Biased Signaling of the G-Protein-Coupled Receptor ß2AR Is Governed by Conformational Exchange Kinetics.

G-protein-coupled receptors (GPCRs) mediate a wide range of human physiological functions by transducing extracellular ligand binding events into intracellular responses. GPCRs can activate parallel, independent signaling pathways mediated by G proteins or ß-arrestins. Whereas "balanced" agonists activate both pathways equally, "biased" agonists dominantly activate one pathway, which is of interest for designing GPCR-targeting drugs because it may mitigate undesirable side effects. Previous studies demonstrated that ß-arrestin activation is associated with transmembrane helix VII (TM VII) of GPCRs. Here, single-molecule fluorescence spectroscopy with the ß2-adrenergic receptor (ß2AR) in the ligand-free state showed that TM VII spontaneously fluctuates between one inactive and one active-like conformation. The presence of the ß-arrestin-biased agonist isoetharine prolongs the dwell time of TM VII in the active-like conformation compared with the balanced agonist formoterol, suggesting that ligands can induce signaling bias by modulating the kinetics of receptor conformational exchange.

Systematic errors in detecting biased agonism: Analysis of current methods and development of a new model-free approach.

Discovering biased agonists requires a method that can reliably distinguish the bias in signalling due to unbalanced activation of diverse transduction proteins from that of differential amplification inherent to the system being studied, which invariably results from the non-linear nature of biological signalling networks and their measurement. We have systematically compared the performance of seven methods of bias diagnostics, all of which are based on the analysis of concentration-response curves of ligands according to classical receptor theory. We computed bias factors for a number of ß-adrenergic agonists by comparing BRET assays of receptor-transducer interactions with Gs, Gi and arrestin. Using the same ligands, we also compared responses at signalling steps originated from the same receptor-transducer interaction, among which no biased efficacy is theoretically possible. In either case, we found a high level of false positive results and a general lack of correlation among methods. Altogether this analysis shows that all tested methods, including some of the most widely used in the literature, fail to distinguish true ligand bias from "system bias" with confidence. We also propose two novel semi quantitative methods of bias diagnostics that appear to be more robust and reliable than currently available strategies.

Biased signaling pathways in ß2-adrenergic receptor characterized by 19F-NMR.

Extracellular ligand binding to G protein-coupled receptors (GPCRs) modulates G protein and ß-arrestin signaling by changing the conformational states of the cytoplasmic region of the receptor. Using site-specific (19)F-NMR (fluorine-19 nuclear magnetic resonance) labels in the ß(2)-adrenergic receptor (ß(2)AR) in complexes with various ligands, we observed that the cytoplasmic ends of helices VI and VII adopt two major conformational states. Changes in the NMR signals reveal that agonist binding primarily shifts the equilibrium toward the G protein-specific active state of helix VI. In contrast, ß-arrestin-biased ligands predominantly impact the conformational states of helix VII. The selective effects of different ligands on the conformational equilibria involving helices VI and VII provide insights into the long-range structural plasticity of ß(2)AR in partial and biased agonist signaling.

Non-cAMP-mediated bronchial arterial vasodilation in response to inhaled beta-agonists.

We studied the dose-dependent effects of inhaled isoetharine HCl, a beta-adrenergic bronchodilator (2.5, 5.0, 10.0, and 20.0 mg), on bronchial blood flow (Qbr) in anesthetized sheep. Isoetharine resulted in a dose-dependent increase in Qbr. With a total dose of 17.5 mg, Qbr increased from baseline values of 22 +/- 3.4 (SE) to 60 +/- 16 ml/min (P < 0.001), an effect independent of changes in cardiac output and systemic arterial pressure. To further study whether synthesis of endogenous nitric oxide (NO) affects beta-agonist-induced increases in Qbr, we administered isoetharine (20 mg) by inhalation before and after the NO-synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME). Intravenous L-NAME (30 mg/kg) rapidly decreased Qbr by approximately 80% of baseline, whereas L-NAME via inhalation (10 mg/kg) resulted in a delayed and smaller (approximately 22%) decrease. Pretreatment with L-NAME via both routes of administration attenuated bronchial arterial vasodilation after subsequent challenge with isoetharine. We conclude that isoetharine via inhalation increases Qbr in a dose-dependent manner and that beta-agonist-induced relaxation of vascular smooth muscle in the bronchial vasculature is partially mediated via synthesis of NO.

Nitric oxide and beta-adrenergic agonist-induced bronchial arterial vasodilation.

In anesthetized sheep, we measured bronchial blood flow (Qbr) by an ultrasonic flow probe to investigate the interaction between inhaled nitric oxide (NO; 100 parts/million) given for 5 min and 5 ml of aerosolized isoetharine (1.49 x 10(-2) M concentration). NO and isoetharine increased Qbr from 26.5 +/- 6.5 to 39.1 (SE) +/- 10.6 and 39.7 +/- 10.7 ml/min, respectively (n = 5). Administration of NO immediately after isoetharine further increased Qbr to 57.3 +/- 15.1 ml/min. NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester hydrochloride (L-NAME; 30 mg/kg, in 20 ml saline given i.v.) decreased Qbr to 14.6 +/- 2.6 ml/min. NO given three times alternately with isoetharine progressively increased Qbr from 14.6 +/- 2.6 to 74.3 +/- 17.0 ml/min, suggesting that NO and isoetharine potentiate vasodilator effects of each other. In three other sheep, after L-NAME three sequential doses of isoetharine increased Qbr from 10.2 +/- 3.4 to 11.5 +/- 5.7, 11.7 +/- 4.7, and 13.3 +/- 5.7 ml/min, respectively, indicating that effects of isoetharine are predominantly mediated through synthesis of NO. When this was followed by three sequential administrations of NO, Qbr increased by 146, 172, and 185%, respectively. Thus in the bronchial circulation, there seems to be a close interaction between adenosine 3',5'-cyclic monophosphate- and guanosine 3',5'-cyclic monophosphate-mediated vasodilation.

Pulmonary dysfunction after primary closure of an abdominal wall defect and its improvement with bronchodilators.

To determine the extent of pulmonary dysfunction following primary closure of an abdominal wall defect, we obtained pulmonary function tests (PFT) in 11 newborn infants with gastroschisis and 6 with large omphaloceles admitted to a newborn ICU in a children's hospital. Patients were 1 to 30 days of age at the time of the PFT; all required endotracheal intubation and mechanical ventilation for operative procedures or for postoperative ventilatory support. Full-term infants (n = 21) undergoing minor surgical procedures provided comparative measurements. Flow-volume curves were obtained with manual inflation of the lungs followed by forced deflation using negative pressure, or by passive expiration, under sedation and pharmacologic paralysis. Deflation flow-volume curves gave measurements of forced vital capacity (FVC) and maximal expiratory flow at 25% of vital capacity from residual volume (MEF25). Modified passive mechanics technique gave passive expiratory curves that provided measurements of respiratory system compliance (Crs) and resistance (Rrs). Tests were done: within 48 h (period A), 3-7 days (period B), and 8-30 days after surgical repair (period C). Pulmonary function testing after nebulized 0.1% isoetharine (a bronchodilator), to test for bronchial reactivity, began midway during the study period in 15 patients. Preoperative and postoperative tests were obtained in 5 patients. Closure of an abdominal wall defect decreased FVC, Crs, and MEF25 by up to 50% of normal, reference values after surgery (P less than 0.05). FVC and MEF25 approached values of normal infants by 4 weeks, whereas Crs remained 50% lower.(ABSTRACT TRUNCATED AT 250 WORDS)

Decreased duration of emergency department treatment of chronic obstructive pulmonary disease exacerbations with the addition of ipratropium bromide to beta-agonist therapy.

STUDY OBJECTIVES: To determine the benefit of the addition of ipratropium bromide to beta-agonist therapy of acute exacerbations of chronic obstructive pulmonary disease. DESIGN: The trial was randomized and double blinded. SETTING: The study was conducted in the emergency department of Parkland Memorial Hospital, a busy, inner-city, county hospital. INTERVENTIONS: Patients were treated in the medicine emergency department with either the standard regimen of nebulized isoetharine, 0.5 mL of a 1% solution (5.0 mg) diluted to 2.0 mL with normal saline every hour (control group) or with the same regimen plus ipratropium bromide, 54 micrograms (three puffs) after the first isoetharine treatment and 36 micrograms (two puffs) after the second and fourth (experimental group). A placebo metered-dose inhaler used in the same manner as the ipratropium blinded the study to both the patients and medical personnel. MEASUREMENTS AND MAIN RESULTS: The group treated with the addition of ipratropium (30) was discharged from the ED an average of 91 minutes (P less than .05) sooner than the control group (25) and required on the average one less isoetharine treatment (P less than .05). The pulmonary functions tested, forced expiratory volume in the first second, and the forced vital capacity were the same in the two groups initially and on discharge, as identical discharge criteria were used in each group. CONCLUSION: The addition of ipratropium to standard beta-agonist treatment of chronic obstructive pulmonary disease exacerbations shortens the duration of treatment required in the ED.

Bronchial reactivity in infants in acute respiratory failure with viral bronchiolitis.

Airway reactivity and the effects of bronchodilators in infants are controversial. We studied the response to bronchodilator treatment in 14 mechanically ventilated infants (mean age, 2.74 months; range, 0.6-5.9) in respiratory failure caused by respiratory syncytial virus (RSV)-associated bronchiolitis. Sixteen infants without lung disease, undergoing elective surgery, provided normal values. Maximum expiratory deflation flow-volume (DFV) curves were produced by manual inflation of the lungs with an anesthesia bag to a predetermined static airway pressure followed by rapid deflation with a negative airway pressure before and after administration of bronchodilator. At baseline, the bronchiolitis group had a forced vital capacity (FVC) of 34.5 +/- 3.6 ml/kg compared with 41.8 +/- 1.5 ml/kg in the normal group; maximum expiratory flow rate at 25% of FVC (MEF25) was 10.2 +/- 2.0 ml/kg/s compared with 27.3 +/- 2.0 ml/kg/s in the normal group. The clinical and radiologic impression was severe lower airway obstruction and air trapping. After administration of bronchodilator, FVC did not increase significantly, but MEF25isov increased by over 30% in 13 of 14 infants. Mean MEF25 increased by 148 +/- 43.2% to 21.7 +/- 3.9 ml/kg/s (P less than 0.02). These findings indicate that during the acute phase of severe RSV-positive bronchiolitis most infants have airway reactivity that responds positively to bronchodilator treatment.

Hypomania related to phenelzine and isoetharine interaction in one patient.

A case is reported of a man with apparent unipolar depression that was responsive to treatment with phenelzine who became hypomanic when isoetharine was added to treat his chronic obstructive pulmonary disease. The role of beta-adrenergic receptors in affective illness is reviewed in light of this case.

Comparison of metaproterenol, isoetharine and salbutamol in the relief of methacholine-induced bronchospasm in dogs.

We evaluated cardiovascular effects and effectiveness of isoetharine, metaproterenol and salbutamol, when administered intratracheally to relieve methacholine-induced bronchospasm in dogs anaesthetized with 50 per cent nitrous oxide, oxygen, halothane and mechanically ventilated. Methacholine 2 micrograms X kg-1 X hour-1 was administrated first followed by halothane (1 MAC) for 30 minutes (control), then metaproterenol, isoetharine or salbutamol. Metaproterenol (15 mg) significantly decreased transpulmonary pressure to 20.1 +/- 0.5 (SE) from 22.5 +/- 1.15 cmH2O (p less than 0.025) after three min and to 15 +/- 0.5 cmH2O (p less than 0.005) after 90 min. Isoetharine (2.5 mg) decreased transpulmonary pressure after five min to 22.1 +/- 1 from 24.5 +/- 1.5 cmH2O (p less than 0.05), and to 21.75 +/- 0.55 mmH2O after 90 min. Salbutamol 25 micrograms X kg-1 decreased transpulmonary pressure to 20.7 +/- 0.75 from 24.25 +/- 1.28 after three min and to 16 +/- .5 after 90 min. The peak effects on airway pressure occurred at 15 min for metaproterenol, 25 min for salbutamol and 20 min for isoetharine. Pulmonary vascular resistance was not significantly changed during halothane anaesthesia alone but decreased significantly after metaproterenol and isoetharine infusion. Heart rate increased ten per cent after metaproterenol, three per cent after isoetharine, and five per cent after salbutamol. No arrhythmias occurred in any group. Cardiac output increased significantly to 3.25 +/- 0.2 from 1.5 +/- 0.17 L X min-1 (p less than 0.025) after metaproterenol to 3.2 +/- .025 from 1.45 +/- .009 after salbutamol and was unchanged after isoetharine. Metaproterenol and salbutamol in the presence of 1 MAC halothane anaesthesia relieved methacholine-induced bronchospasm more rapidly than did isoetharine. The onset of effect was 3 +/- 0.05 min for metaproterenol and salbutamol and 5 +/- 0.01 min for isoetharine. The effect lasted 210 +/- 10.5 min for metaproterenol, 170 +/- 12.5 min for salbutamol and 90 +/- 4.75 min for isoetharine.

A method for comparing the peak intensity and duration of action of aerosolized bronchodilators using bronchoprovocation with methacholine.

The effects of aerosolized metaproterenol (1.30 mg) and isoetharine (1.02 mg) on nonspecific airway responsiveness to inhaled methacholine were quantitated and compared in 6 asthmatic subjects for 4 h after dosing. Both metaproterenol and isoetharine demonstrated bronchodilatation and reduced airway responsiveness during the first hour. Isoetharine had lost its bronchodilator effects and its effects on airway responsiveness by 2.25 h. The effects of metaproterenol on airway responsiveness had decreased by half at 2.25 h, while its bronchodilator effect remained maximal. Metaproterenol had no effect on airway responsiveness by 4 h, despite persisting bronchodilatation. Thus, for metaproterenol, there is disparity between duration of bronchodilation and suppression of airway responsiveness. Because methacholine sensitivity is a measure of nonspecific airway responsiveness, which appears to correlate with the severity of asthma, this methodology may provide more relevant information for assessment of duration of effect and estimation of appropriate dosing intervals for maintenance therapy than do more traditional methods.

Bronchodilator characteristics of nebulized metaproterenol sulfate, isoetharine, and atropine in chronic asthma.

A randomized double-blind comparison of aerosolized META, ISO, ATR, and PI solutions delivered by motorized mist nebulizer was conducted in subjects with known reversible airway obstruction. Vital signs and spirometric parameters were monitored sequentially during each of four 6 hr test periods. Abstention from all medication was required for at least 12 hr before each test period. Baseline percent predicted FEV1 and FEF25-75 among subjects for all test periods were statistically similar. Compared to the other agents tested META treatment (15 mg) resulted in significantly improved global response and higher mean percent increases in FEV1 and FEF25-75 over the entire test period as well as at individual measurement points up to 4 hr for FEV1 and 5 hr for FEF25-75. Peak FEV1 and FEF25-75 values with META treatment were noted at 30 min and persisted above baseline during the entire 6 hr. ATR (2 mg) treatment resulted in later onset of bronchodilatory effect and, as with ISO (125 micrograms), more rapid diminution in measured response. Administration of saline alone resulted in a net 9% increase in FEV1 and 11% in FEF25-75 over the entire test period. Clinically insignificant tremor was noted in 41% of META-treated subjects. Delivery of META by a motorized mist nebulizer provides safe, effective bronchodilation and META is superior to the other agents tested in terms of onset of action, peak reversal of airway obstruction, and duration of effect.

Comparison of bronchodilator effectiveness of fenoterol and isoetharine.

The present study was undertaken to compare efficacy and duration of action of fenoterol and isoetharine in patients who subjectively required urgent therapy. Analysis of the resulting data showed that in this population fenoterol had a longer duration of action and was more effective at the later time periods.

Effect of isoetharine on cardiac conduction in man.

The effects of isoetharine on the His bundle electrogram were studied in 10 patients with heart disease. Recordings were made at varied heart rates using atrial pacing. Isoetharine significantly reduced the AH interval with atrial pacing, but it had no effect on the HV interval. Second degree heart block occurred at higher pacing rates after isoetharine treatment as compared to the control state. The heart rate and blood pressure showed no significant change after isoetharine. The functional and effective refractory period were measured with the use of the extra-stimulus technique. The functional refractory period of the AV node, as well as the effective refractory period of the atrium, significantly decreased after isoetharine. Thus, isoetharine can improve conduction through the atrioventricular node. The drug does have a cardiac effect as measured by its action on the human conduction system.

Volume-adjusted maximal mid-expiratory flow (Iso-volume FEF25-75%): definition of "Significant" responsiveness in healthy, normal subjects.

Iso-volume FEF25-75% was evaluated in 30 non-smoking, healthy subjects (16-58 years) to define the percentage change over baseline required for responsiveness to inhaled bronchodilators using this measurement to be significant. The mean percentage change from baseline for Iso-volume FEF25-75% was 15.3% +/- 17.2% (1 SD). Improvement of greater than 49.6% (mean +/- 2 SD) over baseline is required for the change reflected by the Iso-volume FEF25-75% to be significant.

Respiratory drugs influence lung mucociliary clearance in central and peripheral ciliated airways.

Radiolabelling of mucus and gamma-camera imaging techniques were utilized to differentiate mucociliary function in large central and small peripheral bronchi of man. Lung mucus clearance was analyzed for the entire right lung field and a peripheral region, which was representative of mucus clearance from airways distal to lobar bronchi. On control days, healthy subjects breathed monodisperse particles, average size 4.19 micrometers, and achieved central patterns of deposition with mouth breathing at rest. Matched deposition patterns were achieved on treatment days when isoetharine or isoproterenol influence on mucus clearance was measured and compared to control. Whole and peripheral lung clearances were increased by beta-adrenergic aerosol: isoetharine/control clearance ratios for whole and peripheral lung average 1.47 and 1.50, respectively; similar results were found for isoproterenol with ratios of 1.47 and 1.23, respectively. These data indicated that in healthy subjects the peripheral bronchi have the longest turn-over times for replacement of their mucous linings, and that these airways, like the larger airways of the trachea and main bronchi, can be stimulated by beta-adrenergic agents to increase their base-line flow of mucus.

Airway responses to antigen challenge in allergic rhinitis and allergic asthma.

The density dependence of the maximum expiratory flow-volume curve, functional residual capacity (FRC), and specific airway conductance (SGaw) were determined before and during bronchial provocation with ragweed extract in 27 subjects with ragweed hypersensitivity and a history of either bronchial asthma (16 subjects) or allergic rhinitis (11 subjects). Mean baseline SGaw was significantly lower while mean volume of isoflow (Visov) and FrC were significantly higher in subjects with bronchial asthma. During antigen challenge, 10 of 16 subjects with bronchial asthma (63%) and five of 11 subjects with allergic rhinitis (45%) showed a greater than 35% decrease in SGaw ("reactors"): mean relative decreases in SGaw from baseline were 46% and 53%, respectively. The remaining subjects showed a less than 35% decrease in SGaw ("nonreactors") with mean relative decreases of 9% (allergic asthma) and 6% (allergic rhinitis). Mean Visov increased in all subjects with bronchial asthma and in eight of 11 subjects with allergic rhinitis. A significant increase in FRC (6%) was seen only in the "reactors" with bronchial asthma. Following antigen challenge, the beta adrenergic agonist, isoetharine, increased SGaw and decreased Visov. We conclude that in asymptomatic subjects with ragweed hypersensitivity, (1) central and peripheral airway function is more abnormal in subjects with bronchial asthma than in subjects with allergic rhinitis, (2) subjects of both groups show quantitatively and qualitatively comparable airway responses during antigen challenge with a decrease in SGaw or an increase in Visov, possibly representing increase in central and/or peripheral airflow resistance, respectively, (3) Visov may be a more sensitive indicator of airway response to antigen challenge than SGaw, and (4) the bronchodilator effects of a beta adrenergic agonist on antigen-induced bronchospasm are similar in both groups.