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BACKGROUND Despite overt insulin resistance, adipocytes of genetically obese Zucker rats accumulate the excess of calorie intake in the form of lipids. AIM To investigate whether factors can replace or reinforce insulin lipogenic action by exploring glucose uptake activation by hydrogen peroxide, since it is produced by monoamine oxidase (MAO) and semicarbazide-sensitive amine oxidase (SSAO) in adipocytes. METHODS 3H-2-deoxyglucose uptake (2-DG) was determined in adipocytes from obese and lean rats in response to insulin or MAO and SSAO substrates such as tyramine and benzylamine. 14C-tyramine oxidation and binding of imidazolinic radioligands [3H-Idazoxan, 3H-(2-benzofuranyl)-2-imidazoline] were studied in adipocytes, the liver, and muscle. The influence of in vivo administration of tyramine + vanadium on glucose handling was assessed in lean and obese rats. RESULTS 2-DG uptake and lipogenesis stimulation by insulin were dampened in adipocytes from obese rats, when compared to their lean littermates. Tyramine and benzylamine activation of hexose uptake was vanadate-dependent and was also limited, while MAO was increased and SSAO decreased. These changes were adipocyte-specific and accompanied by a greater number of imidazoline I2 binding sites in the obese rat, when compared to the lean. In vitro, tyramine precluded the binding to I2 sites, while in vivo, its administration together with vanadium lowered fasting plasma levels of glucose and triacylglycerols in obese rats. CONCLUSION The adipocytes from obese Zucker rats exhibit increased MAO activity and imidazoline binding site number. However, probably as a consequence of SSAO down-regulation, the glucose transport stimulation by tyramine is decreased as much as that of insulin in these insulin-resistant adipocytes. The adipocyte amine oxidases deserve more studies with respect to their putative contribution to the management of glucose and lipid handling.

Agmatine (AG), idazoxan (IDZ), and efaroxan (EFR) are imidazoline receptor ligands with beneficial effects in central nervous system disorders. The present study aimed to evaluate the interaction between AG, IDZ, and EFR with an opiate, tramadol (TR), in a conditioned place preference (CPP) paradigm. In the experiment, we used five groups with 8 adult male Wistar rats each. During the condition session, on days 2, 4, 6, and 8, the rats received the drugs (saline, or TR, or IDZ and TR, or EFR and TR, or AG and TR) and were placed in their least preferred compartment. On days 1, 3, 5, and 7, the rats received saline in the preferred compartment. In the preconditioning, the preferred compartment was determined. In the postconditioning, the preference for one of the compartments was reevaluated. TR increased the time spent in the non-preferred compartment. AG decreased time spent in the TR-paired compartment. EFR, more than IDZ, reduced the time spent in the TR-paired compartment, but without statistical significance. AG reversed the TR-induced CPP, while EFR and IDZ only decreased the time spent in the TR-paired compartment, without statistical significance.

Non-selective α2-adrenoreceptor (AR) stimulation delivers favourable sedative, analgesic, muscle relaxant and anxiolytic actions in companion animals, but is associated with cardiovascular and respiratory side effects. Anxiety conditions underscore monoamine disturbances amenable to α2-AR modulation. We investigated sub-chronic (14 day s.c.) treatment with the selective α2C-AR antagonist, ORM-10921 (0.03, 0.1, 0.3 mg/kg/d) on hippocampal noradrenaline (NA), dopamine (DA), serotonin (5-HT) and their turnover levels in stress sensitive Flinders Sensitive Line (FSL) rats versus Flinders Resistant Line (FRL) controls, using high performance liquid chromatography. The effects of ORM-10921 were compared to the non-selective α2-AR antagonist, idazoxan (IDAZ; 3 mg/kg/d), and to imipramine (IMI; 15 mg/kg/d), a reference antidepressant in this model. FSL rats displayed significantly reduced 5-HT (p = 0.03) and DA (p = 0.02) levels vs. FRL controls, while NA levels showed a similar trend. ORM-10921 significantly increased NA (all doses p ≤ 0.02), 5-HT (0.1 and 0.3 mg/kg p ≤ 0.03) and DA levels (all doses p ≤ 0.03), which correlated with decreased monoamine turnover. In contrast, IDAZ significantly elevated NA (p

Extensive clinical and experimental studies established that depression and mood disorders are highly prevalent neuropsychiatric conditions in Alzheimer’s disease (AD). However, its neurochemical basis is not clearly understood. Thus, understanding the neural mechanisms involved in mediating the co-morbidity of depression and AD may be crucial in exploring new pharmacological treatments for this condition. The present study investigated the role of the agmatinergic system in β-amyloid (Aββ1−42) peptide-induced depression using forced swim test (FST) in mice. Following the 28th days of its administration, Aβ1−42 peptide produced depression-like behavior in mice as evidenced by increased immobility time in FST and increased expression of pro-inflammatory cytokines like IL-6 and TNF-α compared to the control animals. The Aβ1−42 peptide-induced depression and neuroinflammatory markers were significantly inhibited by agmatine −, moxonidine, 2-BFI and l -arginine by once-daily administration during day 8–27 of the protocol. The antidepressant-like effect of agmatine in Aβ1−42 peptide in mice was potentiated by imidazoline receptor I1 agonist, moxonidine and imidazoline receptor I2 agonist 2-BFI at their sub-effective doses. On the other hand, it was completely blocked by imidazoline receptor I1 antagonist, efaroxan and imidazoline receptor I2 antagonist, idazoxan Also, agmatine levels were significantly reduced in brain samples of β-amyloid injected mice as compared to the control animals. In conclusion, the present study suggests the importance of endogenous agmatinergic system and imidazoline receptors system in β-amyloid induced a depressive-like behavior in mice. The data projects agmatine as a potential therapeutic target for the AD-associated depression and comorbidities.

Although alterations in the sympathetic nervous system (SNS) with age have been reported, and serious degenerative diseases of the autonomic nervous system such as multiple system atrophy are more likely to strike older people, connections between dysregulated adrenergic receptors and age-associated diseases and phenotypes have not been well studied. Two recent reports suggest that SNS may be more closely connected than previously appreciated. First, low nanomolar concentrations of Alzheimer's disease (AD)-associated Aβ42-amyloid oligomers alter signaling by SNS neurotransmitter norepinephrine (NE) to sufficiently activate kinase GSK3β to hyperphosphorylate tau, a key mediator of neurotoxicity in AD. Connecting beta-amyloid to tau in AD has been a key quest in understanding AD and developing therapeutics. The α2 adrenergic receptor inhibitory drug idazoxan reduces GSK3β activity and tau phosphorylation in AD mice with improved cognitive function, even in the presence of beta-amyloid deposits. In this study, SNS activation in the brain coupled with problematic Aβ42-amyloid oligomers result in serious consequences that can be ameliorated by reducing SNS signaling. A second example of the detrimental effects of increased SNS signaling is the premature graying of hair in response to stress. Secretion of NE resulting from stress causes differentiation of most hair pigment melanocyte stem cells (MeSCs) into melanocytes, rapidly depleting the hair follicle of pigment-producing cells as mature melanocytes undergo apoptosis and MeSCs are eventually eliminated. Blockade of NE SNS signaling preserves hair coloration in stressed animals. Increased SNS activation has serious apparently irreversible effects on homeostasis in both situations. Although neither report directly addresses aging, given that AD and the loss of hair pigmentation have strong age associations, it is of interest to better understand the role that SNS has in promoting age-associated phenotypes generally and determine if tuning the SNS through drug-mediated attenuation of SNS signaling may be of medical benefit.

Adrenergic receptors (AR) in the ventral tegmental area (VTA) modulate local neuronal activity and, as a consequence, dopamine (DA) release in the mesolimbic forebrain. Such modulation has functional significance: intra-VTA blockade of αsub1/sub-AR attenuates behavioral responses to salient environmental stimuli in rat models of drug seeking and conditioned fear as well as phasic DA release in the nucleus accumbens (NAc). In contrast, αsub2/sub-AR in the VTA has been suggested to act primarily as autoreceptors, limiting local noradrenergic input. The regulation of noradrenaline efflux by αsub2/sub-AR could be of clinical interest, as αsub2/sub-AR agonists are proposed as promising pharmacological tools in the treatment of PTSD and substance use disorder. Thus, the aim of our study was to determine the subtype-specificity of αsub2/sub-ARs in the VTA capable of modulating phasic DA release. We used fast scan cyclic voltammetry (FSCV) in anaesthetized male rats to measure DA release in the NAc after combined electrical stimulation and infusion of selected αsub2/sub-AR antagonists into the VTA. Intra-VTA microinfusion of idazoxan - a non-subtype-specific αsub2/sub-AR antagonist, as well as BRL-44408 - a selective αsub2A/sub-AR antagonist, attenuated electrically-evoked DA in the NAc. In contrast, local administration of JP-1302 or imiloxan (αsub2B/sub- and αsub2C/sub-AR antagonists, respectively) had no effect. The effect of BRL-44408 on DA release was attenuated by intra-VTA DA Dsub2/subantagonist (raclopride) pre-administration. Finally, we confirmed the presence of αsub2A/sub-AR protein in the VTA using western blotting. In conclusion, these data specify αsub2A/sub-, but not αsub2B/sub- or αsub2C/sub-AR as the receptor subtype controlling NA release in the VTA.

The duodenal mucosa is regularly exposed to a low osmolality, and recent experiments suggest that hypotonicity increases mucosal permeability in an osmolality-dependent manner. The aim was to examine whether the sympathetic nervous system, via action on α-adrenoceptors, affects the hypotonicity-induced increase in duodenal mucosal permeability. The duodenum of anaesthetised rats was perfused in vivo with a 50 mM NaCl solution in the presence of adrenergic α-adrenoceptor drugs. Studied were the effects on mucosal permeability (blood-to-lumen clearance of 51Cr-EDTA), arterial blood pressure, luminal alkalinisation, transepithelial fluid flux, and motility. Hypotonicity induced a six-fold increase in mucosal permeability, a response that was reversible and repeatable. The α2-adrenoceptor agonist clonidine abolished the hypotonicity-induced increase in mucosal permeability, reduced arterial blood pressure, inhibited duodenal motility, and decreased luminal alkalinisation. The α2-adrenoceptor antagonists, yohimbine and idazoxan, prevented the inhibitory effect of clonidine on the hypotonicity-induced increase in mucosal permeability. The α1-agonist phenylephrine or the α1-antagonist prazosin elicited their predicted effect on blood pressure but did not affect the hypotonicity-induced increase in mucosal permeability. None of the α1- or α2-adrenoceptor drugs changed the hypotonicity-induced net fluid absorption. In conclusion, stimulation of the adrenergic α2-adrenoceptor prevents the hypotonicity-induced increase in mucosal permeability, suggesting that the sympathetic nervous system has the capability to regulate duodenal mucosal permeability.

The duodenal mucosa is regularly exposed to a low osmolality, and recent experiments suggest that hypotonicity increases mucosal permeability in an osmolality-dependent manner. The aim was to examine whether the sympathetic nervous system, via action on α-adrenoceptors, affects the hypotonicity-induced increase in duodenal mucosal permeability. The duodenum of anaesthetised rats was perfused in vivo with a 50 mM NaCl solution in the presence of adrenergic α-adrenoceptor drugs. Studied were the effects on mucosal permeability (blood-to-lumen clearance of 51Cr-EDTA), arterial blood pressure, luminal alkalinisation, transepithelial fluid flux, and motility. Hypotonicity induced a six-fold increase in mucosal permeability, a response that was reversible and repeatable. The α2-adrenoceptor agonist clonidine abolished the hypotonicity-induced increase in mucosal permeability, reduced arterial blood pressure, inhibited duodenal motility, and decreased luminal alkalinisation. The α2-adrenoceptor antagonists, yohimbine and idazoxan, prevented the inhibitory effect of clonidine on the hypotonicity-induced increase in mucosal permeability. The α1-agonist phenylephrine or the α1-antagonist prazosin elicited their predicted effect on blood pressure but did not affect the hypotonicity-induced increase in mucosal permeability. None of the α1- or α2-adrenoceptor drugs changed the hypotonicity-induced net fluid absorption. In conclusion, stimulation of the adrenergic α2-adrenoceptor prevents the hypotonicity-induced increase in mucosal permeability, suggesting that the sympathetic nervous system has the capability to regulate duodenal mucosal permeability.

At present, whether α2-adrenoceptors in the prelimbic cortex (PrL) are involved in Parkinson's disease-related anxiety is unclear. We examined the effects of PrL α2-adrenoceptors on anxiety-like behaviors in rats with unilateral 6-hydroxydopamine lesions of the medial forebrain bundle. Compared to the sham operation, the lesion induced anxiety-like responses as measured by the open field test and elevated plus-maze test. Intra-PrL injection of the α2-adrenoceptor agonist clonidine (1.25, 2.5 or 5 μg/rat) produced anxiolytic effects in sham-operated and lesioned rats. Furthermore, intra-PrL injection of the α2-adrenoceptor antagonist idazoxan (1, 2 or 4 μg/rat) induced anxiogenic effects in two groups of rats. The effective doses produced by clonidine and idazoxan in lesioned rats were higher than those in sham-operated rats. Neurochemical results showed that intra-PrL injection of clonidine (5 μg/rat) or idazoxan (4 μg/rat) decreased or increased dopamine (DA) and noradrenaline (NA) and serotonin (5-HT) levels in the medial prefrontal cortex (mPFC) and amygdala in sham-operated and lesioned rats, respectively. These results suggest that α2-adrenoceptors in the PrL are involved in the regulation of anxiety-like behaviors, which is attributable to changes in DA, NA and 5-HT levels in the mPFC and amygdala after activation and blockade of α2-adrenoceptors.

Pharmacotherapies for fibromyalgia treatment are lacking. This study examined the antinociceptive and antidepressant-like effects of imidazoline I2 receptor (I2R) agonists in a reserpine-induced model of fibromyalgia in rats. Rats were treated for 3 days with vehicle or reserpine. The von Frey filament test was used to assess the antinociceptive effects of I2 receptor agonists, and the forced swim test was used to assess the antidepressant-like effects of these drugs. 2-BFI (3.2-10 mg/kg, intraperitoneally), phenyzoline (17.8-56 mg/kg, intraperitoneally), and CR4056 (3.2-10 mg/kg, intraperitoneally) all dose-dependently produced significant antinociceptive effects, which were attenuated by the I2R antagonist idazoxan. Only CR4056 significantly reduced the immobility time in the forced swim test in both vehicle-treated and reserpine-treated rats. These data suggest that I2R agonists may be useful to treat fibromyalgia-related pain and comorbid depression.

26RFa is an endogenous ligand for the QRFP receptor. We previously found that intracerebroventricular injection of 26RFa produces an analgesic effect in a rat formalin test. In the present study, we directly tested the hypothesis that the analgesic effects of 26RFa in the formalin test are mediated in well-recognized regions of the descending inhibitory pain pathways, such as the rostral ventromedial medulla (RVM), locus coeruleus (LC), and periaqueductal grey (PAG) in rats. Injection cannulae were stereotaxically placed in the RVM, LC, or PAG through a burr hole. 26RFa (15 μg) or saline was delivered in a total volume of 0.5 μL. In a formalin test, 50 μL of 5% formalin was injected subcutaneously into the hind paw. In an antagonist study, idazoxan, an α-2 antagonist, or naloxone, an opioid receptor antagonist, was administered. Microinjection of 26RFa into the RVM had no effect compared with that in saline-injected rats. Microinjection of 26RFa into the LC contralateral, but not ipsilateral, to the formalin injection site significantly decreased the number of flinching behaviors compared with that of saline-injected rats. This effect was antagonized by intrathecal injection of idazoxan. Microinjection of 26RFa into the contralateral, but not ipsilateral, PAG produced an analgesic effect, and this effect was partly antagonized by intraperitoneal naloxone. These data suggest that 26RFa microinjected into the contralateral LC induced noradrenaline release in the spinal cord and produced an analgesic effect. In the contralateral PAG, 26RFa activated the opioid system, and some analgesic effects were mediated by opioid system activation.

金沢大学医薬保健研究域薬学系, Effects of stress on the reward system are well established in the literature. Although previous studies have revealed that stress can reinstate extinguished addictive behaviors related to cocaine, the effects of stress on the rewarding memory of cocaine are not fully understood. Here, we provide evidence that stress potentiates the expression of rewarding memory of cocaine via the activation of brainstem-reward circuitry using a cocaine-induced conditioned place preference (CPP) paradigm combined with restraint stress in rats. The rats exposed to 30-minute restraint stress immediately before posttest exhibited significantly larger CPP scores compared with non-stressed rats. Intra-laterodorsal tegmental nucleus (LDT) microinjection of a β or α2 adrenoceptor antagonist attenuated the stress-induced enhancement of cocaine CPP. Consistent with this observation, intra-LDT microinjection of a β or α2 adrenoceptor agonist before posttest increased cocaine CPP. Additionally, intra-ventral tegmental area (VTA) microinjection of antagonists for the muscarinic acetylcholine, nicotinic acetylcholine or glutamate receptors attenuated the stress-induced enhancement of cocaine CPP. Finally, intra-medial prefrontal cortex (mPFC) microinjection of a D1 receptor antagonist also reduced the stress-induced enhancement of cocaine CPP. These findings suggest a mechanism wherein the LDT is activated by noradrenergic input from the locus coeruleus, leading to the activation of VTA dopamine neurons via both cholinergic and glutamatergic transmission and the subsequent excitation of the mPFC to enhance the memory of cocaine-induced reward value. © 2018 Society for the Study of Addiction., Embargo Period 12 months

Acute kidney injury (AKI) is a frequent and serious complication of sepsis; however, there are currently no effective therapies. Inflammation and oxidative stress are the major mechanisms implicated in lipopolysaccharide (LPS)-induced AKI. Dexmedetomidine (DEX) has been reported to have remarkable anti-inflammatory and antioxidant effects. Here, we examined the renoprotective effects of DEX and potential underlying mechanisms in rats with LPS-induced AKI. We analyzed renal function and structure; serum inflammatory cytokine; renal oxidant and antioxidant levels; and renal expression of glycogen synthase kinase-3β (GSK-3β)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway-related proteins in rats 4 hr after administration of LPS. Pretreatment with DEX improved renal function and significantly reduced the levels of inflammatory cytokines and oxidative stress markers. Treatment with DEX and the GSK-3β inhibitor SB216367 promoted phosphorylation of GSK-3β, induced Nrf2 nuclear translocation, and increased transcription of the Nrf2 target genes heme oxygenase-1 and NAD(P)H quinone oxidoreductase-1, primarily in renal tubules. Alpha-2-adrenergic receptor (α2-AR) antagonist atipamezole and imidazoline I 2 receptor (I 2 R) antagonist idazoxan reversed the effects of DEX. These results suggest that the renoprotective effects of DEX are mediated via α2-AR and I 2 R-dependent pathways that reduce inflammation and oxidative stress through GSK-3β/Nrf2 signaling.

Background Locomotor sensitization to repeated ethanol (EtOH) administration is proposed to play a role in early and recurring steps of addiction. The present study was designed to examine the effect of agmatine on EtOH-induced locomotor sensitization in mice. Methods Mice received daily single intraperitoneal injection of EtOH (2.5 g/kg, 20 v/v) for 7 consecutive days. Following a 3-day EtOH-free phase, the mice were challenged with EtOH on day 11 with a single injection of EtOH. Agmatine (10 to 40 μg/mouse), endogenous agmatine enhancers (l-arginine [80 μg/mouse], arcaine [50 μg/mouse], aminoguanidine [25 μg/mouse]), and imidazoline receptor agonist/antagonists were injected (intracerebroventricular [i.c.v.]) either daily before the injection of EtOH during the 7-day development phase or on days 8, 9, and 10 (EtOH-free phase). The horizontal locomotor activity was determined on days 1, 3, 5, 7, and 11. Results Agmatine (20 to 40 μg/mouse) administration for 7 days (development phase) significantly attenuated the locomotor sensitization response of EtOH challenge on day 11. Further, the agmatine administered only during EtOH-free period (days 8, 9, and 10) also inhibited the enhanced locomotor activity on the 11th day to EtOH challenge as compared to control mice indicating blockade of expression of sensitization. Daily treatment (i.c.v.) with endogenous agmatine enhancers like l-arginine (80 μg/mouse) or arcaine (50 μg/mouse) and aminoguanidine (25 μg/mouse) restrained the development as well as expression of sensitization to EtOH. Imidazoline I1 receptor agonist, moxonidine, and I2 agonist, 2-BFI, not only decreased the development and expression of locomotor sensitization but also potentiated the effect of agmatine when employed in combination. Importantly, I1 receptor antagonist, efaroxan, and I2 antagonist, idazoxan, blocked the effect of agmatine, revealing the involvement of imidazoline receptors in agmatine-mediated inhibition of EtOH sensitization. Conclusions Inhibition of EtOH sensitization by agmatine is mediated through imidazoline receptors and project agmatine and imidazoline agents in the pharmacotherapy of alcohol addiction.

In addition to schizophrenia and bipolar disorder, aripiprazole is approved as an adjunct for major depressive disorder (MDD). Adding aripiprazole to the 5-HT reuptake inhibitor escitalopram reverses the inhibitory action of escitalopram on firing activity of rat 5-HT, norepinephrine (NE) and DA neurons. This study investigated how aripiprazole, escitalopram and their combination affect the net effect of 5-HT and NE neurotransmission in the rat hippocampus. Electrophysiological recordings of hippocampus CA3 pyramidal neurons were conducted in anesthetized Sprague-Dawley rats after 2- and 14-day administration regimens. Aripiprazole and escitalopram (2 and 5 mg/kg/day, respectively) were delivered alone or in combination through subcutaneous injections and implanted osmotic minipumps, respectively. Overall neurotransmission of 5-HT and NE were assessed by determining possible enhancements in tonic activation of 5-HT1A receptors and α1- and α2-adrenoceptors. This was achieved by assessing increases of firing rate of pyramidal neurons due to disinhibition induced by injections of antagonists for these three types of receptors. While neither 2- and 14-day administration of escitalopram nor aripiprazole significantly altered firing rate of pyramidal neurons following injection of 5-HT1A antagonist WAY100635, their combination for 14 days significantly increased this parameter. Fourteen days of the same drug regimens did not change firing following injection of the α1- and α2-adrenoceptor antagonists prazosin and idazoxan, respectively. A synergy between aripiprazole and escitalopram was thus documented by an increase in the tonic activation of 5-HT1A receptors after 14 days of administration that may account, at least in part, for the benefits of this strategy in MDD.

Experimental studies and clinical trials revealed the complex interconnections between imidazoline system and various other mediators such as epinephrine, norepinephrine; thus, explain their involvement in the pathophysiological mechanisms of different motor, behavioral and cognitive disturbances. In this study, we tested the influence induced by idazoxan and efaroxan on the cognitive performances in rats. Groups of 6 adult male Wistar rats were treated intraperitoneally according to the following protocol: group I (Control): distilled water 0.3 ml/100g; group II (IDZ): 3 mg/kg idazoxan and group III (EFR): 1 mg/kg efaroxan. The effects of the imidazoline receptor antagonists on the rats cognitive functions were assessed using the radial-arm maze, in order to count the time spent into the arms, the number of baited arms visited, but previously explored (working memory errors); the time taken to consume all baits and the number of entering in non-baited arms (reference memory errors). The data were expressed as mean +/- standard deviation, and statistically analyzed using SPSS version 17.0 Software for Windows, followed by ANOVA one-way method. The administration of IDZ, as well as of EFR was accompanied by a substantial diminution in the number of working memory errors, and the period of time to consume all baits, statistically significant (p[0.01) compared to control group. The use of these two imidazoline receptors antagonists resulted in a considerable decrease in the reference memory errors number, statistically significant (p[0.01) compared to the group treated with distilled water. The influence of IDZ on the evaluated parameters was more accentuated than the effects induced by EFR in all sessions of testing, in this behavioral experimental model. Our findings indicate that treatment with both imidazoline receptor antagonists, idazoxan and efaroxan was associated by a facilitation of the short-term memory retention, an enhancement of discriminative spatial learning, and an improvement of long-term memory performance in radial arm maze in rats.

Among vertebrate ectotherms, air breathing frequency is generally constrained across warmer temperatures, but decreases during cooling. The brainstem mechanisms that give rise to this ventilatory strategy are unclear. Neuromodulation has recently been shown to stabilize motor circuit output across temperatures. Therefore, we tested the hypothesis that an important neuromodulatory system in respiratory control network, norepinephrine, produces this pattern of respiratory motor activity across temperatures. To this end, we used in vitro brainstem-spinal cord preparations from adult bullfrogs, Lithobates catesbeianus, to assess the role of noradrenergic signaling in shaping the frequency response of the respiratory network during temperature changes. We identified that noradrenergic signaling through the α1 adrenergic receptor constrains motor output from the respiratory network across warm temperatures. In contrast, the α2 adrenergic receptor actively inhibits respiratory motor output during cooling. These results indicate that noradrenergic tuning, rather than passive thermal responses, produces temperature responses of the respiratory circuits.

BACKGROUND: Ventricular-like human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exhibit the electrophysiological characteristics of spontaneous beating. Previous studies demonstrated that dexmedetomidine (DMED), a highly selective and widely used α(2)-adrenoceptor agonist for sedation, analgesia, and stress management, may induce antiarrhythmic effects, especially ventricular tachycardia. However, the underlying mechanisms of the DMED-mediated antiarrhythmic effects remain to be fully elucidated. METHODS: A conventional patch-clamp recording method was used to investigate the direct effects of DMED on spontaneous action potentials, pacemaker currents (I(f)), potassium (K(+)) channel currents (I(K1) and I(Kr)), sodium (Na(+)) channel currents (I(Na)), and calcium (Ca(2+)) channel currents (I(Ca)) in ventricular-like hiPSC-CMs. RESULTS: DMED dose-dependently altered the frequency of ventricular-like spontaneous action potentials with a half-maximal inhibitory concentration (IC(50)) of 27.9 µM (n=6) and significantly prolonged the action potential duration at 90% repolarization (APD(90)). DMED also inhibited the amplitudes of the I(Na) and I(Ca) without affecting the activation and inactivation curves of these channels. DMED decreased the time constant of the Na(+) and Ca(2+) channel activation at potential –40 to –20 mv, and –20 mv. DMED increased the time constant of inactivation of the Na(+) and Ca(2+) channels. However, DMED did not affect the I(K1), I(Kr), I(f), and their current-voltage relationship. The ability of DMED to decrease the spontaneous action potential frequency and the Na(+) and Ca(2+) channel amplitudes, were not blocked by yohimbine, idazoxan, or phentolamine. CONCLUSIONS: DMED could inhibit the frequency of spontaneous action potentials and decrease the I(Na) and I(Ca) of hiPSC-CMs via mechanisms that were independent of the α(2)-adrenoceptor, the imidazoline receptor, and the α(1)-adrenoceptor. These inhibitory effects on hiPSC-CMs may contribute to the antiarrhythmic effects of DMED.

The current rodent study applied in vivo fast-scan cyclic voltammetry (FSCV), paired with a pharmacological approach, to measure the release of the catecholamines (CA) dopamine (DA) and norepinephrine (NE) in the basolateral amygdala (BLA) following locus coeruleus (LC) stimulation. The primary goal was to determine if exposure to either social (social defeat) or non-social (forced swim) stress altered LC-evoked catecholamine release dynamics in the BLA. We used idazoxan (α2 adrenergic receptor antagonist) and raclopride (D2 dopamine receptor antagonist) to confirm the presence of NE and DA, respectively, in the measured CA signal. In non-stressed rats, injection of idazoxan, but not raclopride, resulted in a significant increase in the detected CA signal, indicating the presence of NE but not DA. Following exposure to either stress paradigm, the measured CA release was significantly greater after injection of either drug, suggesting the presence of both NE and DA in the LC-induced CA signal after social or non-social stress. Furthermore, acute administration of alcohol significantly decreased the CA signal in stressed rats, while it did not have an effect in naïve animals. Together, these data reveal that, while LC stimulation primarily elicits NE release in the BLA of control animals, both social and non-social stress unmask a novel dopaminergic component of LC catecholamine signaling. Future studies will be needed to identify the specific neural mechanism(s) responsible for these plastic changes in LC-BLA catecholamine signaling and to assess the possible contribution of these changes to the maladaptive behavioral phenotypes that develop following exposure to these stressors.

Neuropeptide W (NPW) messenger ribonucleic acid (mRNA) and NPBW1 and/or NPBW2 mRNA are expressed in the descending pain inhibitory system. In the present study, we examined whether NPW microinjected into the descending pain inhibitory system, such as the periaqueductal gray (PAG), locus coeruleus (LC), and rostral ventromedial medulla (RVM), produces an analgesic effect using a rat formalin test. Microinjections of NPW into the PAG ipsilateral and contralateral to the formalin-injected side, LC ipsilateral and contralateral to the formalin-injected side, and RVM produced an analgesic effect. In the RVM study, the analgesic effect was antagonized by WAY100135, a 5-HT1A antagonist, and enhanced by prazosin, an α1 antagonist, and SB269970, a 5-HT7 antagonist. Naloxone, an opioid antagonist, also antagonized the effect of NPW in the RVM study. In the ipsilateral LC study, the analgesic effect was antagonized by WAY100135, idazoxan, an α2 antagonist, and naloxone and was enhanced by prazosin and SB269970. In the contralateral LC study, the analgesic effect was antagonized by prazosin, idazoxan, SB269970, and naloxone. The analgesic effect was antagonized by WAY100135, SB269970, idazoxan, and naloxone in the ipsilateral and contralateral PAG studies. These findings strongly suggest that NPBW1/W2 activation by NPW microinjection into the RVM, LC, and PAG affect the descending pain modulatory system and produce anti-nociceptive and pro-nociceptive effects in the rat formalin test.

Paclitaxel, a primary chemotherapeutic agent used to treat numerous solid malignancies, is commonly associated with debilitating peripheral neuropathy. However, a satisfactory gold-standard monotherapy for this neuropathic pain is not currently available. A combination strategy of two or more medications with different properties may achieve more beneficial effects than monotherapy. Thus, we investigated the analgesic efficacies and spinal mechanisms of the combination strategy, including bee venom acupuncture (BVA) and venlafaxine (VLX) against paclitaxel-induced allodynia in mice. Four intraperitoneal infusions of paclitaxel on alternating days (2 mg/kg/day) induced cold and mechanical allodynia for at least 1 week as assessed using acetone and the von Frey hair test, respectively. Co-treatment of BVA (1.0 mg/kg, s.c., ST36) with VLX (40 mg/kg, i.p.) at the medium dose produced a longer-lasting and additive effect than each monotherapy at the highest dose (BVA, 2.5 mg/kg, VLX, 60 mg/kg). Spinal pre-administration of idazoxan (&alpha, 2-adrenergic receptor antagonist, 10 &mu, g), methysergide (mixed 5-HT1/5-HT2 receptor antagonist, 10 &mu, g), or MDL-72222 (5-HT3 receptor antagonist, 10 &mu, g) abolished this analgesia. These results suggest that the combination therapy with BVA and VLX produces long-lasting and additive analgesic effects on paclitaxel-induced allodynia, via the spinal noradrenergic and serotonergic mechanism, providing a promising clinical strategy.

Clozapine is a second generation antipsychotic drug that has proven to be helpful in the management of patients with psychotic disorders that are resistant to other medications. Unfortunately, the majority of patients treated with clozapine develop metabolic dysregulation, including weight gain and insulin resistance. There are few treatments available to effectively counter these side-effects. The goal of the present study was to use an established animal model to better understand the nature of these metabolic side-effects and determine whether existing drugs could be used to alleviate metabolic changes. Adult female rats were treated with a range of doses of clozapine (2, 10 and 20 mg/kg) and subjected to the hyperinsulinemic-euglycemic clamp, to measure whole-body insulin resistance. Clozapine dose-dependently decreased the glucose infusion rate, reflecting pronounced insulin resistance. To reverse the insulin resistance, rats were co-treated with the ganglionic blocker mecamylamine (0.1, 1.0 and 5.0 mg/kg) which dose-dependently reversed the effects of 10 mg/kg clozapine. A 1.0 mg/kg dose of mecamylamine independently reversed the large increase in peripheral epinephrine caused by treatment with clozapine. To study the influence of specific adrenoceptors, rats were treated with multiple doses of α1 (prazosin), α2 (idazoxan), β1 (atenolol) and β2 (butoxamine) adrenoceptor antagonists after the onset of clozapine-induced insulin resistance. Both beta blockers were effective in attenuating the effects of clozapine, while idazoxan had a smaller effect; no change was seen with prazosin. The current results indicate that peripheral catecholamines may play a role in clozapine’s metabolic effects and be a target for future treatments.

Non-selective α

Based on the finding that a central antihypertensive agent with high affinity for I1-type imidazoline receptors ? rilmenidine, shows cytotoxic effects on cultured cancer cell lines, it has been suggested that imidazoline receptors agonists might have a therapeutic potential in the cancer therapy. Nevertheless, potential rilmenidine side effects caused by activation of α-adrenoceptors, or other associated receptors and enzymes, might hinder its therapeutic benefits. Considering that human α-adrenoceptors belong to the rhodopsin-like class A of G-protein-coupled receptors (GPCRs) it is reasonable to assume that imidazolines might have the affinity for other receptors from the same class. Therefore, to investigate possible off-target effects of imidazoline ligands we have prepared a reverse docking protocol on class A GPCRs, using imidazoline ligands and their decoys. To verify our in silico results, three ligands with high scores and three ligands with low scores were tested for antagonistic activity on α2- adrenoceptors.

Made available in DSpace on 2020-12-12T02:02:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-07-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The role of endothelium in the electrical-field stimulation (EFS)-induced contractions of Chelonoidis carbonaria aorta was investigated. Contractions were evaluated in the presence and absence of L-NAME (100 μM), tetrodotoxin (1 μM), phentolamine (10 and 100 μM), phenoxybenzamine (1 and 10 μM), prazosin (100 μM), idazoxan (100 μM), atropine (10 μM), D-tubocurarine (10 μM) or indomethacin (10 μM). EFS-induced contraction was also carried out in endothelium-denuded rings. EFS-induced contraction was investigated by the sandwich assay. Concentration curves to endothelin-1 (0.1–100 nM) and U46619 (0.001–100 μM) were also constructed to calculate both Emax and EC50. EFS at 16 Hz contracted Chelonoidis aorta, which was almost abolished by the endothelium removal. The addition of L-NAME increased the EFS response (2.0 ± 0.4 and 8.3 ± 1.9 mN). In L-NAME treated aortic rings, tetrodotoxin did not change the EFS-response (5.1 ± 1.8 and 4.9 ± 1.7 mN). Indomethacin, atropine and d-tubucurarine also did not affect the EFS-response. Phentolamine at 10 μM did not change the EFS-induced contraction; however, at 100 μM, reduced it (3.9 ± 1 and 1.9 ± 0.3 mN). Prazosin and idazoxan did not change EFS-induced contractions. Phenoxybenzamine at 1 μM reduced by 76% (9.6 ± 3.4 and 2.3 ± 0.8 mN) and at 10 μM by 90% the EFS response. Immunohistochemistry identified tyrosine hydroxylase in the endothelium and brain, whereas S100 protein was found only in brain. In conclusion, endothelium modulates EFS-induced contractions in Chelonoidis aortic rings and this modulation may be due to endothelium-derived catecholamines, possibly dopamine. Superior Institute of Biomedical Sciences Ceará State University (UECE) Faculty of Medical Sciences Department of Pharmacology University of Campinas (UNICAMP) Department of Pharmacology Institute of Biosciences of Botucatu UNESP - São Paulo State University Department of Pharmacology Institute of Biomedical Sciences USP – University of São Paulo Department of Pharmacology Institute of Biosciences of Botucatu UNESP - São Paulo State University FAPESP: 2016/04731-8 FAPESP: 2016/09539-8 FAPESP: 2017/15175-1 FAPESP: 2018/24971-9

Zerumbone has shown great potential in various pathophysiological models of diseases, particularly in neuropathic pain conditions. Further understanding the mechanisms of action is important to develop zerumbone as a potential anti-nociceptive agent. Numerous receptors and pathways function to inhibit and modulate transmission of pain signals. Previously, we demonstrated involvement of the serotonergic system in zerumbone’s anti-neuropathic effects. The present study was conducted to determine zerumbone’s modulatory potential involving noradrenergic, transient receptor potential vanilloid type 1 (TRPV1) and N-methyl-D-aspartate (NMDA) receptors in chronic constriction injury (CCI)-induced in vitro and lipopolysaccharide (LPS)-induced SH-SY5Y in vitro neuroinflammatory models. von Frey filament and Hargreaves plantar tests were used to assess allodynia and hyperalgesia in the chronic constriction injury-induced neuropathic pain mouse model. Involvement of specific adrenoceptors were investigated using antagonists— prazosin (α1-adrenoceptor antagonist), idazoxan (α2-adrenoceptor antagonist), metoprolol (β1-adrenoceptor antagonist), ICI 118,551 (β2-adrenoceptor antagonist), and SR 59230 A (β3-adrenoceptor antagonist), co-administered with zerumbone (10 mg/kg). Involvement of excitatory receptors; TRPV and NMDA were conducted using antagonists capsazepine (TRPV1 antagonist) and memantine (NMDA antagonist). Western blot was conducted to investigate the effect of zerumbone on the expression of α2A-adrenoceptor, TRPV1 and NMDA NR2B receptors in CCI-induced whole brain samples of mice as well as in LPS-induced SH-SY5Y neuroblastoma cells. Pre-treatment with α1- and α2-adrenoceptor antagonists significantly attenuated both anti-allodynic and anti-hyperalgesic effects of zerumbone. For β-adrenoceptors, only β2-adrenoceptor antagonist significantly reversed the anti-allodynic and anti-hyperalgesic effects of zerumbone. β1-adrenoceptor antagonist only reversed the anti-allodynic effect of zerumbone. The anti-allodynic and anti-hyperalgesic effects of zerumbone were both absent when TRPV1 and NMDA receptors were antagonized in both nociceptive assays. Zerumbone treatment markedly decreased the expression of α2A-adrenoceptor, while an up-regulation was observed of NMDA NR2B receptors. Expression of TRPV1 receptors however did not significantly change. The in vitro study, representing a peripheral model, demonstrated the reduction of both NMDA NR2B and TRPV1 receptors while significantly increasing α2A-adrenoceptor expression in contrast to the brain samples. Our current findings suggest that the α1-, α2-, β1- and β2-adrenoceptors, TRPV1 and NMDA NR2B are essential for the anti-allodynic and antihyperalgesic effects of zerumbone. Alternatively, we demonstrated the plasticity of these receptors through their response to zerumbone’s administration.

Electrical field stimulation (EFS) induces contractions of both snake aorta and human umbilical cord vessels (HUCV) which were dependent on the presence of the endothelium. This study aimed to establish the nature of the mediator(s) responsible for EFS‐induced contractions in HUCV. Rings with or without endothelium from human umbilical artery (HUA) or vein (HUV) were mounted in organ bath chambers containing oxygenated, heated Krebs‐Henseleit's solution. Basal release of dopamine (DA), noradrenaline, and adrenaline was measured by LC‐MS‐MS. Cumulative concentration‐response curves were performed with dopamine in the absence and in the presence of L‐NAME or of dopamine antagonists. EFS studies were performed in the presence and absence of L‐NAME, the α‐adrenergic blockers prazosin and idazoxan, and the dopamine antagonists SCH‐23390 and haloperidol. Tyrosine hydroxylase (TH) and dopa‐decarboxylase (DDC) were studied by immunohistochemistry and fluorescence in situ hybridizations. Basal release of dopamine requires an intact endothelium in both HUA and HUV. TH and DDC are present only in the endothelium of both HUA and HUV as determined by immunohistochemistry. Dopamine induced contractions in HUA only in the presence of L‐NAME. Dopamine‐induced contractions in HUV were strongly potentiated by L‐NAME. The EFS‐induced contractions in both HUA and HUV were potentiated by L‐NAME and inhibited by the D2‐like receptor antagonist haloperidol. The α‐adrenergic antagonists prazosin and idazoxan and the D1‐like receptor antagonist SCH‐23390 had no effect on the EFS‐induced contractions of HUA and HUV. Endothelium‐derived dopamine is a major modulator of HUCV reactivity in vitro.

Social play behaviour is a vigorous, highly rewarding social activity abundant in the young of most mammalian species, including humans. Social play is thought to be important for social, emotional and cognitive development, yet its neural underpinnings are incompletely understood. We have previously shown that low doses of methylphenidate suppress social play behaviour through a noradrenergic mechanism of action, and that methylphenidate exerts its effect within the prefrontal cortex, amygdala and habenula. In the present study, we sought to reveal whether these regions work in parallel or in series to mediate the play-suppressant effect of methylphenidate. To that aim, we tested whether infusion of the α2-adrenoceptor antagonist RX821002 into the anterior cingulate cortex, infralimbic cortex, basolateral amygdala or habenula prevents the effect of methylphenidate on social play behaviour, or the psychomotor stimulant effect of methylphenidate. We found that the social play-suppressant effect of methylphenidate was not prevented by infusion of the α2-adrenoceptor antagonist into either region, or by infusion of RX821002 into both the anterior cingulate and infralimbic cortex. By contrast, RX821002 infusion into the anterior cingulate modestly enhanced social play, and infusion of the antagonist into the infralimbic cortex attenuated the psychomotor stimulant effect of methylphenidate. We conclude that there is redundancy in the neural circuitry that mediates the play-suppressant effect of methylphenidate, whereby prefrontal cortical and subcortical limbic mechanisms act in parallel. Moreover, our data support the notion that prefrontal noradrenergic mechanisms contribute to the locomotor enhancing effect of psychostimulant drugs.

Stimulation of α2-adrenoceptor/I1-imidazoline receptors in the rostral ventrolateral medulla decreases the blood pressure via sympathoinhibition. However, alteration of receptor responses in genetically hypertensive rats remains unclear. We examined cardiovascular responses of α2-adrenoceptor/I1-imidazoline receptor agonist and antagonists microinjected into the rostral ventrolateral medulla of conscious spontaneously hypertensive rats and normotensive Wistar Kyoto rats. Injection of 2-nmol clonidine-an α2-adrenoceptor/I1-imidazoline receptor agonist-unilaterally into the rostral ventrolateral medulla decreased the blood pressure, heart rate, and renal sympathetic nerve activity; the responses were significantly enhanced in spontaneously hypertensive rats than in Wistar Kyoto rats. Co-injection of 2-nmol 2-methoxyidazoxan (a selective α2-adrenoceptor antagonist) or 2-nmol efaroxan (an I1-receptor antagonist) with 2 nmol of clonidine attenuated the hypotensive and bradycardic effects of clonidine-only injection. Injection of 2-methoxyidazoxan alone increased the blood pressure and heart rate in spontaneously hypertensive rats, but not in Wistar Kyoto rats. These results suggest enhanced responsiveness of α2-adrenoceptor/I1-imidazoline receptors in the rostral ventrolateral medulla of spontaneously hypertensive rats.

Locus coeruleus (LC)-spinal noradrenergic projections are important to endogenous analgesic mechanisms and can be activated by local glutamate signaling in the LC. The current study examined the local glutamatergic, GABAergic, and noradrenergic influences on glutamate release in the LC and noradrenergic descending inhibition in rats 6 weeks after spinal nerve ligation (SNL). Intra-LC injection of the α2 adrenoceptor antagonist idazoxan or the group 2 metabotropic glutamate receptor (mGluR) antagonist (RS)-α-Methyl-4-tetrazolylphenylglycine (MTPG) increased withdrawal thresholds in SNL animals and this was reversed by the blockade of α-amino-3-hydroxy-5-methyl- 4-isoxazolepropionic acid (AMPA) receptors in the LC or α2-adrenoceptors in the spinal cord, but not in normal animals. Neither blockade of GABA-A nor GABA-B receptors in the LC affected withdrawal thresholds in normal and SNL animals. Intra-LC perfusion of idazoxan increased extracellular glutamate in the LC in SNL animals but not in normal animals. Intra-LC perfusion of MTPG increased extracellular glutamate in the LC in both normal and SNL animals. These results suggest that local noradrenaline and glutamate tonically inhibit glutamate release in the LC after peripheral nerve injury and this may contribute to reduced descending inhibition in response to noxious input during chronic neuropathic pain.