Longitudinal stability of asthma characteristics and biomarkers from the Airways Disease Endotyping for Personalized Therapeutics (ADEPT) study.

BACKGROUND: Asthma is a biologically heterogeneous disease and development of novel therapeutics requires understanding of pathophysiologic phenotypes. There is uncertainty regarding the stability of clinical characteristics and biomarkers in asthma over time. This report presents the longitudinal stability over 12 months of clinical characteristics and clinically accessible biomarkers from ADEPT. METHODS: Mild, moderate, and severe asthma subjects were assessed at 5 visits over 12 months. Assessments included patient questionnaires, spirometry, bronchodilator reversibility, fractional exhaled nitric oxide (FENO), and biomarkers measured in induced sputum. RESULTS: Mild (n = 52), moderate (n = 55), and severe (n = 51) asthma cohorts were enrolled from North America and Western Europe. For all clinical characteristics and biomarkers, group mean data showed no significant change from visit to visit. However, individual data showed considerable variability. FEV1/FVC ratio showed excellent reproducibility while pre-bronchodilator FEV1 and FVC were only moderately reproducible. Of note bronchodilator FEV1 reversibility showed low reproducibility, with the nonreversible phenotype much more reproducible than the reversible phenotype. The 7-item asthma control questionnaire (ACQ7) demonstrated moderate reproducibility for the combined asthma cohorts, but the uncontrolled asthma phenotype (ACQ7 > 1.5) was inconstant in mild and moderate asthma but stable in severe asthma. FENO demonstrated good reproducibility, with the FENO-low phenotype (FENO < 35 ppb) more stable than the FENO-high phenotype (FENO ≥ 35 ppb). Induced sputum inflammatory phenotypes showed marked variability across the 3 sputum samples taken over 6 months. CONCLUSIONS: The ADEPT cohort showed group stability, individual stability in some parameters e.g. low FEV1/FVC ratio, and low FENO, but marked individual variability in other clinical characteristics and biomarkers e.g. type-2 biomarkers over 12 months. This variability is possibly related to seasonal variations in climate and allergen exposure, medication changes and acute exacerbations. The implications for patient selection strategies based on clinical biomarkers may be considerable.

Asthma characteristics and biomarkers from the Airways Disease Endotyping for Personalized Therapeutics (ADEPT) longitudinal profiling study.

BACKGROUND: Asthma is a heterogeneous disease and development of novel therapeutics requires an understanding of pathophysiologic phenotypes. The purpose of the ADEPT study was to correlate clinical features and biomarkers with molecular characteristics, by profiling asthma (NCT01274507). This report presents for the first time the study design, and characteristics of the recruited subjects. METHODS: Patients with a range of asthma severity and healthy non-atopic controls were enrolled. The asthmatic subjects were followed for 12 months. Assessments included history, patient questionnaires, spirometry, airway hyper-responsiveness to methacholine, fractional exhaled nitric oxide (FENO), and biomarkers measured in induced sputum, blood, and bronchoscopy samples. All subjects underwent sputum induction and 30 subjects/cohort had bronchoscopy. RESULTS: Mild (n = 52), moderate (n = 55), severe (n = 51) asthma cohorts and 30 healthy controls were enrolled from North America and Western Europe. Airflow obstruction, bronchodilator response and airways hyperresponsiveness increased with asthma severity, and severe asthma subjects had reduced forced vital capacity. Asthma control questionnaire-7 (ACQ7) scores worsened with asthma severity. In the asthmatics, mean values for all clinical and biomarker characteristics were stable over 12 months although individual variability was evident. FENO and blood eosinophils did not differ by asthma severity. Induced sputum eosinophils but not neutrophils were lower in mild compared to the moderate and severe asthma cohorts. CONCLUSIONS: The ADEPT study successfully enrolled asthmatics across a spectrum of severity and non-atopic controls. Clinical characteristics were related to asthma severity and in general asthma characteristics e.g. lung function, were stable over 12 months. Use of the ADEPT data should prove useful in defining biological phenotypes to facilitate personalized therapeutic approaches.

Esophageal dilation: simple and effective treatment for adults with eosinophilic esophagitis and esophageal rings and narrowing.

The goal of this article is to present the results of the long-term treatment with esophageal dilation of a consecutive series of adults with eosinophilic esophagitis (EoE). EoE in adults is a disease of middle aged white males, with recurrent food impactions and dysphagia. The exact treatment of EoE is unknown due to the uncertainty of the pathogenesis. Currently, the long-term follow-up of adult EoE patients is limited. Sixteen consecutive adult patients (12 males/4 females between ages 27 and 58 years) with EoE underwent a detailed history and baseline upper gastrointestinal endoscopy (EGD) with multiple esophageal biopsies. Thirteen had esophageal dilation. Fifteen were on proton pump inhibitor (PPI) therapy. After dilation, one patient was treated with a restrictive diet. One patient took prn fluticasone. Most of the patients had allergy testing for food and aeroallergens. Follow-up evaluation with similar testing was on average 22 months (range: 12-40 months). Six patients were not available for follow-up. None of the remaining 10 patients had a food impaction; one required further esophageal dilation. Only two patients had intermittent dysphagia. The average dysphagia score decreased from 2.1 to 0.3 (P < 0.002). The average number of eosinophils at follow-up was not significantly different from baseline (120 eosinophils/HPF proximally and 165 eosinophils/HPF distally (P= 0.75). The gross endoscopy findings were unchanged in all patients except one who normalized. A total of 62% and 75% of patients had positive tests for aeroallergens and food allergens, respectively. Over an average of two years, esophageal dilation provided excellent symptomatic relief among 10 adult EoE patients despite no improvement in the mucosal eosinophilia or gross endoscopic appearance.

Regulation of interleukin-1beta and interleukin-1beta inhibitor release by human airway epithelial cells.

In asthma, human airway epithelial cells (HAECs) regulate the intensity of mucosal inflammation, in part, by releasing the pro-inflammatory cytokine interleukin (IL)-1beta. However, the IL-1beta inhibitors, IL-1 receptor antagonist (IL-1RA) and soluble IL-1 receptor type II (sIL-1RII), regulate IL-1beta bioactivity. In order to better understand the control of IL-1beta activity in the airway mucosa, the role(s) of tumour necrosis factor (TNF)-alpha, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) in the release of IL-1beta and its inhibitors by cultured HAECs were examined. HAECs were treated with TNF-alpha (2-200 ng.mL(-1)), dibutyryl cAMP (0.01-1 mM), 8-bromo-cGMP (0.01-1 mM) or vehicle for 24 h, and cytokine levels in the HAEC-conditioned medium were measured by enzyme-linked immunosorbent assay. HAECs produced IL-1beta, IL-1RA and sIL-1RII constitutively, but the inhibitor concentrations greatly exceeded that of IL-1beta (by approximately 100- and approximately 550-fold, respectively). TNF-alpha dose-dependently increased the levels of all IL-1beta cytokine family members. However, over the range of TNF-alpha concentrations studied, IL-1beta concentration increased more than those of its inhibitors. cAMP increased constitutive and TNF-alpha-stimulated IL-1beta release but reduced that of sIL-1RII. In contrast, cGMP had no effect on IL-1beta but reduced IL-1RA and sIL-1RII release. Under basal conditions, the disproportionate release of inhibitors relative to interleukin-1beta by human airway epithelial cells probably prevents interleukin-1beta-mediated biological effects. Tumour necrosis factor-alpha, cyclic adenosine monophosphate and cyclic guanosine monophosphate may potentiate mucosal inflammation by increasing interleukin-1beta levels relative to those of its inhibitors in the airway mucosa.

Effect of tiotropium bromide on circadian variation in airflow limitation in chronic obstructive pulmonary disease.

BACKGROUND: In chronic obstructive pulmonary disease (COPD), the degree of circadian variation in forced expiratory volume in 1 second (FEV1) and the influence of anticholinergic blockade is not known. Tiotropium is a long acting inhaled anticholinergic bronchodilator that increases daytime FEV1 in COPD. We hypothesised that tiotropium would modify the overnight change in FEV1, and this would be unaffected by the timing of drug administration. METHODS: A double blind, randomised, placebo controlled trial was conducted with tiotropium 18 mg once daily in the morning (09.00 hours), evening (21.00 hours), or an identical placebo. Patients with stable COPD (n=121, FEV1=41% predicted) underwent spirometric tests every 3 hours for 24 hours at baseline and after 6 weeks of treatment. RESULTS: There were no significant differences at baseline between the groups. Tiotropium improved mean (SE) FEV1 (over 24 hours) in the morning (1.11 (0.03) l) and evening (1.06 (0.03) l) groups compared with placebo (0.90 (0.03) l), and nocturnal FEV1 (mean of 03.00 and 06.00 hours) in the morning (1.03 (0.03) l) and evening (1.04 (0.03) l) groups compared with placebo (0.82 (0.03) l) at the 6 week visit (p<0.01). FEV1 before morning or evening dosing was similar, while the peak FEV1 moved later in the day with active treatment. The mean percentage change in FEV1 from 09.00 hours to 03.00 hours (the nocturnal decline in FEV1) was -2.8% in the morning group, -1.0% in the evening group, and -12.8% in the placebo group. The magnitude of the peak to trough change in FEV1 was not statistically different. CONCLUSIONS: Tiotropium produced sustained bronchodilation throughout the 24 hour day without necessarily abolishing circadian variation in airway calibre.

Nuclear factor-kappa B augments beta(2)-adrenergic receptor expression in human airway epithelial cells.

Interleukin (IL)-1 beta increases beta(2)-adrenergic receptor (beta(2)-AR) mRNA and density by protein kinase C (PKC)-dependent mechanisms in human airway epithelial cells. The present study examined the role of several nuclear transcription factors in the PKC-activated upregulation of beta(2)-AR expression. BEAS-2B cells were exposed to the PKC activator phorbol 12-myristate 13-acetate (PMA; 0.1 microM for 2-18 h). PMA had no effect on activator protein (AP)-2 or cAMP response element binding protein DNA binding activity but markedly increased nuclear factor (NF)-kappa B and AP-1 binding as assessed by electrophoretic gel mobility shift assay. PMA also increased the activity of a beta(2)-AR promoter-luciferase reporter construct in transiently transfected cells. These effects were inhibited by the PKC inhibitors Ro-31-8220 and calphostin C. Furthermore, with increasing Ro-31-8220, beta(2)-AR promoter-reporter activity correlated closely with both NF-kappa B and AP-1 activities (r > 0.89 for both). Finally, the selective NF-kappa B inhibitor MG-132 dose dependently reduced NF-kappa B binding and beta(2)-AR promoter activity but increased AP-1 binding. We conclude that PKC-induced upregulation of beta(2)-AR expression in human airway epithelial cells appears to be mediated, at least in part, by increases in NF-kappa B activity.

IL-1beta enhances beta2-adrenergic receptor expression in human airway epithelial cells by activating PKC.

Protein kinase C (PKC)-activated signal transduction pathways regulate cell growth and differentiation in many cell types. We have observed that interleukin (IL)-1beta upregulates beta2-adrenergic receptor (beta2-AR) density and beta2-AR mRNA in human airway epithelial cells (e.g., BEAS-2B). We therefore tested the hypothesis that PKC-activated pathways mediate IL-1beta-induced beta-AR upregulation. The role of PKC was assessed from the effects of 1) the PKC activator phorbol 12-myristate 13-acetate (PMA) on beta-AR density, 2) selective PKC inhibitors (calphostin C and Ro-31-8220) on beta-AR density, and 3) IL-1beta treatment on the cellular distribution of PKC isozymes. Recombinant human IL-1beta (0.2 nM for 18 h) increased beta-AR density to 213% of control values (P < 0.001). PMA (1 microM for 18 h) increased beta-AR density to 225% of control values (P < 0.005), whereas Ro-31-8220 and calphostin C inhibited the IL-1beta-induced upregulation of beta-AR in dose-dependent fashion. PKC isozymes detected by Western blotting included alpha, betaII, epsilon, mu, zeta, and lambda/iota. IL-1beta increased PKC-mu immunoreactivity in the membrane fraction and had no effect on the distribution of the other PKC isozymes identified. These data indicate that IL-1beta-induced beta-AR upregulation is mimicked by PKC activators and blocked by PKC inhibitors and appears to involve selective activation of the PKC-mu isozyme. We conclude that signal transduction pathways activated by PKC-mu upregulate beta2-AR expression in human airway epithelial cells.

Chronic effects of inhaled albuterol on beta-adrenoceptor system function in human respiratory cells.

The in vivo effects of beta-adrenergic receptor (betaAR) agonists given chronically by metered-dose inhaler (MDI) on the molecular components of the beta-adrenoceptor system expressed by human respiratory cells are poorly understood. This study examined the effects of inhaled albuterol (180 microg four times daily for 7 days) on betaAR function of airway epithelial cells (AECs) and alveolar macrophages (AMs) freshly isolated from 10 normal subjects. Responses were related to beta2AR genotype in codons 16 and 27, regions which affect chronic responses to beta2-agonists. In AEC, betaAR density and adenosine cyclic 3',5'-phosphate (cAMP) production in response to isoproterenol (ISO) were significantly lower in the albuterol versus placebo treatment arm (p < 0.01 for both). Moreover, in AEC, albuterol treatment increased betaAR-kinase (betaARK) protein immunoreactivity. In contrast, in AM, albuterol tended to decrease betaAR density and cAMP production but changes did not achieve statistical significance (p > 0.20 for both) and had no effect on betaARK immunoreactivity. Changes in betaAR density occurred in all subjects but tended to be greater in subjects with the glycine 16 genotype. In cultured cells exposed to equal concentrations of beta-agonist in vitro, the magnitude of betaAR down-regulation (p < 0.05) and cAMP densensitization (p < 0.05) was greater in AEC than AM. These results indicate that albuterol taken by inhalation in a therapeutically relevant dose for 1 week produces betaAR down-regulation, densensitizes the cAMP response of airway epithelial cells to a beta2-adrenergic agonist, and increases betaARK immunoreactivity. Greater densensitization of AEC than AM in response to chronic albuterol inhalation likely reflects cell type-specific responses.

Brush Biopsy and Culture of Airway Epithelial Cells: ß-Adrenergic Receptor System Function.

Stimulation by catecholamine agonists of the ß-adrenergic coupled adenylyl cyclase (ßAR-AC) system, expressed on human tracheobronchial epithelial cells (ECs), elicits a variety of cellular responses that favorably affect airway function, the intensity of the inflammatory reaction, and even the integrity of the epithelial lining (1-6). For example, ß-agonist-stimulated production of second messenger, cyclic adenosine monophosphate (cAMP), enhances salt and water exchange (2), ciliary beating (3), mucus secretion by goblet cells (1,4), proliferation of airway ECs (5), and protection against free radical induced injury (6).

Salmeterol added to inhaled corticosteroid therapy is superior to doubling the dose of inhaled corticosteroids: a randomized clinical trial.

This randomized, double-blind, double-dummy, parallel group clinical trial compared the efficacy and safety of adding salmeterol xinafoate to concurrent inhaled beclomethasone dipropionate therapy with doubling the dose of beclomethasone dipropionate in patients experiencing symptoms on low-dose beclomethasone. Salmeterol added to low-dose beclomethasone was superior (p < or = 0.05) to doubling the dose of beclomethasone in improving peak expiratory flow (PEF) and forced expiratory volume in 1 sec (FEV1), and in reducing symptoms of asthma, sleep loss, nighttime awakenings, and use of albuterol. Both treatment regimens had comparable safety profiles. In asthma patients inadequately controlled despite the use of low-dose inhaled corticosteroids (i.e., less than 400 microg per day), the addition of salmeterol may be a more effective treatment option than doubling the dose of inhaled corticosteroids.

Variability of electrophrenic diaphragm twitch stimulation over time in normal subjects.

Transcutaneous electrophrenic twitch stimulation is a potentially powerful way to assess diaphragm contractile function in response to interventions which may alter respiratory muscle strength and endurance. At present, the variability of the transdiaphragmatic twitch pressure (Pdi(T)) over a several hour period is not well described. The present study examines the reproducibility of Pdi(T) amplitude and the twitch occlusion technique of assessing maximum transdiaphragmatic pressure (Pdi(max)) in seven normal adults stimulated intermittently every hour for a total 4-h period. In one subject, data were obtained on two occasions separated by a 2-month interval. Among all subjects, the Pdi(T) amplitude expressed as a percentage of the Pdi(max) was highly reproducible over 4 h (coefficient of variation 5.3). Peak Pdi(T) was inversely related to graded voluntary Pdi (r = -0.0996) and the relationship was virtually identical over 4 h (r = - 0.999, P = 0.96). These data show that Pdi(T) at functional residual capacity and the twitch occlusion relationship are highly reproducible.

Dose-response characteristics of nebulized albuterol in the treatment of acutely ill, hospitalized asthmatics.

We investigated the bronchodilator dose-response to nebulized albuterol and the dose of albuterol which produces maximal bronchodilation in the acutely ill, hospitalized asthmatic. Consecutively admitted patients from the emergency room in status asthmaticus who fulfilled the inclusion criteria (age <41 years old and <12 pack-years of smoking) were studied. Albuterol was administered by nebulizer (Puritan-Bennett Raindrop) in repeated 2.5-mg treatments up to a total dose of 10 mg and the bronchodilator response was measured by a computerized spirometer. Twenty-two patients were studied. Baseline spirometry showed a (mean +/- SE) forced expiratory volume in 1 sec (FEV1) of 1.26 +/- 0.14 L (42 +/- 4.0% predicted), which increased significantly (p < 0.05) during albuterol titration to a maximum FEV1 of 1.70 +/- 0.19 L (57 +/- 5% of predicted). After cumulative doses of 2.5, 5.0, 7.5, and 10.0 mg of nebulized albuterol, 27%, 45%, 72%, and 77% of patients, respectively, attained maximum bronchodilation. The remaining 23% of patients did not respond to doses up to 10 mg of albuterol. The maximum FEV1 response to albuterol did not correlate with the initial severity of airflow obstruction (r = 0.36, p > 0.05). Pulse rate and arterial oxygen saturation were not significantly affected by nebulized albuterol up to a total dose of 10 mg. No arrhythmias were noted. In summary, most hospitalized asthmatics (72%) required a cumulative dose of 7.5 mg of nebulized albuterol to achieve maximum bronchodilation and a large fraction (50%) required higher albuterol doses than the standard 2.5 mg. The bronchodilatory response to nebulized albuterol varied widely among patients in status asthmaticus and could not be predicted from the initial severity of airflow obstruction. Because side effects were minimal, it would be reasonable to use 7.5 mg of nebulized albuterol as initial therapy. Alternatively, dose-response titration with albuterol would be advantageous.

Effects of topical corticosteroids on inflammatory mediator-induced eicosanoid release by human airway epithelial cells.

BACKGROUND: Airway epithelial cells are among the first cells to come in contact with aerosolized corticosteroids. However, the relative potencies and time course of action of the several commonly used aerosolized corticosteroids on eicosanoid production by airway epithelial cells are unknown. OBJECTIVES: This study compared the effects of fluticasone, budesonide, and triamcinolone on eicosanoid output by human airway epithelial cells in vitro. We also determined the spectrum of eicosanoids affected and the mechanism for corticosteroid action. METHODS: Cultured BEAS-2B airway epithelial cells (a transformed cell line) were exposed to corticosteroids (1 nmol/L to 1 micromol/L) for 2 to 48 hours and then assayed for basal- and bradykinin (BK)-stimulated eicosanoid output. The eicosanoid profile was identified by HPLC in tritiated arachidonic acid prelabelled cells, and PGE2, the major eicosanoid product, was quantitated by RIA. The effect of corticosteroids on the immunoreactivity of key proteins involved in eicosanoid metabolism (ie, cyclooxygenase [COX], phospholipase A2 [PLA2], and Clara cell protein, a PLA2 inhibitor) was determined by Western blotting. RESULTS: Eicosanoid output was largely confined to prostaglandins with values of 5 +/- 2 and 82 +/- 35 ng PGE2/10(6) cells for basal- and BK stimulation, respectively (n = 8). All 3 corticosteroids inhibited basal- and BK-induced PGE2 output in a dose- and time-dependent manner. Fluticasone and budesonide completely eliminated PGE2 output in nanomolar concentrations in contrast to triamcinolone, which required micromolar concentration. The rank order of potency was: fluticasone = budesonide > triamcinolone. The time course of action for PGE2 inhibition also differed, with budesonide acting more slowly than the other 2 corticosteroids (P = .04). All 3 corticosteroids markedly reduced COX2 with little effect on COX1, cPLA2 (Type IV), or iPLA2 (Type VI) immunoreactivity or their relative distribution in cytosol versus membrane fractions. Clara cell protein immunoreactivity was undetectable in control and corticosteroid-treated cell lysates. CONCLUSION: These results show that in a human airway epithelial cell line, the 3 inhaled corticosteroids commonly used to treat asthma differ in onsets of action as inhibitors of prostaglandin synthesis and vary considerably in potency. All 3 corticosteroids act mechanistically in similar fashion by inhibiting COX2 synthesis.

Tolerance and early outcome results of postprostatectomy three-dimensional conformal radiotherapy.

PURPOSE: Three-dimensional conformal radiotherapy (3D-CRT) has been associated with a reduction in acute and late toxicity among patients treated for localized prostatic cancer. The purpose of this study is to assess the acute and late toxicity of 3D-CRT delivered to patients in the postprostatectomy setting and to analyze which factors predict for durable biochemical control in this group of patients. METHODS AND MATERIALS: Between 1988 and 1994, 42 patients were treated after prostatectomy with three-dimensional conformal radiotherapy. The median time from prostatectomy to radiotherapy was 11 months. Indications for treatment included a rising serum PSA level in 28 patients (65%) and positive surgical margins without a rising PSA level in 14 (35%). Twenty-five patients (60%) had pathologic stage T3 disease, and 32 (74%) had tumor at or close to the surgical margins. The median dose was 64.8 Gy, and the median follow-up time was 2 years. RESULTS: 3D-CRT in the postprostatectomy setting was well tolerated. Three patients (7%) experienced Grade II acute genitourinary toxicity and nine patients (21%) experienced Grade II acute gastrointestinal toxicity during treatment. No patient experienced Grade III or higher acute morbidity. The 2-year actuarial risk for Grade II late genitourinary and gastrointestinal late complications were 5 and 9%, respectively. In patients with existing incontinence, the incidence of worsening stress incontinence 6 months after treatment was 17%, which resolved within 12 months to its preradiotherapy level in four of six cases (66%). The overall 2-year postirradiation PSA relapse-free survival rate was 53%. The 2-year PSA relapse-free survival was 66% for patients with undetectable PSA levels in the immediate postoperative period compared to 26% for those with detectable levels of PSA after surgery (p < 0.006). Furthermore, for patients with preradiotherapy PSA levels of < or = 1.0 ng/ml, the 2-year PSA relapse-free survival was 74% compared to 17% of those with preradiotherapy PSA levels of > 1.0 ng/ml (p < 0.002). The resection margin status, presence of seminal vesicle involvement, Gleason score, and the preprostatectomy PSA level did not impact on PSA relapse-free survival. A Cox proportional hazards regression analysis demonstrated that a preradiotherapy PSA value of > 1 ng/ml (p < 0.002) was the most important covariate predicting for a rising PSA after radiotherapy. CONCLUSIONS: After prostatectomy, three-dimensional conformal radiotherapy is associated with minimal treatment-related morbidity. Patients with postprostatectomy, preradiotherapy PSA levels < or = 1.0 ng/ml, and those patients who had undetectable PSA levels in the immediate postoperative period are more likely to benefit from local adjuvant therapy.

IL-1 beta alters beta-adrenergic receptor adenylyl cyclase system function in human airway epithelial cells.

Inflammatory cells release a variety of cytokines, including interleukin (IL)-1 beta, into the airway in asthma. This study examined the effects of human IL-1 beta on the function of the beta-adrenergic receptor (beta AR)-adenylyl cyclase (AC) system in BEAS-2B cells, a human airway epithelial cell line. IL-1 beta markedly increased beta AR density (Bmax; P < 0.001) primarily by increasing the percentage of the beta 2AR subtype (from 67 to 91%; P < 0.001). Bmax increased monotonically over time in response to 200 pM IL-1 beta and was approximately 2.5-fold greater than control cells between 36 and 42 h. In contrast, the concentration response of Bmax to IL-1 beta given for 18 h was biphasic. Bmax increased with IL-1 beta concentrations from 2 to 200 pM, but, at > 200 pM, it decreased progressively toward control values. IL-1 beta-induced increases in Bmax with IL-1 beta were associated with approximately threefold increases in beta 2 AR mRNA and were blocked by the protein synthesis inhibitor cycloheximide. Despite the marked increase in Bmax, however, IL-1 beta depressed adenosine 3',5'-cyclic monophosphate (cAMP) responses to isoproterenol and forskolin, a direct activator of AC (P < 0.001 by analysis of variance for both). The inhibitory effect of IL-1 beta on cAMP production appeared to be explained by increases in the activity of an inhibitory GTP binding protein because IL-1 beta treatment increased the activity of a pertussis toxin ADP-ribosylated Gi alpha protein by approximately 2.5-fold; and pretreatment of intact cells with pertussis toxin inhibited the effect of IL-1 beta on cAMP production. These data indicate that IL-1 beta-mediated changes in the beta AR-AC system function in airway epithelial cells are complex and involve expression of receptor protein, GTP binding protein, and possibly AC itself. Increases in IL-1 beta may contribute to abnormalities in airway function in subjects with asthma.

Chronic effects of catecholamines on the beta 2-adrenoreceptor system in cultured human airway epithelial cells.

Chronic catecholamine treatment induces beta-adrenergic receptor (beta AR) downregulation, i.e., a loss of total cell receptors. In the human respiratory tract, the mechanism(s) underlying beta AR downregulation remains poorly understood. The present study, therefore, examined the effects of 24 h of exposure to isoproterenol (Iso; 10 nM or 1 microM) on beta AR density and the rate of beta AR degradation, steady-state beta 2-adrenergic receptor (beta 2 AR) mRNA levels, and the content of Gs alpha and Gi alpha proteins in cultured human bronchial epithelial cells (i.e., the BEAS-2B cell line). beta AR density assessed by binding with [125I]iodopindolol decreased in a dose-dependent fashion with 24 h of Iso exposure. With Iso (1 microM), beta AR density decreased by approximately 82%. In contrast, forskolin (100 microM) and dibutyryl adenosine 3',5'-cyclic monophosphate (1 mM), agents that also increase adenosine 3',5'-cyclic monophosphate (cAMP) levels, had no significant effect on beta AR density. Iso exposure also elicited a concomitant decrease in Iso-stimulated cAMP but had no significant effect on the content of the G proteins G alpha i2 and Gs alpha assessed by immunoblotting and toxin-catalyzed ADP ribosylation. Of note, Iso exposure (1 microM) had no effect on steady-state levels of beta 2 AR mRNA measured both by Northern analysis and by reverse transcriptase-polymerase chain reaction. However, beta AR half-life assessed in the presence of the protein synthesis inhibitor cycloheximide was reduced by approximately 60% in Iso-treated cells (i.e., from 37 h in control to 16 h in 1 microM Iso). These results suggest that, in human airway epithelial cells, beta 2 AR downregulation 1) is not primarily driven by intracellular cAMP levels, 2) is not associated with significant decreases in steady-state levels of beta 2 AR mRNA, and 3) is largely posttranslationally regulated by increases in the rate of receptor protein degradation.

Effect of lung volume on the respiratory action of the canine sternomastoid.

We tested the hypothesis that because the resting length of the canine sternomastoid (SM) muscles is relatively insensitive to lung volume change, the SM may maintain its inspiratory force generation regardless of lung volume. The relationships between SM pre- and postcontraction in situ fiber lengths and SM-produced inspiratory pressure generation [i.e., esophageal (Pes)] and rib cage displacements were examined in adult supine anesthetized dogs at residual volume (RV), functional residual capacity, and total lung capacity. SM muscle contraction was produced by isolated bilateral supramaximal electrical stimulation during hyperventilation-induced apnea. In all animals, SM contraction produced negative change in Pes (i.e., an inspiratory action). Passively increasing lung volume from RV to total lung capacity decreased (P < or = 0.01) the SM-produced Pes by -66 +/- 4% but had a relatively small effect on SM in situ pre- and postcontraction fiber length (< 3%). Whereas SM contraction at RV produced a cranial displacement of the sternum and increased the upper rib cage cross-sectional area, passively elevating lung volume diminished the SM-produced expansion of the upper rib cage. Hyperinflation did not increase the impedance of the sternum to cranial displacement during SM contraction, suggesting that hyperinflation caused a dissociation between the mechanical action of the sternum and the upper rib cage. These results suggest that mechanical dissociation of the ribs and sternum may diminish the contribution of the SM to inspiratory volume generation when breathing is done from elevated end-expiratory lung volumes.

Expression and function of the beta-adrenergic receptor coupled-adenylyl cyclase system on human airway epithelial cells.

Beta-adrenergic agonist-mediated activation of the beta receptor coupled-adenylyl cyclase (beta AR-AC) system expressed by human airway epithelial cells alters airway function. However, little is known about the magnitude of expression, subtype, and function of the beta receptor-adenylyl cyclase (beta AR-AC) system in human airway epithelial cells from healthy, nonsmoking subjects. Therefore, we characterized beta AR number and subtype and the cAMP response to isoproterenol (iso) in acutely dissociated human tracheocytes harvested from 22 healthy, nonsmoking adults during fibroptic bronchoscopy. Moreover, because the regulation of beta AR-AC system function in response to beta-agonists or inflammatory mediators released into the airway in asthma is poorly understood, we examined the cAMP response to iso after 30 min exposure of cells to iso or the protein kinase C activator, sn-1,2-dioctanoyl glycerol (diC8). The beta AR-AC system was highly expressed and functional in human airway epithelial cells. Group mean beta AR density (i.e., Bmax), equilibrium dissociation constant (Kd), and the percentage of beta 2AR subtypes assessed by radioligand binding were approximately 8,900 receptors/cell, 45 pM, and approximately 80%, respectively. Mean maximum cAMP production was approximately 42 pmol/10(5) cells, and the mean EC50 of the response to iso was 131 nM. However, Bmax and cAMP responses to iso varied considerably across subjects. For example, Bmax varied ninefold, and the EC50 of the cAMP response varied 39-fold interindividually. The EC50 was inversely related to beta AR density (r = -0.81, p < 0.05), suggesting that sensitivity of the cAMP response to iso was in part dependent on beta AR density. In all experiments, cAMP responses to iso stimulation were markedly desensitized in dose-dependent fashion by 30 min pretreatment with iso or diC8. For example, pretreatment with iso 10 microM or diC8 100 microM reduced maximum cAMP production to 22 and 63% of control values, respectively. These data indicate that: (1) the beta AR-AC system is highly expressed on acutely dissociated airway epithelial cells from normal adult, but beta AR expression and its functional coupling to adenylyl cyclase vary considerably interindividually; and (2) the beta AR-AC system of normal human airway epithelial cells is rapidly desensitized by exposure to beta-adrenergic agonists or activators of PKC.

Eicosanoid production in rabbit tracheal epithelium by adenine nucleotides: mediation by P2-purinoceptors.

Adenosine triphosphate (ATP) acting through epithelial nucleotide receptors exerts multiple physiologic actions on airway mucociliary clearance and caliber. However, the effect of ATP on arachidonate metabolism in the airway remains unknown. In this study, the ability of ATP to regulate eicosanoid production was studied in vitro in full-thickness rabbit tracheal strips and separately in rabbit epithelial explant cultures. In the freshly isolated strips, ATP increased prostaglandin E2 (PGE2) release in a dose-dependent fashion, with an activation threshold at 10 microM ATP and a 3.5-fold increase in PGE2 output at 1 mM ATP. Epithelium removal decreased 1 mM ATP-evoked PGE2 release by 68%. Reverse-phase, high-pressure liquid chromatography (HPLC) of media from 3H-arachidonic acid-incubated epithelial explants exposed to 1 mM ATP demonstrated increased output of the cyclooxygenase products PGE2 and prostaglandin F2a (PGF2a). Other identifiable eicosanoids did not increase. The concentration-response for ATP-induced PGE2 release by explants was similar to that of tracheal strips. PGE2 release by 1 mM ATP was 27% of that elicited by ionomycin (10 microM) and was markedly inhibited by indomethacin (10 microM). Purinoceptor agonist-stimulated PGE2 release by the epithelium yielded a rank order of potency of uridine triphosphate (UTP) > or = ATP > 2-methylthio-ATP (2MeSATP) >> alpha,beta-methyleneadenosine-5'-triphosphate (AMP-CPP) > or = adenosine. These results indicate that ATP, acting primarily through an epithelial P2-purinoceptor similar to the P2a subtype, stimulates eicosanoid metabolism in rabbit airway epithelium via the cyclooxygenase pathway, producing PGE2 as the predominant species.

Comparison of diaphragm strength between healthy adult elderly and young men.

To test the hypothesis that aging is associated with a reduction in the diaphragm's force-generating capacity, we compared the maximum transdiaphragmatic pressure (Pdimax) obtained during voluntary maximal inspiratory efforts in nine young (19-28 yr) and ten elderly (65-75 yr) subjects. The relationship between Pdi and lung volume was compared in the two groups by having subjects make maximal inspiratory maneuvers at specified lung volumes (i.e., 20, 40, and 80% vital capacity). Subjects underwent symptom-limited exercise tests to characterize their aerobic capacities and evaluate the relationship between aerobic capacity and Pdimax. The average Pdimax of the elderly subjects (128 +/- 9 cm H2O) was significantly lower (p < 0.003) than the average Pdimax of the younger subjects (171 +/- 8 cm H2O). In the elderly, Pdi was lower across the range of lung volumes tested (p < 0.001), and Pdimax occurred at similar relative lung volumes (elderly, at 47% total lung capacity [TLC]; young, at 50% TLC) in both groups. The elderly subjects were quite fit based on their VO2max, and there was no significant relationship between Pdimax and VO2max. This study suggests that diaphragm strength is reduced in elderly individuals. This age-related decrease in diaphragm strength may predispose elderly patients to diaphragm fatigue in the presence of conditions that impair inspiratory muscle function or increase ventilatory load.