A3AR Agonists Research Articles

LB787 CLE-400: A potent alpha-2 adrenergic receptor agonist for the treatment of chronic itch

Pruritus (itch) is a major symptom of several dermatological diseases but has limited therapeutic options available. Itch stimuli are transmitted via primary sensory neurons in the skin by activating pruriceptors/nociceptors on small unmyelinated C-fibers, through the spinal cord for final processing in the brain. CLE-400 is an aqueous gel of detomidine that is being developed for the topical treatment of itch. Detomidine is a potent α2-adrenergic receptor (α2-AR) agonist. α2-AR have been shown to be expressed on skin nociceptors and their activation by topical administration of detomidine could lead to inhibition of itch neural signalling.

Guanabenz inhibits alveolar bone resorption in a rat model of periodontitis

Increase of sympathetic activity has been known to exacerbate osteoporosis through promotion of bone resorption. However, it is largely unknown about involvement of sympathetic activity in exacerbation of periodontitis. In this study, we investigated whether α2-adrenergic receptor (α2-AR) agonist guanabenz which decreases sympathetic activity, attenuates alveolar bone resorption in rats having high sympathetic activity with periodontitis. Volumes of residual alveolar bone and attachment levels in periodontium were examined using micro-computed tomography and hematoxylin-eosin staining, respectively. Furthermore, osteoclast numbers per bone surface and osteoclast surface per bone surface were measured using tartrate-resistant acid phosphatase staining. To examine the suppressive effects of guanabenz on pro-inflammatory cytokines, expression levels of tyrosine hydroxylase (TH), TNF-α, IL1-β, and IL-6 in periodontium were measured using immunohistostaining. Administration of guanabenz attenuated loss of alveolar bone and attachment levels in rats having high sympathetic activity. Furthermore, its administration suppressed osteoclast numbers in rats having high sympathetic activity. TH, TNF-α, IL-1β, and IL-6 positive cells in periodontium in rats treated with guanabenz for 12 weeks, were lower than those in control rats having high sympathetic activity. This study demonstrated administration of α2-AR agonist guanabenz attenuates alveolar bone resorption through decrease of sympathetic activity in rats.

Genetic Polymorphism of rs13306146 Affects α2AAR Expression and Associated With Postpartum Depressive Symptoms in Chinese Women Who Received Cesarean Section.

Postpartum depressive symptom (PDS) is a common psychological and mental disorder after giving birth. Our previous studies showing the application of dexmedetomidine, an α2-AR agonist, can significantly improve maternal sleep, as well as relieve and reduce the incidence of PDS. This study investigated the association between α2AAR gene polymorphisms and PDS. A total of 568 cesarean section patients were enrolled; the incidence of PDS is 18.13% (103 with PDS, 465 with non-PDS). The Edinburgh Postpartum Depression Scale score ≥10 was used to diagnose PDS at 42 days after delivery. The single-nucleotide polymorphisms of α2AR were sequenced by pyrosequencing. The effect of rs13306146 A > G polymorphism on α2AR transcription and the regulation of miR-646 on α2AR expression were assessed by dual luciferase reporter assays or gene transfection. Increased stress during pregnancy, poor relationship between mother-in-law and daughter-in-law, spousal relationship, domestic violence, antenatal depression, self-harm ideation, and stressful life events were all associated with increased PDS incidence (p < 0.05). The logistic regression analysis found that the α2AAR rs13306146 polymorphism was associated with PDS after adjusting confounding variables. The transcriptional function of the α2AAR rs13306146 A allele was decreased compared with the G allele, and the α2AAR expression level was correspondingly decreased (p < 0.05), as the strongest binding ability of miR-646 to the α2AAR rs13306146 AA genotype. The effect of α2AAR rs13306146 A > G polymorphism may change the binding ability of miR-646 at the 3′UTR of the α2AAR gene, affecting the expression of α2AAR. This study supports the involvement of the norepinephrine system in the pathogenesis of PDS. Genotypes of α2AAR may be novel and useful biomarkers for PDS.

The α2AR/Caveolin-1/p38MAPK/NF-κB axis explains dexmedetomidine protection against lung injury following intestinal ischaemia-reperfusion.

Intestinal ischaemia‐reperfusion (I/R) injury can result in acute lung injury due to ischaemia and hypoxia. Dexmedetomidine (Dex), a highly selective alpha2‐noradrenergic receptor (α2AR) agonist used in anaesthesia, is reported to regulate inflammation in organs. This study aimed to investigate the role and mechanism of Dex in lung injury caused by intestinal I/R. After establishing a rat model of intestinal I/R, we measured the wet‐to‐dry specific gravity of rat lungs upon treatments with Dex, SB239063 and the α2AR antagonist Atipamezole. Moreover, injury scoring and histopathological studies of lung tissues were performed, followed by ELISA detection on tumour necrosis factor‐α (TNF‐α), interleukin (IL)‐1β and IL‐6 expression. Correlation of Caveolin‐1 (Cav‐1) protein expression with p38, p‐p38, p‐p65 and p65 in rat lung tissues was analysed, and the degree of cell apoptosis in lung tissues after intestinal I/R injury was detected by TUNEL assay. The lung injury induced by intestinal I/R was a dynamic process. Moreover, Dex had protective effects against lung injury by mediating the expression of Cal‐1 and α2A‐AR. Specifically, Dex promoted Cav‐1 expression via α2A‐AR activation and mitigated intestinal I/R‐induced lung injury, even in the presence of Atipamezole. The protective effect of Dex on intestinal I/R‐induced lung injury was also closely related to α2A‐AR/p38 mitogen‐activated protein kinases/nuclear factor‐kappaB (MAPK/NF‐κB) pathway. Dex can alleviate pulmonary inflammation after in intestinal I/R by promoting Cav‐1 to inhibit the activation of p38 and NF‐κB. In conclusion, Dex can reduce pulmonary inflammatory response even after receiving threats from both intestinal I/R injury and Atipamezole.

The insulin receptor family and protein kinase B (Akt) are activated in the heart by alkaline pH and α1-adrenergic receptors.

Insulin and insulin-like growth factor stimulate protein synthesis and cardioprotection in the heart, acting through their receptors (INSRs, IGF1Rs) and signalling via protein kinase B (PKB, also known as Akt). Protein synthesis is increased in hearts perfused at alkaline pHo to the same extent as with insulin. Moreover, α1-adrenergic receptor (α1-AR) agonists (e.g. phenylephrine) increase protein synthesis in cardiomyocytes, activating PKB/Akt. In both cases, the mechanisms are not understood. Our aim was to determine if insulin receptor-related receptors (INSRRs, activated in kidney by alkaline pH) may account for the effects of alkaline pHo on cardiac protein synthesis, and establish if α1-ARs signal through the insulin receptor family. Alkaline pHo activated PKB/Akt signalling to the same degree as insulin in perfused adult male rat hearts. INSRRs were expressed in rat hearts and, by immunoblotting for phosphorylation (activation) of INSRRs/INSRs/IGF1Rs, we established that INSRRs, together with INSRs/IGF1Rs, are activated by alkaline pHo. The INSRR/INSR/IGF1R kinase inhibitor, linsitinib, prevented PKB/Akt activation by alkaline pHo, indicating that INSRRs/INSRs/IGF1Rs are required. Activation of PKB/Akt in cardiomyocytes by α1-AR agonists was also inhibited by linsitinib. Furthermore, linsitinib inhibited cardiomyocyte hypertrophy induced by α1-ARs in cultured cells, reduced the initial cardiac adaptation (24 h) to phenylephrine in vivo (assessed by echocardiography) and increased cardiac fibrosis over 4 days. We conclude that INSRRs are expressed in the heart and, together with INSRs/IGF1Rs, the insulin receptor family provide a potent system for promoting protein synthesis and cardioprotection. Moreover, this system is required for adaptive hypertrophy induced by α1-ARs.

Spinal A3 adenosine receptor activation acutely restores morphine antinociception in opioid tolerant male rats.

Opioids are potent analgesics, but their pain-relieving effects diminish with repeated use. The reduction in analgesic potency is a hallmark of opioid analgesic tolerance, which hampers opioid pain therapy. In the central nervous system, opioid analgesia is critically modulated by adenosine, a purine nucleoside implicated in the beneficial and detrimental actions of opioid medications. Here, we focus on the A3 adenosine receptor (A3 AR) in opioid analgesic tolerance. Intrathecal administration of the A3 AR agonist MRS5698 with daily systemic morphine in male rats attenuated the reduction in morphine antinociception over 7days. In rats with established morphine tolerance, intrathecal MRS5698 partially restored the antinociceptive effects of morphine. However, when MRS5698 was discontinued, these animals displayed a reduced antinociceptive response to morphine. Our results suggest that MRS5698 acutely and transiently potentiates morphine antinociception in tolerant rats. By contrast, in morphine-naïve rats MRS5698 treatment did not impact thermal nociceptive threshold or affect antinociceptive response to a single injection of morphine. Furthermore, we found that morphine-induced adenosine release in cerebrospinal fluid was blunted in tolerant animals, but total spinal A3 AR expression was not affected. Collectively, our findings indicate that spinal A3 AR activation acutely potentiates morphine antinociception in the opioid tolerant state.

DHHC21 deficiency attenuates renal dysfunction during septic injury

Renal dysfunction is one of the most common complications of septic injury. One critical contributor to septic injury-induced renal dysfunction is renal vascular dysfunction. Protein palmitoylation serves as a novel regulator of vascular function. Here, we examined whether palmitoyl acyltransferase (PAT)-DHHC21 contributes to septic injury-induced renal dysfunction through regulating renal hemodynamics. Multispectral optoacoustic imaging showed that cecal ligation and puncture (CLP)-induced septic injury caused impaired renal excretion, which was improved in DHHC21 functional deficient (Zdhhc21dep/dep) mice. DHHC21 deficiency attenuated CLP-induced renal pathology, characterized by tissue structural damage and circulating injury markers. Importantly, DHHC21 loss-of-function led to better-preserved renal perfusion and oxygen saturation after CLP. The CLP-caused reduction in renal blood flow was also ameliorated in Zdhhc21dep/dep mice. Next, CLP promoted the palmitoylation of vascular α1-adrenergic receptor (α1AR) and the activation of its downstream effector ERK, which were blunted in Zdhhc21dep/dep mice. Vasoreactivity analysis revealed that renal arteries from Zdhhc21dep/dep mice displayed reduced constriction response to α1AR agonist phenylephrine compared to those from wild-type mice. Consistently, inhibiting PATs with 2-bromopalmitate caused a blunted vasoconstriction response to phenylephrine in small arteries isolated from human kidneys. Therefore, DHHC21 contributes to impaired renal perfusion and function during septic injury via promoting α1AR palmitoylation-associated vasoconstriction.

Dexmedetomidine Inhibits ASIC Activity via Activation of α2A Adrenergic Receptors in Rat Dorsal Root Ganglion Neurons.

Dexmedetomidine (DEX), a selective α2 adrenergic receptor (α2-AR) agonist, has been shown to have peripheral analgesic effects in a variety of pain conditions. However, the precise molecular mechanisms have not yet been fully elucidated. Acid sensing ion channels (ASICs) are the major player in pain associated with tissue acidosis. Given that both α2-ARs and ASICs exist in dorsal root ganglia (DRG) neurons, we therefore investigated the effects of DEX on the functional activity of ASICs. Herein, whole-cell patch-clamp recordings demonstrated that DEX suppressed ASIC-mediated and acid-evoked currents and action potentials in dissociated rat DRG neurons. DEX shifted downwards concentration-response curve to protons, with a decrease of 35.83 ± 3.91% in the maximal current response to pH 4.5. DEX-induced inhibition of ASIC currents was blocked by the α2A-AR antagonist BRL44408 in DRG neurons. DEX also inhibited ASIC3 currents in CHO cells co-expressing ASIC3 and α2A-ARs, but not in ASIC3 transfected CHO cells without α2A-ARs expression. DEX-induced inhibition of ASIC currents was mimicked by the protein kinase A inhibitor H-89, and blocked by intracellular application of the Gi/o protein inhibitor pertussis toxin and the cAMP analog 8-Br-cAMP. In addition, peripherally administration of DEX dose-dependently relieved nociceptive responses to intraplantar injection of acetic acid in rats through local α2A-ARs. Our results indicated that DEX inhibited the functional activity of ASICs via α2A-ARs and intracellular Gi/o proteins and cAMP/protein kinase A signaling pathway in rat DRG neurons, which was a novel potential mechanism that probably mediated peripheral analgesia of DEX.

The Influence of Anesthesia on the Cardiovascular Effects of Centrally Administered Clonidine

The α2 adrenergic receptor (α2AR) agonists are known to induce hypotensive responses when administered via intracerebroventricular (icv) route in normotensive rats anesthetized with either pentobarbital or urethane. However, the icv injection of α2AR agonists such as clonidine (CLO) produces hypertensive responses in normotensive conscious rats. To date, the effects icv administration of CLO in normotensive rats under ketamine/xylazine anesthesia, a commonly used anesthetic combination in experimental studies, is still unknown. We hypothesized that anesthetic drugs differentially modulate cardiovascular responses to centrally administered CLO. To evaluate this hypothesis, we assessed blood pressure (BP) and heart rate (HR) of normotensive male rats anesthetized with pentobarbital or ketamine/xylazine, both under mechanical ventilation. These animals received CLO (10 μg/5 μL) or dexmedetomidine (DEX; 0.6 μg/5 μL) by icv route. In additional experiments, yohimbine (5 μg/5 μL), a selective α2AR antagonist, was given by icv route 15 min before a second injection of CLO or DEX in ketamine/xylazine anesthetized rats. As widely described, the icv microinjection of CLO decreased the mean arterial pressure (MAP) and HR by 40.73 ± 6.3 mmHg and 134 ± 15 bpm in pentobarbital anesthetized rats (n = 5). On the other hand, in ketamine/xylazine anesthetized rats (n = 6) CLO increased MAP by 29.4 ± 5.1 mmHg and decreased HR by 34 ± 5 bpm, a less pronounced effect when compared with the pentobarbital group (p < 0.0001). Similarly, the icv microinjection of DEX in ketamine/xylazine anesthetized rats (n = 6) increased MAP by 20.8 ± 3.2 mmHg and decreased HR by 51 ± 16 bpm. Notably, yohimbine generated a pattern of hypotensive responses comparable to CLO and DEX. Additionally, after yohimbine administration, both CLO- and DEX-increased MAP were reduced to 9.9 ± 2.3 mmHg and 6.7 ± 1.1 mmHg, respectively (n = 6, p < 0.05), despite the lack of influence of yohimbine in the bradycardia induced by these α2AR agonists in ketamine/xylazine anesthetized rats. This study discloses that in normotensive rats under ketamine/xylazine anesthesia, central administration of CLO produces a hypertensive effect, opposing the findings obtained in pentobarbital-anesthetized rats and resembling the vascular responses described in conscious animals. This data was reproduced by DEX and reduced by yohimbine, suggesting that stimulation of central α2AR takes a preponderant role in this effect. Our results reinforce the relevance regarding the influence of anesthetic choice for studies involving cardiovascular parameters.

Dichotomous effects of smooth muscle TRPV4 channels on vascular contractility

Ca2+ signaling mechanisms in smooth muscle cells (SMCs) determine vascular contractility. Transient receptor potential vanilloid 4 (TRPV4) ion channels have been described as a crucial Ca2+ influx pathway in SMCs from small, resistance-sized arteries. However, the impact of SMC TRPV4 (TRPV4SMC) channel activity on blood pressure remains unknown, mostly due to the lack of studies in SMC-specific knockout mice. We hypothesized that TRPV4SMC channels control the resting blood pressure. Radiotelemetric blood pressure recordings showed reduced daytime and nighttime blood pressures in the inducible, SMC-specific TRPV4 knockout (TRPV4SMC-/-) mice compared to the control mice. The effect of TRPV4SMC-/- on vascular contractility was assessed using two endogenous vasoconstrictor mechanisms- 1) SMC α1 adrenergic receptor (α1AR) stimulation and 2) increased intraluminal pressure. Phenylephrine (α1AR agonist)-induced constriction of pressurized mesenteric arteries (MAs, 80 mm Hg, 1 nM-10 mM phenylephrine) and blood pressure elevation (10 mg/kg phenylephrine, i.p.) were blunted in TRPV4SMC-/- mice, identifying a central role of TRPV4SMC channels in α1AR-induced vasoconstriction and blood pressure elevation. Individual Ca2+ influx signals through TRPV4SMC channels (TRPV4SMC sparklets) were recorded using spinning disk confocal imaging in fluo-4-loaded MAs pressurized to 80 mm Hg. Phenylephrine (1 mM) activated TRPV4SMC sparklets, an effect abolished by protein kinase C (PKC) inhibitor (Go6976, 1 mM). These results indicated that PKC-TRPV4SMC channel signaling is a major contributor to α1AR-induced vasoconstriction. In direct contrast with PE-induced constriction, intraluminal pressure-induced constriction was higher in MAs from TRPV4SMC-/- mice, suggesting that SMC TRPV4 channels limit pressure-induced vasoconstriction. Previous studies have shown that TRPV4SMC channels can activate large-conductance, Ca2+-sensitive K+ (BK) channels, thereby decreasing pressure-induced vasoconstriction. Consistent with these findings, vasoconstriction in response to BK channel inhibitor (paxilline, 1 mM) was reduced in MAs from TRPV4SMC-/- mice, supporting a dilatory TRPV4SMC channel-BK channel signaling under resting conditions. In situ proximity ligation assay revealed co-localization of TRPV4SMC channels with both α1ARs and BK channels. Overall, TRPV4SMC channels increase the resting blood pressure but have divergent effects on vascular contractility. TRPV4SMC channels contribute to α1AR-induced vasoconstriction but limit pressure-induced vasoconstriction. Therefore, selective vasoactive stimulus-TRPV4SMC channel coupling may determine the impact of TRPV4SMC channels on vascular resistance.

Design and Synthesis of 2,6-Disubstituted-4'-Selenoadenosine-5'-N,N-Dimethyluronamide Derivatives as Human A3 Adenosine Receptor Antagonists.

A new series of 4′-selenoadenosine-5′-N,N-dimethyluronamide derivatives as highly potent and selective human A3 adenosine receptor (hA3AR) antagonists, is described. The highly selective A3AR agonists, 4′-selenoadenosine-5′-N-methyluronamides were successfully converted into selective antagonists by adding a second N-methyl group to the 5′-uronamide position. All the synthesized compounds showed medium to high binding affinity at the hA3AR. Among the synthesized compounds, 2-H-N6-3-iodobenzylamine derivative 9f exhibited the highest binding affinity at hA3AR. (Ki = 22.7 nM). The 2-H analogues generally showed better binding affinity than the 2-Cl analogues. The cAMP functional assay with 2-Cl-N6-3-iodobenzylamine derivative 9l demonstrated hA3AR antagonist activity. A molecular modelling study suggests an important role of the hydrogen of 5′-uronamide as an essential hydrogen bonding donor for hA3AR activation.

PDRN, a natural bioactive compound, blunts inflammation and positively reprograms healing genes in an “in vitro” model of oral mucositis

Oral mucositis is a side effect hard to treat following high dose chemotherapy or radiotherapy. Adenosine A2A receptor stimulation blocks NF-κB and boosts the Wnt/β-catenin signaling, thus blunting inflammation and triggering growth factor codifying genes. Polydeoxyribonucleotide (PDRN) is a registered drug that activates the A2A receptor. Therefore, the aim of this study was to evaluate PDRN effects in an “in vitro” model of oral mucositis induced by prompting an inflammatory phenotype in human gingival fibroblasts (GF) and human oral mucosal epithelial cells (EC). GF and EC were stimulated with LPS (2 μg/ml) alone or in combination with i) PDRN (100 μg/ml); ii) PDRN plus ZM241385 (1 μM) as an A2AR antagonist; iii) CGS21680 (1 μM) as an A2AR agonist. LPS boosted NF-κB, TNF-α and IL-6 expression, decreased IL-10 levels and downregulated both Wnt/β-catenin, VEGF and EGF expression. PDRN reverted the LPS-induced phenotype as well as CGS21680. Co-incubation with ZM241385 abolished PDRN effects, thus confirming A2A receptor involvement in PDRN mechanism of action. These results suggest that PDRN efficacy may be due to a “dual mode” of action: NF-κB inhibition and Wnt/β-catenin signaling activation. However, these interesting findings need to be confirmed by animal and clinical studies.

Alpha and beta adrenoceptors activate interleukin-6 transcription through different pathways in cultured astrocytes from rat spinal cord

In brain astrocytes, noradrenaline (NA) has been shown to up-regulate IL-6 production via β-adrenoceptors (ARs). However, the underlying intracellular mechanisms for this regulation are not clear, and it remains unknown whether α-ARs are involved. In this study, we investigated the AR-mediated regulation of IL-6 mRNA levels in the cultured astrocytes from rat spinal cord. NA, the α1-agonist phenylephrine, and the β-agonist isoproterenol increased IL-6 mRNA levels. The phenylephrine-induced IL-6 increase was accompanied by an increase in ERK phosphorylation, and these effects were blocked by inhibitors of PKC and ERK. The isoproterenol-induced IL-6 increase was accompanied by an increase in CREB phosphorylation, and these effects were blocked by a PKA inhibitor. Our results indicate that IL-6 increases by α1- and β-ARs are mediated via the PKC/ERK and cAMP/PKA/CREB pathways, respectively. Moreover, conditioned medium collected from astrocytes treated with the α2-AR agonist dexmedetomidine, increased IL-6 mRNA in other astrocytes. In this study, we elucidate that α1- and α2-ARs, in addition to β-ARs, promote IL-6 transcription through different pathways in spinal cord astrocytes.

Functional interplay between adenosine A2A receptor and NMDA preconditioning in fear memory and glutamate uptake in the mice hippocampus

N-methyl D-aspartate (NMDA) administered at subtoxic dose plays a protective role against neuronal excitotoxicity, a mechanism described as preconditioning. Since the activation of adenosinergic receptors influences the achievement of NMDA preconditioning in the hippocampus, we evaluated the potential functional interplay between adenosine A1 and A2A receptors (A1R and A2AR) activities and NMDA preconditioning. Adult male Swiss mice received saline (NaCl 0.9 g%, i.p.) or a nonconvulsant dose of NMDA (75 mg/kg, i.p.) and 24 h later they were treated with the one of the ligands: A1R agonist (CCPA, 0.2 mg/kg, i.p.) or antagonist (DPCPX, 3 mg/kg, i.p.), A2AR agonist (CGS21680, 0.05 mg/kg, i.p.) or antagonist (ZM241385, 0.1 mg/kg, i.p.) and subjected to contextual fear conditioning task. Binding properties and content of A2AR and glutamate uptake were assessed in the hippocampus of mice subjected to NMDA preconditioning. Treatment with CGS21680 increased the time of freezing during the exposure of animals to the new environment. NMDA preconditioning did not affect the freezing time of mice per se, but it prevented the response observed after the activation of A2AR. Furthermore, the activation of A2AR by CGS21680 after the preconditioning blocked the increase of glutamate uptake induced by NMDA preconditioning. The immunodetection of A2AR in total hippocampal homogenates showed no significant differences evoked by NMDA preconditioning and did not alter A2AR maximum binding for the selective ligand [3H]CGS21680. These results demonstrate changes in A2AR functionality in mice following NMDA preconditioning.

Dexmedetomidine provides protection to neurons against OGD/R-induced oxidative stress and neuronal apoptosis

Dexmedetomidine, a potent α2-adrenoceptor (α2-AR) agonist, is extensively used in the operating room (OR) and intensive care unit (ICU) and has been applied for the treatment of several diseases. Western blotting has been routinely used to investigate the protein levels of α-adrenergic receptor (α-AR), apoptosis related proteins (Bcl-2, Bax and Cleaved Caspase 3) and a range of proteins associated with the Nrf2/ARE pathway (Nrf2, HO-1, NQO-1, SOD) in neurons. The CCK-8 assay was used to determine cell survival rates while the Co-IP assay was used to investigate the binding ability between α2-AR and Nrf2. The TUNEL assay was used to detect cell apoptosis in neurons. OGD/R treatment reduced the level of α2-AR protein in neurons and reduced neuronal survival in a time-dependent manner. However, treatment with dexmedetomidine led to the increased protein expression of α2-AR in OGD/R-treated neurons and enhanced survival rate of OGD/R-treated neurons. From a mechanistic point-of-view, Nrf2 can effectively bind with α2-AR. Silencing Nrf2 reversed the effects of dexmedetomidine on cell viability, oxidative stress, and neuronal apoptosis in OGD/R-treated neurons. The activation of α2-AR by dexmedetomidine had a protective effect in neurons against OGD/R-triggered oxidative stress and neuronal apoptosis by modulating the Nrf2/ARE pathway.

Combined Therapy of A1AR Agonists and A2AAR Antagonists in Neuroinflammation.

Alzheimer’s, Parkinson’s, and multiple sclerosis are neurodegenerative diseases related by neuronal degeneration and death in specific areas of the central nervous system. These pathologies are associated with neuroinflammation, which is involved in disease progression, and halting this process represents a potential therapeutic strategy. Evidence suggests that microglia function is regulated by A1 and A2A adenosine receptors (AR), which are considered as neuroprotective and neurodegenerative receptors, respectively. The manuscript’s aim is to elucidate the role of these receptors in neuroinflammation modulation through potent and selective A1AR agonists (N6-cyclopentyl-2′- or 3′-deoxyadenosine substituted or unsubstituted in 2 position) and A2AAR antagonists (9-ethyl-adenine substituted in 8 and/or in 2 position), synthesized in house, using N13 microglial cells. In addition, the combined therapy of A1AR agonists and A2AAR antagonists to modulate neuroinflammation was evaluated. Results showed that A1AR agonists were able, to varying degrees, to prevent the inflammatory effect induced by cytokine cocktail (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and interferon (IFN)-γ), while A2AAR antagonists showed a good ability to counteract neuroinflammation. Moreover, the effect achieved by combining the two most effective compounds (1 and 6) in doses previously found to be non-effective was greater than the treatment effect of each of the two compounds used separately at maximal dose.

Intervention of Tyrosine Hydroxylase Expression Alters Joint Inflammation and Th17/Treg Imbalance in Collagen-Induced Arthritis.

Neuroendocrine dysregulation has been associated with rheumatoid arthritis (RA). Tyrosine hydroxylase (TH), a rate-limiting enzyme for synthesis of neuroendocrine hormones such as epinephrine, is also expressed in T lymphocytes and regulates balance between helper T (Th) 17 cells and regulatory T (Treg) cells. Herein, we aimed to show that TH expression in joints alleviates joint inflammation and Th17/Treg imbalance in collagen-induced arthritis (CIA), an animal model of RA, and these effects may be implemented by the mechanism of epinephrine action on α1-adrenoreceptor (α1-AR) in T cells. CIA was prepared by intradermal injection of collagen type II in tail base of DBA1/J mice. On the 33rd day post-immunization, lentiviral vectors encoding TH or TH shRNA were injected into ankle joints of CIA mice. Limb inflammation of the mice was assessed beginning from day 21 until day 69 post-immunization by measurement of limb swelling, erythema and rigidity. Th17 and Treg differentiation and function in ankle joints were assessed on day 69 post-immunization by test of the expression of Th17 transcriptional factor ROR-γt and the levels of proinflammatory cytokines interleukin (IL)-17 and IL-22 as well as the expression of Treg transcriptional factor Foxp3 and the levels of antiinflammatory cytokines transforming growth factor (TGF)-β1 and IL-10. T cells were obtained from the spleen of mice that had been immunized with collagen type II 41 day earlier and treated with epinephrine or α1-AR agonist phenylephrine in vitro. Flow cytometry was used to analyze the percentages of CD25-IL-17+ cells and CD25+Foxp3+ cells in CD4+ T cells. TH gene overexpression in ankle joints of CIA mice reduced limb inflammation and Th17-related transcription factor expression and inflammatory cytokine production but increased Treg-related antiinflammatory cytokine production in the joints. In contrast, TH gene silence in ankle joints of CIA mice enhanced limb inflammation and Th17 cell activity but decreased Treg cell function in the joints. Epinephrine upregulated α1-AR expression in T cells derived from CIA mice. Both epinephrine and phenylephrine reduced CIA-induced Th17 transcription factor expression and inflammatory cytokine production but enhanced Treg antiinflammatory cytokine production in vitro. Upregulating TH expression in joints alleviates joint inflammation and Th17/Treg imbalance in CIA at least partially by enhancing epinephrine action on α1-AR in T cells.

ADENOSINE A3 RECEPTOR INTERACTS WITH GASDERMIN D TO MODULATE INTESTINAL EPITHELIAL CELL PYROPTOSIS IN ULCERATIVE COLITIS

Abstract Background Adenosine A3 receptor (A3AR) plays a role in intestinal inflammation, but little is known about its mechnisms in intestinal inflammation such as ulcerative colitis (UC). Pyroptosis, characterized by Gasdermin D (GSDMD) activation, is implicated in the pathogenesis of UC. We investigated the role of A3AR in GSDMD-mediated pyroptosis in UC and its underlying molecular mechanisms. Methods The expression of A3AR in colonic mucosa of patients with UC were examined. A3AR agonist was used to study the role of A3AR in ex vivo colonic explants of UC patients. In addition, human intestinal epithelial cells Caco-2 were used to further verify the effect of A3AR on pyroptosis induced by LPS+ATP. RT-qPCR and western blotting were used to detect the expression levels of pyroptosis-associated factors including NLRP3, caspase-1, gasdermin-D N-terminal domain (GSDMD-NT), IL-1β and IL-18 in colonic tissues and Caco-2 cells. Immunofluorescence was used to detect the protein expression in tissues and cells. Enzyme-linked immunosorbent assay was used to determine the levels of IL-1β and IL-18 in tissue and cell culture supernatants. Lactate dehydrogenase (LDH) release assay and propidium iodide (PI) staining were used to measure cell pyroptosis. Molecular interactions beween A3AR and GSDMD were investigated using Co-IP and GST pull-down assays. Results A3AR expression was significantly reduced in colonic mucosa of patients with active UC, and colonic epithelial cells were the main cell subpopulation with down-regulated A3AR expression. Pyroptosis-associated factors, including Caspase-1, NLRP3, and GSDMD-NT, were upregulated in UC colonic tissues. The expression of A3AR and GSDMD-NT was negatively correlated. A3AR agonist reduced the production of cytokines (IL-1β and IL-18) and attenuates the expression levels of NLRP3 and GSDMD-NT in the colonic tissues of patients with UC. Furthermore, A3AR overexpression alleviated pyroptosis with reduced LDH release, PI-stained cell number and decreased expressions of GSDMD-NT, NLRP3, caspase-1, IL-1β, and IL-18 in the LPS+ATP-stimulated Caco-2 cells, whereas the opposite occurred in cells treated with small interfeing RNA (siRNA) targeting A3AR. Knockdown of GSDMD in Caco-2 cells significantly blocked the effects of A3AR overexpression or down-regulation on pyroptosis, suggesting that A3AR acts through the GSDMD-mediated pyroptosis pathway. Co-IP and GST pull-down assays showed that A3AR interacted with GSDMD. Conclusion A3AR modulates intestinal inflammation in UC through GSDMD-mediated intestinal epithelial cell pyroptosis. We propose a novel mechanism by which A3AR-GSDMD interaction affects UC through pyroptosis, suggesting that A3AR is a potential target for the treatment of UC.

Role of A2-adrenoceptors in Rat Heart with the Model of Myocardial Infarction

Chronic adrenergic and angiotensin-energic stimulation of the heart muscle is one of the main causes of the onset and development of heart failure. α2-AR is widespread in the cardiovascular system and can be further applied and introduced into the heart's regenerative cell therapy. This study's objective was to study the effect of α2-AR stimulation on the performance of an isolated heart in rats with a model of myocardial infarction. To study the mechanisms of MI, experiments were carried out according to the classical technique of H. Selye - ligation of the anterior branch of the left coronary artery and perfusion of a Langendorff heart with a model of myocardial infarction. We studied the parameters of myocardial activity by changes in heart rate, as well as the left ventricle pressure (LVP), and the CF (dp/dtmax)/(dp/dtmin) of the left ventricle. Coronary dilator activity was assessed by measuring the outflow of the perfusion solution through the coronary arteries. The amplitude of the left ventricle's pressure wave after application of the α2-AR agonist clonidine hydrochloride at a concentration of 10-6 M to the perfused solution increased by 44% (p≤0.05) from the initial values. The rate of contraction and the rate of relaxation of the left ventricular myocardium also increased by 20% (p≤0.05) and 37% (p≤0.05), respectively. The activation of α2-AR in adult rats' isolated hearts with a model of myocardial infarction caused a decrease in heart rate by 18% (p≤0.05) and CF by 2% (p≤0.05).

Arbutin effectively ameliorates the symptoms of Parkinson's disease: the role of adenosine receptors and cyclic adenosine monophosphate.

An antagonistic communication exists between adenosinergic and dopaminergic signaling in the basal ganglia, which suggests that the suppression of adenosine A2A receptors-cyclic adenosine monophosphate pathway may be able to restore the disrupted dopamine transmission that results in motor symptoms in Parkinson's disease (PD). Arbutin is a natural glycoside that possesses antioxidant, anti-inflammatory, and neuroprotective properties. The purpose of this study was to investigate whether arbutin could ameliorate the symptoms of PD and to examine the underlying mechanism. In this study, Swiss albino mouse models of PD were established by the intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine for 4 successive days, with the concurrent intraperitoneal administration of arbutin (50 and 100 mg/kg) for 7 days. The results showed that arbutin significantly reduced lipid peroxidation, total nitrite levels, and inflammation in the substantia nigra and striatum of PD mouse models. In addition, arbutin decreased the activity of endogenous antioxidants, reduced the levels of dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, and γ-aminobutyric acid, and minimized neurodegeneration in the striatum. Arbutin also reduced the abnormal performance of PD mouse models in the open field test, bar test, pole test, and rotarod test. The therapeutic efficacy of arbutin was similar to that of madopar. The intraperitoneal injection of the A2AR agonist CGS21680 (0.5 mg/kg) attenuated the therapeutic effects of arbutin, whereas the intraperitoneal injection of forskolin (3 mg/kg) enhanced arbutin-mediated improvements. These findings suggest that arbutin can improve the performance of PD mouse models by inhibiting the function of the A2AR and enhancing the effects of cyclic adenosine monophosphate. This study was approved by the Institutional Animal Ethics Committee (1616/PO/Re/S/12/CPCSEA) on November 17, 2019 (approval No. IAEC/2019/010).