Trafficking of α1B-adrenergic receptor mediated by inverse agonist in living cells

AIM The project is aimed at understanding the action of inverse agonist at single molecule level and capturing the real time picture of molecular behavior of α1B-adrenergic receptor (AR) mediated by inverse agonist in living cells by single molecule detection (SMD). METHODS The location and distribution of α1B-AR was detected by laser confocal and whole cell ^3H-prazosin binding assay. Dynamic imaging of BODIPY-FL-labeled prazosin (Praz), specific antagonist of (1-AR, was observed in α1B-AR stably expressed human embryonic kidney 293 (HEK293) living cells. The detection of real-time dynamic behaviors of AR was achieved by using fluorescence-labeled AR and its ligand combined with SMD techniques. RESULTS α1B-AR was predominantly distributed on the cell surface and 8.2% of the total receptors were located in cytosol.     

Anti-nociception mediated by a κ opioid receptor agonist is blocked by a δ receptor agonist

BACKGROUND AND PURPOSE

The opioid receptor family comprises four structurally homologous but functionally distinct sub-groups, the μ (MOP), δ (DOP), κ (KOP) and nociceptin (NOP) receptors. As most opioid agonists are selective but not specific, a broad spectrum of behaviours due to activation of different opioid receptors is expected. In this study, we examine whether other opioid receptor systems influenced KOP-mediated antinociception.

EXPERIMENTAL APPROACH

We used a tail withdrawal assay in C57Bl/6 mice to assay the antinociceptive effect of systemically administered opioid agonists with varying selectivity at KOP receptors. Pharmacological and genetic approaches were used to analyse the interactions of the other opioid receptors in modulating KOP-mediated antinociception.

KEY RESULTS

Etorphine, a potent agonist at all four opioid receptors, was not anti-nociceptive in MOP knockout (KO) mice, although etorphine is an efficacious KOP receptor agonist and specific KOP receptor agonists remain analgesic in MOP KO mice. As KOP receptor agonists are aversive, we considered KOP-mediated antinociception might be a form of stress-induced analgesia that is blocked by the anxiolytic effects of DOP receptor agonists. In support of this hypothesis, pretreatment with the DOP antagonist, naltrindole (10 mg·kg⁻¹), unmasked etorphine (3 mg·kg⁻¹) antinociception in MOP KO mice. Further, in wild-type mice, KOP-mediated antinociception by systemic U50,488H (10 mg·kg⁻¹) was blocked by pretreatment with the DOP agonist SNC80 (5 mg·kg⁻¹) and diazepam (1 mg·kg⁻¹).

CONCLUSIONS AND IMPLICATIONS

Systemic DOP receptor agonists blocked systemic KOP antinociception, and these results identify DOP receptor agonists as potential agents for reversing stress-driven addictive and depressive behaviours mediated through KOP receptor activation.

LINKED ARTICLES

This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2     

High-affinity σ1 protein agonist reduces clinical and pathological signs of experimental autoimmune encephalomyelitis

Background and Purpose

Selective agonists of the sigma-1 receptor (σ1 protein) are generally reported to protect against neuronal damage and modulate oligodendrocyte differentiation. Human and rodent lymphocytes possess saturable, high-affinity binding sites for compounds binding to the σ1 protein and potential immunomodulatory properties have been described for σ1 protein ligands. Experimental autoimmune encephalomyelitis (EAE) is recognized as a valuable model of the inflammatory aspects of multiple sclerosis (MS). Here, we have assessed the role of a σ1 protein agonist, containing the tetrahydroisoquinoline-hydantoin structure, in EAE.

Experimental Approach

EAE was induced in SJL/J female mice by active immunization with myelin proteolipid protein (PLP)_139–151 peptide. The σ1 protein agonist was injected i.p. at the time of immunization (day 0). Disease severity was assessed clinically and by histopathological evaluation of the CNS. Phenotyping of B-cell subsets and regulatory T-cells were performed by flow cytometry in spleen and cervical lymph nodes.

Key Results

Prophylactic treatment of EAE mice with the σ1 protein agonist prevented mononuclear cell accumulation and demyelination in brain and spinal cord and increased T2 B-cells and regulatory T-cells, resulting in an overall reduction in the clinical progression of EAE.

Conclusions and Implications

This σ1 protein agonist, containing the tetrahydroisoquinoline-hydantoin structure, decreased the magnitude of inflammation in EAE. This effect was associated with increased proportions of B-cell subsets and regulatory T-cells with potential immunoregulatory functions. Targeting of the σ1 protein might thus provide new therapeutic opportunities in MS.     

β2-Adrenoceptor agonist activity of higenamine

Higenamine was included in the World Anti-Doping Agency (WADA) Prohibited Substances and Methods List as a β₂-adrenoceptor agonist in 2017, thereby resulting in its prohibition both in and out of competition. The present mini review describes the physiology and pharmacology of adrenoceptors, summarizes the literature addressing the mechanism of action of higenamine and extends these findings with previously unpublished in silico and in vitro work. Studies conducted in isolated in vitro systems, whole-animal preparations and a small number of clinical studies suggest that higenamine acts in part as a β₂-adrenoceptor agonist. In silico predictive tools indicated that higenamine and possibly a metabolite have a high probability of interacting with the β₂-receptor as an agonist. Stable expression of human β₂-receptors in Chinese hamster ovary (CHO) cells to measure agonist activity not only confirmed the activity of higenamine at β₂ but also closely agreed with the in silico prediction of potency for this compound. These data confirm and extend literature findings supporting the inclusion of higenamine in the Prohibited List.     

Pharmacological profiles of a novel α1-adrenoceptor agonist,PNO-49 B,at α1-adrenoceptor subtypes

The effects of a newly synthesized compound, PNO-49B, (R)-(-)-3-(2-amino-l-hydroxyethyl)-4-fluo-romethanesulfonanilide hydrochloride, on ₁-adrenocepfor subtypes were examined in various tissues in which the following distribution of ₁-adrenoceptor subtypes has been suggested: dog carotid artery (_1B), dog mesenteric artery (_1N), rabbit thoracic aorta (_1B + _1L), rat liver (₁B), rat vas deferens (_1A + _1L), rat cerebral cortex (_1A + _1B) and rat thoracic aorta (controversial subtype).PNO-49 B (0.1–100 M) produced concentration-dependent contractions in dog mesenteric artery, rabbit thoracic aorta, rat thoracic aorta and rat vas deferens; and the maximal amplitudes of contraction were almost the same as or slighly less than those of noradrenaline. By contrast, the maximal response to PNO-49 B in dog carotid artery was markedly smaller than the response to noradrenaline. In rabbit thoracic aorta, the contractile response to PNO-49 B was not affected by inactivation of the _1B subtype with chloroethylclonidine (CEC), although the response to noradrenaline was attenuated by that treatment. The dissociation constants (K_A) of PNO-49 B were not different among the rat thoracic aorta, dog carotid and mesenteric arteries and rabbit thoracic aorta (CEC-pretreated). The contractile responses to PNO-49 B were inhibited competitively by prazosin, HV723 (-ethyl-3,4,5-trimethoxy--(3-((2-(2-methoxy-phenoxy)-ethyl)))-amino(propyl)benzeneacetonitrile fumarate) and by WB4101 (2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane). The estimated pA₂ values were high for prazosin and WB4101 in rat thoracic aorta and for HV 723 in dog mesenteric artery, whereas the pA₂ values for these three antagonists in rabbit thoracic aorta were low and were not altered by pretreatment with CEC. The binding of [³H]-prazosin to membranes prepared from rat vas deferens and liver was inhibited by PNO-49 B in a concentration-dependent manner. The resulting pK₁ value for the liver was approximately 1.5 log units lower (one thirtieth in affinity) than the values for the epididymal and prostatic portions of the vas deferens. PNO-49B also inhibited biphasically [³H]prazosin binding to prazosin-high affinity sites of rat cerebral cortex membranes, and the low but high affinity sites for PNO-49B was abolished by CEC-pretreatment. PNO-49B had no effect on the prejunctional ₂-adrenoceptors in rat vas deferens (prostatic portion) nor on the -adrenoceptors in rat atria. The contractile response to PNO-49 B in rat thoracic aorta was not inhibited by cimetidine, pyrilamine or ketanserin.These results indicate that PNO-49B is an ₁-adrenoceptor agonist with a lower affinity and/or efficacy at the _1B subtype as compared with other ₁-subtypes.     

Attenuation of scopolamine-induced impairment of spontaneous alternation behaviour by antagonist but not inverse agonist and agonist β-carbolines

Mice were tested in a simple automated Y-maze. Total number of arm entries and alternation behaviour were measured. The latter is thought to reflect working memory capacity at a rudimentary level. During an 8-min session, vehicle-treated mice performed 32.4±7.4 arm entries, 51.0±12.4% of which were organized in alternations (triplets). The two variables showed a negative correlation. Scopolamine (1.0 mg/kg) significantly enhanced activity, reduced alternation behaviour and diminished the correlation between the two variables. The effects of benzodiazepine receptor inverse agonist, antagonist and agonist -carbolines on this spontaneous behaviour and on the effects of scopolamine were examined. The effects of inverse agonists and agonists on locomotor activity were complex in interaction with both vehicle and scopolamine. The scopolamine-induced reduction of alternation behaviour was significantly reversed by the antagonist ZK 93426 but not by inverse agonists; furthermore, partial agonists and agonists showed no effects. It is hypothesized that the interaction of antagonist -carbolines with scopolamine is based on a direct GABA-ergic control of cholinergic neurotransmission, and suggests an ability of antagonist -carbolines to antagonize amnestic properties of scopolamine.     

How can 1 + 1 = 3? β2-adrenergic and glucocorticoid receptor agonist synergism in obstructive airway diseases

For a long time it was believed that β(2)-adrenergic receptor agonists used in the treatment of obstructive airway diseases worked primarily on airway smooth muscle cells, causing relaxation, whereas glucocorticoids primarily improved airway function via their anti-inflammatory action, indicating that their clinical synergism occurred at the organism rather than the cellular level. However, it is now becoming clear that both drug classes can affect airway function at multiple levels, including an integrated effect on several cell types. This article summarizes data on the molecular interaction between the two receptor systems, particularly with relevance to phenomena of β(2)-adrenergic receptor desensitization and glucocorticoid insensitivity in the airways. These molecular interactions may contribute to the observed clinical synergism between both drug classes in the treatment of obstructive airway diseases.     

Chronic food restriction in rats augments the central rewarding effect of cocaine and the δ1 opioid agonist, DPDPE, but not the δ2 agonist, deltorphin-II

RATIONALE: There is some, albeit conflicting, evidence that 5-HT3 receptors might be involved in the actions of abused stimulants. Most studies have focussed on examinations of 5-HT3 antagonists; this might be due to a lack of high-affinity 5-HT3 agonists that readily penetrate the blood-brain barrier. OBJECTIVES: N-(3-Chlorophenyl) guanidine (MD-354) is a member of a novel class of 5-HT3 ligands developed in our laboratories. We have previously demonstrated that MD-354 can exert agonist effects and now further explore this action. METHODS: Rats (n=9) were trained to discriminate 2 mg/kg MD-354 from saline vehicle in a two-lever drug discrimination task (VI-15 s schedule of reinforcement). The actions of agents with 5-HT3 character were evaluated. The emetic and antiemetic actions of MD-354 were also examined using the shrew as test subject. RESULTS: Various agents with demonstrated 5-HT3 agonist properties substituted for the MD-354 stimulus (MD-354 ED50=0.5 mg/kg): quipazine (ED50=0.2 mg/kg), meta-chlorophenylbiguanide (mCPBG, ED50=1.4 mg/kg), 2-methyl 5-HT (ED50=4.5 mg/kg), 1-(2-naphthyl)biguanide (2-NBG, ED50=1.9 mg/kg), and N-(2-naphthyl)guanidine (2-NG, ED50=0.7 mg/kg). Administration of the training dose of MD-354 in combination with the 5-HT3 antagonists zacopride and tropisetron resulted in stimulus antagonism (AD50=0.02 mg/kg); administered alone, however, zacopride engendered 81% MD-354-appropriate responding (ED50=0.03 mg/kg). MD-354 was shown to produce an emetic effect in the shrew at very high doses (i.e., 40 mg/kg); however, when administered in combination with cisplatin, MD-354 behaved as an antiemetic agent at 10 mg/kg. CONCLUSION: Taken together, the results indicate that MD-354 is a 5-HT3 agonist and that it might be an agent with partial agonist activity.     

Novel κ-opioid receptor agonist MB-1C-OH produces potent analgesia with less depression and sedation

Aim:

To characterize the pharmacological profiles of a novel κ-opioid receptor agonist MB-1C-OH.

Methods:

[³H]diprenorphine binding and [³⁵S]GTPγS binding assays were performed to determine the agonistic properties of MB-1C-OH. Hot plate, tail flick, acetic acid-induced writhing, and formalin tests were conducted in mice to evaluate the antinociceptive actions. Forced swimming and rotarod tests of mice were used to assess the sedation and depression actions.

Results:

In [³H]diprenorphine binding assay, MB-1C-OH did not bind to μ- and δ-opioid receptors at the concentration of 100 μmol/L, but showed a high affinity for κ-opioid receptor (K_i=35 nmol/L). In [³⁵S]GTPγS binding assay, the compound had an E_max of 98% and an EC₅₀ of 16.7 nmol/L for κ-opioid receptor. Subcutaneous injection of MB-1C-OH had no effects in both hot plate and tail flick tests, but produced potent antinociception in the acetic acid-induced writhing test (ED₅₀=0.39 mg/kg), which was antagonized by pretreatment with a selective κ-opioid receptor antagonist Nor-BNI. In the formalin test, subcutaneous injection of MB-1C-OH did not affect the flinching behavior in the first phase, but significantly inhibited that in the second phase (ED₅₀=0.87 mg/kg). In addition, the sedation or depression actions of MB-1C-OH were about 3-fold weaker than those of the classical κ agonist (−)U50,488H.

Conclusion:

MB-1C-OH is a novel κ-opioid receptor agonist that produces potent antinociception causing less sedation and depression.     

Development of a bivalent dopamine D₂ receptor agonist

Bivalent D? agonists may function as useful molecular probes for the discovery of novel neurological therapeutics. On the basis of our recently developed bivalent dopamine D? receptor antagonists of type 1, the bivalent agonist 2 was synthesized when a spacer built from 22 atoms was employed. Compared to the monovalent control compound 6 containing a capped spacer, the bis-aminoindane derivative 2 revealed substantial steepening of the competition curve, indicating a bivalent binding mode. Dimer-specific Hill slopes were not a result of varying functional properties because both the dopaminergic 2 and the monovalent control agent 6 proved to be D? agonists substantially inhibiting cAMP accumulation and inducing D? receptor internalization. Investigation of the heterobivalent ligands 8 and 9, containing an agonist and a phenylpiperazine-based antagonist pharmacophore, revealed moderate steepening of the displacement curves and antagonist to very weak partial agonist properties.     

The haemodynamic effect of the 5HT1 agonist BMS-180048: a class effect of triptans?

AIMS: To investigate the effects of an intravenous infusion of BMS-180048, a novel 5HT1-like agonist, on the systemic, pulmonary and coronary circulations in patients undergoing diagnostic cardiac catheterisation. METHODS: Ten patients (mean age 55 years (range 41-65)) were studied during diagnostic cardiac catheterisation. The haemodynamic response to an intravenous (i.v.) infusion for 30 min of BMS-180048 (0.56 mg kg(-1) h(-1) for 10 min and 0.39 mg kg(-1) h(-1) for 20 min) was assessed via a 7F Swan Ganz catheter and thermodilution cardiac output system. Quantitative coronary angiography was performed at 10 min intervals. RESULTS: BMS-180048 caused a significant increase in systemic arterial systolic blood pressure (rise of 32.5 mmHg, 95% CI 24,44.5) P=0.009), pulmonary artery systolic (12.2 mmHg, 95% CI 6.8,18.5; P=0.009) and diastolic pressures (8.5 mmHg, 95% CI 5.0,13.8; P=0.009), right atrial pressure (4 mmHg, 95% CI 1.5,5.2; P=0.013) and pulmonary capillary wedge pressure (9.5 mmHg 95% CI 5.2,14.0; P=0.09). There was no significant change in cardiac output (0.1 l min(-1), 95% CI -0.17,0.57, P>0.05). Mean coronary artery diameter in the proximal coronary segments decreased by 0.73 mm (95% CI -1.22,-0.15; P=0.03) at 35 min. The corresponding reduction in middle segments was 0.26 mm (95% CI -0.395,-0.08; P=0.02). There was a non-significant trend to constriction in the most distal segments of 0.28 mm (95% CI -0.68,0.015); P=0.06). One patient experienced chest pain with ECG changes suggestive of ischaemia. CONCLUSIONS: BMS-180048 displayed a cardiovascular profile similar to that previously reported for sumatriptan. These changes appear to reflect a class effect of these agents.     

B-HT 920 acts as an α 1-adrenoceptor agonist in the rabbit aorta under certain in vitro conditions

Summary We have investigated the interaction of the ₂-adrenoceptor agonist B-HT 920 with the -adrenoceptors in the rabbit aorta using various experimental conditions.In standard Krebs-Henseleit solution B-HT 920 behaved as an antagonist when tested against the ₁-agonist phenylephrine. However, it behaved as a partial agonist at -receptors when subcontractile concentrations of various spasmogens (angiotensin II, serotonin, prostaglandin F₂ ) were applied, although no change in the affinity of the receptor to B-HT 920 was observed. By means of the selective antagonists prazosin and rauwolscine it was established that B-HT 920 activated ₁-renoceptors. The same agonistic effects of B-HT 920 were obtained after pretreatment of the animal with reserpine or in the presence of ouabain. The various treatments used (except reserpine) did not influence the contractile response to phenylephrine.The contractile response to B-HT 920 was found to be highly susceptible to the calcium entry blocker nitrendipine whereas the response to phenylephrine was not.It is concluded that spasmogens modulate the responsiveness of -receptors to certain agonists, possibly by causing a depolarization of the cell membrane and, thereby, sensitization of a mechanism involved in excitation-contraction coupling, conceivably a calcium gating mechanism.This study was supported by a grant of the Deutsche Forschungsgemeinschaft     

Dorsal hand vein responses to the α₁-adrenoceptor agonist phenylephrine do not predict responses to the α₂-adrenoceptor agonist dexmedetomidine

Significant inter-individual variability exists in responses of human dorsal hand veins to activation of α-adrenoceptors. Simultaneous graded infusions of the α?- and α?-adrenoceptor agonists phenylephrine (3.66-8000 ng/min) and dexmedetomidine (0.0128-1000 ng/min) were given into dorsal veins of both hands and responses of 75 subjects were analyzed to assess whether a subject's sensitivity to phenylephrine (ED(50)) predicts his sensitivity to dexmedetomidine. Individual ED(50) estimates of dexmedetomidine and phenylephrine ranged between 0.06-412 and 14.2-7450 ng/min and exhibited only a weak positive relationship (r2 =0.074, P=0.018). Finger temperature, body mass index, age and phenylephrine sensitivity together accounted for about 30% of dexmedetomidine ED(50) variation (r2 =0.315, P<0.001). The large inter-individual variability observed in the responses of dorsal hand veins to both α?- and α?-adrenoceptor agonists is not explained by some common factors; instead, dorsal hand vein responsivity is separately determined for both receptor mechanisms.     

Modulation of agonist binding by guanine nucleotides in CHO cells expressing muscarinic m1 – m5 receptors

In membranes prepared from CHO-m2 cells, inhibition of [³H]-N-methylscopolamine ([³H]NMS) binding by several muscarinic agonists resulted in competition curves with Hill slopes significantly different from unity. Addition of 5-guanylylimidodiphosphate (Gpp(NH)p) led to an increase in the IC₅₀ value of the agonists with significant steepening of the inhibition curves. The shift in potency induced by Gpp(NH)p differed among the agonists with a rank order of oxotremorine-M = carbachol > oxotremorine > McN-A-343 = pilocarpine. In CHO-m4 membranes, Gpp(NH)p was less efficacious than in CHO-m2 membranes whereas no effect of the guanine nucleotide was found in membranes prepared from CHO-m1, -m3, and-m5 cells. No major differences in the effect of Gpp(NH)p among agonists were found in CHO-m4 cells. Atropine binding was not affected by the guanine nucleotide. Together, these results indicate that coupling of G-proteins to muscarinic receptors linked to inhibition of cyclic adenosine monophosphate (cAMP) (m2 and m4) but not of those linked to phosphoinositol turnover (m1, m3 and m5) can be perturbed by Gpp(NH)p. The differential effects observed with Gpp(NH)p between agonist binding to m2 and m4 receptors appear to be receptor-specific and may reflect differences in the G proteins activated by these receptors in CHO cells.     

The long-acting β2-adrenoceptor agonist olodaterol attenuates pulmonary inflammation

Background and Purpose

β₂-adrenoceptor agonists are widely used in the management of obstructive airway diseases. Besides their bronchodilatory effect, several studies suggest inhibitory effects on various aspects of inflammation. The aim of our study was to determine the efficacy of the long-acting β₂-adrenoceptor agonist olodaterol to inhibit pulmonary inflammation and to elucidate mechanism(s) underlying its anti-inflammatory actions.

Experimental Approach

Olodaterol was tested in murine and guinea pig models of cigarette smoke- and LPS-induced lung inflammation. Furthermore, effects of olodaterol on the LPS-induced pro-inflammatory mediator release from human parenchymal explants, CD11b adhesion molecule expression on human granulocytes TNF-α release from human whole blood and on the IL-8-induced migration of human peripheral blood neutrophils were investigated.

Key Results

Olodaterol dose-dependently attenuated cell influx and pro-inflammatory mediator release in murine and guinea pig models of pulmonary inflammation. These anti-inflammatory effects were observed at doses relevant to their bronchodilatory efficacy. Mechanistically, olodaterol attenuated pro-inflammatory mediator release from human parenchymal explants and whole blood and reduced expression of CD11b adhesion molecules on granulocytes, but without direct effects on IL-8-induced neutrophil transwell migration.

Conclusions and Implications

This is the first evidence for the anti-inflammatory efficacy of a β₂-adrenoceptor agonist in models of lung inflammation induced by cigarette smoke. The long-acting β₂-adrenoceptor agonist olodaterol attenuated pulmonary inflammation through mechanisms that are separate from direct inhibition of bronchoconstriction. Furthermore, the in vivo data suggest that the anti-inflammatory properties of olodaterol are maintained after repeated dosing for 4 days.Tables of Links