Adrenoceptor Activation Research Articles

Activating astrocytic α2A adrenoceptors in hippocampus reduces glutamate toxicity to attenuate sepsis-associated encephalopathy in mice

BackgroundGlutamate metabolism disorder is an important mechanism of sepsis-associated encephalopathy (SAE). Astrocytes regulate glutamate metabolism. In septic mice, α2A adrenoceptor (α2A-AR) activation in the central nervous system provides neuroprotection. α2A-ARs are expressed abundantly in hippocampal astrocytes. This study was performed to determine whether hippocampal astrocytic α2A-AR activation confers neuroprotection against SAE and whether this protective effect is astrocyte specific and achieved by the modulation of glutamate metabolism. MethodsMale C57BL/6 mice with and without α2A-AR knockdown were subjected to cecal ligation and puncture (CLP). They were treated with intrahippocampal guanfacine (an α2A-AR agonist) or intraperitoneal dexmedetomidine in the presence or absence of dihydrokainic acid [DHK; a glutamate transporter 1 (GLT-1) antagonist] and/or UCPH-101 [a glutamate/aspartate transporter (GLAST) antagonist]. Hippocampal tissue was collected for the measurement of astrocyte reactivity, GLT-1 and GLAST expression, and glutamate receptor subunit 2B (GluN2B) phosphorylation. In vivo real-time extracellular glutamate concentrations in the hippocampus were measured by ultra-performance liquid chromatography tandem mass spectrometry combined with microdialysis, and in vivo real-time hippocampal glutamatergic neuron excitability was assessed by calcium imaging. The mice were subjected to the Barnes maze and fear conditioning tests to assess their learning and memory. Golgi staining was performed to assess changes in the hippocampal synaptic structure. In vitro, primary astrocytes with and without α2A-AR knockdown were stimulated with lipopolysaccharide (LPS) and treated with guanfacine or dexmedetomidine in the presence or absence of 8-bromo- cyclic adenosine monophosphate (8-Br-cAMP, a cAMP analog). LPS-treated primary and BV2 microglia were also treated with guanfacine or dexmedetomidine. Astrocyte reactivity, PKA catalytic subunit, GLT-1 an GLAST expression were determined in primary astrocytes. Interleukin-1β, interleukin-6 and tumor necrosis factor-alpha in the medium of microglia culture were measured. ResultsCLP induced synaptic injury, impaired neurocognitive function, increased astrocyte reactivity and reduced GLT-1 and GLAST expression in the hippocampus of mice. The extracellular glutamate concentration, phosphorylation of GluN2B at Tyr-1472 and glutamatergic neuron excitability in the hippocampus were increased in the hippocampus of septic mice. Intraperitoneal dexmedetomidine or intrahippocampal guanfacine administration attenuated these effects. Hippocampal astrocytes expressed abundant α2A-ARs; expression was also detected in neurons but not microglia. Specific knockdown of α2A-ARs in hippocampal astrocytes and simultaneous intrahippocampal DHK and UCPH-101 administration blocked the neuroprotective effects of dexmedetomidine and guanfacine. Intrahippocampal administration of DHK or UCPH-101 alone had no such effect. In vitro, guanfacine or dexmedetomidine inhibited astrocyte reactivity, reduced PKA catalytic subunit expression, and increased GLT-1 and GLAST expression in primary astrocytes but not in primary astrocytes that received α2A-AR knockdown or were treated with 8-Br-cAMP. Guanfacine or dexmedetomidine inhibited microglial reactivity in BV2 but not primary microglia. ConclusionsOur results suggest that neurocognitive protection against SAE after hippocampal α2A-AR activation is astrocyte specific. This protection may involve the inhibition of astrocyte reactivity and alleviation of glutamate neurotoxicity, thereby reducing synaptic injury. The cAMP/protein kinase A (PKA) signaling pathway is a potential cellular mechanism by which activating α2A-AR modulates astrocytic function.

β2-Adrenoceptor Activation Favor Acquisition of Tumorigenic Properties in Non-Tumorigenic MCF-10A Breast Epithelial Cells.

Noradrenaline and adrenaline, and their cognate receptors, are currently accepted to participate in cancer progression. They may also participate in cancer initiation, although their role in this phase is much less explored. The aim of this work was to study the influence of adrenergic stimulation in several processes related to breast cancer carcinogenesis, using several adrenergic agonists in the MCF-10A non-tumorigenic breast cells. Activation of the β-adrenoceptors promoted an epithelial phenotype in MCF-10A cells, revealed by an increased expression of the epithelial marker E-cadherin and a decrease in the mesenchymal markers, N-cadherin and vimentin. MCF-10A cell motility and migration were also impaired after the β-adrenoceptors activation. Concomitant with this effect, β-adrenoceptors decrease cell protrusions (lamellipodia and filopodia) while increasing cell adhesion. Activation of the β-adrenoceptors also decreases MCF-10A cell proliferation. When the MCF-10A cells were cultured under low attachment conditions, activation the of β- (likely β2) or of α2-adrenoceptors had protective effects against cell death, suggesting a pro-survival role of these adrenoceptors. Overall, our results showed that, in breast cells, adrenoceptor activation (mainly through β-adrenoceptors) may be a risk factor in breast cancer by inducing some cancer hallmarks, providing a mechanistic explanation for the increase in breast cancer incidences that may be associated with conditions that cause massive adrenergic stimulation, such as stress.

Structural and Functional Impact of Adrenoceptor Beta-1 Gene Polymorphism in Patients with Hypertrophic Cardiomyopathy and Response to Beta-Blocker Therapy.

Hypertrophic cardiomyopathy (HCM) is a genetically inherited cardiac disorder with diverse clinical presentations. Adrenergic activity, primarily mediated through beta-adrenoceptors, plays a central role in the clinical course of HCM. Adrenergic stimulation increases cardiac contractility and heart rate through beta-1 adrenoceptor activation. Beta-blocker drugs are recommended as the primary treatment for symptomatic HCM patients to mitigate these effects. This prospective study aimed to investigate the impact of common ADRB-1 gene polymorphisms, specifically serine-glycine at position 49 and arginine-glycine at position 389, on the clinical and structural aspects of HCM. Additionally, the study explored the association between these genetic variations and the response to beta-blocker therapy in HCM patients. A cohort of 147 HCM patients was enrolled, and comprehensive assessments were performed. The findings revealed that the Ser49Gly polymorphism significantly influenced ventricular ectopic beats, with beta-blocker therapy effectively reducing them in Ser49 homozygous patients. Moreover, natriuretic peptide levels decreased, particularly in Ser49 homozygotes, indicating improved cardiac function. Left ventricular outflow obstruction, a hallmark of HCM, was also reduced following beta-blocker treatment in all patient groups. In contrast, the Arg389Gly polymorphism did not significantly impact baseline parameters or beta-blocker response. These results emphasize the role of the Ser49Gly polymorphism in the ADRB-1 gene in shaping the clinical course and response to beta-blocker therapy in HCM patients. This insight may enable a more personalized approach to managing HCM by considering genetic factors in treatment decisions. Further research with larger populations and longer follow-up periods is needed to confirm and expand upon these findings.

Age-dependent effects of the β3 adrenoceptor agonist CL316,243 on human and rat detrusor muscle strips

Motility of detrusor smooth muscle includes adrenergic relaxation and cholinergic contraction. Since the latter may be deregulated in overactive bladder (OAB) pathophysiology, anticholinergics are the standard therapy but occasionally less tolerated due to side effects such as dry mouth and constipation. β3 adrenoceptor agonists also alleviate OAB symptoms by relaxing the detrusor muscle. Their age dependence, however, is far from understood. To address this issue, we induced contractions with KCl (60 mM) and carbachol (from 10 nM to 100 μM) in the presence of the β3 adrenoceptor agonist CL316,243 (from 0.1 to 10 μM) in both human and rat muscle strips. Our results confirmed that both contractions were attenuated by β3 adrenoceptor activation in both species, but with differing age dependence. In humans, specimens from mid-life subjects showed a significantly more pronounced effect of CL316,243 in attenuating carbachol-induced contractions than those from aged subjects (Cohen’s d of maximal attenuation: 1.82 in mid-life versus 0.13 in aged) without altering EC50. Conversely, attenuation of KCl responses by CL316,243 increased during ageing (Spearman correlation coefficient = -0.584, P<0.01). In rats, both KCl- and carbachol-induced contractions were significantly more attenuated by CL316,243 in samples from adolescent as compared to aged samples. Immunohistochemistry in human detrusor sections proved β3 adrenoreceptor abundance to remain unaltered during ageing. In conclusion, our findings suggest differential age-dependent changes in human β3 adrenoceptor-dependent attenuation of detrusor contraction in terms of electromechanical versus pharmacomechanical coupling; they may help understand the differential responsiveness of OAB patients to β3 agents.

GV16 acupoint stimulation with bee venom reduces peripheral hypersensitivity via activation of α2 adrenoceptors in a nitroglycerin-induced migraine mouse model

BackgroundPeripheral hypersensitivities develop in the face and hindpaws of mice with nitroglycerin (NTG)-induced migraine. We evaluated whether diluted bee venom (DBV) injections at acupoints prevented these peripheral hypersensitivities and c-Fos expression in the trigeminal nucleus caudalis (TNC). MethodsNTG (10 mg/kg, intraperitoneal, i.p.) was administered every other day for nine days. DBV (0.1 mg/kg) was subcutaneously injected into the ST36 (Zusanli), LI4 (Hegu), or GV16 (Fengfu) acupoints 75 min after each NTG injection. Mice were pretreated with naloxone (5 mg/kg, i.p.) or yohimbine (5 mg/kg, i.p.) 30 min before the DBV injections. ResultsNTG injection caused facial cold allodynia, hindpaw mechanical allodynia, and increased c-Fos-immunoreactive (ir) cells in the TNC. Repetitive DBV injections at GV16, but not the ST36, or LI4 acupoints, suppressed NTG-induced hindpaw mechanical allodynia and facial cold allodynia. The number of c-Fos-ir cells also decreased in response to DBV injections at the GV16 acupoint. Remarkably, pretreatment with yohimbine reversed the anti-allodynic effects of DBV injections and attenuated the decreased c-Fos expression in response to GV16 DBV treatment. Naloxone did not block the effects of GV16 DBV stimulation. ConclusionThese findings demonstrate that repetitive DBV treatment at the GV16 acupoint relieves NTG-induced facial and hindpaw hypersensitivities and decreases in c-Fos expression in the TNC via activation of the alpha-2 adrenoceptors, but not the opioid receptors.

Dexmedetomidine relieves inflammatory pain by enhancing GABAergic synaptic activity in pyramidal neurons of the anterior cingulate cortex

Pyramidal neuron (Pyn) hyperactivity in the anterior cingulate cortex (ACC) is involved in the modulation of pain. Previous studies indicate that the activation of α2 adrenoceptors (α2-ARs) by dexmedetomidine (DEX) is a safe and effective means of alleviating multiple types of pain. Here, we showed that systemically administered DEX can ameliorate the inflammatory pain induced by hindpaw injection of formalin (FA) and further examined the molecular and synaptic mechanisms of this DEX-elicited antinociceptive effect. We found that FA caused an increase in c-Fos expression in contralateral layer 2/3 (L2/3) ACC, and that intra-ACC infusion of DEX could also relieve phase 2 inflammatory pain behavior. DEX elicited an increase in the amplitude and frequency of miniature inhibitory post-synaptic currents (mIPSCs) and evoked IPSC amplitude, as well as a reduction in the hyperexcitability and both paired-pulse and excitation/inhibition ratios in contralateral L2/3 ACC Pyns of FA mice. These electrophysiological effects were associated with the upregulation of GABA A receptor (GABAAR) subunits. The interaction of phosphorylated Akt (p-Akt) with GABAAR subunits increased in the ACC following administration of DEX. These results suggest that DEX treatment reduces hyperactivity and enhances GABAergic inhibitory synaptic transmission in ACC Pyns, which produces analgesic effects by increasing GABAAR levels and activating the Akt signaling pathway.

PO-02-147 THE ANTIARRHYTHMIC EFFECTS OF MIRABEGRON IN ISOLATED RABBIT HEARTS

β-3 adrenoceptor (AR) activation opposes the effects of β-1 and β-2 AR activation and rescues the effects of excessive catecholamines. BRL37344 (beta3-AR agonist) is antiarrhythmic in canine models. Mirabegron is a β-3 agonist approved by the FDA for the treatment of overactive bladder. A previous study showed that isoproterenol activates the small conductance Ca-activated K current more in female than in male ventricles. To test the hypothesis that mirabegron reduces the proarrhythmic effects of isoproterenol more effectively in female than in male ventricles. We performed optical mapping studies in 6 male and 6 female Langendorff-perfused rabbit hearts first at baseline, then sequentially after isoproterenol (ISO, 100 nM), mirabegron (1000 nM), apamin (100 nM) administration and washout in study 1. In study 2 (5 males and 5 females), we studied at baseline, then after adding mirabegron (250 nM, 500 nM, 750 nM, and 1000 nM), followed by isoproterenol and washout. All hearts underwent 10 attempts of ventricular fibrillation (VF) induction with a ventricular burst (20 Hz for 10 s) pacing at each stage of the experiment. The number of phase singularities (PSs) was determined per episode of VF (PSs/VF). In study 1, mirabegron reduced the VF inducibility (number of VF after 10 attempts) during isoproterenol infusion by an average of -2.75 (p = 0.026). When VF was induced, the PSs/VF increased by 2.4±2.1 in males and 6.6±3.3 in females (p=0.03, Figure A). Mirabegron significantly reduced the PSs/VF in both sexes and eliminated the sex differences. Apamin given after mirabegron in the presence of isoproterenol did not further change PSs/VF (p=0.63) (Figure B). The conduction velocity (CV), estimated by the inverse of the total activation time (1/s), was 16±2.7 for all hearts at baseline and 35±10 after ISO, respectively (p=0.0006). ISO did not increase CV differentially by sex (p=0.93). Mirabegron reduced the CV by an average of 15.6 (from 34±11.4 to 18±3.9) in females, which was significantly (p=0.043) more than 5.1 (from 35±8.7 to 31±9.3) in males. Figure C shows a summary of study 2. Mirabegron significantly reduced PSs/VF at 750 nM in female (p=0.015 vs. baseline) and at 1000 nM in male (p=0.033 vs. baseline) hearts. The addition of ISO did not increase PSs/VF. During ISO infusion, mirabegron reduces the CV and the PSs/VF more in females than males during VF. These data suggest that the β3 agonist is a sex-specific antiarrhythmic agent.

Moxonidine Increases Uptake of Oxidised Low-Density Lipoprotein in Cultured Vascular Smooth Muscle Cells and Inhibits Atherosclerosis in Apolipoprotein E-Deficient Mice.

This study aimed to investigate the effect of the sympatholytic drug moxonidine on atherosclerosis. The effects of moxonidine on oxidised low-density lipoprotein (LDL) uptake, inflammatory gene expression and cellular migration were investigated in vitro in cultured vascular smooth muscle cells (VSMCs). The effect of moxonidine on atherosclerosis was measured by examining aortic arch Sudan IV staining and quantifying the intima-to-media ratio of the left common carotid artery in apolipoprotein E-deficient (ApoE-/-) mice infused with angiotensin II. The levels of circulating lipid hydroperoxides in mouse plasma were measured by ferrous oxidation-xylenol orange assay. Moxonidine administration increased oxidised LDL uptake by VSMCs via activation of α2 adrenoceptors. Moxonidine increased the expression of LDL receptors and the lipid efflux transporter ABCG1. Moxonidine inhibited mRNA expression of inflammatory genes and increased VSMC migration. Moxonidine administration to ApoE-/- mice (18 mg/kg/day) decreased atherosclerosis formation in the aortic arch and left common carotid artery, associated with increased plasma lipid hydroperoxide levels. In conclusion, moxonidine inhibited atherosclerosis in ApoE-/- mice, which was accompanied by an increase in oxidised LDL uptake by VSMCs, VSMC migration, ABCG1 expression in VSMCs and lipid hydroperoxide levels in the plasma.

Carotid dysfunction in senescent female mice is mediated by increased α1A-adrenoceptor activity and COX-derived vasoconstrictor prostanoids.

α-Adrenergic receptors are crucial regulators of vascular hemodynamics and essential pharmacological targets for cardiovascular diseases. With aging, there is an increase in sympathetic activation, which could contribute to the progression of aging-associated cardiovascular dysfunction, including stroke. Nevertheless, there is little information directly associating adrenergic receptor dysfunction in the blood vessels of aged females. This study determined the role of a-adrenergic receptors in carotid dysfunction of senescent female mice (accelerated-senescence prone, SAMP8), compared with a nonsenescent (accelerated-senescence prone, SAMR1). Vasoconstriction to phenylephrine (Phe) was markedly increased in common carotid artery of SAMP8 [area under the curve (AUC), 527 ± 53] compared with SAMR1 (AUC, 334 ± 30, P = 0.006). There were no changes in vascular responses to the vasoconstrictor agent U46619 or the vasodilators acetylcholine (ACh) and sodium nitroprusside (NPS). Hyperactivity to Phe in female SAMP8 was reduced by cyclooxygenase-1 and cyclooxygenase-2 inhibition and associated with augmented ratio of TXA2/PGI2 release (SAMR1, 1.1 ± 0.1 vs. SAMP8, 2.1 ± 0.3, P = 0.007). However, no changes in cyclooxygenase expression were seen in SAMP8 carotids. Selective α1A-receptor antagonism markedly reduced maximal contraction, whereas α1D antagonism induced a minor shift in Phe contraction in SAMP8 carotids. Ligand binding analysis revealed a threefold increase of α-adrenergic receptor density in smooth muscle cells (VSMCs) of SAMP8 vs. SAMR1. Phe rapidly increased intracellular calcium (Cai2+) in VSMCs via the α1A-receptor, with a higher peak in VSMCs from SAMP8. In conclusion, senescence intensifies vasoconstriction mediated by α1A-adrenergic signaling in the carotid of female mice by mechanisms involving increased Cai2+ and release of cyclooxygenase-derived prostanoids.NEW & NOTEWORTHY The present study provides evidence that senescence induces hyperreactivity of α1-adrenoceptor-mediated contraction of the common carotid. Impairment of α1-adrenoceptor responses is linked to increased Ca2+ influx and release of COX-derived vasoconstrictor prostanoids, contributing to carotid dysfunction in the murine model of female senescence (SAMP8). Increased reactivity of the common carotid artery during senescence may lead to morphological and functional changes in arteries of the cerebral microcirculation and contribute to cognitive decline in females. Because the elderly population is growing, elucidating the mechanisms of aging- and sex-associated vascular dysfunction is critical to better direct pharmacological and lifestyle interventions to prevent cardiovascular risk in both sexes.

Locus Coeruleus and Noradrenergic Pharmacology in Neurodegenerative Disease.

Adrenoceptors (ARs) throughout the brain are stimulated by noradrenaline originating mostly from neurons of the locus coeruleus, a brainstem nucleus that is ostensibly the earliest to show detectable pathology in neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. The α1-AR, α2-AR, and β-AR subtypes expressed in target brain regions and on a range of cell populations define the physiological responses to noradrenaline, which includes activation of cognitive function in addition to modulation of neurometabolism, cerebral blood flow, and neuroinflammation. As these heterocellular functions are critical for maintaining brain homeostasis and neuronal health, combating the loss of noradrenergic tone from locus coeruleus degeneration may therefore be an effective treatment for both cognitive symptoms and disease modification in neurodegenerative indications. Two pharmacologic approaches are receiving attention in recent clinical studies: preserving noradrenaline levels (e.g., via reuptake inhibition) and direct activation of target adrenoceptors. Here, we review the expression and role of adrenoceptors in the brain, the preclinical studies which demonstrate that adrenergic stimulation can support cognitive function and cerebral health by reversing the effects of noradrenaline depletion, and the human data provided by pharmacoepidemiologic analyses and clinical trials which together identify adrenoceptors as promising targets for the treatment of neurodegenerative disease.

β3 adrenoceptor agonist mirabegron protects against right ventricular remodeling and drives Drp1 inhibition.

The right ventricular (RV) function determines the prognosis of patients with pulmonary hypertension (PH). Metabolic disorders have been observed in the RV myocardium in PH. Activation of the β3 adrenoceptor improves cardiac function and restores cardiac metabolic efficiency in rodents with heart failure; however, its role in the RV remains uncertain. Experimental PH was induced by monocrotaline (MCT) in rats. Mirabegron, a selective β3 adrenoceptor agonist, was given to MCT rats daily from the day after MCT injection at the dose of 10 mg/kg. In vivo echocardiography and RV catheterization were performed to assess RV hemodynamics, structure, and function. RV fibrosis and hypertrophy were assessed by Sirius Red (SR) and wheat germ agglutinin (WGA) staining respectively. Western blotting was performed to examine the markers of RV fibrosis and hypertrophy, as well as the levels of the key molecules and their phosphorylated forms. The molecular changes were confirmed in the cardiac hypertrophy model of angiotensin II (Ang II) treated H9c2 cardiomyocytes using western blotting. The overloaded RV had increased β3 adrenoceptor expression, which was further increased by mirabegron. Mirabegron reduced RV pressure and reduced RV structural and functional deterioration in MCT rats. Mirabegron decreased cardiac fibrosis and hypertrophy in the overloaded RV. Mirabegron suppressed dynaminrelated protein 1 (Drp1) and promoted AMP-activated protein kinase (AMPK) signaling in the overloaded RV and Ang II treated cardiomyocytes. The β3 adrenoceptor agonist mirabegron reduced RV hypertrophy and fibrosis in PH rats. The treatment effect involved Drp1 inhibition and AMPK activation.

Overlooked switch from transient sedation to sustained excitement in the Biphasic effects of Ephedra Herb extract administered orally to mice

Ethnopharmacological relevanceIn our previous study, we reported that Ephedra Herb extract (EHE) increased the locomotor activity of mice in the open-field test and reduced the immobility time in the forced swim test. Ephedrine alkaloids (EAs) are thought to be responsible for the adverse effects of Ephedra Herb. However, there are no reports to verify that the adverse effects of Ephedra Herb are caused by the amount of EAs in the herb. Therefore, we investigated whether these adverse effects of EHE are caused by the amounts of ephedrine (Eph) and pseudoephedrine (Pse) in the herbal extract. In a preliminary study of the time course analysis of the open field test, we newly observed that EHE evoked switching from transient sedation to sustained excitement. Aim of the studyWe aimed to confirm whether EHE evokes switching from transient sedation to sustained excitement, investigate whether these actions of EHE are caused by the amount of ephedrine (Eph) and pseudoephedrine (Pse) in the herbal extract, and clarify the molecular mechanism of the transient sedative effect. Materials and methodsThe locomotor activity of mice was tested using the open-field test. The immobility times were measured using a forced swim test, and the motor dysfunction in mice was tested using the rotarod test. ResultsEHE, Eph, and Pse induced transient motionlessness between 0 and 15 min after oral administration, however, they did not induce depression-like behavior and motor dysfunction in mice, suggesting that the motionlessness induced by EHE, Eph, or Pse resulted from sedation. The α2a adrenoceptor inhibitor, atipamezole, decreased their sedative effects. Thus, immediately after EHE administration, the transient sedative effect is mediated through the activation of the α2a adrenoreceptor by Eph and Pse. EHE and Eph increased total locomotor activity for 15–120 min after oral administration; however, Pse had no effect. Therefore, the slow-onset and sustained excitatory effects of EHE are mediated by Eph. ConclusionsWe discovered for the first time that EHE evokes diphasic action by switching from transient sedation to sustained excitement. The transient sedation was evoked by the Eph and Pse in the herbal extract via activation of the α2a adrenoceptor and the sustained excitement was caused by the Eph in the herbal extract.

Adrenergic receptors control frequency-dependent switching of the exocytosis mode between “full-collapse” and “kiss-and-run” in murine motor nerve terminal

AimsNeurotransmitter release from the synaptic vesicles can occur through two modes of exocytosis: “full-collapse” or “kiss-and-run”. Here we investigated how increasing the nerve activity and pharmacological stimulation of adrenoceptors can influence the mode of exocytosis in the motor nerve terminal. MethodsRecording of endplate potentials with intracellular microelectrodes was used to estimate acetylcholine release. A fluorescent dye FM1–43 and its quenching with sulforhodamine 101 were utilized to visualize synaptic vesicle recycling. Key findingsAn increase in the frequency of stimulation led to a decrease in the rate of FM1–43 unloading despite the higher number of quanta released. High frequency activity promoted neurotransmitter release via the kiss-and-run mechanism. This was confirmed by experiments utilizing (I) FM1–43 dye quencher, that is able to pass into the synaptic vesicle via fusion pore, and (II) loading of FM1–43 by compensatory endocytosis. Noradrenaline and specific α2-adrenoreceptors agonist, dexmedetomidine, controlled the mode of synaptic vesicle recycling at high frequency activity. Their applications favored neurotransmitter release via full-collapse exocytosis rather than the kiss-and-run pathway. SignificanceAt the diaphragm neuromuscular junctions, neuronal commands are translated into contractions necessary for respiration. During stress, an increase in discharge rate of the phrenic nerve shifts the exocytosis from the full-collapse to the kiss-and-run mode. The stress-related molecule, noradrenaline, restricts neurotransmitter release in response to a high frequency activity, and prevents the shift in the mode of exocytosis through α2-adrenoceptor activation. This may be a component of the mechanism that limits overstimulation of the respiratory system during stress.

Characterisation of bis(4-aminoquinoline)s as α1A adrenoceptor allosteric modulators

The development of sub-type selective α1 adrenoceptor ligands has been hampered by the high sequence similarity of the amino acids forming the orthosteric binding pocket of the three α1 adrenoceptor subtypes, along with other biogenic amine receptors. One possible approach to overcome this issue is to target allosteric sites on the α1 adrenoceptors. Previous docking studies suggested that one of the quinoline moieties of a bis(4-aminoquinoline), comprising a 9-carbon methylene linker attached via the amine groups, could interact with residues outside of the orthosteric binding site while, simultaneously, the other quinoline moiety bound within the orthosteric site. We therefore hypothesized that this compound could act in a bitopic manner, displaying both orthosteric and allosteric binding properties. To test this proposition, we investigated the allosteric activity of a series of bis(4-aminoquinoline)s with linker lengths ranging from 2 to 12 methylene units (designated C2–C12). A linear trend of increasing [3H]prazosin dissociation rate with increasing linker length between C7 and C11 was observed, confirming their action as allosteric modulators. These data suggest that the optimal linker length for the bis(4-aminoquinoline)s to occupy the allosteric site of the α1A adrenoceptor is between 7 and 11 methylene units. In addition, the ability of C9 bis(4-aminoquinoline) to modulate the activation of the α1A adrenoceptor by norepinephrine was subsequently examined, showing that C9 acts as a non-competitive antagonist. Our findings indicate that the bis(4-aminoquinolines) are acting as allosteric modulators of orthosteric ligand binding, but not efficacy, in a bitopic manner.

α-1 Adrenoceptor Activation in the Dorsal Raphe Nucleus Decreases Food Intake in Fasted Rats.

The dorsal raphe (DR) nucleus is involved in a myriad of physiological functions, such as the control of sleep-wake cycle, motivation, pain, energy balance, and food intake. We have previously demonstrated that in ad libitum fed rats the intra-DR administration of phenylephrine, an α-1 receptor agonist, does not affect food intake, whereas clonidine, an α-2 receptor agonist, potently stimulates food intake. These results indicated that in fed rats an increased adrenergic tonus blocked food intake, since the activation of α-2 auto-receptors, which decreases pre-synaptic release of adrenaline/noradrenaline, affected food intake. Thus, in this study we assessed whether the response to adrenergic stimuli would differ after overnight fasting, a situation of low adrenergic activity in the DR. Intra-DR administration of adrenaline and noradrenaline blocked food intake evoked by overnight fasting. Similarly, phenylephrine administration decreased hunger-induced food intake. These changes in food intake were accompanied by changes in other behaviors, such as increased immobility time and feeding duration. On the other hand, intra-DR administration of clonidine did not affect food-intake or associated behaviors. These results further support the hypothesis that in fed animals, increased adrenergic tonus in DR neurons inhibiting feeding, while in fasted rats the adrenergic tonus decreases and favors food intake. These data indicate a possible mechanism through which adrenergic input to the DRN contributes to neurobiology of feeding.

Adrenergic α2 receptors are implicated in seizure-induced respiratory arrest in DBA/1 mice

AimsSudden unexpected death in epilepsy (SUDEP) is a serious and underestimated public health burden. Both clinical and animal studies show that seizure-induced respiratory arrest (S-IRA) is the primary cause of death in SUDEP. Our previous studies demonstrated that atomoxetine, a norepinephrine reuptake inhibitor (NRI), suppresses S-IRA in DBA/1 mice, suggesting that noradrenergic neurotransmission modulates S-IRA. However, it remains unclear which adrenoceptors are implicated in S-IRA in DBA/1 mice. Materials and methodsNaïve DBA/1 mice exhibit a low incidence of S-IRA, but after primed by acoustic stimulation, they become consistently susceptible to S-IRA. Atomoxetine, adrenoceptor agonists, antagonists or vehicle was intraperitoneally (i.p.) administered alone or in combination, and the effects of drug treatments on S-IRA incidence and seizure behaviors were examined. Key findingsThe incidence of S-IRA in primed DBA/1 mice was significantly reduced by clonidine, an α2 adrenoceptor agonist, as compared with that of the vehicle control. However, compared with the vehicle control, S-IRA was not altered by cirazoline, an α1 agonist. Consistent with previous reports, atomoxetine reduced S-IRA in primed DBA/1 mice. The suppressing effect of atomoxetine on S-IRA was prevented by injection of an α2 adrenoceptor antagonist, yohimbine or atipamezole, but not by prazosin, an α1 antagonist. Administration of α1 or α2 antagonists alone did not promote the incidence of S-IRA in nonprimed DBA/1 mice. SignificanceThese data demonstrate that noradrenergic neurotransmission modulates S-IRA predominantly via α2 adrenoceptors in DBA/1 mice, indicating that selective activation of α2 adrenoceptors can potentially prevent SUDEP.

Selective activation of adrenoceptors potentiates IKs current in pulmonary vein cardiomyocytes through the protein kinase A and C signaling pathways

Delayed rectifier K+ current (IKs) is a key contributor to repolarization of action potentials. This study investigated the mechanisms underlying the adrenoceptor-induced potentiation of IKs in pulmonary vein cardiomyocytes (PVC). PVC were isolated from guinea pig pulmonary vein. The action potentials and IKs current were recorded using perforated and conventional whole-cell patch-clamp techniques. The expression of IKs was examined using immunocytochemistry and Western blotting. KCNQ1, a IKs pore-forming protein was detected as a signal band approximately 100 kDa in size, and its immunofluorescence signal was found to be mainly localized on the cell membrane. The IKs current in PVC was markedly enhanced by both β1- and β2-adrenoceptor stimulation with a negative voltage shift in the current activation, although the potentiation was more effectively induced by β2-adrenoceptor stimulation than β1-adrenoceptor stimulation. Both β-adrenoceptor-mediated increases in IKs were attenuated by treatment with the adenylyl cyclase (AC) inhibitor or protein kinase A (PKA) inhibitor. Furthermore, the IKs current was increased by α1-adrenoceptor agonist but attenuated by the protein kinase C (PKC) inhibitor. PVC exhibited action potentials in normal Tyrode solution which was slightly reduced by HMR-1556 a selective IKs blocker. However, HMR-1556 markedly reduced the β-adrenoceptor-potentiated firing rate. The stimulatory effects of β- and α1-adrenoceptor on IKs in PVC are mediated via the PKA and PKC signal pathways. HMR-1556 effectively reduced the firing rate under β-adrenoceptor activation, suggesting that the functional role of IKs might increase during sympathetic excitation under in vivo conditions.

Chronic Administrations of Guanfacine on Mesocortical Catecholaminergic and Thalamocortical Glutamatergic Transmissions.

It has been established that the selective α2A adrenoceptor agonist guanfacine reduces hyperactivity and improves cognitive impairment in patients with attention-deficit/hyperactivity disorder (ADHD). The major mechanisms of guanfacine are considered to involve the activation of the postsynaptic α2A adrenoceptor of glutamatergic pyramidal neurons in the frontal cortex, but the effects of chronic guanfacine administration on catecholaminergic and glutamatergic transmissions associated with the orbitofrontal cortex (OFC) are yet to be clarified. The actions of guanfacine on catecholaminergic transmission, the effects of acutely local and systemically chronic (for 7 days) administrations of guanfacine on catecholamine release in pathways from the locus coeruleus (LC) to OFC, the ventral tegmental area (VTA) and reticular thalamic-nucleus (RTN), from VTA to OFC, from RTN to the mediodorsal thalamic-nucleus (MDTN), and from MDTN to OFC were determined using multi-probe microdialysis with ultra-high performance liquid chromatography. Additionally, the effects of chronic guanfacine administration on the expression of the α2A adrenoceptor in the plasma membrane fraction of OFC, VTA and LC were examined using a capillary immunoblotting system. The acute local administration of therapeutically relevant concentrations of guanfacine into the LC decreased norepinephrine release in the OFC, VTA and RTN without affecting dopamine release in the OFC. Systemically, chronic administration of therapeutically relevant doses of guanfacine for 14 days increased the basal release of norepinephrine in the OFC, VTA, RTN, and dopamine release in the OFC via the downregulation of the α2A adrenoceptor in the LC, OFC and VTA. Furthermore, systemically, chronic guanfacine administration did not affect intrathalamic GABAergic transmission, but it phasically enhanced thalamocortical glutamatergic transmission. The present study demonstrated the dual actions of guanfacine on catecholaminergic transmission—acute attenuation of noradrenergic transmission and chronic enhancement of noradrenergic transmission and thalamocortical glutamatergic transmission. These dual actions of guanfacine probably contribute to the clinical effects of guanfacine against ADHD.

Effect of β1 /β2 -adrenoceptor blockade on β3 -adrenoceptor activity in the rat cremaster muscle artery.

The physiological role of vascular β3 -adrenoceptors is not fully understood. Recent evidence suggests cardiac β3 -adrenoceptors are functionally effective after down-regulation of β1 /β2 -adrenoceptors. The functional interaction between the β3 -adrenoceptor and other β-adrenoceptor subtypes in rat striated muscle arteries was investigated. Studies were performed in cremaster muscle arteries isolated from male Sprague-Dawley rats. β-adrenoceptor expression was assessed through RT-PCR and immunofluorescence. Functional effects of β3 -adrenoceptor agonists and antagonists and other β-adrenoceptor ligands were measured using pressure myography. All three β-adrenoceptor subtypes were present in the endothelium of the cremaster muscle artery. The β3 -adrenoceptor agonists mirabegron and CL 316,243 had no effect on the diameter of pressurized (70 mmHg) cremaster muscle arterioles with myogenic tone, while the β3 -adrenoceptor agonist SR 58611A and the nonselective β-adrenoceptor agonist isoprenaline caused concentration-dependent dilation. In the presence of β1/2 -adrenoceptor antagonists nadolol (10 μM), atenolol (1 μM) and ICI 118,551 (0.1 μM) both mirabegron and CL 316,243 were effective in causing vasodilation and the potency of SR 58611A was enhanced, while responses to isoprenaline were inhibited. The β3 -adrenoceptor antagonist L 748,337 (1 μM) inhibited vasodilation caused by β3 -adrenoceptor agonists (in the presence of β1/2 -adrenoceptor blockade), but L 748,337 had no effect on isoprenaline-induced vasodilation. All three β-adrenoceptor subtypes were present in the endothelium of the rat cremaster muscle artery, but β3 -adrenoceptor mediated vasodilation was only evident after blockade of β1/2 -adrenoceptors. This suggests constitutive β1/2 -adrenoceptor activity inhibits β3 -adrenoceptor function in the endothelium of skeletal muscle resistance arteries.

Exercise Training Stabilizes RyR2-Dependent Ca2+ Release in Post-infarction Heart Failure.

Aim: Dysfunction of the cardiac ryanodine receptor (RyR2) is an almost ubiquitous finding in animal models of heart failure (HF) and results in abnormal Ca2+ release in cardiomyocytes that contributes to contractile impairment and arrhythmias. We tested whether exercise training (ET), as recommended by current guidelines, had the potential to stabilize RyR2-dependent Ca2+ release in rats with post-myocardial infarction HF.Materials and Methods: We subjected male Wistar rats to left coronary artery ligation or sham operations. After 1 week, animals were characterized by echocardiography and randomized to high-intensity interval ET on treadmills or to sedentary behavior (SED). Running speed was adjusted based on a weekly VO2max test. We repeated echocardiography after 5 weeks of ET and harvested left ventricular cardiomyocytes for analysis of RyR2-dependent systolic and spontaneous Ca2+ release. Phosphoproteins were analyzed by Western blotting, and beta-adrenoceptor density was quantified by radioligand binding.Results: ET increased VO2max in HF-ET rats to 127% of HF-SED (P < 0.05). This coincided with attenuated spontaneous SR Ca2+ release in left ventricular cardiomyocytes from HF-ET but also reduced Ca2+ transient amplitude and slowed Ca2+ reuptake during adrenoceptor activation. However, ventricular diameter and fractional shortening were unaffected by ET. Analysis of Ca2+ homeostasis and major proteins involved in the regulation of SR Ca2+ release and reuptake could not explain the attenuated spontaneous SR Ca2+ release or reduced Ca2+ transient amplitude. Importantly, measurements of beta-adrenoceptors showed a normalization of beta1-adrenoceptor density and beta1:beta2-adrenoceptor ratio in HF-ET.Conclusion: ET increased aerobic capacity in post-myocardial infarction HF rats and stabilized RyR2-dependent Ca2+ release. Our data show that these effects of ET can be gained without major alterations in SR Ca2+ regulatory proteins and indicate that future studies should include upstream parts of the sympathetic signaling pathway.