High affinity [3H]formoterol binding sites in lung: characterization and autoradiographic mapping

doi:10.1016/0922-4106(94)90023-X

European Journal of Pharmacology: Molecular Pharmacology

Volume 269, Issue 1, 15 September 1994, Pages 35-41

https://doi.org/10.1016/0922-4106(94)90023-X Get rights and content

Abstract

Agonist binding to the β₂-adrenoceptors and its mapping were studied using the newly developed radioligand [³H]formoterol. The results of [³H]formoterol saturation binding and formoterol inhibition of [³H]formoterol binding were consistent with binding to a single class of receptors (K_d = 1.34 ± 0.15 nM, B_max = 154.9 ± 8.0 fmol/mg protein in guinea pig lung membranes, n = 8; K_d = 1.05 ± 0.17 nM, B_max = 67.8 ± 8.1 fmol/mg protein in human lung membranes, n = 5) and competition assays with other agonists and antagonists disclosed only a single class of site. The nonhydrolyzable GTP analogue GTPγS caused a reduction in both K_d and B_max, indicating that the receptors labelled by [³H]formoterol are coupled to a guanine nucleotide binding regulatory protein. Receptor mapping of [³H]formoterol binding sites shows that β₂-adrenoceptors were widely distributed in both guinea pig and human lung, with dense labelling over airway epithelium and uniformly over alveolar walls, and sparse labelling of airway and vascular smooth muscle. In addition, submucosal glands were also sparsely labelled in human bronchus. The distribution of β₂-adrenoceptors was similar to the pattern previously described with non-selective radiolabelled antagonists in the presence of selective β₁-adrenoceptor antagonists.

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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.
The use of inhaled formoterol in the treatment of asthma
2006, Annals of Allergy, Asthma and Immunology
To discuss the clinical efficacy and safety of formoterol when used to relieve symptoms of asthma and prevent exercise-induced bronchoconstriction (EIB).
A PubMed search was performed for articles published between 1997 and 2005 with the keywords formoterol, asthma, and long-acting β₂-adrenergic agonist, with cross-referencing to identify peer-reviewed journal articles.
Published articles on the clinical use of formoterol for asthma or EIB were included as well as articles detailing the pharmacologic properties of the drug. To present a thorough review of the literature, published studies based on patient number, study design, or other measures of study quality were not excluded.
Formoterol is the only long-acting β₂-adrenergic agonist that combines a rapid onset of action (within 3 minutes) with a long duration of effect (approximately 12 hours). Clinically, as recommended by asthma treatment guidelines, formoterol in conjunction with inhaled corticosteroids (ICSs) is a preferred treatment for moderate to severe persistent asthma. Significant clinical data support the use of formoterol in combination with ICSs for the treatment of asthma, with studies demonstrating improved pulmonary function and symptom scores and decreased need for maintenance ICSs and short-acting β₂-adrenergic agonists (SABAs) as relief medication. Recent studies also demonstrate that use of formoterol as needed as relief medication is associated with a prolonged time to exacerbation, improved pulmonary function, and decreased asthma symptoms. When used as monotherapy, formoterol provides protection against EIB. Clinical data also demonstrate that formoterol is safe and well tolerated even in high doses, with an adverse event profile similar to that of SABAs.
Overall, formoterol is safe and effective as adjunct controller therapy for moderate and severe persistent asthma and as monotherapy for EIB.
Receptor imaging in the thorax with PET
2004, European Journal of Pharmacology
This review focuses on positron emission tomography (PET)-imaging of receptors in the sympathetic and the parasympathetic systems of heart and lung and highlights the human applications of PET. For the α-adrenoceptor, only [¹¹C]GB67 (N2-{6-[(4-amino-6,7-dimethoxy-2-quinazolinyl)(methyl)amino]hexyl}-N2-[¹¹C]methyl-2-furamide hydrochloride) has been developed. Its potential for application in patients needs to be assessed. For both the β-adrenergic and the muscarinic systems, potent PET radioligands have been prepared and evaluated in patients. It has been possible to measure receptor densities quantitatively in human heart {[¹¹C]MQNB: [¹¹C]methylquinuclidinyl benzilate, [¹¹C]CGP12177: S-(3′-t-butylamino-2′-hydroxypropoxy)-benzimidazol-2-[¹¹C]one and [¹¹C]CGP12388: (S)-4-(3-(2′-[¹¹C]isopropylamino)-2-hydroxypropoxy)-2H-benzimidazol-2-one} and qualitatively in lung {[¹¹C]VC002: N-[¹¹C]-methyl-piperidin-4-yl-2-cyclohexyl-2-hydroxy-2-phenylacetate and [¹¹C]CGP12177}. Besides these subtype nonselective radioligands, the development of compounds that are selective for one subtype are ongoing and have not found successful application in humans yet.
Pharmacological and biochemical characterization of the beta-adrenergic signal transduction pathway in different segments of the respiratory tract
2003, Biochemical Pharmacology
Citation Excerpt :
On the other hand, our data confirm in more detail the data from earlier investigations in guinea-pigs, showing that β-adrenoceptors are unevenly distributed throughout the airways with the highest density in the lung and the lowest density in the trachea [15,38]. In earlier and recent studies it was unanimously demonstrated that in most mammalian species, the β2-adrenoceptors as well as β2-adrenoceptor mRNA both predominate in peripheral lung, including airway smooth muscle with the ratio of about 80:20 (β2:β1) [12,39], and in airway epithelium [4]. However, there is still a long-standing debate to what extent functional responses (relaxation) to β-adrenergic agonists or to obstructive disease states in the respiratory tract are mediated in relation to the β-adrenergic receptor subtype concentration and the subsequent β-adrenergic signal transduction [40].
Although in the respiratory system there is great therapeutic interest in manipulating and understanding the β-adrenoceptor–G-protein–adenylate cyclase (AC) signal transduction pathway, little is known on segmental differences among lung, bronchus, and trachea with regard to the receptor concentration and interaction to G-proteins and coupling to AC. In this study, patterns of distribution and absolute quantities of β-adrenoceptor subtypes β₁ and β₂ were determined in membranes of equine lung parenchyma, bronchial and tracheal epithelium with the underlying smooth muscle by saturation and competition binding assays using the radioligand (−)-[ $^{125} I$ ]-iodocyanopindolol (ICYP). Additionally, the functional coupling of β-adrenoceptors to G-proteins (assessed by β-agonist competition binding in the presence and absence of GTP) as well as the coupling efficiency and biochemical activities of AC was investigated in each region. The specific ICYP binding was rapid, reversible, saturable with time and of high affinity. The radioligand binding identified more total β-adrenoceptors in the lung than in bronchus or trachea (428±19, 162.4±4.8, 75.6±1.2 fmol/mg protein, respectively) with about 40% of receptors in the high affinity state. The β₂-adrenoceptor subtype predominated in all segments (approximately 74–80%), as the highly selective β₂-adrenoceptor antagonist ICI 118,551 was about 10,000 times more potent in inhibiting ICYP binding than was the β₁-selective adrenoceptor antagonist CGP 20712A, and β-adrenoceptor agonists inhibited ICYP binding with an order of potency: (−)-isoprenaline>(−)-adrenaline>(−)-noradrenaline. The dissociation constant (K_d) was higher in the trachea than in bronchus or lung (13.0±0.9 pM vs. 20.0±2.3 pM vs. 30.8±4.4 pM, P<0.05, respectively). The β₂-adrenoceptor-mediated AC response was tissue-dependent; stimulants acting on β-adrenoceptor (isoproterenol), G-protein (GTP, NaF) and AC (forskolin, Mn²⁺) enhanced AC responses in all three regions, but the AC activity was higher in tracheal crude membranes than in bronchus or lung ( $trachea ⋙ bronchus > lung$ ), hence, the number of β₂-adrenoceptors correlated inversely with the amount of AC. We conclude that (1) the stoichiometry of components within the pulmonary β-adrenoceptor–G-protein complex is segment-dependent, and (2) the receptor number or AC activity is possibly the rate-limiting factor in the β-adrenoceptor–G-protein–AC-mediated physiological responses. Thus, it is speculated that this could have important therapeutic consequences in β-adrenoceptor agonist-induced receptor regulation in bronchial asthma.
Effects of β-agonists on airway epithelial cells
2002, Journal of Allergy and Clinical Immunology
β-Adrenergic receptor (βAR) agonists exert a variety of effects on airway epithelial cells. Among their best known actions is their ability to increase ciliary beat frequency, mediated by cyclic adenosine monophosphate (cAMP) production, stimulation of protein kinase A (PKA), and phosphorylation of an outer dynein arm light chain. Submucosal glands express βARs, and β-agonists may stimulate secretion of mucus from airways, although human data are controversial. β-Agonists may also affect ion transport across epithelial cells by opening apical ion channels such as the cystic fibrosis transmembrane regulator. This effect, likely to occur in submucosal glands, can influence water fluxes across the airway epithelium and may have profound influences on mucus hydration. βAR activation can increase intracellular calcium in some ciliated cells, thereby stimulating ciliary beating and possibly influencing transepithelial ion transport. βAR-mediated activation of cAMP-dependent protein kinase accelerates epithelial cell migration, thereby enhancing epithelial wound repair. β-Agonists reduce the ultrastructural damage seen with infection and potentiate secretion of certain cytokines from epithelial cells while inhibiting secretion of others. Finally, β-agonists may have effects on airway epithelial cells that are mediated through βARs but do not require cAMP production. The signaling mechanisms of some β-agonist effects are not well understood but are important to our understanding of airway epithelial cell growth, differentiation, and function. (J Allergy Clin Immunol 2002;110:S275-81.)
A randomized, 12-week, double-blind, placebo-controlled study comparing formoterol dry powder inhaler with albuterol metered-dose inhaler
2001, Annals of Allergy, Asthma and Immunology
Formoterol is a β₂-adrenergic agent which, when inhaled, produces rapid and long-lasting bronchodilatation.
The aim of this study was to compare the efficacy, safety, and tolerability of formoterol powder for inhalation delivered via the Aerolizer device with placebo and with albuterol delivered via metered-dose inhaler in patients with mild to moderate persistent asthma.
In a multicenter, double-blind, parallel-group study, 541 patients were randomized at 26 trial sites to receive either formoterol, 12 μg twice daily; formoterol, 24 μg twice daily; albuterol, 180 μg four times daily; or a placebo for 12 weeks. The effects of each treatment on lung function, asthma symptoms, and frequency of rescue albuterol use were evaluated. Adverse effects and clinical laboratory parameters were also evaluated.
The bronchodilatory effects of formoterol were rapid in onset and persisted for 12 hours. Both formoterol doses were more effective than placebo and albuterol for objective measures of lung function. Morning and evening peak expiratory flow rates were more improved with formoterol, and formoterol provided significantly greater improvements in asthma symptom scores compared with both albuterol and placebo. Overall, patients taking formoterol used significantly less rescue medication than did those taking albuterol or placebo. Nocturnal awakenings occurred less often with formoterol than with placebo or albuterol. The therapeutic effects of formoterol were maintained over the entire 12 weeks of treatment. Adverse events were similar for all treatment groups, and clinical laboratory data were unremarkable.
Rapid-onset, long-acting formoterol, administered via the Aerolizer inhaler, is an effective and safe treatment for patients with mild to moderate persistent asthma.

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High affinity [3H]formoterol binding sites in lung: characterization and autoradiographic mapping

Abstract

Life Sci.

Eur. J. Pharmacol.

Eur. J. Pharmacol.

J. Biol. Chem.

Eur. J. Pharmacol. (Mol. Pharmacol. Section)

Comput. Prog. Biomed.

Anal. Biochem.

Binding of the novel ligand [4,5-3H-leu10]substance P to high-affinity NK-1 receptors on guinea pig lung membranes: modulation by GTP analogs and sulfhydryl modifying agents

J. Pharmacol. Exp. Ther.

Localization of beta-adrenoceptors in mammalian lung by light microscopic autoradiography

Nature

A new approach to the treatment of asthma

N. Engl. J. Med.

The β-agonist dilemma (editorial)

N. Engl. J. Med.

Autoradiographic visualization of beta-adrenoceptor subtypes in human lung

Am. Rev. Respir. Dis.

Inhibition of IgE-dependent histamine release from human dispersed lung mast cells by anti-allergic drugs and salbutamol

Br. J. Pharmacol.

A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase-coupled beta-adrenergic receptors

J. Biol. Chem.

High affinity [³H]formoterol binding sites in lung: characterization and autoradiographic mapping

Binding of the novel ligand [4,5-³H-leu¹⁰]substance P to high-affinity NK-1 receptors on guinea pig lung membranes: modulation by GTP analogs and sulfhydryl modifying agents