Bitter Taste Receptors (TAS2Rs) in Human Lung Macrophages: Receptor Expression and Inhibitory Effects of TAS2R Agonists.

BACKGROUND: Bitter-taste receptors (TAS2Rs) are involved in airway relaxation but are also expressed in human blood leukocytes. We studied TAS2R expression and the effects of TAS2R agonists on the lipopolysaccharide (LPS)-induced cytokine release in human lung macrophages (LMs). METHODS: Lung macrophages were isolated from patients undergoing surgery for carcinoma. We used RT-qPCR to measure transcripts of 16 TAS2Rs (TAS2Rs 3/4/5/7/8/9/10/14/19/20/31/38/39/43/45 and 46) in unstimulated and LPS-stimulated (10 ng.mL-1) LMs. The macrophages were also incubated with TAS2R agonists for 24 h. Supernatant levels of the cytokines TNF-α, CCL3, CXCL8 and IL-10 were measured using ELISAs. RESULTS: The transcripts of all 16 TAS2Rs were detected in macrophages. The addition of LPS led to an increase in the expression of most TAS2Rs, which was significant for TAS2R7 and 38. Although the promiscuous TAS2R agonists, quinine and denatonium, inhibited the LPS-induced release of TNF-α, CCL3 and CXCL8, diphenidol was inactive. Partially selective agonists (dapsone, colchicine, strychnine, and chloroquine) and selective agonists [erythromycin (TAS2R10), phenanthroline (TAS2R5), ofloxacin (TAS2R9), and carisoprodol (TAS2R14)] also suppressed the LPS-induced cytokine release. In contrast, two other agonists [sodium cromoglycate (TAS2R20) and saccharin (TAS2R31 and 43)] were inactive. TAS2R agonists suppressed IL-10 production - suggesting that this anti-inflammatory cytokine is not involved in the inhibition of cytokine production. CONCLUSION: Human LMs expressed TAS2Rs. Experiments with TAS2R agonists' suggested the involvement of TAS2Rs 3, 4, 5, 9, 10, 14, 30, 39 and 40 in the inhibition of cytokine production. TAS2Rs may constitute new drug targets in inflammatory obstructive lung disease.

Epinephrine Dose Has a Preventive Effect on the Occurrence of Stress Ulcer-Induced Gastrointestinal Bleeding in Critically Ill Patients.

BACKGROUND: Epinephrine may impair splanchnic blood flow, but the impact of epinephrine dose on the occurrence of clinically significant gastrointestinal bleeding (CSGB) caused by stress ulcer remains unclear. We investigated the effect of epinephrine dose on the occurrence of stress ulcer-related CSGB in intensive care unit (ICU) patients. METHODS: In this prospective, observational, cohort study conducted in a French teaching hospital, 40 consecutive ICU patients receiving epinephrine infusion in whom a stress ulcer was diagnosed by an upper gastrointestinal endoscopy were included, from February 2010 to July 2015. The effects of epinephrine dose, and other covariates, on the occurrence of stress ulcer-related CSGB were analyzed using a multiple logistic regression model for repeated measures: At each observation, each patient serves as his own control. RESULTS: A total of 1484 time-dependent epinephrine dose modifications were available for analysis. The median epinephrine dose rate was 0.8 (0-9.5) mg/h, and the median epinephrine cumulative dose was 44.8 (2.6-2343) mg. Epinephrine, expressed as the average dose per day at time t, had a significant protective effect on the occurrence of stress ulcer (odds ratio 0.22; 95% confidence interval (CI), 0.12-0.38; p < 0.0001, for a log10 increase of epinephrine dose). Enteral feeding had also a protective effect (odds ratio 0.55; 95% CI 0.41-0.72; p < 0.0001, for a log10 increase of kcal/day). Only renal replacement therapy increased the occurrence of stress ulcer in the model. CONCLUSIONS: An increase in the average dose of epinephrine per day increased the time to occurrence of stress ulcer in critically ill patients.

The impact of low-frequency, low-force cyclic stretching of human bronchi on airway responsiveness.

BACKGROUND: In vivo, the airways are constantly subjected to oscillatory strain (due to tidal breathing during spontaneous respiration) and (in the event of mechanical ventilation) positive pressure. This exposure is especially problematic for the cartilage-free bronchial tree. The effects of cyclic stretching (other than high-force stretching) have not been extensively characterized. Hence, the objective of the present study was to investigate the functional and transcriptional response of human bronchi to repetitive mechanical stress caused by low-frequency, low-force cyclic stretching. METHODS: After preparation and equilibration in an organ bath, human bronchial rings from 66 thoracic surgery patients were stretched in 1-min cycles of elongation and relaxation over a 60-min period. For each segment, the maximal tension corresponded to 80% of the reference contraction (the response to 3 mM acetylcholine). The impact of cyclic stretching (relative to non-stretched controls) was examined by performing functional assessments (epithelium removal and incubation with sodium channel agonists/antagonists or inhibitors of intracellular pathways), biochemical assays of the organ bath fluid (for detecting the release of pro-inflammatory cytokines), and RT-PCR assays of RNA isolated from tissue samples. RESULTS: The application of low-force cyclic stretching to human bronchial rings for 60 min resulted in an immediate, significant increase in bronchial basal tone, relative to non-cyclic stretching (4.24 ± 0.16 g vs. 3.28 ± 0.12 g, respectively; p < 0.001). This cyclic stimulus also increased the affinity for acetylcholine (-log EC50: 5.67 ± 0.07 vs. 5.32 ± 0.07, respectively; p p < 0.001). Removal of airway epithelium and pretreatment with the Rho-kinase inhibitor Y27632 and inward-rectifier K+ or L-type Ca2+ channel inhibitors significantly modified the basal tone response. Exposure to L-NAME had opposing effects in all cases. Pro-inflammatory pathways were not involved in the response; cyclic stretching up-regulated the early mRNA expression of MMP9 only, and was not associated with changes in organ bath levels of pro-inflammatory mediators. CONCLUSION: Low-frequency, low-force cyclic stretching of whole human bronchi induced a myogenic response rather than activation of the pro-inflammatory signaling pathways mediated by mechanotransduction.

Effectiveness of a load-imposing device for cyclic stretching of isolated human bronchi: a validation study.

BACKGROUND: Mechanical ventilation may induce harmful effects in the airways of critically ill patients. Nevertheless, the effects of cyclic stretching caused by repetitive inflation-deflation of the bronchial compartment have not been well characterized in humans. The objective of the present study was to assess the effectiveness of a load-imposing device for the cyclic stretching of human bronchi. METHODS: Intact bronchial segments were removed from 128 thoracic surgery patients. After preparation and equilibration in an organ bath, bronchi were stretched repetitively and cyclically with a motorized transducer. The peak force imposed on the bronchi was set to 80% of each individual maximum contraction in response to acetylcholine and the minimal force corresponded to the initial basal tone before stretching. A 1-min cycle (stretching for 15 sec, relaxing for 15 sec and resting for 30 sec) was applied over a time period ranging from 5 to 60 min. The device's performance level was assessed and the properties of the stretched bronchi were compared with those of paired, non-stretched bronchi. RESULTS: Despite the intrinsic capacities of the device, the targets of the tension adjustments remained variable for minimal tension (156-178%) while the peak force set point was unchanged (87-115%). In the stretched bronchi, a time-dependent rise in basal tone (P < .05 vs. non-stretched) was apparent after as little as 5 min of cyclic stretching. The stretch-induced rise in basal tone continued to increase (P < .01) after the stretching had ended. Only 60 min of cyclic stretching was associated with a significant (P < .05) increase in responsiveness to acetylcholine, relative to non-stretched bronchi. CONCLUSIONS: Low-frequency, low-force, cyclic loading of human bronchi is associated with elevated basal tone and acetylcholine responsiveness. The present experimental model is likely to be a useful tool for future investigations of the bronchial response to repetitive stress during mechanical ventilation.

Wnt/ß-catenin signaling modulates human airway sensitization induced by ß2-adrenoceptor stimulation.

BACKGROUND: Regular use of ß2-agonists may enhance non-specific airway responsiveness. The wingless/integrated (Wnt) signaling pathways are responsible for several cellular processes, including airway inflammation and remodeling while cAMP-PKA cascade can activate the Wnt signaling. We aimed to investigate whether the Wnt signaling pathways are involved in the bronchial hyperresponsiveness induced by prolonged exposure to ß2-adrenoceptor agonists in human isolated airways. METHODS: Bronchi were surgically removed from 44 thoracic surgery patients. After preparation, bronchial rings and primary cultures of bronchial epithelial cells were incubated with fenoterol (0.1 µM, 15 hours, 37 °C), a ß2-agonist with high intrinsic efficacy. The effects of inhibitors/blockers of Wnt signaling on the fenoterol-induced airway sensitization were examined and the impact of fenoterol exposure on the mRNA expression of genes interacting with Wnt signaling or cAMP-PKA cascade was assessed in complete bronchi and in cultured epithelial cells. RESULTS: Compared to paired controls, fenoterol-sensitization was abolished by inhibition/blockage of the Wnt/ß-catenin signaling, especially the cell-surface LRP5/6 co-receptors or Fzd receptors (1 µM SFRP1 or 1 µM DKK1) and the nuclear recruitment of TCF/LEF transcriptions factors (0.3 µM FH535). Wnt proteins secretion did not seem to be involved in the fenoterol-induced sensitization since the mRNA expression of Wnt remained low after fenoterol exposure and the inactivator of Wnt secretion (1 µM IWP2) had no effect on the fenoterol-sensitization. Fenoterol exposure did not change the mRNA expression of genes regulating Wnt signaling or cAMP-PKA cascade. CONCLUSIONS: Collectively, our pharmacological investigations indicate that fenoterol-sensitization is modulated by the inhibition/blockage of canonical Wnt/ß-catenin pathway, suggesting a phenomenon of biased agonism in connection with the ß2-adrenoceptor stimulation. Future experiments based on the results of the present study will be needed to determine the impact of prolonged fenoterol exposure on the extra- and intracellular Wnt signaling pathways at the protein expression level.

Effect of severe acidosis on vasoactive effects of epinephrine and norepinephrine in human distal mammary artery.

OBJECTIVE: Acidosis is a very common pathologic process in perioperative management. However, how to correct severe acidosis to improve the efficacy of vasoconstrictors in hemodynamically unstable patients is still debated. The present study investigated whether severe extracellular acidosis influences the vasoactive properties of vasoconstrictors on human isolated arteries. METHODS: Segments of intact distal internal mammary arteries were removed from 41 patients undergoing artery bypass grafting. The arterial rings were washed in Krebs-Henseleit solution and suspended in an organ bath. The rings were set at a pretension equivalent of 100 mm Hg, and the relaxation response to 10 µM acetylcholine was verified. Concentration-response curves for epinephrine, norepinephrine, methoxamine (α1A/D-adrenoceptor agonist), phenylephrine (equipotent agonist of α1A/B-adrenoceptors), and clonidine (α2-adrenoceptor agonist) were achieved under control conditions (pH 7.40) and under acidic conditions by substitution of the Krebs-Henseleit solution with a modified solution. RESULTS: Decreasing the pH from 7.40 to 7.20, 7.0, or 6.80 did not significantly alter the potency and efficacy of epinephrine and norepinephrine, although the standardized effect size was sometimes large. Severe acidosis (pH 6.80) did not significantly change the potency and efficacy of phenylephrine and clonidine, although it increased the efficacy and potency of methoxamine (P < .001 and P = .04 vs paired control conditions, respectively). CONCLUSIONS: Extracellular acidosis did not impair the vasoactive properties of epinephrine and norepinephrine in human medium-size arteries until pH 6.80. The results of the present study also suggest that acidosis might potentiate arterial responsiveness to vasoconstrictors, mostly by way of the α1D-adrenoceptor.

Growth-arrest-specific 6 (GAS6) protein in ARDS patients: determination of plasma levels and influence of PEEP setting.

BACKGROUND: Growth-arrest-specific protein 6 (GAS6) is a vitamin K-dependent protein expressed by endothelial cells and leukocytes participating in cell survival, migration and proliferation and involved in many pathological situations. The aim of our study was to assess its implication in ARDS and its variation according to PEEP setting, considering that different cyclic stresses could alter GAS6 plasma levels. METHODS: Our subjects were enrolled in the ExPress study comparing a minimal alveolar distention (low-PEEP) ventilatory strategy to a maximal alveolar recruitment (high-PEEP) strategy in ARDS. Plasma GAS6, interleukin-8 (IL-8), and vascular endothelial growth factor (VEGF) levels were measured at day 0 and day 3 by enzyme-linked immunosorbent assay in blood samples prospectively collected during the study for a subset of 52 subjects included in 8 centers during year 2005. RESULTS: We found that GAS6 plasma level was elevated in the whole population at day 0: median 106 ng/mL IQR 77-139 ng/mL, with significant correlations with IL-8, the Simplified Acute Physiology Score II and the Organ Dysfunction and Infection scores. Statistically significant decreases in GAS6 and IL-8 plasma levels were observed between day 0 and day 3 in the high-PEEP group (P = .02); while there were no differences between day 0 and day 3 in the low-PEEP group. CONCLUSIONS: GAS6 plasma level is elevated in ARDS patients. The high-PEEP strategy is associated with a decrease in GAS6 and IL-8 plasma levels at day 3, without significant differences in day 28 mortality between the 2 groups. (Clinicaltrials.gov NCT00188058).

Impact of organizational culture on preventability assessment of selected adverse events in the ICU: evaluation of morbidity and mortality conferences.

PURPOSE: To determine whether organizational culture is associated with preventability assessment of reported adverse events (AE) in intensive care units (ICU). DESIGN: Blind review of time randomly distributed case notes written in the form of structured abstracts by the nurses who participated in recently implemented morbidity and mortality conferences from December 2006 to June 2010 in a 18-bed ICU in France. Ninety-five abstracts summarizing the discussions of 95 AE involving 95 patients were reviewed by two external blinded pairs (each comprised of one senior intensivist and one psychologist). METHODS: A score for each organizational culture style was determined, with the highest scorer being considered the dominant style present in the abstract. RESULTS: Reliability of the classification and quantification of culture traits between pairs was very good or good for 13 dimensions and moderate for two others. The two pairs deemed 32/95 and 43/95 of AE preventable (κ = 0.59). Concordance was very good (κ = 0.85) between the external pairs for evaluation of the dominant culture style. The Cochran-Armitage trend test indicated an increasing trend for change of the dominant organizational culture style over time: the team-satisfaction-oriented culture took a leading role (p = 0.02), while the people-security-oriented culture decreased dramatically (p < 0.001). The task-security-oriented culture was significantly associated with a preventable judgment, while the people-security-oriented culture was significantly associated with an unpreventable judgment (p < 0.001). CONCLUSIONS: This study demonstrated a strong relationship between preventability assessment of AE reported by caregivers and their organizational culture in the ICU.

Intranasal drug delivery: an efficient and non-invasive route for systemic administration: focus on opioids.

Intranasal administration is a non-invasive route for drug delivery, which is widely used for the local treatment of rhinitis or nasal polyposis. Since drugs can be absorbed into the systemic circulation through the nasal mucosa, this route may also be used in a range of acute or chronic conditions requiring considerable systemic exposure. Indeed, it offers advantages such as ease of administration, rapid onset of action, and avoidance of first-pass metabolism, which consequently offers for example an interesting alternative to intravenous, subcutaneous, oral transmucosal, oral or rectal administration in the management of pain with opioids. Given these indisputable interests, fentanyl-containing formulations have been recently approved and marketed for the treatment of breakthrough cancer pain. This review will outline the relevant aspects of the therapeutic interest and limits of intranasal delivery of drugs, with a special focus on opioids, together with an in-depth discussion of the physiological characteristics of the nasal cavity as well as physicochemical properties (lipophilicity, molecular weight, ionisation) and pharmaceutical factors (absorption enhancers, devices for application) that should be considered for the development of nasal drugs.

Airway response to acute mechanical stress in a human bronchial model of stretch.

INTRODUCTION: Lung inflation may have deleterious effects on the alveoli during mechanical ventilation. However, the consequences of stretch during excessive lung inflation on basal tone and responsiveness of human bronchi are unknown. This study was undertaken to devise an experimental model of acute mechanical stretch in isolated human bronchi and to investigate its effect on airway tone and responsiveness. METHODS: Bronchi were removed from 48 thoracic surgery patients. After preparation and equilibration in an organ bath, bronchial rings were stretched for 5 min using a force (2.5 × basal tone) that corresponded to airway-inflation pressure > 30 cm H2O. The consequences of stretch were examined by using functional experiments, analysis of organ-bath fluid, and ribonucleic acid (RNA) isolation from tissue samples. RESULTS: Following removal of the applied force the airways immediately developed an increase in basal tone (P < 0.0001 vs. paired controls) that was sustained and it did so without significantly increasing responsiveness to acetylcholine. The spontaneous tone was abolished with a Rho-kinase inhibitor and epithelium removal, a leukotriene antagonist or nitric oxide synthase inhibitors reduced it, whereas indomethacin, sensory nerve inhibitors or antagonists for muscarinic, endothelin and histamine receptors had no effect. Stretch enhanced leukotriene-E4 production during the immediate spontaneous contraction of human bronchi (P < 0.05). Moreover, stretch up-regulated the early mRNA expression of genes involved in wingless-type mouse mammary tumor virus integration-site family (WNT)-signaling and Rho-kinase pathways. CONCLUSIONS: Stretching human bronchi for only 5 min induces epithelial leukotriene release via nitric oxide synthase activation and provokes a myogenic response dependent on Rho-kinase and WNT-signaling pathways. From a clinical perspective, these findings highlight the response of human airway to acute mechanical stress during excessive pulmonary inflation.

Impact of morbidity and mortality conferences on analysis of mortality and critical events in intensive care practice.

BACKGROUND: Morbidity and mortality conferences are a tool for evaluating care management, but they lack a precise format for practice in intensive care units. OBJECTIVES: To evaluate the feasibility and usefulness of regular morbidity and mortality conferences specific to intensive care units for improving quality of care and patient safety. METHODS: For 1 year, a prospective study was conducted in an 18-bed intensive care unit. Events analyzed included deaths in the unit and 4 adverse events (unexpected cardiac arrest, unplanned extubation, reintubation within 24-48 hours after planned extubation, and readmission to the unit within 48 hours after discharge) considered potentially preventable in optimal intensive care practice. During conferences, events were collectively analyzed with the help of an external auditor to determine their severity, causality, and preventability. RESULTS: During the study period, 260 deaths and 100 adverse events involving 300 patients were analyzed. The adverse events rate was 16.6 per 1000 patient-days. Adverse events occurred more often between noon and 4 pm (P = .001).The conference consensus was that 6.1% of deaths and 36% of adverse events were preventable. Preventable deaths were associated with iatrogenesis (P = .008), human errors (P < .001), and failure of unit management factors or communication (P = .003). Three major recommendations were made concerning standardization of care or prescription and organizational management, and no similar incidents have recurred. CONCLUSION: In addition to their educational value, regular morbidity and mortality conferences formatted for intensive care units are useful for assessing quality of care and patient safety.

beta2-Agonist modulates epithelial gene expression involved in the T- and B-cell chemotaxis and induces airway sensitization in human isolated bronchi.

Regular use of beta(2)-adrenoceptor agonists may enhance non-specific airway responsiveness and inflammation. In earlier experimental studies, we showed that prolonged in vitro fenoterol exposure induced airway sensitization via perturbed epithelial regulation of bronchoconstriction. The aim of the present work was to examine the involvement of inflammatory mediator genes and proinflammatory cells and to investigate the role of the bronchial epithelium in these untoward effects. Bronchial tissues were surgically removed from 17 ex-smokers. Bronchial rings and primary cultures of bronchial epithelial cells were incubated with 0.1microM fenoterol for 15h. Levels of mRNA-expression were analyzed using a real-time quantitative reverse transcription-polymerase chain reaction array. Bronchial rings were contracted with endothelin-1 and immune cell infiltration was assessed by immunohistochemistry. Compared to paired controls, fenoterol up-regulated the mRNAs of cytokines/proteins implicated in the recruitment of T and B cells or the activation and proliferation of bronchial epithelial cells (CCL20/MIP-3alpha, FOXA2, PPAR-gamma) in isolated bronchi and in cultured epithelial cells. Fenoterol exposure significantly enhanced CD8(+)-T and differentiated CD138(+)-B-cells infiltration into the bronchi, especially the subepithelial area. Increase in CD8 or CD138 labeling-intensity strongly correlated with rise in maximal contraction to endothelin-1 induced by fenoterol exposure. In summary, our results show that fenoterol modulates the T and B cells chemotaxis possibly via the epithelial chemokine secretion in isolated bronchi from ex-smokers. They also suggest that the infiltration of resident T and B cells into the subepithelial area is associated with an increase in airway responsiveness due to fenoterol exposure.

Ultrasonographic diagnostic criterion for severe diaphragmatic dysfunction after cardiac surgery.

BACKGROUND: Severe diaphragmatic dysfunction can prolong mechanical ventilation after cardiac surgery. An ultrasonographic criterion for diagnosing severe diaphragmatic dysfunction defined by a reference technique such as transdiaphragmatic pressure measurements has never been determined. METHODS: Twenty-eight patients requiring mechanical ventilation > 7 days postoperatively were studied. Esophageal and gastric pressures were measured to calculate transdiaphragmatic pressure during maximal inspiratory effort and the Gilbert index, which evaluates the diaphragm contribution to respiratory pressure swings during quiet ventilation. Ultrasonography allowed measuring right and left hemidiaphragmatic excursions during maximal inspiratory effort. Best E is the greatest positive value from either hemidiaphragm. Twenty cardiac surgery patients with uncomplicated postoperative course were also evaluated with ultrasonography preoperatively and postoperatively. Measurements were performed in semirecumbent position. RESULTS: Transdiaphragmatic pressure during maximal inspiratory effort was below normal value in 27 of the 28 patients receiving prolonged mechanical ventilation (median, 39 cm H(2)O; interquartile range [IQR] 28 cm H(2)O). Eight patients had Gilbert indexes 0 (30 mm; IQR, 10 mm; vs 19 mm; IQR, 7 mm, respectively; p = 0.001). Best E < 25 mm had a positive likelihood ratio of 6.7 (95% confidence interval [CI], 2.4 to 19) and a negative likelihood ratio of 0 (95% CI, 0 to 1.1) for having a Gilbert index

Role of nitric oxide synthase/arginase balance in bronchial reactivity in patients with chronic obstructive pulmonary disease.

Competition between nitric oxide synthases (NOSs) and arginases for their common substrate l-arginine could be involved in the regulation of cholinergic airway reactivity and subsequent airway remodeling. The aims of this study were to evaluate the relationships between the expression of this enzymatic balance and the effects of NOS and arginase inhibition on bronchoconstrictive response to acetylcholine of patients without and with early chronic obstructive pulmonary disease (COPD). Twenty-two human bronchi [15 COPD (9 GOLD-0, 6 GOLD-1, -2-A), 7 nonsmokers] were investigated for immunohistochemistry and modulation of acetylcholine-induced airway constriction. Significantly increased expression of NOS2 in immunoblots of bronchial tissue and staining in smooth muscle cells was evidenced in patients with COPD compared with control subjects, whereas no modification of arginase expression was evidenced. Forced expiratory volume in 1 s (FEV1) and NOS2 expression were negatively correlated (rho=-0.54, P=0.027). Pharmacological experiments demonstrated that resting tension was elevated in COPD compared with control subjects (2,243+/-154 vs. 1,574+/-218 mg, P=0.03) and was positively correlated with the expression of NOS2 (rho=0.61, P=0.044), whereas constrictor response to acetylcholine was similar [active tension, sensitivity (-logEC10), and reactivity (slope)]. The sole effect of the specific arginase inhibitor Nomega-hydroxy-nor-L-arginine (1 microM) was to decrease sensitivity in COPD patients, whereas 1 mM NG-nitro-L-arginine methyl ester unexpectedly decreased resting tension because of a non-cGMP-dependent effect. In conclusion, an upregulation of NOS2 expression in COPD patients is involved in airway tone regulation and functional airflow limitation, whereas increased arginase activity is involved in airway sensitivity.

Phosphodiesterase 4 inhibitors modulate beta2-adrenoceptor agonist-induced human airway hyperresponsiveness.

Chronic exposure of human isolated bronchi to beta2-adrenergic agonists, especially fenoterol, potentiates smooth muscle contraction in response to endothelin-1 (ET-1), a peptide implicated in chronic inflammatory airway diseases. 5'-Cyclic adenosine monophosphate (cAMP) pathways are involved in fenoterol-induced hyperresponsiveness. The present study investigated whether chronic elevation of intracellular cAMP by other pathways than beta2-adrenoceptor stimulation provokes bronchial hyperresponsiveness. Samples from eighteen human bronchi were sensitized to ET-1 by prolonged incubation with 0.1 microM fenoterol (15 h, 21 degrees C), or, under similar conditions, were incubated with a selective type-3 phosphodiesterase inhibitor (1 microM siguazodan), two selective type-4 phosphodiesterase inhibitors (0.1 microM rolipram and 0.1 microM cilomilast), a combination of fenoterol and rolipram (0.1 microM each) or of fenoterol and cilomilast (0.1 microM each). Rolipram and cilomilast, but not siguazodan, induced hyperresponsiveness (p < 0.01 and p < 0.05 vs. paired controls, respectively) similar to the fenoterol effect. Fenoterol-induced bronchial hyperresponsiveness was significantly enhanced by coincubation with cilomilast (p < 0.05 vs. fenoterol alone) but not with rolipram. Our results suggest that prolonged activation of intracellular cAMP through phosphodiesterase 4 inhibition induces hyperresponsiveness to ET-1 in human isolated bronchi. However, differences in subcellular localization of phosphodiesterase 4 may provoke divergent responsiveness patterns when human bronchi are continuously exposed to selective phosphodiesterase inhibitors with or without beta2-adrenergic agonists.

Protease-activated receptor 2 in regulation of bronchomotor tone: effect of tobacco smoking.

Protease-activated receptors are G protein-coupled receptors activated by serine-proteases. Protease-activated receptor 2 is involved in the regulation of airway smooth muscle tone but its effects vary according to species and experimental conditions. We determined the effects of protease-activated receptor 2 activation on smooth muscle tone and airway reactivity to histamine in guinea pigs and smoking or non-smoking humans. The effects of trypsin and protease-activated receptor activating peptide on the isometric tension and response to histamine of guinea pig tracheal and human bronchial rings were studied. Human tissues were obtained from 6 smokers and 4 non-smokers. We assessed the effects of epithelial removal, inhibitors of cyclooxygenases, nitric oxide synthases, neutral endopeptidase and antagonists of acetylcholine, histamine, bradykinin and tachykinin receptors. Bronchomotor responses to protease-activated receptor 2 activation were variable in guinea pig, in half of animals PAR2 activation induced smooth muscle relaxation through the epithelial release of prostanoids but not of nitric oxide. In human airways, protease-activated receptor 2 activation reduced responsiveness to histamine in bronchial rings from smokers but increased responsiveness in bronchi from non-smokers. This study demonstrates an influence of tobacco smoking on the effect of protease-activated receptor 2 activation on airway responsiveness in humans, with an increased protection against histamine-induced contractions, probably through an increased epithelial release of prostanoids. The role of airway protease-activated receptor 2 may be to maintain smooth muscle tone homeostasis.

Clinically significant gastrointestinal bleeding in critically ill patients with and without stress-ulcer prophylaxis.

OBJECTIVE: To compare the rates of clinically significant gastrointestinal bleeding and the number of blood units and endoscopies required for gastrointestinal hemorrhage between patients receiving or not receiving stress-ulcer prophylaxis. DESIGN: Historical observational study comparing two consecutive periods: with (phase 1) and without stress-ulcer prophylaxis (phase 2). DESIGN AND SETTING: A 17-bed intensive care unit in a university teaching hospital. PATIENTS. In phase 1 there were 736 patients and in phase 2737. Those in the two phases were comparable in age and reason for admission; clinically significant gastrointestinal bleeding rates did not differ between the two phases, but patients in phase 2 were more severely ill. MEASUREMENTS AND RESULTS: Comparable numbers of blood units were transfused per bleeding patient in the two phases, especially for patients with significant gastrointestinal bleeding. During each phase 19 fibroscopies were performed for significant bleeding, and two patients required surgery. The clinically significant gastrointestinal bleeding rate and outcome did not differ in patients with at least one risk factor. Total expenditures directly related to gastrointestinal bleeding were similar during the two phases; the total cost incurred by stress-ulcer prophylaxis was estimated at 6700. CONCLUSIONS: Our results suggest that stress-ulcer prophylaxis does not influence the clinically significant gastrointestinal bleeding rate in intensive care unit patients or the cost of its management.

In vitro sensitization of human bronchus by beta2-adrenergic agonists.

Incubation of human distal bronchi from 48 patients for 15 h with 10(-7) M fenoterol induced sensitization characterized by an increase in maximal contraction to endothelin-1 (ET-1) and acetylcholine (ACh). Incubation of human bronchi with 10(-6), 3 x 10(-6), and 10(-5) M forskolin (an adenyl cyclase activator) reproduced sensitization to ET-1 and ACh. The sensitizing effect of fenoterol was inhibited by coincubation with gliotoxine (a nuclear factor-kappaB inhibitor), dexamethasone, indomethacin (a cyclooxygenase inhibitor), GR-32191 (a TP prostanoid receptor antagonist), MK-476 (a cysteinyl leukotriene type 1 receptor antagonist), SR-140333 + SR-48968 + SR-142801 (neurokinin types 1, 2, and 3 tachykinin receptor antagonists) with or without HOE-140 (a bradykinin B(2) receptor antagonist), SB-203580 (an inhibitor of the 38-kDa mitogen-activated protein kinase, p38(MAPK)), or calphostin C (a protein kinase C blocker). Our results suggest that chronic exposure to fenoterol induces proinflammatory effects mediated by nuclear factor-kappaB and pathways involving leukotrienes, prostanoids, bradykinin, tachykinins, protein kinase C, and p38(MAPK), leading to the regulation of smooth muscle contraction to ET-1 and ACh.