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.

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.

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.

Noradrenaline increases intracellular Ca2+ concentration in epithelial cells via α2-adrenoceptors in isolated mouse ileal crypts.

The stimulation of α2-adrenoceptors caused a transient increase of intracellular calcium concentration ([Ca2+]i) monitored by ratiometry using Fura-2 in epithelial cells including enterochromaffin cells in isolated mouse ileal crypts, while stimulation of α1-and ß-adrenoceptors had no effect. The effect of noradrenaline was suppressed by α2-adrenoceptor antagonists, but not by α1-and ß-adrenoceptor antagonists, and partially suppressed by Ni2+ and nicardipine, but not by ω-conotoxin and ω-agatoxin. These results suggest that noradrenaline causes an increase of [Ca2+]i by the influx of extracellular Ca2+ through certain Ca2+ channels via α2-adrenoceptors in epithelial cells of mouse ileal crypts.

Effect of dexmedetomidine on cardiorespiratory regulation in spontaneously breathing adult rats.

PURPOSE: We examined the cardiorespiratory effect of dexmedetomidine, an α2- adrenoceptor/imidazoline 1 (I1) receptor agonist, in spontaneously breathing adult rats. METHODS: Male rats (226-301 g, n = 49) under isoflurane anesthesia had their tail vein cannulated for drug administration and their tail artery cannulated for analysis of mean arterial pressure (MAP), pulse rate (PR), and arterial blood gases (PaO2, PaCO2, pH). After recovery, one set of rats received normal saline for control recording and was then divided into three experimental groups, two receiving dexmedetomidine (5 or 50 µg·kg-1) and one receiving normal saline (n = 7 per group). Another set of rats was divided into four groups receiving dexmedetomidine (50 µg·kg-1) followed 5 min later by 0.5 or 1 mg∙kg-1 atipamezole (selective α2-adrenoceptor antagonist) or efaroxan (α2-adrenoceptor/I1 receptor antagonist) (n = 6 or 8 per group). Recordings were performed 15 min after normal saline or dexmedetomidine administration. RESULTS: Compared with normal saline, dexmedetomidine (5 and 50 µg·kg-1) decreased respiratory frequency (fR, p = 0.04 and < 0.01, respectively), PR (both p < 0.01), and PaO2 (p = 0.04 and < 0.01), and increased tidal volume (both p = 0.049). Dexmedetomidine at 5 µg·kg-1 did not significantly change minute ventilation (V'E) (p = 0.87) or MAP (p = 0.24), whereas dexmedetomidine at 50 µg·kg-1 significantly decreased V'E (p = 0.03) and increased MAP (p < 0.01). Only dexmedetomidine at 50 µg·kg-1 increased PaCO2 (p < 0.01). Dexmedetomidine (5 and 50 µg·kg-1) significantly increased blood glucose (p < 0.01), and dexmedetomidine at 50 µg·kg-1 increased hemoglobin (p = 0.04). Supplemental atipamezole or efaroxan administration similarly prevented the 50 µg·kg-1 dexmedetomidine-related cardiorespiratory changes. PRINCIPAL CONCLUSION: These results suggest that dexmedetomidine-related hypoventilation and hypertension are observed simultaneously and occur predominantly through activation of α2-adrenoceptors, but not I1 receptors, in spontaneously breathing adult rats.

Suppression of P2X3 receptor-mediated currents by the activation of α2A -adrenergic receptors in rat dorsal root ganglion neurons.

AIMS: The α2 -adrenergic receptor (α2 -AR) agonists have been shown to be effective in the treatment of various pain. For example, dexmedetomidine (DEX), a selective α2A -AR agonist, can be used for peripheral analgesia. However, it is not yet fully elucidated for the precise molecular mechanisms. P2X3 receptor is a major receptor processing nociceptive information in primary sensory neurons. Herein, we show that a functional interaction of α2A -ARs and P2X3 receptors in dorsal root ganglia (DRG) neurons could contribute to peripheral analgesia of DEX. METHODS: Electrophysiological recordings were carried out on rat DRG neurons, and nociceptive behavior was quantified in rats. RESULTS: The activation of α2A -ARs by DEX suppressed P2X3 receptor-mediated and α,ß-methylene-ATP (α,ß-meATP)-evoked inward currents in a concentration-dependent and voltage-independent manner. Pre-application of DEX shifted the α,ß-meATP concentration-response curve downwards, with a decrease of 50.43 ± 4.75% in the maximal current response of P2X3 receptors to α,ß-meATP in the presence of DEX. Suppression of α,ß-meATP-evoked currents by DEX was blocked by the α2A -AR antagonist BRL44408 and prevented by intracellular application of the Gi/o protein inhibitor pertussis toxin, the adenylate cyclase activator forskolin, and the cAMP analog 8-Br-cAMP. DEX also suppressed α,ß-meATP-evoked action potentials through α2A -ARs in rat DRG neurons. Finally, the activation of peripheral α2A -ARs by DEX had an analgesic effect on the α,ß-meATP-induced nociception. CONCLUSIONS: These results suggested that activation of α2A -ARs by DEX suppressed P2X3 receptor-mediated electrophysiological and behavioral activity via a Gi/o proteins and cAMP signaling pathway, which was a novel potential mechanism underlying analgesia of peripheral α2A -AR agonists.

Effect of Clonidine Hydrochloride on Isolated Newborn Rat Heart.

The concentration dependenies of the chronotropic response and changes in blood supply to the isolated heart of 7-day-old newborn rats induced by application of α2-adrenergic receptor agonist clonidine hydrochloride in concentrations of 10-9-10-6 M were revealed. The minimum concentration of α2-adrenergic receptor agonist caused tachycardia, while higher concentrations led to bradycardia. The maximum effect manifesting in a decrease in coronary flow was recorded at the minimum concentration of the agonist, while the highest concentration had no effect on the coronary flow. When comparing these results with those obtained in control adult rats, we found that the most pronounced differences in the chronotropic effects were observed after addition of the minimum concentration of the α2-adrenergic receptor agonist: bradycardia in adult rats and tachycardia in newborns. The maximum differences in coronary flow parameters were observed after addition of α2-adrenergic receptor agonist in the maximum concentration that induced a two-phase response in adult rats and had no effect on the blood supply in newborns.

Interaction between α1B - and other α1 - and α2 -adrenoceptors in producing contractions of mouse spleen.

We have investigated the interaction of α1 - and α2 -adrenoceptor subtypes in producing isometric contractions to NA in mouse whole spleen. The α1 -adrenoceptor antagonist prazosin (10-8  M) or the α2 -adrenoceptor antagonist yohimbine (10-6  M) alone produced only small shifts in NA potency in wild type (WT) mice, but the combination produced a large shift in NA potency. In spleen from α1A/D -KO mice, the effects of prazosin and the combination of prazosin and yohimbine were similar to their effects in WT mice. Hence, in α1A/D -KO mice, in which the only α1 -adrenoceptor present is the α1B -adrenoceptor, prazosin still antagonized contractions to NA. The α1A -adrenoceptor antagonist RS100329 (3 × 10-9 M) produced significant shifts in the effects of higher concentrations of NA (EC50 and EC75 levels) and the α1D -adrenoceptor antagonist BMY7378 (3 × 10-8 M) produced significant shifts in the effects of lower concentrations of NA (EC25 and EC50 levels). The effects of BMY7378 and RS00329 demonstrate α1D -adrenoceptor and α1A -adrenoceptor components and suggest that the α1B -adrenoceptor interacts with an α1D -adrenoceptor, and to a lesser extent an α1A -adrenoceptor, at low, and an α1A -adrenoceptor at high, NA concentrations. This study demonstrates the complex interaction between α1 - and α2 -adrenoceptor subtypes in producing contractions of mouse spleen and may have general implications for α-adrenoceptor mediated control of smooth muscle.

Norepinephrine May Oppose Other Neuromodulators to Impact Alzheimer's Disease.

While much of biomedical research since the middle of the twentieth century has focused on molecular pathways inside the cell, there is increasing evidence that extracellular signaling pathways are also critically important in health and disease. The neuromodulators norepinephrine (NE), serotonin (5-hydroxytryptamine, 5HT), dopamine (DA), acetylcholine (ACH), and melatonin (MT) are extracellular signaling molecules that are distributed throughout the brain and modulate many disease processes. The effects of these five neuromodulators on Alzheimer's disease (AD) are briefly examined in this paper, and it is hypothesized that each of the five molecules has a u-shaped (or Janus-faced) dose-response curve, wherein too little or too much signaling is pathological in AD and possibly other diseases. In particular it is suggested that NE is largely functionally opposed to 5HT, ACH, MT, and possibly DA in AD. In this scenario, physiological "balance" between the noradrenergic tone and that of the other three or four modulators is most healthy. If NE is largely functionally opposed to other prominent neuromodulators in AD, this may suggest novel combinations of pharmacological agents to counteract this disease. It is also suggested that the majority of cases of AD and possibly other diseases involve an excess of noradrenergic tone and a collective deficit of the other four modulators.

Noradrenergic Signaling Disengages Feedforward Transmission in the Nucleus Accumbens Shell.

The nucleus accumbens shell (NAcSh) receives extensive monoaminergic input from multiple midbrain structures. However, little is known how norepinephrine (NE) modulates NAc circuit dynamics. Using a dynamic electrophysiological approach with optogenetics, pharmacology, and drugs acutely restricted by tethering (DART), we explored microcircuit-specific neuromodulatory mechanisms recruited by NE signaling in the NAcSh of parvalbumin (PV)-specific reporter mice. Surprisingly, NE had little direct effect on modulation of synaptic input at medium spiny projection neurons (MSNs). In contrast, we report that NE transmission selectively modulates glutamatergic synapses onto PV-expressing fast-spiking interneurons (PV-INs) by recruiting postsynaptically-localized α2-adrenergic receptors (ARs). The synaptic effects of α2-AR activity decrease PV-IN-dependent feedforward inhibition onto MSNs evoked via optogenetic stimulation of cortical afferents to the NAcSh. These findings provide insight into a new circuit motif in which NE has a privileged line of communication to tune feedforward inhibition in the NAcSh.SIGNIFICANCE STATEMENT The nucleus accumbens (NAc) directs reward-related motivational output by integrating glutamatergic input with diverse neuromodulatory input from monoamine centers. The present study reveals a synapse-specific regulatory mechanism recruited by norepinephrine (NE) signaling within parvalbumin-expressing interneuron (PV-IN) feedforward inhibitory microcircuits. PV-IN-mediated feedforward inhibition in the NAc is instrumental in coordinating NAc output by synchronizing the activity of medium spiny projection neurons (MSNs). By negatively regulating glutamatergic transmission onto PV-INs via α2-adrenergic receptors (ARs), NE diminishes feedforward inhibition onto MSNs to promote NAc output. These findings elucidate previously unknown microcircuit mechanisms recruited by the historically overlooked NE system in the NAc.

Dexmedetomidine and Clonidine Attenuate Sevoflurane-Induced Tau Phosphorylation and Cognitive Impairment in Young Mice via α-2 Adrenergic Receptor.

BACKGROUND: Anesthetic sevoflurane induces tau phosphorylation and cognitive impairment in young mice. The underlying mechanism and the targeted interventions remain largely unexplored. We hypothesized that dexmedetomidine and clonidine attenuated sevoflurane-induced tau phosphorylation and cognitive impairment by acting on α-2 adrenergic receptor. METHODS: Six-day-old mice received anesthesia with 3% sevoflurane 2 hours daily on postnatal days 6, 9, and 12. Alpha-2 adrenergic receptor agonist dexmedetomidine and clonidine were used to treat the mice with and without the α-2 adrenergic receptor antagonist yohimbine. Mouse hippocampi were harvested and subjected to western blot analysis. The New Object Recognition Test and Morris Water Maze were used to measure cognitive function. We analyzed the primary outcomes by using 2- and 1-way analysis of variance (ANOVA) and Mann-Whitney U test to determine the effects of sevoflurane on the amounts of phosphorylated tau, postsynaptic density-95, and cognitive function in young mice after the treatments with dexmedetomidine, clonidine, and yohimbine. RESULTS: Both dexmedetomidine and clonidine attenuated the sevoflurane-induced increase in phosphorylated tau amount (94 ± 16.3% [dexmedetomidine plus sevoflurane] versus 240 ± 67.8% [vehicle plus sevoflurane], P < .001; 125 ± 13.5% [clonidine plus sevoflurane] versus 355 ± 57.6% [vehicle plus sevoflurane], P < .001; mean ± standard deviation), sevoflurane-induced reduction in postsynaptic density-95 (82 ± 6.6% [dexmedetomidine plus sevoflurane] versus 31 ± 12.4% [vehicle plus sevoflurane], P < .001; 95 ± 6.4% [clonidine plus sevoflurane] versus 62 ± 18.4% [vehicle plus sevoflurane], P < .001), and cognitive impairment in the young mice. Interestingly, yohimbine reversed the effects of dexmedetomidine and clonidine on attenuating the sevoflurane-induced changes in phosphorylated tau, postsynaptic density-95, and cognitive function. CONCLUSIONS: Dexmedetomidine and clonidine could inhibit the sevoflurane-induced tau phosphorylation and cognitive impairment via activation of α-2 adrenergic receptor. More studies are needed to confirm the results and to determine the clinical relevance of these findings.

Triple reuptake inhibition of serotonin, norepinephrine, and dopamine increases the tonic activation of α2-adrenoceptors in the rat hippocampus and dopamine levels in the nucleus accumbens.

Clinical studies have shown the therapeutic efficacy of an increase in dopamine (DA) transmission in treatment of major depressive disorder (MDD). In the present study, we investigated whether blockade of DA transporters in addition to serotonin (5-HT) and norepinephrine (NE) produced additional adaptations of monoaminergic systems. In vivo electrophysiological recordings were carried out in male anesthetized rats. Vehicle, the 5-HT reuptake inhibitor escitalopram, the NE/DA reuptake blocker nomifensine and their combination (triple reuptake inhibition; TRI) were delivered for 2 or 14 days. Firing activity of NE, 5-HT and DA neurons was assessed. Tonic activation of 5-HT1A receptors and α1- and α2-adrenoceptors was determined in the hippocampus and extracellular DA levels in the nucleus accumbens (NAc). Unlike escitalopram, nomifensine and TRI administration increased the tonic activation of α2-adrenoceptors in the hippocampus despite decreasing NE neuronal firing activity after 2 and 14 days of administration. The firing activity of 5-HT neurons was increased after prolonged nomifensine and TRI regimens, while addition of nomifensine to escitalopram prevented the early 2-day suppression of firing by 5-HT reuptake inhibition. The tonic activation of 5-HT1A receptors was enhanced only with escitalopram. Whereas escitalopram and nomifensine decreased firing activity of DA neurons after a 2-day administration, their combination normalized it to baseline level after 14 days; this was accompanied by a robust increase in extracellular DA levels in the NAc. In summary, these results indicate that TRI increases NE and DA but not 5-HT transmission, suggesting a differential efficacy profile in MDD patients.

α 2-Adrenoceptors: Challenges and Opportunities-Enlightenment from the Kidney.

It was indeed a Don Quixote-like pursuit of the mechanism of essential hypertension when we serendipitously discovered α 2-adrenoceptors (α 2-ARs) in skin-lightening experiments in the frog. Now α 2-ARs lurk on the horizon involving hypertension causality, renal denervation for hypertension, injury from falling in the elderly and prazosin's mechanism of action in anxiety states such as posttraumatic stress disorder (PTSD). Our goal here is to focus on this horizon and bring into clear view the role of α 2-AR-mediated mechanisms in these seemingly unrelated conditions. Our narrative begins with an explanation of how experiments in isolated perfused kidneys led to the discovery of a sodium-retaining process, a fundamental mechanism of hypertension, mediated by α 2-ARs. In this model system and in the setting of furosemide-induced sodium excretion, α 2-AR activation inhibited adenylate cyclase, suppressed cAMP formation, and caused sodium retention. Further investigations led to the realization that renal α 2-AR expression in hypertensive animals is elevated, thus supporting a key role for kidney α 2-ARs in the pathophysiology of essential hypertension. Subsequent studies clarified the molecular pathways by which α 2-ARs activate prohypertensive biochemical systems. While investigating the role of α 1-adrenoceptors (α 1-ARs) versus α 2-ARs in renal sympathetic neurotransmission, we noted an astonishing result: in the kidney α 1-ARs suppress the postjunctional expression of α 2-ARs. Here, we describe how this finding relates to a broader understanding of the role of α 2-ARs in diverse disease states. Because of the capacity for qualitative and quantitative monitoring of α 2-AR-induced regulatory mechanisms in the kidney, we looked to the kidney and found enlightenment.

Activation of α-adrenoceptors depresses synaptic transmission of myelinated afferents and inhibits pathways mediating primary afferent depolarization (PAD) in the in vitro mouse spinal cord.

Somatosensory afferent transmission strength is controlled by several presynaptic mechanisms that reduce transmitter release at the spinal cord level. We focused this investigation on the role of α-adrenoceptors in modulating sensory transmission in low-threshold myelinated afferents and in pathways mediating primary afferent depolarization (PAD) of neonatal mouse spinal cord. We hypothesized that the activation of α-adrenoceptors depresses low threshold-evoked synaptic transmission and inhibits pathways mediating PAD. Extracellular field potentials (EFPs) recorded in the deep dorsal horn assessed adrenergic modulation of population monosynaptic transmission, while dorsal root potentials (DRPs) recorded at root entry zone assessed adrenergic modulation of PAD. We found that noradrenaline (NA) and the α1-adrenoceptor agonists phenylephrine and cirazoline depressed synaptic transmission (by 15, 14 and 22%, respectively). DRPs were also depressed by NA, phenylephrine and cirazoline (by 62, 30, and 64%, respectively), and by the α2-adrenoceptor agonist clonidine, although to a lower extent (20%). We conclude that NA depresses monosynaptic transmission of myelinated afferents onto deep dorsal horn neurons via α1-adrenoceptors and inhibits interneuronal pathways mediating PAD through the activation of α1- and α2-adrenoceptors. The functional significance of these modulatory actions in shaping cutaneous and muscle sensory information during motor behaviors requires further study.

Dexmedetomidine alleviates H2O2-induced oxidative stress and cell necroptosis through activating of α2-adrenoceptor in H9C2 cells.

Oxidative stress induced necroptosis is important in myocardial ischemia/reperfusion injury. Dexmedetomidine (Dex), an α2-adrenoceptor (α2-AR) agonist, has protective effect on oxidative stress induced cell apoptosis, but effects of Dex and Dex-mediated α2-AR activation on oxidant induced necroptosis was unclear. H9C2 cardiomyocytes were pre-treated with or without Dex and α2-AR antagonist yohimbine hydrochloride (YOH) before being exposed to H2O2 to induce oxidative cellular damage. Cell viability and lactate dehydrogenase (LDH) were detected by ELISA kits, protein expressions of Heme Oxygenase 1(HO-1), receptor interacting protein kinase 1 (RIPK1) and receptor interacting protein kinase 3 (RIPK3) were observed by WB, and TUNEL was used to detected cell apoptosis. H2O2 significantly decreased cell viability and increased LDH release and necroptotic and apoptotic cell deaths (all p < 0.05, H2O2 vs. Control). Dex preconditioning alleviated these injuries induced by H2O2. Dex preconditioning significantly increased expression of protein HO-1 and decreased expressions of proteins RIPK1 and RIPK3 induced by H2O2, while all these protective effects of Dex were reversed by YOH (all p < 0.05, Dex + H2O2 vs. H2O2; and YOH + Dex + H2O2 vs. Dex + H2O2). However, YOH did not prevent this protective effect of Dex against H2O2 induced apoptosis (YOH + Dex + H2O2 vs. Dex + H2O2, p > 0.05). These findings indicated that Dex attenuates H2O2 induced cardiomyocyte necroptotic and apoptotic cell death respectively dependently and independently of α2-AR activation.

Involvement of monoaminergic targets in the antidepressant- and anxiolytic-like effects of the synthetic alkamide riparin IV: Elucidation of further mechanisms through pharmacological, neurochemistry and computational approaches.

Despite recent advances, current antidepressants have considerable limitations: late onset of action and the high profile of refractoriness. Biomedical research with natural products has gained growing interest in the last years, and had provide useful candidates for new antidepressants. Riparins are a group of natural alkamides obtained from Aniba riparia, which had marked neuroactive effects, mainly as antidepressant and antinociceptive agents. We made modifications of the basic structure of riparins, originating a synthetic alkamide, also known as riparin IV (RipIV). RipIV demonstrated a superior analgesic effect than its congeners and a marked antidepressant-like effect. However, the basic mechanism for the central effects of RipIV remains unknown. Here, we aimed to investigate the participation of monoaminergic neurotransmission targets in the antidepressant-like effects of RipIV. To do this, we applied a combined approach of experimental (classical pharmacology and neurochemistry) and computer-aided techniques. Our results demonstrated that RipIV presented antidepressant- and anxiolytic-like effects without modifying locomotion and motor coordination of mice. Also, RipIV increased brain monoamines and their metabolite levels. At the higher dose (100 mg/kg), RipIV increased serotonin concentrations in all studied brain areas, while at the lower one (50 mg/kg), it increased mainly dopamine and noradrenaline levels. When tested with selective receptor antagonists, RipIV antidepressant effect showed dependence of the activation of multiple targets, including D1 and D2 dopamine receptors, 5-HT2A/2, 5-HT3 receptors and α2 adrenergic receptors. Molecular docking demonstrated favorable binding conformation and affinity of RipIV to monoamine oxidase B (MAO-B), serotonin transporter (SERT), α1 receptor, D2 receptor, dopamine transporter (DAT) and at some extent GABA-A receptor. RipIV also presented a computationally predicted favorable pharmacokinetic profile. Therefore, this study demonstrated the involvement of monoaminergic targets in the mechanism of RipIV antidepressant-like action, and provide evidence of it as a promising new antidepressant.

Dexmedetomidine modulates neuroinflammation and improves outcome via alpha2-adrenergic receptor signaling after rat spinal cord injury.

BACKGROUND: Spinal cord injury induces inflammatory responses that include the release of cytokines and the recruitment and activation of macrophages and microglia. Neuroinflammation at the lesion site contributes to secondary tissue injury and permanent locomotor dysfunction. Dexmedetomidine (DEX), a highly selective α2-adrenergic receptor agonist, is anti-inflammatory and neuroprotective in both preclinical and clinical trials. We investigated the effect of DEX on the microglial response, and histological and neurological outcomes in a rat model of cervical spinal cord injury. METHODS: Anaesthetised rats underwent unilateral (right) C5 spinal cord contusion (75 kdyne) using an impactor device. The locomotor function, injury size, and inflammatory responses were assessed. The effect of DEX was also studied in a microglial cell culture model. RESULTS: DEX significantly improved the ipsilateral upper-limb motor dysfunction (grooming and paw placement; P<0.0001 and P=0.0012), decreased the injury size (P<0.05), spared white matter (P<0.05), and reduced the number of activated macrophages (P<0.05) at the injury site 4 weeks post-SCI. In DEX-treated rats after injury, tissue RNA expression indicated a significant downregulation of pro-inflammatory markers (e.g. interleukin [IL]-1ß, tumour necrosis factor-α, interleukin (IL)-6, and CD11b) and an upregulation of anti-inflammatory and pro-resolving M2 responses (e.g. IL-4, arginase-1, and CD206) (P<0.05). In lipopolysaccharide-stimulated cultured microglia, DEX produced a similar inflammation-modulatory effect as was seen in spinal cord injury. The benefits of DEX on these outcomes were mostly reversed by an α2-adrenergic receptor antagonist. CONCLUSIONS: DEX significantly improves neurological outcomes and decreases tissue damage after spinal cord injury, which is associated with modulation of neuroinflammation and is partially mediated via α2-adrenergic receptor signaling.

Chronic fluoxetine reverses the effects of chronic corticosterone treatment on α2-adrenoceptors in the rat frontal cortex but not locus coeruleus.

Disruption of the hypothalamic-pituitary-adrenal axis is an established finding in patients with anxiety and/or depression. Chronic corticosterone administration in animals has been proposed as a model for the study of these stress-related disorders and the antidepressant action. Alterations of the central noradrenergic system and specifically of inhibitory α2-adrenoceptors seem to be part of the pathophysiology of depression and contribute to the antidepressant activity. The present study evaluates in male rats the effect of chronic corticosterone treatment during 35 days (16-20 mg kg-1 day-1) on the sensitivity of α2-adrenoceptors expressed in the somatodendritic and terminal noradrenergic areas locus coeruleus (LC) and prefrontal cortex (PFC), respectively. Further, the effect of chronic fluoxetine treatment (5 mg kg-1, i.p., since the 15th day) on the sensitivity of α2-adrenoceptors was examined under control conditions and in corticosterone-treated rats. The α2-adrenoceptor functionality was analysed in vitro by agonist-mediated [35S]GTPγS binding stimulation and in vivo through the modulation of noradrenaline (NA) release evaluated by dual-probe microdialysis. The concentration-effect curves of the [35S]GTPγS binding stimulation by the agonist UK14304 (5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine) demonstrated a desensitization of cortical α2-adrenoceptors induced by corticosterone (-logEC50 = 6.7 ±â€¯0.2 vs 8.2 ±â€¯0.3 in controls) that was reverted by fluoxetine treatment (-logEC50 = 7.5 ±â€¯0.3). Local administration of the α2-adrenoceptor antagonist RS79948 ((8aR,12aS,13aS)-5,8,8a,9,10,11,12,12a,13,13a-decahydro-3-methoxy-12-(ethylsulfonyl)-6H-isoquino[2,1-g][1,6]naphthyridine) (0.1-100 µmol L-1) into the LC induced a concentration-dependent NA increase in the PFC of the control group (Emax = 191 ±â€¯30%) but non-significant effect was observed in corticosterone-treated rats (Emax = 133 ±â€¯46%), reflecting a desensitization of α2-adrenoceptors that control the firing of noradrenergic neurons. Fluoxetine treatment did not alter the corticosterone-induced desensitization in this area (Emax = 136 ±â€¯19%). No effect of fluoxetine on α2-adrenoceptor functionality was observed in control animals (Emax = 223 ±â€¯30%). In PFC, the local administration of RS79948 increased NA in controls (Emax = 226 ±â€¯27%) without effect in the corticosterone group (Emax = 115 ±â€¯26%), suggesting a corticosterone-induced desensitization of terminal α2-adrenoceptors. Fluoxetine administration prevented the desensitization induced by corticosterone in the PFC (Emax = 233 ±â€¯33%) whereas desensitized α2-adrenoceptors in control animals (Emax = -24 ±â€¯10%). These data indicate that chronic corticosterone increases noradrenergic activity by acting at different α2-adrenoceptor subpopulations. Treatment with the antidepressant fluoxetine seems to counteract these changes by acting mainly on presynaptic α2-adrenoceptors expressed in terminal areas.

The adenosine A2A receptor antagonist SCH58261 reduces macrophage/microglia activation and protects against experimental autoimmune encephalomyelitis in mice.

Multiple sclerosis (MS) is a chronic autoimmune inflammatory disease of the central nervous system (CNS) affecting more than 2.5 million individuals worldwide. In the present study, myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) mice were treated with adenosine receptor A2A antagonist SCH58261 at different periods of EAE development. The administration of SCH58261 at 11-28 days post-immunization (d.p.i.) with MOG improved the neurological deficits. This time window corresponds to the therapeutic time window for MS treatment. SCH58261 significantly reduced the CNS neuroinflammation including reduced local infiltration of inflammatory cells, demyelination, and the numbers of macrophage/microglia in the spinal cord. Importantly, SCH58261 ameliorated the EAE-induced neurobehavioral deficits. By contrast, the SCH58261 treatment was ineffective when administered at the beginning of the onset of EAE (i.e., 1-10 d.p.i). The identification of the effective therapeutic window of A2A receptor antagonist provide insight into the role of A2A receptor signaling in EAE, and support SCH58261 as a candidate for the treatment of MS in human.