Emotionally significant stimuli, including potential threats from the external environment, trigger an increase in body temperature, a response known as emotional hyperthermia. Sympathetically-mediated brown adipose tissue (BAT) thermogenesis contributes substantially to this hyperthermic response. The systemic administration of α2-adrenergic agonists is known to inhibit both febrile and shivering responses. In the present study, we investigated whether systemic administration of clonidine, a α2-adrenoceptor agonist, attenuates the emotional hyperthermia evoked in conscious unrestrained rats suddenly confronted with a second (intruder) rat, itself confined to a small cage. Pre-implanted thermistors were used to measure BAT and body temperature in conscious, freely moving, male Sprague-Dawley rats. The rats were pre-treated with intraperitoneally administered vehicle (Ringer solution) or clonidine (1, 10 and 100 µg/kg). Clonidine, in a dose-dependent manner, reduced the intruder-elicited increases in BAT (log-dose linear regression F(1,16) = 9.52, R2 = 0.37, P < 0.01) and body temperature (F(1,16) = 6.48, R2 = 0.29, P < 0.05). We also investigated, in anesthetized rats, whether systemic clonidine administration inhibits BAT sympathetic nerve discharge evoked via activation of neurons in the lateral habenula (LHb) - a nucleus involved in the regulation of emotional hyperthermia. In anesthetized rats, clonidine abolished the BAT sympathetic nerve discharges elicited via bicuculline-mediated disinhibition of the LHb. These results suggest that activation of central α2-adrenergic receptors attenuates the process of emotional hyperthermia by reduction of BAT thermogenesis.

Rewarming from accidental hypothermia is often complicated by hypothermia-induced cardiac dysfunction, calling for immediate pharmacologic intervention. Studies show that although cardiac pharmacologic support is applied when rewarming these patients, a lack of updated treatment recommendations exist. Mainly due to lack of clinical and experimental data, neither of the international guidelines includes information about pharmacologic cardiac support at temperatures below 30 °C. However, core temperature of accidental hypothermia patients is often reduced below 30 °C. Few human studies exploring effects of adrenergic drugs during hypothermia have been published, and therefore prevailing information is collected from pre-clinical studies. The most prominent finding in these studies is an apparent depressive effect of adrenaline on cardiac function when used in doses which elevate cardiac output during normothermia. Also noradrenaline and isoprenaline largely lacked positive cardiac effects during hypothermia, while dopamine is a more promising drug for supporting cardiac function during rewarming. Data and information from these studies are in support of the prevailing notion; not to use adrenergic drugs at core temperatures below 30 °C.

The role of adrenergic innervation in the regulation of lacrimal gland (LG) ductal fluid secretion is unknown. The Aim of the present study was to investigate the effect of adrenergic stimulation on fluid secretion in isolated LG duct segments and to study the underlying intracellular mechanisms.

The β3 -adrenergic receptor (β3 -AR) is found in several tissues such as adipose tissue and urinary bladder. It is a therapeutic target because it plays a role in thermogenesis, lipolysis, and bladder relaxation. Two β3 -AR agonists are used clinically: mirabegron 1 and vibegron 2, which are indicated for overactive bladder syndrome. However, these drugs show adverse effects, including increased blood pressure in mirabegron patients. Hence, new β3 -AR agonists are needed as starting points for drug development. Previous pharmacophore modeling studies of the β3 -AR did not involve experimental in vitro validation. Therefore, this study aimed to conduct prospective virtual screening and confirm the biological activity of virtual hits. Ligand-based pharmacophore modeling was performed since no 3D structure of human β3 -AR is yet available. A dataset consisting of β3 -AR agonists was prepared to build and validate the pharmacophore models. The best model was employed for prospective virtual screening, followed by physicochemical property filtering and a docking evaluation. To confirm the activity of the virtual hits, an in vitro assay was conducted, measuring cAMP levels at the cloned β3 -AR. Out of 35 tested compounds, 4 compounds were active in CHO-K1 cells expressing the human β3 -AR, and 8 compounds were active in CHO-K1 cells expressing the mouse β3 -AR.

Fragment-based drug discovery has emerged as an alternative to conventional lead identification and optimization strategies generally supported by biophysical detection techniques. Membrane targets like G protein-coupled receptors (GPCRs), however, offer challenges in lack of generic immobilization or stabilization methods for the dynamic, membrane-bound supramolecular complexes. Also modeling of different functional states of GPCRs proved to be a challenging task. Here we report a functional cell-based high concentration screening campaign for the identification of adrenergic α2C receptor agonists compared with the virtual screening of the same ligand set against an active-like homology model of the α2C receptor. The conventional calcium mobilization-based assay identified active fragments with a similar incidence to several other reported fragment screens on GPCRs. 16 out of 3071 screened fragments turned out as specific ligands of α2C, two of which were identified by virtual screening as well and several of the hits possessed surprisingly high affinity and ligand efficiency. Our results indicate that in vitro biological assays can be utilized in the fragment hit identification process for GPCR targets.

In previous works it was shown that catecholamine-induced hypodipsia is mediated by alpha1-adrenergic receptors while food intake (FI) inhibition supposes also beta-adrenergic participation. We used sodium nitroprusside (N) as a vasodilator, alone or mixed with various adrenergic agonists and measured FI and water intake (WI) in rats either deprived food and water overnight or in postprandial conditions after only 1 hour of deprivation in day time. N injected alone had no effect after overnight deprivation but diminished significantly norepinephrine (NE)-induced inhibition of both intakes, while epinephrine (E) inhibited only FI. In day time, N stimulated 30 min FI by 60% and WI by 84% in male but not in female rats. Isoproterenol (I) stimulated only WI (by 155%), while phenylephrine (P) and E inhibited it by 55%. In the presence of N, I increased WI even more (by 220%) but reduced FI. P + N and E + N increased FI by 41% and 128% as compared with P and E, respectively. Only P-induced inhibition of WI was canceled in presence of N. The results show that N, probably due to nitric oxide production, may induce hyperphagia and hyperdipsia in 1 hour-deprived male rats and also that catecholamine effects on FI and WI are differently modulated by N.

The autonomic nervous system, consisting of sympathetic and parasympathetic branches, plays an important role in regulating metabolic homeostasis. The sympathetic nervous system (SNS) regulates hepatic lipid metabolism by regulating adrenergic receptor activation, resulting in the stimulation of hepatic very-low-density lipoprotein-triglyceride (TG) production in vivo. However, only a few studies on the relationship between SNS and hepatic steatosis have been reported. Here, we investigate the effect of adrenergic receptor agonists on hepatic steatosis in mice fed a high-fat diet (HFD). The α-adrenergic receptor agonist phenylephrine (10 mg/kg/d) or the β-adrenergic receptor agonist isoproterenol (30 mg/kg/d) was coadministered with HFD to male mice. After five weeks, hepatic steatosis, TG levels, and hepatic fat metabolism-related biomarkers were examined. HFD treatment induced hepatic steatosis, and cotreatment with phenylephrine, but not isoproterenol, attenuated this effect. Phenylephrine administration upregulated the mRNA levels of hepatic peroxisome proliferator-activated receptor alpha and its target genes (such as carnitine palmitoyltransferase 1) and increased hepatic β-hydroxybutyrate levels. Additionally, phenylephrine treatment increased the expression of the autophagosomal marker LC3-II but decreased that of p62, which is selectively degraded during autophagy. These results indicate that phenylephrine inhibits hepatic steatosis through stimulation of β-oxidation and autophagy in the liver.

Vesicular monoamine transporter 2 (VMAT2) is responsible for packing monoamine neurotransmitters into synaptic vesicles for storage and subsequent neurotransmission. VMAT2 inhibitors are approved for symptomatic treatment of tardive dyskinesia and Huntington's chorea, but despite being much-studied inhibitors their exact binding site and mechanism behind binding and inhibition of monoamine transport are not known. Here we report the identification of several approved drugs, notably β2-adrenergic agonists salmeterol, vilanterol and formoterol, β2-adrenergic antagonist carvedilol and the atypical antipsychotic ziprasidone as inhibitors of rat VMAT2. Further, plausible binding modes of the established VMAT2 inhibitors reserpine and tetrabenazine and hit compounds salmeterol and ziprasidone were identified using molecular dynamics simulations and functional assays using VMAT2 wild-type and mutants. Our findings show VMAT2 as a potential off-target of treatments with several approved drugs in use today and can also provide important first steps in both drug repurposing and therapy development targeting VMAT2 function.

In a cell line, stably expressing α1A-adrenoceptors fused to the mCherry red fluorescent protein, noradrenaline, methoxamine, and oxymetazoline induced concentration-dependent increases in intracellular calcium. All of these agents increase α1A-adrenoceptor phosphorylation and internalization. Transient co-expression of these receptors with Rab proteins tagged with the enhanced Green Fluorescent Protein was employed to estimate α1A-adrenoceptor-Rab interaction using Förster Resonance Energy Transfer. Noradrenaline and methoxamine increased α1A-adrenoceptor interaction with Rab5 and Rab7 but did not modify it with Rab9. Oxymetazoline induced adrenoceptor interaction with Rab5 and Rab9 and only an insignificant increase in Rab7 signal. Phorbol myristate acetate increased α1A-adrenoceptor interaction with Rab5 and Rab9 but did not modify it with Rab7. The agonists and the active phorbol ester, all of which induce receptor phosphorylation and internalization, favor receptor interaction with Rab5, i.e., association with early endosomes. Cell stimulation with phorbol myristate acetate induced the α1A-adrenoceptors to interact with the late endosomal marker, Rab9, suggesting that the receptors are directed to slow recycling endosomes once they have transited to the Trans-Golgi network to be retrieved to the plasma membrane. The agonists noradrenaline and methoxamine likely induce a faster recycling and might direct some of the adrenoceptors toward degradation and/or very slow recycling to the plasma membrane. Oxymetazoline produced a mixed pattern of interaction with the Rab proteins. These data indicate that α1A-adrenoceptor agonists can trigger different vesicular traffic and receptor fates within the cells.

Millions of patients suffer from asthma worldwide. However, the first-line drugs used to treat asthma, namely, the beta-adrenergic receptors agonists (β-agonists), are not recommended for use as monotherapy because of their severe dose-related side effects. This limitation has prompted the search for new therapies, which can be used in conjunction with β--agonists so that lower doses can be administered. Sinigrin is a major compound found in many antiasthmatic medicinal plants. In this study, we explored the antiasthmatic activity of sinigrin when used in combination with β-agonists and its underlying mechanism. Sinigrin enhanced the asthma-relieving effects of isoproterenol and reduced the effective isoproterenol dose in an acute-asthma model in guinea pigs. Mechanistically, sinigrin enhanced the cAMP levels induced by β-agonists by inhibiting PDE4. The resulting increase in cAMP levels stimulated the activity of the downstream effector protein kinase A, which would be expected to ultimately induce the relaxation of airway smooth muscle. In conclusion, sinigrin enhances the asthma-relieving effects of β-agonists by regulating the cAMP signaling pathway and represents a potential add-on drug to β-agonists for the treatment of asthma.

The purpose of this study was to carry an extended literature review to compare antimuscarnics with beta adrenergic agonists (mirabegron) in treatment of overactive bladder.

Pulmonary edema (PE) is an issue widely noted in acute exposure to hypoxia as seen in high altitude climbers, yet the etiology of this is not defined. Previous studies in rats showed that both hypoxia and strong sympathetic activation may induce PE. As acute exposure to hypoxia is accompanied by sympathetic activation, we assume that this may impair pulmonary circulation and contribute to the development of hypoxic PE. The aim of the present study was to investigate the effects of adrenergic agonists and antagonists as models for overstimulation and suppression, respectively, of sympathetic activity on cardiovascular function and formation of PE in hypoxic rats. Norepinephrine or adrenergic blockers were infused to rats exposed to normobaric hypoxia with 10% O2 over time intervals up to 24 h. Normoxic and hypoxic controls received 0.9% NaCl infusion. We evaluated hemodynamic function and lung histology. A significant decrease of left ventricular systolic function was observed after 6 h of hypoxia. This effect was less pronounced with α-adrenergic blockade but was more severe with combined α-plus β-adrenergic blockade. Norepinephrine delayed the onset of hypoxic left ventricular depression but did not reduce its degree. Significant PE developed after 16 h of hypoxia. It regressed under α- but not with β-adrenergic blockade, and was aggravated by combining hypoxia with norepinephrine. Almost half of the animals exposed to hypoxia over 16-24 h suffered cardiorespiratory arrest during the experiment and presented with signs of acute right ventricular failure. They had significantly elevated serum catecholamine concentrations and significantly stronger PE than the others. Notably, most of them had received norepinephrine or combined adrenergic blockade. Mild changes in serum catecholamine concentrations indicated that hypoxic sympathoadrenergic activation was only weak. Hence, it was not sufficient to prevent left ventricular depression. However, the results show that α-adrenergic mechanisms contribute to the formation of hypoxic PE. Adrenergic blockade but also sympathetic overactivity may induce pulmonary congestion, PE and acute right ventricular failure indicating that a fine balance of sympathetic activation under hypoxic conditions is crucial. This has important implications for climbers to high altitude as well as for patients suffering from hypoxia.

The biased agonism of the G protein-coupled receptors (GPCRs), where in addition to a traditional G protein-signaling pathway a GPCR promotes intracellular signals though β-arrestin, is a novel paradigm in pharmacology. Biochemical and biophysical studies have suggested that a GPCR forms a distinct ensemble of conformations signaling through the G protein and β-arrestin. Here we report on the dynamics of the β2 adrenergic receptor bound to the β-arrestin and G protein-biased agonists and the empty receptor to further characterize the receptor conformational changes caused by biased agonists. We use conventional and accelerated molecular dynamics (aMD) simulations to explore the conformational transitions of the GPCR from the active state to the inactive state. We found that aMD simulations enable monitoring of the transition within the nanosecond time scale while capturing the known microscopic characteristics of the inactive states, such as the ionic lock, the inward position of F6.44, and water clusters. Distinct conformational states are shown to be stabilized by each biased agonist. In particular, in simulations of the receptor with the β-arrestin-biased agonist N-cyclopentylbutanepherine, we observe a different pattern of motions in helix 7 when compared to simulations with the G protein-biased agonist salbutamol that involves perturbations of the network of interactions within the NPxxY motif. Understanding the network of interactions induced by biased ligands and the subsequent receptor conformational shifts will lead to development of more efficient drugs.

Do sedatives engage natural sleep pathways? It is usually assumed that anesthetic-induced sedation and loss of righting reflex (LORR) arise by influencing the same circuitry to lesser or greater extents. For the α2 adrenergic receptor agonist dexmedetomidine, we found that sedation and LORR were in fact distinct states, requiring different brain areas: the preoptic hypothalamic area and locus coeruleus (LC), respectively. Selective knockdown of α2A adrenergic receptors from the LC abolished dexmedetomidine-induced LORR, but not sedation. Instead, we found that dexmedetomidine-induced sedation resembled the deep recovery sleep that follows sleep deprivation. We used TetTag pharmacogenetics in mice to functionally mark neurons activated in the preoptic hypothalamus during dexmedetomidine-induced sedation or recovery sleep. The neuronal ensembles could then be selectively reactivated. In both cases, non-rapid eye movement sleep, with the accompanying drop in body temperature, was recapitulated. Thus, α2 adrenergic receptor-induced sedation and recovery sleep share hypothalamic circuitry sufficient for producing these behavioral states.

Asthma is an inflammatory disease of the airway that poses a major threat to human health. With increase industrialization in the developed and developing countries, the incidence of asthma is on the rise. The β2-adrenergic receptor is an important target in designing anti-asthmatic drugs. The synthetic agonists of the β2-adrenergic receptor used over the years proved effective, but with indispensable side effects, thereby limiting their therapeutic use on a long-term scale. Inverse agonists of this receptor, although initially contraindicated, had been reported to have long-term beneficial effects. Phytochemicals from Agemone mexicana were screened against the human β2-adrenergic receptor in the agonist, inverse agonist, covalent agonist, and the antagonist conformations. Molecular docking of the phyto-constituents showed that the plant constituents bind better to the inverse agonist bound conformation of the protein, and revealed two flavanones; eriodictyol and hesperitin, with lower free energy (ΔG) values and higher affinities to the inverse agonist bound receptor than the co-crystallized ligand. Eriodictyol and hesperitin bind with the glide score of -10.684 and - 9.958 kcal/mol respectively, while the standard compound ICI-118551, binds with glide score of -9.503 kcal/mol. Further interaction profiling at the protein orthosteric site and ADME/Tox screening confirmed the drug-like properties of these compounds.

Trace amine-associated receptors (TAAR) are rhodopsin-like G-protein-coupled receptors (GPCR). TAAR are involved in modulation of neuronal, cardiac and vascular functions and they are potentially linked with neurological disorders like schizophrenia and Parkinson's disease. Subtype TAAR1, the best characterized TAAR so far, is promiscuous for a wide set of ligands and is activated by trace amines tyramine (TYR), phenylethylamine (PEA), octopamine (OA), but also by thyronamines, dopamine, and psycho-active drugs. Unfortunately, effects of trace amines on signaling of the two homologous β-adrenergic receptors 1 (ADRB1) and 2 (ADRB2) have not been clarified yet in detail. We, therefore, tested TAAR1 agonists TYR, PEA and OA regarding their effects on ADRB1/2 signaling by co-stimulation studies. Surprisingly, trace amines TYR and PEA are partial allosteric antagonists at ADRB1/2, whereas OA is a partial orthosteric ADRB2-antagonist and ADRB1-agonist. To specify molecular reasons for TAAR1 ligand promiscuity and for observed differences in signaling effects on particular aminergic receptors we compared TAAR, tyramine (TAR) octopamine (OAR), ADRB1/2 and dopamine receptors at the structural level. We found especially for TAAR1 that the remarkable ligand promiscuity is likely based on high amino acid similarity in the ligand-binding region compared with further aminergic receptors. On the other hand few TAAR specific properties in the ligand-binding site might determine differences in ligand-induced effects compared to ADRB1/2. Taken together, this study points to molecular details of TAAR1-ligand promiscuity and identified specific trace amines as allosteric or orthosteric ligands of particular β-adrenergic receptor subtypes.

Chinese hamster ovary cell constructs expressing either the β 1-, β 2- or β 3-adrenergic receptor (AR) were used to determine whether a novel β-AR modulator, lubabegron fumarate (LUB; Experior, Elanco Animal Health) might exert greater potency for a specific β-AR subtype. EC50 values calculated based on cAMP accumulation in dose response curves indicate that LUB is highly selective for the β 3-AR subtype, with an EC50 of 6 × 10-9 M, with no detectible agonistic activity at the β 2-AR. We hypothesized that the accumulation of lipolytic markers would reflect the agonist activity at each of the β-receptor subtypes of the specific ligand; additionally, there would be differences in receptor subtype expression in subcutaneous (s.c.) and intrmuscular (i.m.) adipose tissues. Total RNA was extracted from adipose tissue samples and relative mRNA levels for β 1-, β2-, and β 3-AR were measured using real-time quantitative polymerase chain reaction. Fresh s.c. and i.m. adipose tissue explants were incubated with isoproterenol hydrochloride (ISO; β-AR pan-agonist), dobutamine hydrochloride (DOB; specific β 1-AA), salbutamol sulfate (SAL; specific β 2-AA), ractopamine hydrochloride (RAC), zilpaterol hydrochloride (ZIL), BRL-37344 (specific β 3-agonist), or LUB for 30 min following preincubation with theophylline (inhibitor of phosphodiesterase). Relative mRNA amounts for β 1-, β 2-, and β 3-AR were greater (P < 0.05) in s.c. than in i.m. adipose tissue. The most abundant β-AR mRNA in both adipose tissues was the β 2-AR (P < 0.05), with the β 1- and β 3-AR subtypes being minimally expressed in i.m. adipose tissue. ISO, RH, and ZH stimulated the release of glycerol and nonesterified fatty acid (NEFA) from s.c. adipose tissue, but these β-AR ligands did not alter concentrations of these lipolytic markers in i.m. adipose tissue. LUB did not affect glycerol or NEFA concentrations in s.c. or i.m. adipose tissue, but attenuated (P < 0.05) the accumulation of cAMP mediated by the β 1- and β 2-AR ligands DOB and SAL in s.c. adipose tissue. Collectively, these data indicate that bovine i.m. adipose tissue is less responsive than s.c. adipose tissue to β-adrenergic ligands, especially those that are agonists at the β 1- and β3-receptor subtypes. The minimal mRNA expression of the β 1- and β 3 subtypes in i.m. adipose tissue likely limits the response potential to agonists for these β-AR subtypes.

Old age, the most important risk factor for Alzheimer's disease (AD), is associated with thermoregulatory deficits. Brown adipose tissue (BAT) is the main thermogenic driver in mammals and its stimulation, through β3 adrenergic receptor (β3AR) agonists or cold acclimation, counteracts metabolic deficits in rodents and humans. Studies in animal models show that AD neuropathology leads to thermoregulatory deficits, and cold-induced tau hyperphosphorylation is prevented by BAT stimulation through cold acclimation. Since metabolic disorders and AD share strong pathogenic links, we hypothesized that BAT stimulation through a β3AR agonist could exert benefits in AD as well.

Asthma and other airway obstructive disorders are characterized by heightened inflammation and excessive airway epithelial cell reactive oxygen species (ROS), which give rise to a highly oxidative environment. After decades of use, β2-adrenergic receptor (β2AR) agonists remain at the forefront of treatment options for asthma, however, chronic use of β2-agonists leads to tachyphylaxis to the bronchorelaxant effects, a phenomenon that remains mechanistically unexplained. We have previously demonstrated that β2AR agonism increases ROS generation in airway epithelial cells, which upholds proper receptor function via feedback oxidation of β2AR cysteine thiolates to Cys-S-sulfenic acids (Cys-SOH). Our previous results also demonstrate that prevention of normal redox cycling of this post-translational oxi-modification back to the thiol prevents proper receptor function. Given that Cys-S-sulfenic acids can be irreversibly overoxidized to Cys-S-sulfinic (Cys-SO2H) or S-sulfonic (Cys-SO3H) acids, which are incapable of further participation in redox reactions, we hypothesized that β2-agonist tachyphylaxis may be explained by hyperoxidation of β2AR to S-sulfinic acids. Here, using airway epithelial cell lines and primary small airway epithelial cells from healthy and asthma-diseased donors, we show that β2AR agonism generates H2O2 in a receptor and NAPDH oxidase-dependent manner. We also demonstrate that acute and chronic receptor agonism can facilitate β2AR S-sulfination, and that millimolar H2O2 concentrations are deleterious to β2AR-mediated cAMP formation, an effect that can be rescued to a degree in the presence of the cysteine-donating antioxidant N-acetyl-L-cysteine. Our results reveal that the oxidative state of β2AR may contribute to receptor functionality and may, at least in part, explain β2-agonist tachyphylaxis.

Extensive evidence demonstrates functional interactions between the adrenergic and opioid systems in a diversity of tissues and organs. While some effects are due to receptor and second messenger cross-talk, recent research has revealed an extracellular, allosteric opioid binding site on adrenergic receptors that enhances adrenergic activity and its duration. The present research addresses whether opioid receptors may have an equivalent extracellular, allosteric adrenergic binding site that has similar enhancing effects on opioid binding. Comparison of adrenergic and opioid receptor sequences revealed that these receptors share very significant regions of similarity, particularly in some of the extracellular and transmembrane regions associated with adrenergic binding in the adrenergic receptors. Five of these shared regions from the mu opioid receptor (muOPR) were synthesized as peptides and tested for binding to adrenergic, opioid and control compounds using ultraviolet spectroscopy. Adrenergic compounds bound to several of these muOPR peptides with low micromolar affinity while acetylcholine, histamine and various adrenergic antagonists did not. Similar studies were then conducted with purified, intact muOPR with similar results. Combinations of epinephrine with methionine enkephalin or morphine increased the binding of both by about half a log unit. These results suggest that muOPR may be allosterically enhanced by adrenergic agonists.