GPCR signaling is highly compartmentalized in human cardiomyocytes and severely remodeled in atrial fibrillation

Atrial fibrillation (AF) has been linked to the remodeling of membrane receptors and alterations in downstream cAMP-dependent regulation. However, to date, no study has elucidated how the increase on cAMP upon different G-protein-coupled receptors (GPCRs) can lead to different physiological compartmentalized responses. The aim of this study was to investigate the compartmentally specific effects of GPCRs on cAMP levels in human atrial myocytes (HAMs) from patients with AF and control patients without AF (Ctl), and how these compartmentalized effects are altered in AF. HAMs were isolated from 60 AF and 76 Ctl patient tissues. Cells were transduced with adenoviruses (Epac1-camps, pm-Epac1-camps and Epac1-JNC) and cultured for 48 hours to express the FRET-based cAMP sensor in the cytosolic, membrane, and RYR2 nanodomains. Förster-resonance energy transfer (FRET) was used to measure cAMP levels in 525 HAMs stimulated with isoprenaline (100 µM), serotonin (100 µM), or the A2AR agonist CGS (200 nM). A desensitization to β-adrenergic receptor stimulation was exclusively found in the cytosol of AF myocytes, while no difference was seen in the RYR2 or LTCC compartment. Similar effects were observed upon serotonin stimulation with a significant desensitization in the cytosol, and no difference in the RYR2 compartment. In response to A2ARs stimulation AF myocytes displayed a significantly higher cytosolic increase in cAMP levels. However, no response was seen in the LTCC compartment in response to serotonin or A2AR stimulation. Collectively, our data show that cAMP levels are highly compartmentalized and differentially regulated by GPCRs. Furthermore, these results provide a mechanistic insight for the previously reported functional effects seen upon stimulation of these three receptors.

Sake yeast induces the sleep-promoting effects under the stress-induced acute insomnia in mice

Sleep deprivation induces adverse effects on the health, productivity, and performance. The individuals who could not get enough sleep temporarily experience the symptoms of an induced acute insomnia. This study investigated the efficacy of sake yeast in treatment of acute insomnia in mice. The results of this study showed that sake yeast induced a significant dose-dependent wake reduction, a rapid eye movement (REM) and a non-REM (NREM) sleep enhancement during the first 6 h after the oral administration of sake yeast with locomotor activity and core body temperature decreases under the stressful environment in a new cage. In fact, the wake amounts at 3 h and 6 h were significantly reduced after the oral administration of sake yeast compared with the vehicle. The NREM sleep amounts at 3 h and 6 h significantly increased after the administration of sake yeast compared with the vehicle. The REM amount at 6 h significantly increased after the administration of sake yeast compared with the vehicle, but not at 3 h. The previous study suggested that the sleep-promoting effects of sake yeast could be referred from the activating effect of adenosine A2A receptor (A2AR). In conclusion, the sake yeast is a potential therapeutic agent for acute insomnia, being a A2AR agonist with stress-reducing and anti-anxiety properties, constituting a promising adjuvant treatment strategy for acute insomnia to traditional pharmacotherapy.

Secondary Burn Progression Mitigated by an Adenosine 2A Receptor Agonist

Background Current burn therapy is largely supportive with limited therapies to curb secondary burn progression. Adenosine 2A receptor (A2AR) agonists have anti-inflammatory effects with decreased inflammatory cell infiltrate and release of pro-inflammatory mediators. Using a porcine comb burn model, we examined whether A2AR agonists could mitigate burn progression. Study Design Eight full-thickness comb burns (4 prongs with 3 spaces per comb) per pig were generated with the following specifications: temperature 115° C, 3 kg force, and 30 second application time. In a randomized fashion, animals (4 per group) were then treated with A2AR agonist (ATL-1223, 3 ng/kg/min, intravenous infusion over 6 hours) or vehicle control. Necrotic interspace development was the primary outcome and additional histologic assessments were conducted. Results Analysis of unburned interspaces (72 per group) revealed that ATL-1223 treatment decreased the rate of necrotic interspace development over the first 4 days following injury (p<0.05). Treatment significantly decreased dermal neutrophil infiltration at 48 hours following burn (14.63±4.30 vs 29.71±10.76 neutrophils/high-power field, p=0.029). Additionally, ATL-1223 treatment was associated with fewer interspaces with evidence of microvascular thrombi through post-burn day 4 (18.8% vs 56.3%, p=0.002). Two weeks following insult, the depth of injury at distinct burn sites (adjacent to interspaces) was significantly reduced by ATL-1223 treatment (2.91±0.47 vs 3.28±0.58 mm, p=0.038). Conclusion This work demonstrates the ability of an A2AR agonist to mitigate burn progression through dampening local inflammatory processes. Extended dosing strategies may yield additional benefit and improve cosmetic outcome in those with severe injury.

Adenosine A2A receptor signaling promotes FoxO associated autophagy in chondrocytes

Autophagy, a homeostatic pathway upregulated during cellular stress, is decreased in osteoarthritic chondrocytes and this reduction in autophagy is thought to contribute to the development and progression of osteoarthritis (OA). The adenosine A2A receptor (A2AR) is a potent anti-inflammatory receptor and deficiency of this receptor leads to the development of OA in mice. Moreover, treatment using liposomally conjugated adenosine or a specific A2AR agonist improved joint scores significantly in both rats with post-traumatic OA (PTOA) and mice subjected to a high fat diet obesity induced OA. Importantly, A2AR ligation is beneficial for mitochondrial health and metabolism in vitro in primary and the TC28a2 human cell line. An additional set of metabolic, stress-responsive, and homeostatic mediators include the Forkhead box O transcription factors (FoxOs). Data has shown that mouse FoxO knockouts develop early OA with reduced cartilage autophagy, indicating that FoxO-induced homeostasis is important for articular cartilage. Given the apparent similarities between A2AR and FoxO signaling, we tested the hypothesis that A2AR stimulation improves cartilage function through activation of the FoxO proteins leading to increased autophagy in chondrocytes. We analyzed the signaling pathway in the human TC28a2 cell line and corroborated these findings in vivo in a metabolically relevant obesity-induced OA mouse model. We found that A2AR stimulation increases activation and nuclear localization of FoxO1 and FoxO3, promotes an increase in autophagic flux, improves metabolic function in chondrocytes, and reduces markers of apoptosis in vitro and reduced apoptosis by TUNEL assay in vivo. A2AR ligation additionally enhances in vivo activation of FoxO1 and FoxO3 with evidence of enhanced autophagic flux upon injection of the liposome-associated A2AR agonist in a mouse obesity-induced OA model. These findings offer further evidence that A2AR may be an excellent target for promoting chondrocyte and cartilage homeostasis.

Adenosine A1-A2A Receptor-Receptor Interaction: Contribution to Guanosine-Mediated Effects

Guanosine, a guanine-based purine nucleoside, has been described as a neuromodulator that exerts neuroprotective effects in animal and cellular ischemia models. However, guanosine’s exact mechanism of action and molecular targets have not yet been identified. Here, we aimed to elucidate a role of adenosine receptors (ARs) in mediating guanosine effects. We investigated the neuroprotective effects of guanosine in hippocampal slices from A2AR-deficient mice (A2AR−/−) subjected to oxygen/glucose deprivation (OGD). Next, we assessed guanosine binding at ARs taking advantage of a fluorescent-selective A2AR antagonist (MRS7396) which could engage in a bioluminescence resonance energy transfer (BRET) process with NanoLuc-tagged A2AR. Next, we evaluated functional AR activation by determining cAMP and calcium accumulation. Finally, we assessed the impact of A1R and A2AR co-expression in guanosine-mediated impedance responses in living cells. Guanosine prevented the reduction of cellular viability and increased reactive oxygen species generation induced by OGD in hippocampal slices from wild-type, but not from A2AR−/− mice. Notably, while guanosine was not able to modify MRS7396 binding to A2AR-expressing cells, a partial blockade was observed in cells co-expressing A1R and A2AR. The relevance of the A1R and A2AR interaction in guanosine effects was further substantiated by means of functional assays (i.e., cAMP and calcium determinations), since guanosine only blocked A2AR agonist-mediated effects in doubly expressing A1R and A2AR cells. Interestingly, while guanosine did not affect A1R/A2AR heteromer formation, it reduced A2AR agonist-mediated cell impedance responses. Our results indicate that guanosine-induced effects may require both A1R and A2AR co-expression, thus identifying a molecular substrate that may allow fine tuning of guanosine-mediated responses.

A2AR Transmembrane 2 Peptide Administration Disrupts the A2AR-A2AR Homoreceptor but not the A2AR-D2R Heteroreceptor Complex: Lack of Actions on Rodent Cocaine Self-Administration

It was previously demonstrated that rat adenosine A2AR transmembrane V peptide administration into the nucleus accumbens enhances cocaine self-administration through disruption of the A2AR-dopamine (D2R) heteroreceptor complex of this region. Unlike human A2AR transmembrane 4 (TM4) and 5 (TM5), A2AR TM2 did not interfere with the formation of the A2AR-D2R heteroreceptor complex in cellular models using BRET1 assay. A2AR TM2 was proposed to be part of the of the receptor interface of the A2AR homomer instead and was therefore tested in the current article for effects on rat cocaine self-administration using rat A2AR synthetic TM2 peptide bilaterally injected into the nucleus accumbens. The injected A2AR TM2 peptide failed to significantly counteract the inhibitory action of the A2AR agonist CGS 21680 (0.1 mg/Kg) on cocaine self-administration. In line with these results, the microinjected A2AR TM2 peptide did not reduce the number of proximity ligation assay blobs identifying A2AR-D2R heteroreceptor complexes in the nucleus accumbens. In contrast, the A2AR TM2 peptide significantly reduced the number of A2AR-A2AR homoreceptor complexes in the nucleus accumbens. As to effects on the receptor–receptor interactions in the A2AR-D2R heteroreceptor complexes, the A2AR TM2 peptide did not alter the significant increase in the D2R Ki, high values produced by the A2AR agonist CGS 21680 ex vivo in the ventral striatum. The results indicate that the accumbal A2AR-A2AR homomeric complexes are not involved in mediating the A2AR agonist-induced inhibition of cocaine self-administration.

OSU-6162, a Sigma1R Ligand in Low Doses, Can Further Increase the Effects of Cocaine Self-Administration on Accumbal D2R Heteroreceptor Complexes

Cocaine was previously shown to act at the Sigma1R which is a target for counteracting cocaine actions. It therefore becomes of interest to test if the monoamine stabilizer (–) OSU-6162 (OSU-6162) with a nanomolar affinity for the Sigma1R can acutely modulate in low doses the effects of cocaine self-administration. In behavioral studies, OSU-6162 (5 mg/kg, s.c.) did not significantly change the number of active lever pressing and cocaine infusions. However, a trend to reduce cocaine readouts was found after 3 days of treatment. In contrast, in maintenance of cocaine self-administration, the proximity ligation assay performed on brains from rats pretreated with OSU-6162 showed highly significant increases in the density of the D2R-Sigma1R heteroreceptor complexes in the shell of the nucleus accumbens versus OSU-6162 induced increases in this region of yoked saline rats. In cocaine self-administration, highly significant increases were also induced by OSU-6162 in the A2AR-D2R heteroreceptor complexes in the nucleus accumbens shell versus vehicle-treated rats. Furthermore, ex vivo, the A2AR agonist CGS21680 (100 nM) produced a marked and significant increase of the D2R Ki high values in the OSU-6162-treated versus vehicle-treated rats under maintenance of cocaine self-administration. These results indicate a substantial increase in the inhibitory allosteric A2AR-D2R interactions following cocaine self-administration upon activation by the A2AR agonist ex vivo. The current results indicate that OSU-6162 via its high affinity for the Sigma1R may increase the number of accumbal shell D2R-Sigma1R and A2AR-D2R heteroreceptor complexes associated with further increases in the antagonistic A2AR-D2R interactions in cocaine self-administration.

Abstract 13: Adenosine Receptor Activation During Extracorporeal Cardiopulmonary Resuscitation Improves Survival After Cardiac Arrest in Swine

Introduction: Despite advances in resuscitation protocols, including the addition of Extracorporeal Cardiopulmonary Resuscitation (ECPR), survival after cardiac arrest remains less than 40% and novel methods are needed to attenuate global injury and improve outcomes. ATL1223, an adenosine 2A receptor (A2AR) agonist has been shown to attenuate organ specific reperfusion injury by modulating the interaction of inflammatory cells. Hypothesis: A2AR activation during ECPR will improve survival and decrease the burden of injury in a large animal model of cardiac arrest. Methods: Adult swine underwent 20 minutes of circulatory arrest followed by defibrillation and 6 hours of ECPR. Animals were randomized to receive saline control (n=5) or the A2AR agonist Regadenoson (0.144 and 14.4mcg/kg/hr, n=5/group). Animals were subsequently weaned from ECPR and monitored for 24 hours. Clinical and biochemical endpoints were compared between groups. Results: The administration of Regadenoson increased survival after cardiac arrest compared to saline controls (10/10, 100% vs 2/5, 40%, p=0.02, Figure 1). Anesthetic administration (p=0.41), fluid resuscitation (p=0.54), and epinephrine required to maintain target arterial pressure (p=0.08) were similar for all subjects. Biochemical markers of organ damage, including creatinine (p=0.87), aspartate aminotransferase (p=0.89), and troponin I (p=0.38), were similar among groups (Figure 2). Conclusions: In a clinically relevant model of cardiac arrest treated with ECPR, selective A2AR agonism increased survival from 40% to 100% at 24 hours. These results suggest A2AR activation is a promising therapeutic target after cardiac arrest.

The Role of CD39/CD73/Ado/A2AR Axis and HIF-1α in Chronic Lymphocytic Leukemia

Introduction: CD39/CD73/ADO/A2A system plays important roles in tumor growth and therapy. Extracellular adenosine (ADO) receptor A2A (A2AR) is the dominant receptor expressed on chronic lymphocytic leukemia (CLL) cells. ADO has been shown to protect CLL cells from spontaneous and drug-induced apoptosis through A2AR activation. Overexpression of hypoxia inducible factor-1α (HIF-1α), an important mediator controlling the expression of a wide variety of apoptotic genes, has been observed in bone marrow leukemic cells from CLL patients. However, the reciprocal action of A2AR and HIF-1α in CLL remains elusive. This study was aimed to explore the molecular mechanisms underlying the relationship between A2AR activation and HIF-1α in CLL. Materials and Methods: Peripheral blood (PB) and bone marrow (BM) samples were collected from 30 healthy volunteers (control) and 20 patients who were diagnosed with CLL for the first time at the Hematology Department in our hospital. CD39/CD73/ADO/A2AR axis-related protein and HIF-1α protein expressions in CLL PB and BM tissues were examined by Western-blot. CD39, CD73, A2AR and HIF-1α expression on CLL cells were also determined by FACS. CLL cell line (Lym-2) was purchased from ATCC and incubated in DMEM medium supplemented with 10% FBS. The protein expressions of CD39, CD73 and A2AR on Lym-2 cells were confirmed by Western-blot. To study the impacts of A2AR activation and inactivation on CLL cells, CLL cells were treated with A2AR agonist CGS21680 or antagonist SCH58261, followed by determination of cell proliferation and HIF-1α protein expression. Results: The protein expressions of CD39, CD73, A2AR and HIF-1α were higher in CLL group than those in control group (BM: CD39 1.471 vs 0.926, CD73 1.097 vs 0.489, A2AR 1.139 vs 0.342 and HIF-1α 0.940 vs 0.362, P<0.05 Figure 1A; PB : CD39 1.809 vs 1.331, CD73 1.039 vs 0.653, A2AR 1.738 vs 1.119 and HIF-1α 1.336 vs 1.010, P<0.05 Figure 1B). Moreover, CD73 and HIF-1α protein expression was found to have relationship with disease stage. Patients who belonged to stage IV (Rai) and stage B/C (Binet) exhibited higher levels of CD73 and HIF-1α than patients who belonged to stage I to III (Rai) and stage A (Binet) . Data from FACS analysis showed that the proportion of CD39+CLL cells was higher than CD73+CLL cells and HIF-1α+CLL cells (P<0.05, Figure 2A). When compared to control group, the proportions of CD39+cells and CD73+ cells were significant higher in CLL group (Figure 2B). Western-blot results showed that Lym-2 cells expressed CD39, CD73 and A2AR simultaneously (Figure 3A). The results from CLL cells and A2AR agonist/antagonist incubation showed that when CGS21680 concentration was of ≥ 10uM, cell proliferation was promoted obviously (Figure 3B). On the contrary, SCH58261 could inhibit CLL cells proliferation when its concentration was of ≥5uM at a concentration dependent manner (Figure 3C). Moreover, HIF-1α protein expression was also affected by A2AR agonist at a concentration dependent manner(0uM 0.851, 10uM 1.116, 20uM 1.420, 50uM 1.41, Figure 3D). Conclusion: Our study showed that ADO produced by CD39 and CD73 could affect CLL cells proliferation and HIF-1α expression through A2AR-mediated mechanisms. The exploration of the molecular mechanisms underlying the relationship between A2AR activation and HIF-1α could help us to find a novel approach to CLL therapy. Disclosures: No relevant conflicts of interest to declare. Disclosures No relevant conflicts of interest to declare.