Dexmedetomidine attenuates lipopolysaccharide-induced renal cell fibrotic phenotypic changes by inhibiting necroinflammation via activating α2-adrenoceptor: A combined randomised animal and in vitro study.

BACKGROUND: Acute kidney injury (AKI) was reported to be one of the initiators of chronic kidney disease (CKD) development. Necroinflammation may contribute to the progression from AKI to CKD. Dexmedetomidine (Dex), a highly selective α2-adrenoreceptor (AR) agonist, has cytoprotective and "anti-" inflammation effects. This study was designed to investigate the anti-fibrotic properties of Dex in sepsis models. METHODS: C57BL/6 mice were randomly treated with an i.p. injection of lipopolysaccharides (LPS) (10 mg/kg) alone, LPS with Dex (25 µg/kg), or LPS, Dex and Atipamezole (Atip, an α2-adrenoreceptor antagonist) (500 µg/kg) (n=5/group). Human proximal tubular epithelial cells (HK2) were also cultured and then exposed to LPS (1 µg/ml) alone, LPS and Dex (1 µM), transforming growth factor-beta 1 (TGF-ß1) (5 ng/ml) alone, TGF-ß1 and Dex, with or without Atip (100 µM) in culture media. Epithelial-mesenchymal transition (EMT), cell necrosis, necroptosis and pyroptosis, and c-Jun N-terminal kinase (JNK) phosphorylation were then determined. RESULTS: Dex treatment significantly alleviated LPS-induced AKI, myofibroblast activation, NLRP3 inflammasome activation, and necroptosis in mice. Atip counteracted its protective effects. Dex attenuated LPS or TGF-ß1 induced EMT and also prevented necrosis, necroptosis, and pyroptosis in response to LPS stimulation in the HK2 cells. The anti-EMT effects of Dex were associated with JNK phosphorylation. CONCLUSIONS: Dex reduced EMT following LPS stimulation whilst simultaneously inhibiting pyroptosis and necroptosis via α2-AR activation in the renal tubular cells. The "anti-fibrotic" and cytoprotective properties and its clinical use of Dex need to be further studied.

Role of noradrenergic and dopaminergic systems in the antinociceptive effect of N-(3-(phenylselanyl)prop-2-yn-1-yl)benzamide in mice.

Pain has a negative impact on public health, reducing quality of life. Unfortunately, current treatments are not fully effective and have adverse effects. Therefore, there is a need to develop new analgesic compounds. Due to promising results regarding the antinociceptive effect of N-(3-(phenylselanyl)prop-2-in-1-yl)benzamide (SePB), this study aimed to evaluate the participation of the dopaminergic and noradrenergic systems in this effect in mice, as well as its toxicity. To this, the antagonists sulpiride (D2/D3 receptor antagonist, 5 mg/kg), SCH-23390 (D1 receptor antagonist, 0.05 mg/kg), prazosin (α1 adrenergic receptor antagonist, 0.15 mg/kg), yohimbine (α2-adrenergic receptors, 0.15 mg/kg) and propranolol (non-selective ß-adrenergic antagonist, 10 mg/kg) were administered intraperitoneally to mice 15 min before SePB (10 mg/kg, intragastrically), except for propranolol (20 min). After 26 min of SePB administration, the open field test was performed for 4 min to assess locomotor activity, followed by the tail immersion test to measure the nociceptive response. For the toxicity test, animals received a high dose of 300 mg/kg of SePB. SePB showed an increase in the latency for nociceptive response in the tail immersion test, and this effect was prevented by SCH-23390, yohimbine and propranolol, indicating the involvement of D1, α2 and ß-adrenergic receptors in the antinociceptive mechanism of the SePB effect. No changes were observed in the open field test, and the toxicity assessment suggested that SePB has low potential to induce toxicity. These findings contribute to understanding SePB's mechanism of action, with a focus on the development of new alternatives for pain treatment.

Novel Scaffold Agonists of the α2A Adrenergic Receptor Identified via Ensemble-Based Strategy.

The α2A adrenergic receptor (α2A-AR) serves as a critical molecular target for sedatives and analgesics. However, α2A-AR ligands with an imidazole ring also interact with an imidazoline receptor as well as other proteins and lead to undesirable effects, motivating us to develop more novel scaffold α2A-AR ligands. For this purpose, we employed an ensemble-based ligand discovery strategy, integrating long-term molecular dynamics (MD) simulations and virtual screening, to identify new potential α2A-AR agonists with novel scaffold. Our results showed that compounds SY-15 and SY-17 exhibited significant biological effects in the preliminary evaluation of protein kinase A (PKA) redistribution assays. They also reduced levels of intracellular cyclic adenosine monophosphate (cAMP) in a dose-dependent manner. Upon treatment of the cells with 100 µM concentrations of SY-15 and SY-17, there was a respective decrease in the intracellular cAMP levels by 63.43% and 53.83%. Subsequent computational analysis was conducted to elucidate the binding interactions of SY-15 and SY-17 with the α2A-AR. The binding free energies of SY-15 and SY-17 calculated by MD simulations were -45.93 and -71.97 kcal/mol. MD simulations also revealed that both compounds act as bitopic agonists, occupying the orthosteric site and a novel exosite of the receptor simultaneously. Our findings of integrative computational and experimental approaches could offer the potential to enhance ligand affinity and selectivity through dual-site occupancy and provide a novel direction for the rational design of sedatives and analgesics.

Limited bedding and nesting increases ethanol drinking in female rats.

Prenatal opioid exposure (POE) and postnatal adverse experiences are early life adversities (ELA) that often co-occur and increase problematic alcohol (EtOH) drinking during adolescence. We investigated the relationship between POE, postnatal adversity, and adolescent EtOH drinking in rats. We also sought to determine whether ELAs affect alpha-adrenoceptor density in the brain because the noradrenergic system is involved in problematic alcohol drinking and its treatment. We hypothesized that the combination of POE and postnatal adversity will increase alcohol drinking in rats compared to rats with exposure to either adversity alone or to control. We also predicted that POE and postnatal adversity would increase α1-adrenoceptor density and decrease α2-adrenoceptor density in brain to confer a stress-responsive phenotype. Pregnant rats received morphine (15 mg/kg/day) or saline via subcutaneous minipumps from gestational day 9 until birth. Limited bedding and nesting (LBN) procedures were introduced from postnatal day (PD) 3-11 to mimic early life adversity-scarcity. Offspring rats (PD 31-33) were given opportunities to drink EtOH (20 %, v/v) using intermittent-access, two-bottle choice (with water) procedures. Rats given access to EtOH were assigned into sub-groups that were injected with either yohimbine (1 mg/kg, ip) or vehicle (2 % DMSO, ip) 30 min prior to each EtOH access session to determine the effects of α2-adrenoceptor inhibition on alcohol drinking. We harvested cortices, brainstems, and hypothalami from EtOH-naïve littermates on either PD 30 or PD 70 and conducted radioligand receptor binding assays to quantify α1- and α2-adrenoceptor densities. Contrary to our hypothesis, only LBN alone increased EtOH intake in female adolescent rats compared to female rats with POE. Neither POE nor LBN affected α1- or α2-adrenoceptor densities in the cortex, brainstem, or hypothalamus of early- or late-aged adolescent rats. These results suggest a complex interaction between ELA type and sex on alcohol drinking.

LC-derived excitatory synaptic transmission to dorsal raphe serotonin neurons is inhibited by activation of alpha2-adrenergic receptors.

In the central nervous system, noradrenaline transmission controls the degree to which we are awake, alert, and attentive. Aberrant noradrenaline transmission is associated with pathological forms of hyper- and hypo-arousal that present in numerous neuropsychiatric disorders often associated with dysfunction in serotonin transmission. In vivo, noradrenaline regulates the release of serotonin because noradrenergic input drives the serotonin neurons to fire action potentials via activation of excitatory α1-adrenergic receptors (α1-AR). Despite the critical influence of noradrenaline on the activity of dorsal raphe serotonin neurons, the source of noradrenergic afferents has not been resolved and the presynaptic mechanisms that regulate noradrenaline-dependent synaptic transmission have not been described. Using an acute brain slice preparation from male and female mice and electrophysiological recordings from dorsal raphe serotonin neurons, we found that selective optogenetic activation of locus coeruleus terminals in the dorsal raphe was sufficient to produce an α1-AR-mediated excitatory postsynaptic current (α1-AR-EPSC). Activation of inhibitory α2-adrenergic receptors (α2-AR) with UK-14,304 eliminated the α1-AR-EPSC via presynaptic inhibition of noradrenaline release, likely via inhibition of voltage-gated calcium channels. In a subset of serotonin neurons, activation of postsynaptic α2-AR produced an outward current through activation of GIRK potassium conductance. Further, in vivo activation of α2-AR by systemic administration of clonidine reduced the expression of c-fos in the dorsal raphe serotonin neurons, indicating reduced neural activity. Thus, α2-AR are critical regulators of serotonin neuron excitability.

Nicotine Diminishes Alpha2-Adrenergic Receptor-Dependent Protection Against Oxidative Stress in H9c2 Cardiomyocytes.

Introduction: Nicotine is a major component of cigarette smoke with various detrimental cardiovascular effects, including increased oxidative stress in the heart. Agonism of α2-adrenergic receptors (ARs), such as with dexmedetomidine, has been documented to exert cardioprotective effects against oxidative stress and related apoptosis and necroptosis. α2-ARs are membrane-residing G protein-coupled receptors (GPCRs) that primarily activate Gi/o proteins. They are also subjected to GPCR-kinase (GRK)-2-dependent desensitization, which entails phosphorylation of the agonist-activated receptor by GRK2 to induce its decoupling from G proteins, thus terminating α2AR-mediated G protein signaling. Objective: In the present study, we sought to examine the effects of nicotine on α2AR signaling and effects in H9c2 cardiomyocytes exposed to H2O2 to induce oxidative cellular damage. Methods and Results: As expected, treatment of H9c2 cardiomyocytes with H2O2 significantly decreased cell viability and increased oxidative stress, as assessed by reactive oxygen species (ROS)-associated fluorescence levels (DCF assay) and superoxide dismutase activity. Both H2O2 effects were partly rescued by α2AR activation with brimonidine in control cardiomyocytes but not in cells pretreated with nicotine for 24 hours, in which brimonidine was unable to reduce H2O2-induced cell death and oxidative stress. This was due to severe α2AR desensitization, manifested as very low Gi protein activation by brimonidine, and accompanied by GRK2 upregulation in nicotine-treated cardiomyocytes. Finally, pharmacological inhibition of adenylyl cyclase (AC) blocked H2O2-dependent oxidative damage in nicotine-pretreated H9c2 cardiomyocytes, indicating that α2AR activation protects against oxidative injury via its classic coupling to Gai-mediated AC inhibition. Discussion/Conclusions: Nicotine can negate the cardioprotective effects of α2AR agonists against oxidative injury, which may have important implications for patients treated with this class of drugs that are chronic tobacco smokers.

Alpha-2a adrenergic receptor activation in genetic absence epilepsy: An absence status model?

OBJECTIVE: The objective of the study was to propose a candidate animal model of absence status epilepticus induced by specific alpha-2a adrenergic receptor (α2AR) activation. We also aim to investigate the responsiveness of this model to classical anti-status or anti-absence medications. METHODS: An α2AR agonist, dexmedetomidine (DEX), was injected intracerebroventricularly into adult rats with genetic absence epilepsy, and their electroencephalography (EEG) was recorded. The total duration, number, and mean duration of each spike-and-wave discharges (SWDs) were evaluated. The blocks of absence status events were classified as the initial and second sets of absence statuses. Ethosuximide (ETX) was administered as a pretreatment to another group of rats and later injected with 2.5 µg DEX. In addition, ETX, valproic acid (VPA), diazepam (DIAZ), and atipamezole (ATI) were administered after induced status-like events following DEX administration. Power spectral characteristics and coherence analysis were performed on the EEG to assess the absence status events and sleep. RESULTS: The 2.5 µg dose of DEX increased the total SWD duration and induced continuous SWDs up to 26 min. Following the initial absence status event, sleep was induced; then, the second period of absence status-like activities were initiated. ETX pretreatment blocked the occurrence of absence status-like activities. Power spectral density analyses revealed that DEX-induced post-sleep activities had higher power in delta frequency band (1-4 Hz) and attenuated power of 7 Hz harmonics (14 and 21 Hz) than the pre-injection seizure. The mean duration of SWDs were decreased in all the groups, but occasional prolonged activities were seen in ETX or VPA-injected rats but not with DIAZ or ATI. SIGNIFICANCE: This study presents an absence status epilepticus animal model that is activated by α2AR activation to investigate the pathophysiological role of absence status. Unlike other agents ATI switched off the second set of absence statuses to normal SWDs, without sedation or lethargy, can show it may preferentially block absence status-like activity. THE PLAIN LANGUAGE SUMMARY: This study proposes a rat model for prolonged seizures, resembling absence status epilepticus. Activating the brain's alpha-2a adrenergic receptor with dexmedetomidine induced seizures lasting up to 26 minutes. Ethosuximide pretreatment and post-treatment with valproic acid, diazepam, and atipamezole decreased induced seizures. The findings suggest this model is valuable for studying absence status epilepticus. In addition, atipamezole normalized abnormal seizures without sedation, hinting at its potential for targeted treatment and further research.

Inhibiting Intracellular α2C-Adrenoceptor Surface Translocation Using Decoy Peptides: Identification of an Essential Role of the C-Terminus in Receptor Trafficking.

The G protein-coupled α2-adrenoceptor subtype C (abbreviated α2C-AR) has been implicated in peripheral vascular conditions and diseases such as cold feet-hands, Raynaud's phenomenon, and scleroderma, contributing to morbidity and mortality. Microvascular α2C-adrenoceptors are expressed in specialized smooth muscle cells and mediate constriction under physiological conditions and the occlusion of blood supply involving vasospastic episodes and tissue damage under pathological conditions. A crucial step for receptor biological activity is the cell surface trafficking of intracellular receptors, triggered by cAMP-Epac-Rap1A GTPase signaling, which involves protein-protein association with the actin-binding protein filamin-2, mediated by critical amino acid residues in the last 14 amino acids of the receptor carboxyl (C)-terminus. This study assessed the role of the C-terminus in Rap1A GTPase coupled receptor trafficking by domain-swapping studies using recombinant tagged receptors in transient co-transfections and compared with wild-type receptors using immunofluorescence microscopy. We further tested the biological relevance of the α2C-AR C-terminus, when introduced as competitor peptides, to selectively inhibit intracellular α2C-AR surface translocation in transfected as well as in microvascular smooth muscle cells expressing endogenous receptors. These studies contribute to establishing proof of principle to target intracellular α2C-adrenoceptors to reduce biological activity, which in clinical conditions can be a target for therapy.

Identification of potential agonist-like molecules for α2-adrenergic receptor by multi-layer virtual screening to combat sinusitis.

Sinusitis is one of the most common respiratory inflammatory conditions and a significant health issue that affects millions of people worldwide with a global prevalence of 10-15%. The side effects of available drug regimens of sinus infection demand the urgent development of new drug candidates to combat sinusitis. With the aim of identifying new drug-like candidates to control sinus, we have conducted multifold comprehensive screening of drug-like molecules targeting α2-adrenergic receptor (α2-AR), which serve as the primary drug target in sinusitis. By structure-based virtual screening of in-house compound's database, ten molecules (CP1-CP10) with agonistic effects for α2-AR were selected, and their binding mechanism with critical residues of α2-AR and their physicochemical properties were studied. Moreover, the process of receptor activation by these compounds and the conformational changes in α2-AR caused by these molecules, were further explored by molecular dynamic simulation. The MM-PBSA estimated free energies of compounds are higher than that of reference agonist (ΔGTOTAL = -39.0 kcal/mol). Among all, CP2-CP3, CP7-CP8 and CP6 have the highest binding free energies of -78.9 kcal/mol, -77.3 kcal/mol, -75.60 kcal/mol, -64.8 kcal/mol, and -61.6 kcal/mol, respectively. While CP4 (-55.0 kcal/mol), CP5 (-49.2 kcal/mol), CP9 (-54.8 ± 0.07 kcal/mol), CP10 (-56.7 ± 0.10 kcal/mol) and CP1 (-46.0 ± 0.08 kcal/mol) also exhibited significant binding free energies. These energetically favorable binding energies indicate strong binding affinity of our compounds for α2-AR as compared to known partial agonist. Therefore, these molecules can serve as excellent drug-like candidates for sinusitis.

Measuring neuron-regulated immune cell physiology via the alpha-2 adrenergic receptor in an ex vivo murine spleen model.

The communication between the nervous and immune systems plays a crucial role in regulating immune cell function and inflammatory responses. Sympathetic neurons, which innervate the spleen, have been implicated in modulating immune cell activity. The neurotransmitter norepinephrine (NE), released by sympathetic neurons, influences immune cell responses by binding to adrenergic receptors on their surface. The alpha-2 adrenergic receptor (α2AR), expressed predominantly on sympathetic neurons, has received attention due to its autoreceptor function and ability to modulate NE release. In this study, we used fast-scan cyclic voltammetry (FSCV) to provide the first subsecond measurements of NE released in the white pulp region of the spleen and validated it with yohimbine, a known antagonist of α2AR. For further application of FSCV in neuroimmunology, we investigated the extent to which subsecond NE from sympathetic neurons is important for immune cell physiology and cytokine production, focusing on tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), and interleukin-6 (IL-6). Our findings provide insights into the regulatory mechanisms underlying sympathetic-immune interactions and show the significance of using FSCV, a traditional neurochemistry technique, to study these neuroimmune mechanisms.

Sympathetic activity regulates epithelial proliferation and wound healing via adrenergic receptor α2A.

Innervation of the intestinal mucosa by the sympathetic nervous system is well described but the effects of adrenergic receptor stimulation on the intestinal epithelium remain equivocal. We therefore investigated the effect of sympathetic neuronal activation on intestinal cells in mouse models and organoid cultures, to identify the molecular routes involved. Using publicly available single-cell RNA sequencing datasets we show that the α2A isoform is the most abundant adrenergic receptor in small intestinal epithelial cells. Stimulation of this receptor with norepinephrine or a synthetic specific α2A receptor agonist promotes epithelial proliferation and stem cell function, while reducing differentiation in vivo and in intestinal organoids. In an anastomotic healing mouse model, adrenergic receptor α2A stimulation resulted in improved anastomotic healing, while surgical sympathectomy augmented anastomotic leak. Furthermore, stimulation of this receptor led to profound changes in the microbial composition, likely because of altered epithelial antimicrobial peptide secretion. Thus, we established that adrenergic receptor α2A is the molecular delegate of intestinal epithelial sympathetic activity controlling epithelial proliferation, differentiation, and host defense. Therefore, this receptor could serve as a newly identified molecular target to improve mucosal healing in intestinal inflammation and wounding.

Dexmedetomidine modulates neuronal activity of horizontal limbs of diagonal band via α2 adrenergic receptor in mice.

BACKGROUND AND OBJECTIVES: Dexmedetomidine (DEX) is widely used in clinical sedation which has little effect on cardiopulmonary inhibition, however the mechanism remains to be elucidated. The basal forebrain (BF) is a key nucleus that controls sleep-wake cycle. The horizontal limbs of diagonal bundle (HDB) is one subregions of the BF. The purpose of this study was to examine whether the possible mechanism of DEX is through the α2 adrenergic receptor of BF (HDB). METHODS: In this study, we investigated the effects of DEX on the BF (HDB) by using whole cell patch clamp recordings. The threshold stimulus intensity, the inter-spike-intervals (ISIs) and the frequency of action potential firing in the BF (HDB) neurons were recorded by application of DEX (2 µM) and co-application of a α2 adrenergic receptor antagonist phentolamine (PHEN) (10 µM). RESULTS: DEX (2 µM) increased the threshold stimulus intensity, inhibited the frequency of action potential firing and enlarged the inter-spike-interval (ISI) in the BF (HDB) neurons. These effects were reversed by co-application of PHEN (10 µM). CONCLUSION: Taken together, our findings revealed DEX decreased the discharge activity of BF (HDB) neuron via α2 adrenergic receptors.

ADRA2A and IRX1 are putative risk genes for Raynaud's phenomenon.

Raynaud's phenomenon (RP) is a common vasospastic disorder that causes severe pain and ulcers, but despite its high reported heritability, no causal genes have been robustly identified. We conducted a genome-wide association study including 5,147 RP cases and 439,294 controls, based on diagnoses from electronic health records, and identified three unreported genomic regions associated with the risk of RP (p < 5 × 10-8). We prioritized ADRA2A (rs7090046, odds ratio (OR) per allele: 1.26; 95%-CI: 1.20-1.31; p < 9.6 × 10-27) and IRX1 (rs12653958, OR: 1.17; 95%-CI: 1.12-1.22, p < 4.8 × 10-13) as candidate causal genes through integration of gene expression in disease relevant tissues. We further identified a likely causal detrimental effect of low fasting glucose levels on RP risk (rG = -0.21; p-value = 2.3 × 10-3), and systematically highlighted drug repurposing opportunities, like the antidepressant mirtazapine. Our results provide the first robust evidence for a strong genetic contribution to RP and highlight a so far underrated role of α2A-adrenoreceptor signalling, encoded at ADRA2A, as a possible mechanism for hypersensitivity to catecholamine-induced vasospasms.

Expression and localization of α2A-adrenergic receptor in the rat post-natal developing cochlea.

Lots of adrenergic receptors (ARs) are widely present across the auditory pathways and are positioned to affect auditory and vestibular functions. However, noradrenergic regulation in the cochlea has not been well characterized. In this study, a rat model of noise-induced hearing loss was developed to investigate the expression of α2A-adrenergic receptor (AR) after acoustic trauma, then, we investigated the expression of α2A-AR in the developing rat cochlea using immunofluorescence, qRT-PCR, and Western blotting. We found that the expression of α2A-AR significantly increased in rats exposed to noise compared with controls. Immunofluorescence analysis demonstrated that α2A-AR is localized on hair cells (HCs), spiral ganglion neurons (SGNs), and the stria vascularis (SV) in the postnatal developing cochlea from post-natal day (P) 0 to P28. Furthermore, we observed α2A-AR mRNA reached a maximum level at P14 and P28 when compared with P0, while no significant differences in α2A-AR protein levels at the various stages when compared with P0. This study provides direct evidence for the expression of α2A-AR in HCs, SGNs, and the SV of the cochlea, indicating that norepinephrine might play a vital role in hearing function within the cochlea through α2A-AR.

ADRA2B and HTR1A: An Updated Study of the Biogenic Amine Receptors Reveals Novel Conserved Motifs Which Play Key Role in Mental Disorders.

Mental disorders are strongly connected with several psychiatric conditions including depression, bipolar disorder, schizophrenia, eating disorder, and suicides. There are many biological conditions and pathways that define these complicated illnesses. For example, eating disorders are complex mental health conditions that require the intervention of geneticists, psychiatrists, and medical experts in order to alleviate their symptoms. A patient with suicidal ideation should first be identified and consequently monitored by a similar team of specialists. Both genetics and epigenetics can shed light on eating disorders and suicides as they are found in the main core of such investigations. In the present study, an analysis has been performed on two specific members of the GPCR family toward drawing conclusions regarding their functionality and implementation in mental disorders. Specifically, evolutionary and structural studies on the adrenoceptor alpha 2b (ADRA2B) and the 5-hydroxytryptamine receptor 1A (HTR1A) have been carried out. Both receptors are classified in the biogenic amine receptors sub-cluster of the GPCRs and have been connected in many studies with mental diseases and malnutrition conditions. The major goal of this study is the investigation of conserved motifs among biogenic amine receptors that play an important role in this family signaling pathway, through an updated evolutionary analysis and the correlation of this information with the structural features of the HTR1A and ADRA2B. Furthermore, the structural comparison of ADRA2B, HTR1A, and other members of GPCRs related to mental disorders is performed.

Tumour immune rejection triggered by activation of α2-adrenergic receptors.

Immunotherapy based on immunecheckpoint blockade (ICB) using antibodies induces rejection of tumours and brings clinical benefit in patients with various cancer types1. However, tumours often resist immune rejection. Ongoing efforts trying to increase tumour response rates are based on combinations of ICB with compounds that aim to reduce immunosuppression in the tumour microenvironment but usually have little effect when used as monotherapies2,3. Here we show that agonists of α2-adrenergic receptors (α2-AR) have very strong anti-tumour activity when used as monotherapies in multiple immunocompetent tumour models, including ICB-resistant models, but not in immunodeficient models. We also observed marked effects in human tumour xenografts implanted in mice reconstituted with human lymphocytes. The anti-tumour effects of α2-AR agonists were reverted by α2-AR antagonists, and were absent in Adra2a-knockout (encoding α2a-AR) mice, demonstrating on-target action exerted on host cells, not tumour cells. Tumours from treated mice contained increased infiltrating T lymphocytes and reduced myeloid suppressor cells, which were more apoptotic. Single-cell RNA-sequencing analysis revealed upregulation of innate and adaptive immune response pathways in macrophages and T cells. To exert their anti-tumour effects, α2-AR agonists required CD4+ T lymphocytes, CD8+ T lymphocytes and macrophages. Reconstitution studies in Adra2a-knockout mice indicated that the agonists acted directly on macrophages, increasing their ability to stimulate T lymphocytes. Our results indicate that α2-AR agonists, some of which are available clinically, could substantially improve the clinical efficacy of cancer immunotherapy.

Distribution of α2-Adrenergic Receptors in the Living Human Brain Using [11C]yohimbine PET.

The neurofunctional basis of the noradrenergic (NA) system and its associated disorders is still very incomplete because in vivo imaging tools in humans have been missing up to now. Here, for the first time, we use [11C]yohimbine in a large sample of subjects (46 healthy volunteers, 23 females, 23 males; aged 20-50) to perform direct quantification of regional alpha 2 adrenergic receptors' (α2-ARs) availability in the living human brain. The global map shows the highest [11C]yohimbine binding in the hippocampus, the occipital lobe, the cingulate gyrus, and the frontal lobe. Moderate binding was found in the parietal lobe, thalamus, parahippocampus, insula, and temporal lobe. Low levels of binding were found in the basal ganglia, the amygdala, the cerebellum, and the raphe nucleus. Parcellation of the brain into anatomical subregions revealed important variations in [11C]yohimbine binding within most structures. Strong heterogeneity was found in the occipital lobe, the frontal lobe, and the basal ganglia, with substantial gender effects. Mapping the distribution of α2-ARs in the living human brain may prove useful not only for understanding the role of the NA system in many brain functions, but also for understanding neurodegenerative diseases in which altered NA transmission with specific loss of α2-ARs is suspected.

Conformationally Selective 2-Aminotetralin Ligands Targeting the alpha2A- and alpha2C-Adrenergic Receptors.

Many important physiological processes are mediated by alpha2A- and alpha2C-adrenergic receptors (α2Rs), a subtype of class A G protein-coupled receptors (GPCRs). However, α2R signaling is poorly understood, and there are few approved medications targeting these receptors. Drug discovery aimed at α2Rs is complicated by the high degree of binding pocket homology between α2AR and α2CR, which confounds ligand-mediated selective activation or inactivation of signaling associated with a particular subtype. Meanwhile, α2R signaling is complex and it is reported that activating α2AR is beneficial in many clinical contexts, while activating α2CR signaling may be detrimental to these positive effects. Here, we report on a novel 5-substituted-2-aminotetralin (5-SAT) chemotype that, depending on substitution, has diverse pharmacological activities at α2Rs. Certain lead 5-SAT analogues act as partial agonists at α2ARs, while functioning as inverse agonists at α2CRs, a novel pharmacological profile. Leads demonstrate high potency (e.g., EC50 < 2 nM) at the α2AR and α2CRs regarding Gαi-mediated inhibition of adenylyl cyclase and production of cyclic adenosine monophosphate (cAMP). To help understand the molecular basis of 5-SAT α2R multifaceted functional activity, α2AR and α2CR molecular models were built from the crystal structures and 1 µs molecular dynamics (MD) simulations and molecular docking experiments were performed for a lead 5-SAT with α2AR agonist and α2CR inverse agonist activity, i.e., (2S)-5-(2'-fluorophenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine (FPT), in comparison to the FDA-approved (for opioid withdrawal symptoms) α2AR/α2CR agonist lofexidine. Results reveal several interactions between FPT and α2AR and α2CR amino acids that may impact the functional activity. The computational data in conjunction with experimental in vitro affinity and function results provide information to understand ligand stabilization of functionally distinct GPCR conformations regarding α2AR and α2CRs.

Upregulations of α1 adrenergic receptors and noradrenaline synthases in the medial prefrontal cortex are associated with emotional and cognitive dysregulation induced by post-weaning social isolation in male rats.

Early-life social isolation induces emotional and cognitive dysregulation, such as increased aggression and anxiety, and decreases neuron excitability in the medial prefrontal cortex (mPFC). The noradrenergic system in the mPFC regulates emotion and cognitive function via α1 or α2A adrenergic receptors, depending on noradrenaline levels. However, social isolation-induced changes in the mPFC noradrenergic system have not been reported. Here, male Wistar rats received post-weaning social isolation for nine consecutive weeks and were administered behavioral tests (novel object recognition, elevated plus maze, aggression, and forced swimming, sequentially). Protein expression levels in the mPFC noradrenergic system (α1 and α2A adrenergic receptors, tyrosine hydroxylase, and dopamine-ß-hydroxylase used as indices of noradrenaline synthesis and release) were examined through western blotting. Social isolation caused cognitive dysfunction, anxiety-like behavior, and aggression, but not behavioral despair. Socially-isolated rats exhibited increased protein levels of the α1 adrenergic receptor, tyrosine hydroxylase, and dopamine-ß-hydroxylase in the mPFC; there was no significant difference between the groups in the α2A adrenergic receptor expression levels. Preferential activation of the α1 adrenergic receptor caused by high noradrenaline concentration in the mPFC may be involved in social isolation-induced emotional and cognitive regulation impairments. Targeting the α1 adrenergic receptor signaling pathway is a potential therapeutic strategy for psychiatric disorders with symptomatic features such as emotional and cognitive dysregulation.

Noradrenaline released from locus coeruleus axons contracts cerebral capillary pericytes via α2 adrenergic receptors.

Noradrenaline (NA) release from locus coeruleus axons generates vascular contractile tone in arteriolar smooth muscle and contractile capillary pericytes. This tone allows neuronal activity to evoke vasodilation that increases local cerebral blood flow (CBF). Much of the vascular resistance within the brain is located in capillaries and locus coeruleus axons have NA release sites closer to pericytes than to arterioles. In acute brain slices, NA contracted pericytes but did not raise the pericyte cytoplasmic Ca2+ concentration, while the α1 agonist phenylephrine did not evoke contraction. Blocking α2 adrenergic receptors (α2Rs, which induce contraction by inhibiting cAMP production), greatly reduced the NA-evoked pericyte contraction, whereas stimulating α2Rs using xylazine (a sedative) or clonidine (an anti-hypertensive drug) evoked pericyte contraction. Noradrenaline-evoked pericyte contraction and capillary constriction are thus mediated via α2Rs. Consequently, α2Rs may not only modulate CBF in health and pathological conditions, but also contribute to CBF changes evoked by α2R ligands administered in research, veterinary and clinical settings.