Increased ENaC-mediated liquid absorption across vitamin-D deficient human airway epithelia.

Vitamin D deficiency is a risk factor for exacerbation of obstructive airway disease, a hallmark of which is mucus dehydration and plugging. Calcitriol (the active form of vitamin D) deficiency in cultured human airway epithelia resulted in increased SCNN1G and ATP1B1 mRNAs encoding subunits of ENaC and the Na-K pump compared with supplemented epithelia. These drive the absorption of airway surface liquid. Consistently, calcitriol-deficient epithelia absorbed liquid faster than supplemented epithelia. Calcitriol deficiency also increased amiloride-sensitive Isc and Gt without altering Na-K pump activity, indicating the changes in amiloride-sensitivity arose from ENaC. ENaC activity can be regulated by trafficking, proteases, and channel abundance. We found the effect was likely not induced by changes to endocytosis of ENaC given that calcitriol did not affect the half-lives of amiloride-sensitive Isc and Gt. Furthermore, trypsin nominally increased Isc produced by epithelia ± calcitriol, suggesting calcitriol did not affect proteolytic activation of ENaC. Consistent with mRNA and functional data, calcitriol deficiency resulted in increased γENaC protein. These data indicate that the vitamin D receptor response controls ENaC function and subsequent liquid absorption, providing insight into the relationship between vitamin D deficiency and respiratory disease.NEW & NOTEWORTHY It is unknown why calcitriol (active vitamin D) deficiency worsens pulmonary disease outcomes. Results from mRNA, immunoblot, Ussing chamber, and absorption experiments indicate that calcitriol deficiency increases ENaC activity in human airway epithelia, decreasing apical hydration. Given that epithelial hydration is required for mucociliary transport and airway innate immune function, the increased ENaC activity observed in calcitriol-deficient epithelia may contribute to respiratory pathology observed in vitamin D deficiency.

Urban Particulate Matter Impairment of Airway Surface Liquid-Mediated Coronavirus Inactivation.

Air pollution particulate matter (PM) is associated with SARS-CoV-2 infection and severity, although mechanistic studies are lacking. We tested whether airway surface liquid (ASL) from primary human airway epithelial cells is antiviral against SARS-CoV-2 and human alphacoronavirus 229E (CoV-229E) (responsible for common colds), and whether PM (urban, indoor air pollution [IAP], volcanic ash) affected ASL antiviral activity. ASL inactivated SARS-CoV-2 and CoV-229E. Independently, urban PM also decreased SARS-CoV-2 and CoV-229E infection, and IAP PM decreased CoV-229E infection. However, in combination, urban PM impaired ASL's antiviral activity against both viruses, and the same effect occurred for IAP PM and ash against SARS-CoV-2, suggesting that PM may enhance SARS-CoV-2 infection.

A Single-Cell Atlas of Large and Small Airways at Birth in a Porcine Model of Cystic Fibrosis.

Lack of CFTR (cystic fibrosis transmembrane conductance regulator) affects the transcriptome, composition, and function of large and small airway epithelia in people with advanced cystic fibrosis (CF); however, whether lack of CFTR causes cell-intrinsic abnormalities present at birth versus inflammation-dependent abnormalities is unclear. We performed a single-cell RNA-sequencing census of microdissected small airways from newborn CF pigs, which recapitulate CF host defense defects and pathology over time. Lack of CFTR minimally affected the transcriptome of large and small airways at birth, suggesting that infection and inflammation drive transcriptomic abnormalities in advanced CF. Importantly, common small airway epithelial cell types expressed a markedly different transcriptome than corresponding large airway cell types. Quantitative immunohistochemistry and electrophysiology of small airway epithelia demonstrated basal cells that reach the apical surface and a water and ion transport advantage. This single cell atlas highlights the archetypal nature of airway epithelial cells with location-dependent gene expression and function.

FXYD3 facilitates Na+ and liquid absorption across human airway epithelia by increasing the transport capacity of the Na/K ATPase.

Na/K ATPase activity is essential for ion transport across epithelia. FXYD3, a γ subunit of the Na/K ATPase, is expressed in the airway, but its function remains undetermined. Single-cell RNA sequencing and immunohistochemistry revealed that FXYD3 localizes within the basolateral membrane of all airway epithelial cells. To study FXYD3 function, we reduced FXYD3 expression using siRNA. After permeabilizing the apical membrane with nystatin, epithelia pretreated with FXYD3-targeting siRNA had lower ouabain-sensitive short-circuit currents than control epithelia. FXYD3-targeting siRNA also reduced amiloride-sensitive short-circuit currents and liquid absorption across intact epithelia. These data are consistent with FXYD3 facilitating Na+ and liquid absorption. FXYD3 may be needed to maintain the high rates of Na+ and fluid absorption observed for airway and other FXYD3-expressing epithelia.

V-Type ATPase Mediates Airway Surface Liquid Acidification in Pig Small Airway Epithelial Cells.

In a newborn pig cystic fibrosis (CF) model, the ability of gland-containing airways to fight infection was affected by at least two major host-defense defects: impaired mucociliary transport and a lower airway surface liquid (ASL) pH. In the gland-containing airways, the ASL pH is balanced by CFTR (CF transmembrane conductance regulator) and ATP12A, which, respectively, control HCO3- transport and proton secretion. We found that, although porcine small airway tissue expressed lower amounts of ATP12A, the ASL of epithelial cultures from CF distal small airways (diameter < 200 µm) were nevertheless more acidic (compared with non-CF airways). Therefore, we hypothesized that gland-containing airways and small airways control acidification using distinct mechanisms. Our microarray data suggested that small airway epithelia mediate proton secretion via ATP6V0D2, an isoform of the V0 d subunit of the H+-translocating plasma membrane V-type ATPase. Immunofluorescence of small airways verified the expression of the V0 d2 subunit isoform at the apical surface of Muc5B+ secretory cells, but not ciliated cells. Inhibiting the V-type ATPase with bafilomycin A1 elevated the ASL pH of small airway cultures, in the presence or absence of HCO3-, and decreased ASL viscosity. These data suggest that, unlike large airways, which are acidified by ATP12A activity, small airways are acidified by V-type ATPase, thus identifying V-type ATPase as a novel therapeutic target for small airway diseases.

Motile cilia of human airway epithelia contain hedgehog signaling components that mediate noncanonical hedgehog signaling.

Differentiated airway epithelia produce sonic hedgehog (SHH), which is found in the thin layer of liquid covering the airway surface. Although previous studies showed that vertebrate HH signaling requires primary cilia, as airway epithelia mature, the cells lose primary cilia and produce hundreds of motile cilia. Thus, whether airway epithelia have apical receptors for SHH has remained unknown. We discovered that motile cilia on airway epithelial cells have HH signaling proteins, including patched and smoothened. These cilia also have proteins affecting cAMP-dependent signaling, including Gαi and adenylyl cyclase 5/6. Apical SHH decreases intracellular levels of cAMP, which reduces ciliary beat frequency and pH in airway surface liquid. These results suggest that apical SHH may mediate noncanonical HH signaling through motile cilia to dampen respiratory defenses at the contact point between the environment and the lung, perhaps counterbalancing processes that stimulate airway defenses.

TNFα and IL-17 alkalinize airway surface liquid through CFTR and pendrin.

The pH of airway surface liquid (ASL) is a key factor that determines respiratory host defense; ASL acidification impairs and alkalinization enhances key defense mechanisms. Under healthy conditions, airway epithelia secrete base ([Formula: see text]) and acid (H+) to control ASL pH (pHASL). Neutrophil-predominant inflammation is a hallmark of several airway diseases, and TNFα and IL-17 are key drivers. However, how these cytokines perturb pHASL regulation is uncertain. In primary cultures of differentiated human airway epithelia, TNFα decreased and IL-17 did not change pHASL. However, the combination (TNFα+IL-17) markedly increased pHASL by increasing [Formula: see text] secretion. TNFα+IL-17 increased expression and function of two apical [Formula: see text] transporters, CFTR anion channels and pendrin Cl-/[Formula: see text] exchangers. Both were required for maximal alkalinization. TNFα+IL-17 induced pendrin expression primarily in secretory cells where it was coexpressed with CFTR. Interestingly, significant pendrin expression was not detected in CFTR-rich ionocytes. These results indicate that TNFα+IL-17 stimulate [Formula: see text] secretion via CFTR and pendrin to alkalinize ASL, which may represent an important defense mechanism in inflamed airways.

Paracellular bicarbonate flux across human cystic fibrosis airway epithelia tempers changes in airway surface liquid pH.

KEY POINTS: Cl- and HCO3- had similar paracellular permeabilities in human airway epithelia. PCl /PNa of airway epithelia was unaltered by pH 7.4 vs. pH 6.0 solutions. Under basal conditions, calculated paracellular HCO3- flux was secretory. Cytokines that increased airway surface liquid pH decreased or reversed paracellular HCO3- flux. HCO3- flux through the paracellular pathway may counterbalance effects of cellular H+ and HCO3- secretion. ABSTRACT: Airway epithelia control the pH of airway surface liquid (ASL), thereby optimizing respiratory defences. Active H+ and HCO3- secretion by airway epithelial cells produce an ASL that is acidic compared with the interstitial space. The paracellular pathway could provide a route for passive HCO3- flux that also modifies ASL pH. However, there is limited information about paracellular HCO3- flux, and it remains uncertain whether an acidic pH produced by loss of cystic fibrosis transmembrane conductance regulator anion channels or proinflammatory cytokines might alter the paracellular pathway function. To investigate paracellular HCO3- transport, we studied differentiated primary cultures of human cystic fibrosis (CF) and non-CF airway epithelia. The paracellular pathway was pH-insensitive at pH 6.0 vs. pH 7.4 and was equally permeable to Cl- and HCO3- . Under basal conditions at pH ∼6.6, calculated paracellular HCO3- flux was weakly secretory. Treating epithelia with IL-17 plus TNFα alkalinized ASL pH to ∼7.0, increased paracellular HCO3- permeability, and paracellular HCO3- flux was negligible. Applying IL-13 increased ASL pH to ∼7.4 without altering paracellular HCO3- permeability, and calculated paracellular HCO3- flux was absorptive. These results suggest that HCO3- flux through the paracellular pathway counterbalances, in part, changes in the ASL pH produced via cellular mechanisms. As the pH of ASL increases towards that of basolateral liquid, paracellular HCO3- flux becomes absorptive, tempering the alkaline pH generated by transcellular HCO3- secretion.

Early pathogenesis of cystic fibrosis gallbladder disease in a porcine model.

Hepatobiliary disease causes significant morbidity in people with cystic fibrosis (CF), yet this problem remains understudied. We previously found that newborn CF pigs have microgallbladders with significant luminal obstruction in the absence of infection and consistent inflammation. In this study, we sought to better understand the early pathogenesis of CF pig gallbladder disease. We hypothesized that loss of CFTR would impair gallbladder epithelium anion/liquid secretion and increase mucin production. CFTR was expressed apically in non-CF pig gallbladder epithelium but was absent in CF. CF pig gallbladders lacked cAMP-stimulated anion transport. Using a novel gallbladder epithelial organoid model, we found that Cl- or HCO3- was sufficient for non-CF organoid swelling. This response was absent for non-CF organoids in Cl-/HCO3--free conditions and in CF. Single-cell RNA-sequencing revealed a single epithelial cell type in non-CF gallbladders that coexpressed CFTR, MUC5AC, and MUC5B. Despite CF gallbladders having increased luminal MUC5AC and MUC5B accumulation, there was no significant difference in the epithelial expression of gel-forming mucins between non-CF and CF pig gallbladders. In conclusion, these data suggest that loss of CFTR-mediated anion transport and fluid secretion contribute to microgallbladder development and luminal mucus accumulation in CF.

Lung function of primary cooks using LPG or biomass and the effect of particulate matter on airway epithelial barrier integrity.

BACKGROUND: Cooks exposed to biomass fuel experience increased risk of respiratory disease and mortality. We sought to characterize lung function and environmental exposures of primary cooking women using two fuel-types in southeastern India, as well as to investigate the effect of particulate matter (PM) from kitchens on human airway epithelial (HAE) cells in vitro. METHODS: We assessed pre- and post-bronchodilator lung function on 25 primary female cooks using wood biomass or liquified petroleum gas (LPG), and quantified exposures from 34 kitchens (PM2.5, PM < 40 µm, black carbon, endotoxin, and PM metal and bacterial content). We then challenged HAE cells with PM, assessing its cytotoxicity to small-airway cells (A549) and its effect on: transepithelial conductance and macromolecule permeability (NuLi cells), and antimicrobial activity (using airway surface liquid, ASL, from primary HAE cells). RESULTS: Lung function was impaired in cooks using both fuel-types. 60% of participants in both fuel-types had respiratory restriction (post bronchodilator FEV1/FVC>90). The remaining 40% in the LPG group had normal spirometry (post FEV1/FVC = 80-90), while only 10% of participants in the biomass group had normal spirometry, and the remaining biomass cooks (30%) had respiratory obstruction (post FEV1/FVC<80). Significant differences were found in environmental parameters, with biomass kitchens containing greater PM2.5, black carbon, zirconium, arsenic, iron, vanadium, and endotoxin concentrations. LPG kitchens tended to have more bacteria (p = 0.14), and LPG kitchen PM had greater sulphur concentrations (p = 0.02). In vitro, PM induced cytotoxicity in HAE A549 cells in a dose-dependent manner, however the effect was minimal and there were no differences between fuel-types. PM from homes of participants with a restrictive physiology increased electrical conductance of NuLi HAE cells (p = 0.06) and decreased macromolar permeability (p ≤ 0.05), while PM from homes of those with respiratory obstruction tended to increase electrical conductance (p = 0.20) and permeability (p = 0.07). PM from homes of participants with normal spirometry did not affect conductance or permeability. PM from all homes tended to inhibit antimicrobial activity of primary HAE cell airway surface liquid (p = 0.06). CONCLUSIONS: Biomass cooks had airway obstruction, and significantly greater concentrations of kitchen environmental contaminants than LPG kitchens. PM from homes of participants with respiratory restriction and obstruction altered airway cell barrier function, elucidating mechanisms potentially responsible for respiratory phenotypes observed in biomass cooks.

Widespread airway distribution and short-term phenotypic correction of cystic fibrosis pigs following aerosol delivery of piggyBac/adenovirus.

Cystic fibrosis (CF) is a common genetic disease caused by mutations in the gene coding for cystic fibrosis transmembrane conductance regulator (CFTR). Although CF affects multiple organ systems, chronic bacterial infections and inflammation in the lung are the leading causes of morbidity and mortality in people with CF. Complementation with a functional CFTR gene repairs this defect, regardless of the disease-causing mutation. In this study, we used a gene delivery system termed piggyBac/adenovirus (Ad), which combines the delivery efficiency of an adenoviral-based vector with the persistent expression of a DNA transposon-based vector. We aerosolized piggyBac/Ad to the airways of pigs and observed widespread pulmonary distribution of vector. We quantified the regional distribution in the airways and observed transduction of large and small airway epithelial cells of non-CF pigs, with ∼30-50% of surface epithelial cells positive for GFP. We transduced multiple cell types including ciliated, non-ciliated, basal, and submucosal gland cells. In addition, we phenotypically corrected CF pigs following delivery of piggyBac/Ad expressing CFTR as measured by anion channel activity, airway surface liquid pH, and bacterial killing ability. Combining an integrating DNA transposon with adenoviral vector delivery is an efficient method for achieving functional CFTR correction from a single vector administration.

Gel-forming mucins form distinct morphologic structures in airways.

Gel-forming mucins, the primary macromolecular components of airway mucus, facilitate airway clearance by mucociliary transport. In cystic fibrosis (CF) altered mucus properties impair mucociliary transport. Airways primarily secrete two closely related gel-forming mucins, MUC5B and MUC5AC. However, their morphologic structures and associations in airways that contain abundant submucosal glands and goblet cells are uncertain. Moreover, there is limited knowledge about mucins in airways not affected by inflammation, infection, or remodeling or in CF airways. Therefore, we examined airways freshly excised from newborn non-CF pigs and CF pigs before secondary manifestations develop. We found that porcine submucosal glands produce MUC5B, whereas goblet cells produce predominantly MUC5AC plus some MUC5B. We found that MUC5B emerged from submucosal gland ducts in the form of strands composed of multiple MUC5B filaments. In contrast, MUC5AC emerged from goblet cells as wispy threads and sometimes formed mucin sheets. In addition, MUC5AC often partially coated the MUC5B strands. Compared with non-CF, MUC5B more often filled CF submucosal gland ducts. MUC5AC sheets also accumulated in CF airways overlying MUC5B strands. These results reveal distinct morphology and interactions for MUC5B and MUC5AC and suggest that the two mucins make distinct contributions to mucociliary transport. Thus, they provide a framework for understanding abnormalities in disease.

A Novel AAV-mediated Gene Delivery System Corrects CFTR Function in Pigs.

Cystic fibrosis is an autosomal-recessive disease that is caused by a mutant CFTR (cystic fibrosis transmembrane conductance regulator) gene and is characterized by chronic bacterial lung infections and inflammation. Complementation with functional CFTR normalizes anion transport across the airway surface. Adeno-associated virus (AAV) is a useful vector for gene therapy because of its low immunogenicity and ability to persist for months to years. However, because its episomal expression may decrease after cell division, readministration of the AAV vector may be required. To overcome this, we designed an integrating AAV-based CFTR-expressing vector, termed piggyBac (PB)/AAV, carrying CFTR flanked by the terminal repeats of the piggyBac transposon. With codelivery of the piggyBac transposase, PB/AAV can integrate into the host genome. Because of the packaging constraints of AAV, careful consideration was required to ensure that the vector would package and express its CFTR cDNA cargo. In this short-term study, PB/AAV-CFTR was aerosolized to the airways of CF pigs in the absence of the transposase. Two weeks later, transepithelial Cl- current was restored in freshly excised tracheal and bronchial tissue. Additionally, we observed an increase in tracheal airway surface liquid pH and bacterial killing in comparison with untreated CF pigs. Airway surface liquid from primary airway cells cultured from treated CF pigs exhibited increased pH correlating with decreased viscosity. Together, these results show that complementing CFTR in CF pigs with PB/AAV rescues the anion transport defect in a large-animal CF model. Delivery of this integrating viral vector system to airway progenitor cells could lead to persistent, life-long expression in vivo.

Electrolyte transport properties in distal small airways from cystic fibrosis pigs with implications for host defense.

While pathological and clinical data suggest that small airways are involved in early cystic fibrosis (CF) lung disease development, little is known about how the lack of cystic fibrosis transmembrane conductance regulator (CFTR) function contributes to disease pathogenesis in these small airways. Large and small airway epithelia are exposed to different airflow velocities, temperatures, humidity, and CO2 concentrations. The cellular composition of these two regions is different, and small airways lack submucosal glands. To better understand the ion transport properties and impacts of lack of CFTR function on host defense function in small airways, we adapted a novel protocol to isolate small airway epithelial cells from CF and non-CF pigs and established an organotypic culture model. Compared with non-CF large airways, non-CF small airway epithelia cultures had higher Cl(-) and bicarbonate (HCO3 (-)) short-circuit currents and higher airway surface liquid (ASL) pH under 5% CO2 conditions. CF small airway epithelia were characterized by minimal Cl(-) and HCO3 (-) transport and decreased ASL pH, and had impaired bacterial killing compared with non-CF small airways. In addition, CF small airway epithelia had a higher ASL viscosity than non-CF small airways. Thus, the activity of CFTR is higher in the small airways, where it plays a role in alkalinization of ASL, enhancement of antimicrobial activity, and lowering of mucus viscosity. These data provide insight to explain why the small airways are a susceptible site for the bacterial colonization.

Phosphatidylinositol 4,5-bisphosphate degradation inhibits the Na+/bicarbonate cotransporter NBCe1-B and -C variants expressed in Xenopus oocytes.

KEY POINTS: We previously reported that the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2 ) directly stimulates heterologously expressed electrogenic Na(+)/bicarbonate cotransporter NBCe1-A in an excised macropatch from the Xenopus oocyte, and indirectly stimulates NBCe1-B and -C in the intact oocyte primarily through inositol 1,4,5-trisphosphate/Ca(2+). In the current study, we expand on a previous observation that PIP2 may also directly stimulate NBCe1 in the intact oocyte. In this study on oocytes, we co-expressed either NBCe1-B or -C and a voltage-sensitive phosphatase (VSP), which depletes PIP2 without changing inositol 1,4,5-trisphosphate, and monitored NBCe1-mediated currents with the two-electrode voltage-clamp technique or pHi changes using Vm/pH-sensitive microelectrodes. Activating VSP inhibited NBCe1-B and -C outward currents and NBCe1-mediated pHi increases, and changes in NBCe1 activity paralleled changes in surface PIP2. This study is a quantitative assessment of PIP2 itself as a regulator of NBCe1-B and -C in the intact cell, and represents the first use of VSP to characterize the PIP2 sensitivity of a transporter. These data combined with our previous work demonstrate that NBCe1-B and -C are regulated by two PIP2-mediated signalling pathways. Specifically, a decrease in PIP2 per se can inhibit NBCe1, whereas hydrolysis of PIP2 to inositol 1,4,5-trisphosphate/Ca(2+) can stimulate the transporter. ABSTRACT: The electrogenic Na(+)/bicarbonate cotransporter (NBCe1) of the Slc4 gene family is a powerful regulator of intracellular pH (pHi) and extracellular pH (pHo), and contributes to solute reabsorption and secretion in many epithelia. Using Xenopus laevis oocytes expressing NBCe1 variants, we have previously reported that the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) directly stimulates NBCe1-A in an excised macropatch, and indirectly stimulates NBCe1-B and -C in the intact oocyte primarily through inositol 1,4,5-trisphosphate (InsP3)/Ca(2+). In the current study, we used the two-electrode voltage-clamp technique alone or in combination with pH/voltage-sensitive microelectrodes or confocal fluorescence imaging of plasma membrane PIP2 to characterize the PIP2 sensitivity of NBCe1-B and -C in whole oocytes by co-expressing a voltage-sensitive phosphatase (VSP) that decreases PIP2 and bypasses the InsP3/Ca(2+) pathway. An oocyte depolarization that activated VSP only transiently stimulated the NBCe1-B/C current, consistent with an initial rapid depolarization-induced NBCe1 activation, and then a subsequent slower VSP-mediated NBCe1 inhibition. Upon repolarization, the NBCe1 current decreased, and then slowly recovered with an exponential time course that paralleled PIP2 resynthesis as measured with a PIP2-sensitive fluorophore and confocal imaging. A subthreshold depolarization that minimally activated VSP caused a more sustained increase in NBCe1 current, and did not lead to an exponential current recovery following repolarization. Similar results were obtained with oocytes expressing a catalytically dead VSP mutant at all depolarized potentials. Depleting endoplasmic reticulum Ca(2+) did not inhibit the NBCe1 current recovery following repolarization from VSP activation, demonstrating that changes in InsP3/Ca(2+) were not responsible. This study demonstrates for the first time that depleting PIP2 per se inhibits NBCe1 activity. The data in conjunction with previous findings implicate a dual PIP2 regulatory pathway for NBCe1 involving both PIP2 itself and generated InsP3/Ca(2+).

Pancreatic duct organoid swelling is chloride-dependent.

Pancreatic secretions become viscous and acidic in Cystic fibrosis (CF), highlighting the role of CFTR in pancreatic fluid and bicarbonate secretion. Forskolin-induced swelling (FIS) assay developed in intestinal organoids measures residual CFTR function. It is not known whether FIS reflects bicarbonate secretion in pancreas, an organ that secretes near-isotonic NaHCO3 levels. To investigate this, we generated pancreatic duct organoids from CF and non-CF pigs. Epithelial and ductal origin was confirmed with epithelial markers, ion transporters and lack of acinar, islet cell markers. CF organoids were small with no identifiable lumen; CFTR was expressed only in non-CF organoids. Utilizing FIS, organoid size increased only in response to chloride, not bicarbonate. This report highlights pancreatic duct organoids isolated for the first time from CF pigs and evidence for chloride and not bicarbonate driving pancreatic organoid swelling. These organoids would be useful to test chloride permeability of CFTR mutations that cause CF pancreatic disease.

Pancreatic enzymes digest obstructive meconium from cystic fibrosis pig intestines.

Introduction: Meconium ileus (MI) is a life-threatening obstruction of the intestines affecting ∼15% of newborns with cystic fibrosis (CF). Current medical treatments for MI often fail, requiring surgical intervention. MI typically occurs in newborns with pancreatic insufficiency from CF. Meconium contains mucin glycoprotein, a potential substrate for pancreatic enzymes or mucolytics. Our study aim was to determine whether pancreatic enzymes in combination with mucolytic treatments dissolve obstructive meconium using the CF pig model. Methods: We collected meconium from CF pigs at birth and submerged it in solutions with and without pancreatic enzymes, including normal saline, 7% hypertonic saline, and the reducing agents N-acetylcysteine (NAC) and dithiothreitol (DTT). We digested meconium at 37 °C with agitation, and measured meconium pigment release by spectrophotometry and residual meconium solids by filtration. Results and discussion: In CF pigs, meconium appeared as a solid pigmented mass obstructing the ileum. Meconium microscopically contained mucus glycoprotein, cellular debris, and bile pigments. Meconium fragments released pigments with maximal absorption at 405 nm after submersion in saline over approximately 8 h. Pancreatic enzymes significantly increased pigment release and decreased residual meconium solids. DTT did not improve meconium digestion and the acidic reducing agent NAC worsened digestion. Pancreatic enzymes digested CF meconium best at neutral pH in isotonic saline. We conclude that pancreatic enzymes digest obstructive meconium from CF pigs, while hydrating or reducing agents alone were less effective. This work suggests a potential role for pancreatic enzymes in relieving obstruction due to MI in newborns with CF.

CFTR-rich ionocytes mediate chloride absorption across airway epithelia.

The volume and composition of a thin layer of liquid covering the airway surface defend the lung from inhaled pathogens and debris. Airway epithelia secrete Cl- into the airway surface liquid through cystic fibrosis transmembrane conductance regulator (CFTR) channels, thereby increasing the volume of airway surface liquid. The discovery that pulmonary ionocytes contain high levels of CFTR led us to predict that ionocytes drive secretion. However, we found the opposite. Elevating ionocyte abundance increased liquid absorption, whereas reducing ionocyte abundance increased secretion. In contrast to other airway epithelial cells, ionocytes contained barttin/Cl- channels in their basolateral membrane. Disrupting barttin/Cl- channel function impaired liquid absorption, and overexpressing barttin/Cl- channels increased absorption. Together, apical CFTR and basolateral barttin/Cl- channels provide an electrically conductive pathway for Cl- flow through ionocytes, and the transepithelial voltage generated by apical Na+ channels drives absorption. These findings indicate that ionocytes mediate liquid absorption, and secretory cells mediate liquid secretion. Segregating these counteracting activities to distinct cell types enables epithelia to precisely control the airway surface. Moreover, the divergent role of CFTR in ionocytes and secretory cells suggests that cystic fibrosis disrupts both liquid secretion and absorption.

PIP2 hydrolysis stimulates the electrogenic Na+-bicarbonate cotransporter NBCe1-B and -C variants expressed in Xenopus laevis oocytes.

Electrogenic Na(+)-bicarbonate cotransporter NBCe1 variants contribute to pH(i) regulation, and promote ion reabsorption or secretion by many epithelia. Most Na(+)-coupled bicarbonate transporter (NCBT) families such as NBCe1 contain variants with differences primarily at the cytosolic N and/or C termini that are likely to impart on the transporters different modes of regulation. For example, N-terminal regions of NBCe1 autoregulate activity. Our group previously reported that cytosolic phosphatidylinositol 4,5-bisphosphate (PIP(2)) stimulates heterologously expressed rat NBCe1-A in inside-out macropatches excised from Xenopus laevis oocytes. In the current study on whole oocytes, we used the two-electrode voltage-clamp technique, as well as pH- and voltage-sensitive microelectrodes, to characterize the effect of injecting PIP(2) on the activity of heterologously expressed NBCe1-A, -B, or -C. Injecting PIP(2) (10 µM estimated final) into voltage-clamped oocytes stimulated NBC-mediated, HCO(3)(-)-induced outward currents by >100% for the B and C variants, but not for the A variant. The majority of this stimulation involved PIP(2) hydrolysis and endoplasmic reticulum (ER) Ca(2+) release. Stimulation by PIP(2) injection was mimicked by injecting IP(3), but inhibited by either applying the phospholipase C (PLC) inhibitor U73112 or depleting ER Ca(2+) with prolonged thapsigargin/EGTA treatment. Stimulating the activity of store-operated Ca(2+) channels (SOCCs) to trigger a Ca(2+) influx mimicked the PIP(2)/IP(3) stimulation of the B and C variants. Activating the endogenous G(q) protein-coupled receptor in oocytes with lysophosphatidic acid (LPA) also stimulated the B and C variants in a Ca(2+)-dependent manner, although via an increase in surface expression for the B variant. In simultaneous voltage-clamp and pH(i) studies on NBCe1-C-expressing oocytes, LPA increased the NBC-mediated pH(i)-recovery rate from a CO(2)-induced acid load by ∼80%. Finally, the general kinase inhibitor staurosporine completely inhibited the IP(3)-induced stimulation of NBCe1-C. In summary, injecting PIP(2) stimulates the activity of NBCe1-B and -C expressed in oocytes through an increase in IP(3)/Ca(2+) that involves a staurosporine-sensitive kinase. In conjunction with our previous macropatch findings, PIP(2) regulates NBCe1 through a dual pathway involving both a direct stimulatory effect of PIP(2) on at least NBCe1-A, as well as an indirect stimulatory effect of IP(3)/Ca(2+) on the B and C variants.

Anion Transport Across Human Gallbladder Organoids and Monolayers.

Fluid and anion secretion are important functions of the biliary tract. It has been established that cAMP regulates Na+ absorption through NHE3. However, mechanisms of gallbladder anion transport are less defined. We created organoids and organoid-derived monolayers from human gallbladder tissue to measure organoid swelling and transepithelial electrophysiology. In our in vitro models, forskolin-stimulation caused organoid swelling and increased transepithelial anion transport. Full organoid swelling required Cl-while changes in short-circuit current were HCO3 --dependent. Organoids and monolayers from an individual homozygous for the cystic fibrosis-causing ΔF508 CFTR mutation had no apical expression of CFTR and minimal changes in transepithelial current and conductance with forskolin treatment. However, organoid swelling remained intact. Dilution potential studies revealed that forskolin treatment increased the paracellular permeability to anions relative to cations. These data suggest a novel paracellular contribution to forskolin-stimulated fluid transport across the gallbladder epithelium.