Modelling dysregulated Na+ absorption in airway epithelial cells with mucosal nystatin treatment.

In cystic fibrosis (CF), the absence of functional CFTR leads to dysregulated Na(+) absorption across airway epithelia. We established an in vitro model of dysregulated Na(+) absorption by treating polarized normal human bronchial epithelial cells (HBEs) with nystatin (Nys), a polyene antibiotic that enables monovalent cations to permeate biological membranes. Acute mucosal Nys produced a rapid increase in short circuit current (I(sc)) that reflected increased transepithelial Na(+) absorption and required Na(+)/K(+)ATPase activity. The acute increase in I(sc) was associated with increased mucosal liquid absorption. Prolonged mucosal Nys treatment resulted in sustained Na(+) hyperabsorption, associated with increased mucosal liquid absorption in comparison with naïve (nontreated, kept under air-liquid interface conditions) or vehicle-treated cultures. Nys treatment was not toxic. Increased lactate accumulation in Nys-treated culture media suggested a higher metabolic rate associated with the higher energy demand for Na(+) transport. After chronic Nys treatment, the increased I(sc) was rapidly lost when the cultures were mounted in Ussing chambers, indicating that Nys could be rapidly removed from the apical membrane. Importantly, chronic Nys treatment promoted sustained mucosal liquid depletion and caused mucus dehydration, compaction, and adhesion to the apical surface of Nys-treated cultures. We conclude that mucosal Nys treatment of HBEs provides a simple in vitro model to recapitulate the Na(+) and volume hyperabsorptive features of CF airway epithelia.

Mucin granules are in close contact with tubular elements of the endoplasmic reticulum.

Live cell imaging methods were used to characterize goblet cells expressing a MUC5AC domain fused to enhanced green fluorescent protein that labels the granule lumen. Golgi complex and endosome/lysosome elements largely resided in the periphery of the granular mass. On the contrary, a tubular meshwork of endoplasmic reticulum (ER) was in close contact with the mucin granules. This meshwork could be identified in fixed native human bronchial goblet cells labeled with an anti-calreticulin antibody. The potential biological significance of this ER network for mucin secretion is discussed.

Chronic airway infection/inflammation induces a Ca2+i-dependent hyperinflammatory response in human cystic fibrosis airway epithelia.

Hyperinflammatory responses to infection have been postulated as a component of cystic fibrosis (CF) lung disease. Studies have linked intracellular calcium (Ca(2+)(i)) mobilization with inflammatory responses in several systems. We have reported that the pro-inflammatory mediator bradykinin (BK) promotes larger Ca(2+)(i) signals in CF compared with normal bronchial epithelia, a response that reflects endoplasmic reticulum (ER)/Ca(2+) store expansion induced by chronic luminal airway infection/inflammation. The present study investigated whether CF airway epithelia were hyperinflammatory and, if so, whether the hyperinflammatory CF phenotype was linked to larger Ca(2+) stores in the ER. We found that DeltaF508 CF bronchial epithelia were hyperinflammatory as defined by an increased basal and mucosal BK-induced interleukin (IL)-8 secretion. However, the CF hyperinflammation expressed in short-term (6-11-day-old) primary cultures of DeltaF508 bronchial epithelia was lost in long-term (30-40-day-old) primary cultures of DeltaF508 bronchial epithelia, indicating this response was independent of mutant cystic fibrosis transmembrane conductance regulator. Exposure of 30-40-day-old cultures of normal airway epithelia to supernatant from mucopurulent material (SMM) from CF airways reproduced the increased basal and mucosal BK-stimulated IL-8 secretion of short-term CF cultures. The BK-triggered increased IL-8 secretion in SMM-treated cultures was mediated by an increased Ca(2+)(i) mobilization consequent to an ER expansion associated with increases in protein synthesis (total, cytokines, and antimicrobial factors). The increased ER-dependent, Ca(2+)(i)-mediated hyperinflammatory epithelial response may represent a general beneficial airway epithelial adaptation to transient luminal infection. However, in CF airways, the Ca(2+)(i)-mediated hyperinflammation may be ineffective in promoting the eradication of infection in thickened mucus and, consequently, may have adverse effects in the lung.

Cystic fibrosis airway epithelial Ca2+ i signaling: the mechanism for the larger agonist-mediated Ca2+ i signals in human cystic fibrosis airway epithelia.

In cystic fibrosis (CF) airways, abnormal epithelial ion transport likely initiates mucus stasis, resulting in persistent airway infections and chronic inflammation. Mucus clearance is regulated, in part, by activation of apical membrane receptors coupled to intracellular calcium (Ca(2+)(i)) mobilization. We have shown that Ca(2+)(i) signals resulting from apical purinoceptor (P2Y(2)-R) activation are increased in CF compared with normal human airway epithelia. The present study addressed the mechanism for the larger apical P2Y(2)-R-dependent Ca(2+)(i) signals in CF human airway epithelia. We show that the increased Ca(2+)(i) mobilization in CF was not specific to P2Y(2)-Rs because it was mimicked by apical bradykinin receptor activation, and it did not result from a greater number of P2Y(2)-R or a more efficient coupling between P2Y(2)-Rs and phospholipase C-generated inositol 1,4,5-trisphosphate. Rather, the larger apical P2Y(2)-R activation-promoted Ca(2+)(i) signals in CF epithelia resulted from an increased density and Ca(2+) storage capacity of apically confined endoplasmic reticulum (ER) Ca(2+) stores. To address whether the ER up-regulation resulted from ER retention of misfolded DeltaF508 CFTR or was an acquired response to chronic luminal airway infection/inflammation, three approaches were used. First, ER density was studied in normal and CF sweat duct human epithelia expressing high levels of DeltaF508 CFTR, and it was found to be the same in normal and CF epithelia. Second, apical ER density was morphometrically analyzed in airway epithelia from normal subjects, DeltaF508 homozygous CF patients, and a disease control, primary ciliary dyskinesia; it was found to be greater in both CF and primary ciliary dyskinesia. Third, apical ER density and P2Y(2)-R activation-mobilized Ca(2+)(i), which were investigated in airway epithelia in a long term culture in the absence of luminal infection, were similar in normal and CF epithelia. To directly test whether luminal infection/inflammation triggers an up-regulation of the apically confined ER Ca(2+) stores, normal airway epithelia were chronically exposed to supernatant from mucopurulent material from CF airways. Supernatant treatment expanded the apically confined ER, resulting in larger apical P2Y(2)-R activation-dependent Ca(2+)(i) responses, which reproduced the increased Ca(2+)(i) signals observed in CF epithelia. In conclusion, the mechanism for the larger Ca(2+)(i) signals elicited by apical P2Y(2)-R activation in CF airway epithelia is an expansion of the apical ER Ca(2+) stores triggered by chronic luminal airway infection/inflammation. Greater ER-derived Ca(2+)(i) signals may provide a compensatory mechanism to restore, at least acutely, mucus clearance in CF airways.

The mitochondrial barriers segregate agonist-induced calcium-dependent functions in human airway epithelia.

In airway epithelia, purinergic receptor (P2Y2-R) stimulation of intracellular calcium (Ca2+i)-regulated ion transport is restricted to the membrane domain ipsilateral to receptor activation, implying compartmentalization of Ca2+i signaling. Because mitochondria can spatially restrict cellular Ca2+i signals, immunocytochemical, electron microscopic, and fluorescent studies of mitochondria localization were performed in human airway epithelia. Although concentrated at the apical domain, mitochondria were found distributed at both the apical and the basolateral poles and in close association with the endoplasmic reticulum. The role of mitochondria in locally restricting P2Y2-R-induced Ca2+i signals was investigated by measuring changes in mitochondrial Ca2+ (Ca2+m) in human airway epithelial monolayers. P2Y2-R activation induced Ca2+m accumulation in mitochondria confined to the domain ipsilateral to P2Y2-R stimulation, which was blocked by mitochondrial uncoupling with 1 microM CCCP and 2.5 microg/ml oligomycin. The role of mitochondria in restricting the cellular cross-talk between basolateral P2Y2-R-dependent Ca2+i mobilization and apical membrane Ca2+-activated Cl- secretion was investigated in studies simultaneously measuring Ca2+i and Cl- secretion in cystic fibrosis human airway epithelial monolayers. Activation of basolateral P2Y2-Rs produced similar increases in Ca2+i in monolayers without and with pretreatment with uncouplers, whereas Ca2+i-activated Cl- secretion was only efficiently triggered in mitochondria-uncoupled conditions. We conclude that (a) mitochondria function as a Ca2+i-buffering system in airway epithelia, compartmentalizing Ca2+i-dependent functions to the membrane ipsilateral to receptor stimulation; and (b) the mitochondria provide structural barriers that protect the airway epithelia against nonspecific activation of Ca2+i-modulated functions associated with Ca2+i signals emanating from the apical or the basolateral membrane domains.