 31‐8220 for PKC and diphenyleneiodonium (DPI) for the ROS‐generating NADPH oxidase. Pre‐treating cells with the ERK1/2 inhibitor PD98059, but not p38‐MAPK inhibitor SB203580, nor PI3 kinase inhibitor, wortmannin, abolished the response. PMA treatment induced the phosphorylation of ERK1/2 with a time‐course consistent with a role in the cAMP response. This phosphorylation was abolished by the ERK1/2 inhibitor PD98059 and the PKC inhibitor Ro 31‐8220, but not the NADPH oxidase inhibitor DPI. These results reveal a role for ERK1/2 in the signaling mechanism of PKC‐ and ROS‐dependent stimulation of cAMP production in human eosinophils, and show that ERK1/2 phosphorylation is upstream of ROS production in the signaling pathway. Supported by grant #MR 01/04 from RA, Kuwait University.

Identification of a Specific Assembly of the G Protein Golf as a Critical and Regulated Module of Dopamine and Adenosine-Activated cAMP Pathways in the Striatum

In the principal neurons of striatum (medium spiny neurons, MSNs), cAMP pathway is primarily activated through the stimulation of dopamine D1 and adenosine A2A receptors, these receptors being mainly expressed in striatonigral and striatopallidal MSNs, respectively. Since cAMP signaling pathway could be altered in various physiological and pathological circumstances, including drug addiction and Parkinson’s disease, it is of crucial importance to identify the molecular components involved in the activation of this pathway. In MSNs, cAMP pathway activation is not dependent on the classical Gs GTP-binding protein but requires a specific G protein subunit heterotrimer containing Gαolf/β2/γ7 in particular association with adenylyl cyclase type 5. This assembly forms an authentic functional signaling unit since loss of one of its members leads to defects of cAMP pathway activation in response to D1 or A2A receptor stimulation, inducing dramatic impairments of behavioral responses dependent on these receptors. Interestingly, D1 receptor (D1R)-dependent cAMP signaling is modulated by the neuronal levels of Gαolf, indicating that Gαolf represents the rate-limiting step in this signaling cascade and could constitute a critical element for regulation of D1R responses. In both Parkinsonian patients and several animal models of Parkinson’s disease, the lesion of dopamine neurons produces a prolonged elevation of Gαolf levels. This observation gives an explanation for the cAMP pathway hypersensitivity to D1R stimulation, occurring despite an unaltered D1R density. In conclusion, alterations in the highly specialized assembly of Gαolf/β2/γ7 subunits can happen in pathological conditions, such as Parkinson’s disease, and it could have important functional consequences in relation to changes in D1R signaling in the striatum.

Adenosine elicits an eNOS-independent reduction in arterial blood pressure in conscious mice that involves adenosine A2A receptors

Adenosine plays an important role in the regulation of heart rate (HR) and vascular reactivity. However, the mechanisms underlying the acute effect of adenosine on arterial blood pressure in conscious mice are unclear. Therefore, this study investigated the effect of the nucleoside on mean arterial blood pressure (MAP) and HR in conscious mice. Chronic indwelling catheters were placed in C57Bl/6J (WT) and endothelial nitric oxide synthase knockout (eNOS(-/-)) mice for continuous measurements of MAP and HR. Using PCR and myograph analysis, involvement of adenosine receptors was investigated in human and mouse renal blood vessels. Bolus infusion of 0.5 mg kg(-1) adenosine elicited significant transient decreases in MAP (99.3 ± 2.3 to 70.4 ± 4.5 mmHg) and HR (603.2 ± 18.3 to 364.3 ± 49.2 min(-1)), which were inhibited by the A(2A) receptor antagonist ZM 241385. Activation of adenosine A(2A) receptors with CGS 21680 (0.02 mg kg(-1)) caused a significant reduction in MAP from 99.6 ± 1.2 to 73.1 ± 3.6 mmHg accompanied by tachycardia (610.5 ± 9.3 to 677.5 ± 9.5 min(-1)). The reduction in MAP observed after adenosine or CGS 21680 administrations was not significantly different in WT and eNOS(-/-) mice. In isolated human and mouse intrarenal arteries, adenosine caused a relaxation dependent on A(2A) adenosine receptor activation. A(2A) receptors were present in both human and mouse arteries whereas A(1) and A(2B) receptors were only present in mouse arteries. In conclusion, acute adenosine administration and selective stimulation of adenosine A(2A) receptors results in an immediate, transient eNOS-independent reduction in MAP. A(2A) receptor activation causes relaxation of human and mouse arteries.

Monoclonal antibody–assisted stimulation of adenosine A 2A receptors induces simultaneous downregulation of CXCR4 and CCR5 on CD4 + T-cells

Immunocompetent cells express various G-protein–coupled receptors that transduce extracellular signals across the plasma membrane. Among them, CXCR4 and CCR5 chemokines receptors and adenosine A 2A receptors (A 2AR) are involved in inflammatory processes. Considering that A 2AR activation may have incidence on CXCR4 and CCR5 protein expression through heterologous desensitization process, we tested Adonis, an agonist-like monoclonal antibody to A 2AR on CD4 + CEM T-cells. We found that Adonis inhibited the CEM cell growth, upregulated A 2AR and downregulated CXCR4 and CCR5 without modifying the CD4 expression. By reducing the expression of CXCR4 and CCR5 chemokines receptors utilized as entry co-receptors by HIV-1 during viral infection of CD4 expressing cells, Adonis stimulation of A 2AR appears as a valuable means to treat infected cells.

Mitochondrial metabolism revisited: a route to cardioprotection

This editorial refers to ‘Inhibition of the malate–aspartate shuttle by pre-ischaemic aminooxyacetate loading of the heart induces cardioprotection’ by N.B. Stottrup et al. , pp. 257–266, this issue. Ischaemic preconditioning confers strong protection against myocardial ischaemia–reperfusion injury.1,2 Although its therapeutic application in the case of acute coronary syndromes is not possible in general, the cellular and molecular processes leading to preconditioning protection may be of interest for the designing of cardioprotective therapies. Many cellular processes have been shown to be involved in preconditioning, including the stimulation of adenosine, bradykinin, and opioid membrane receptors,3 signalling cascades including PKCe and PKG,4,5 nitric oxide,6 and pro-survival kinases such as Akt.7 Mitochondria, a platform of cell signalling and a switch for cell death and survival, are generally regarded to play a key role in transmission and amplification of preconditioning signalling.8 It appears that many signalling processes involved in ischaemic preconditioning converge at the mitochondria.7,9 Stottrup et al. 10 show that by inhibiting the malate–aspartate shuttle …

The role of plasma adenosine deaminase in chemoattractant-stimulated oxygen radical production in neutrophils

Objectives Adenosine deaminase (ADA) has a role in many immunity mediated disorders, such as asthma, tuberculosis and coronary artery disease. This study aims to investigate the ability of plasma ADA to modulate reactive oxygen species (ROS) production in neutrophils, and examine the involvement of adenosine and the cyclic AMP signaling pathway in this process. Methods Neutrophils were stimulated, in the absence or presence of plasma, with the chemotactic peptide fMLP (formyl-methionyl-leucyl-phenylalanine), and the ROS production was determined with luminol-enhanced chemiluminescence. Activity of ADA was measured spectrophotometrically. Results Plasma dose-dependently amplified the ROS generation in fMLP-stimulated neutrophils. In parallel, incubation of neutrophils in plasma elevated the total ADA-activity approximately 10 times from 1.3 U/ml to 12 U/ml. Inhibition of ADA, or type IV phosphodiesterases, significantly lowered the plasma-mediated ROS production. Furthermore, the high-affinity adenosine A 1 receptor antagonists DPCPX and 8-phenyltheophylline markedly inhibited the plasma-induced respiratory burst in neutrophils, suggesting an A 1 receptor–mediated mechanism. Conclusions This study suggests that plasma ADA amplifies the release of toxic oxygen radicals from neutrophils through a downregulation of the inhibitory adenosine/cAMP-system and an enhanced activation of the stimulatory adenosine A 1-receptor. This mechanism has probably a crucial role in regulating neutrophil function and in the defence against microbial infections. However, a sustained neutrophil activation could also contribute to inflammatory disorders such as atherosclerosis.

Lipopolysaccharide and TNF-α modify adenosine A 2A receptor expression and function in equine monocytes

Stimulation of adenosine A 2A receptors results in anti-inflammatory effects in a variety of cell types. Lipopolysaccharide (LPS) and pro-inflammatory cytokines, such as TNF-α and IL-1, have been reported to up-regulate the expression of adenosine A 2A receptors and thereby enhance the functional activity of adenosine A 2A receptors in human and murine monocyte/macrophage cell lines and in monocytes/macrophages isolated from those species. In this study, we investigated the effects of LPS and TNF-α on the expression and functional activity of adenosine A 2A receptors in isolated equine peripheral blood monocytes. The results of this study indicate that LPS and TNF-α up-regulate the transcription of adenosine A 2A receptors for up to 24 h; the response to LPS was of greater magnitude than the response to TNF-α. In this study, incubation with LPS, but not with TNF-α, resulted in down-regulation of adenosine A 3 receptor mRNA expression. Furthermore, incubation of these cells with LPS significantly increases the surface density of adenosine A 2A receptors, and incubation with low concentrations of either LPS or TNF-α significantly increases the potency of the adenosine A 2A receptor agonist, ATL313, to inhibit LPS-induced production of TNF-α. These findings suggest that the increased expression of adenosine A 2A receptors and the enhanced functional potency of adenosine A 2A receptor agonists after exposure to pro-inflammatory substances such as LPS or TNF-α may render adenosine A 2A receptor agonists particularly important in the treatment of the systemic inflammatory response syndrome that occurs secondary to endotoxemia and bacterial infections in adult horses and neonatal foals.

Improvement in coronary endothelial function is independently associated with a slowed progression of coronary artery calcification in type 2 diabetes mellitus

To examine a relationship between alterations of structure and function of the arterial wall in response to glucose-lowering therapy in type 2 diabetes mellitus (DM) after a 1-year follow-up (FU). In DM (n = 22) and in healthy controls (n = 17), coronary artery calcification (CAC) was assessed with electron beam tomography and carotid intima-media thickness (IMT) with ultrasound, whereas coronary function was determined with positron emission tomography-measured myocardial blood flow (MBF) at rest, during cold pressor testing (CPT), and during adenosine stimulation at baseline and after FU. The decrease in plasma glucose in DM after a mean FU of 14 +/- 1.9 months correlated with a lower progression of CAC and carotid IMT (r = 0.48, P < or = 0.036 and r = 0.46, P < or = 0.055) and with an improvement in endothelium-related DeltaMBF to CPT and to adenosine (r = 0.46, P < or = 0.038 and r = 0.36, P < or = 0.056). After adjusting for metabolic parameters by multivariate analysis, the increases in DeltaMBF to CPT after glucose-lowering treatment remained a statistically significant independent predictor of the progression of CAC (P < or = 0.001 by one-way analysis of variance). In DM, glucose-lowering treatment may beneficially affect structure and function of the vascular wall, whereas the observed improvement in endothelium-related coronary artery function may also mediate direct preventive effects on the progression of CAC.

Vasopressin is a major vasoconstrictor involved in hindlimb vascular responses to stimulation of adenosine A1 receptors in the nucleus of the solitary tract

Our previous study showed that stimulation of adenosine A(1) receptors located in the nucleus of the solitary tract (NTS) exerts counteracting effects on the iliac vascular bed: activation of the adrenal medulla and beta-adrenergic vasodilation versus vasoconstriction mediated by neural and unknown humoral factors. In the present study we investigated the relative contribution of three major potential humoral vasoconstrictors: vasopressin, angiotensin II, and norepinephrine in this response. In urethane-chloralose anesthetized rats we compared the integral changes in iliac vascular conductance evoked by microinjections into the NTS of the selective A(1) receptor agonist N(6)-cyclopentyladenosine (CPA; 330 pmol in 50 nl) in intact (Int) animals and following: V(1) vasopressin receptor blockade (VX), angiotensin II AT(1) receptor blockade (ATX), bilateral adrenalectomy + ganglionic blockade (ADX + GX; which eliminated the potential increases in circulating norepinephrine and epinephrine), ADX + GX + VX and ADX + GX + VX + ATX. In Int animals, stimulation of NTS A(1) adenosine receptors evoked typical variable responses with prevailing pressor and vasoconstrictor effects. VX reversed the responses to depressor ones. ATX did not significantly alter the responses. ADX + GX accentuated pressor and vasoconstrictor responses, whereas ADX + GX + VX and ADX + GX + VX + ATX virtually abolished the responses. Stimulation of NTS A(1) adenosine receptors increased circulating vasopressin over fourfold (26.4 + or - 10.4 vs. 117.0 + or - 19 pg/ml). These data strongly suggest that vasopressin is a major vasoconstrictor factor opposing beta-adrenergic vasodilation in iliac vascular responses triggered by stimulation of NTS A(1) adenosine receptors, whereas angiotensin II and norepinephrine do not contribute significantly to the vasoconstrictor responses.