Rapid nongenomic actions of inhaled corticosteroids on long-acting β(2)-agonist transport in the airway.

Corticosteroids inhibit organic cation transporters (OCTs) that play an important role in drug absorption, tissue distribution and elimination. Corticosteroid sensitivity of bronchodilator trafficking in the airway tissue, however, is poorly understood. To assess the effects of inhaled corticosteroids on airway absorption and disposal mechanisms of long-acting β(2)-agonists, human airway epithelial and smooth muscle cell uptake of tritiated formoterol and salmeterol was measured in vitro.

Decreased soluble adenylyl cyclase activity in cystic fibrosis is related to defective apical bicarbonate exchange and affects ciliary beat frequency regulation.

Human airway cilia contain soluble adenylyl cyclase (sAC) that produces cAMP upon HCO(3)(-)/CO(2) stimulation to increase ciliary beat frequency (CBF). Because apical HCO(3)(-) exchange depends on cystic fibrosis transmembrane conductance regulator (CFTR), malfunctioning CFTR might impair sAC-mediated CBF regulation in cells from patients with cystic fibrosis (CF). By Western blot, sAC isoforms are equally expressed in normal and CF airway epithelial cells, but CBF decreased more in CF than normal cells upon increased apical HCO(3)(-)/CO(2) exposure in part because of greater intracellular acidification from unbalanced CO(2) influx (estimated by 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) fluorescence).

Reactive oxygen species and hyaluronidase 2 regulate airway epithelial hyaluronan fragmentation.

Hyaluronidase 2 (Hyal2) is a hyaluronan (HA)-degrading enzyme found intracellularly or/and anchored to the plasma membrane through glycosylphosphatidylinositol (GPI). Normal human bronchial epithelial cells (NHBE) grown at the air-liquid interphase (ALI), treated with PI-specific phospholipase C (PI-PLC), exhibited increased Hyal activity in secretions and decreased protein and activity on the apical membrane, confirming that GPI-anchored Hyal2 is expressed in NHBE cells and it remains active in its soluble form. We have reported that HA degradation was mediated by reactive oxygen species (ROS) in human airways.

Hyaluronidase expression and activity is regulated by pro-inflammatory cytokines in human airway epithelial cells.

Hyaluronan (HA) is present at the apical surface of airway epithelium as a high-molecular-weight polymer. Since HA depolymerization initiates a cascade of events that results in kinin generation and growth factor processing, in the present work we used primary cultures of human bronchial epithelial (HBE) cells grown at the air-liquid interface (ALI) to assess hyaluronidase (Hyal) activity by HA zymography, gene expression by quantitative real-time PCR, and localization by confocal microscopy. Because TNF-alpha and IL-1beta induce Hyals in other cells, we tested their effects on Hyals expression and activity.

The effect of corticosteroids on the disposal of long-acting beta2-agonists by airway smooth muscle cells.

Background: Organic cation transporters (OCTs) have an important role in tissue distribution and elimination of cationic drugs. Carrier-mediated disposal of cationic bronchodilators in the airway tissue, however, is incompletely understood.

Objectives: We sought to assess the uptake of long-acting beta(2)-agonist bronchodilators by bronchial and vascular smooth muscle cells.

Soluble adenylyl cyclase is localized to cilia and contributes to ciliary beat frequency regulation via production of cAMP.

Ciliated airway epithelial cells are subject to sustained changes in intracellular CO(2)/HCO(3)(-) during exacerbations of airway diseases, but the role of CO(2)/HCO(3)(-)-sensitive soluble adenylyl cyclase (sAC) in ciliary beat regulation is unknown. We now show not only sAC expression in human airway epithelia (by RT-PCR, Western blotting, and immunofluorescence) but also its specific localization to the axoneme (Western blotting and immunofluorescence). Real time estimations of [cAMP] changes in ciliated cells, using FRET between fluorescently tagged PKA subunits (expressed under the foxj1 promoter solely in ciliated cells), revealed CO(2)/HCO(3)(-)-mediated cAMP production.

Real-time analysis of cAMP-mediated regulation of ciliary motility in single primary human airway epithelial cells.

Airway ciliary beat frequency regulation is complex but in part influenced by cyclic adenosine monophosphate (cAMP)-mediated changes in cAMP-dependent kinase activity, yet the cAMP concentration required for increases in ciliary beat frequency and the temporal relationship between ciliary beat frequency and cAMP changes are unknown. A lentiviral gene transfer system was developed to express a fluorescence resonance energy transfer (FRET)-based cAMP sensor in ciliated cells. Expression of fluorescently tagged cAMP-dependent kinase subunits from the ciliated-cell-specific foxj1 promoter enhanced expression in fully differentiated ciliated human airway epithelial cells, and permitted simultaneous measurements of ciliary beat frequency and cAMP (represented by the FRET ratio).

Epithelial organic cation transporters ensure pH-dependent drug absorption in the airway.

Most inhaled beta(2)-adrenergic agonist and anticholinergic bronchodilators have low lipid solubility because of their transient or permanent positive net charge at physiologic pH. Airway absorption of these cationic drugs is incompletely understood. We examined carrier-mediated mechanisms of cationic drug uptake by human airway epithelia.