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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.sup.-] 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.sup.-] channels in their basolateral membrane. Disrupting barttin/[Cl.sup.-] channel function impaired liquid absorption, and overexpressing barttin/[Cl.sup.-] channels increased absorption. Together, apical CFTR and basolateral barttin/[Cl.sup.-] channels provide an electrically conductive pathway for [Cl.sup.-] flow through ionocytes, and the transepithelial voltage generated by apical [Na.sup.+] 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., Introduction Pulmonary ionocytes are a rare cell type accounting for approximately 1% of airway epithelial cells. Ionocytes have attracted attention because, although rare, in airway epithelia they contain about half [...]

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. [ABSTRACT FROM AUTHOR]

Without cystic fibrosis transmembrane conductance regulator-mediated (CFTR-mediated) HC[O.sub.3]-secretion, airway epithelia of newborns with cystic fibrosis (CF) produce an abnormally acidic airway surface liquid (ASL), and the decreased pH impairs respiratory host defenses. However, within a few months of birth, ASL pH increases to match that in non-CF airways. Although the physiological basis for the increase is unknown, this time course matches the development of inflammation in CF airways. To learn whether inflammation alters CF ASL pH, we treated CF epithelia with TNF-[alpha] and IL-17 (TNF-[alpha]+IL-17), 2 inflammatory cytokines that are elevated in CF airways. TNF-[alpha]+IL-17 markedly increased ASL pH by upregulating pendrin, an apical Cl/HC[O.sub.3]-exchanger. Moreover, when CF epithelia were exposed to TNF-[alpha]+IL-17, clinically approved CFTR modulators further alkalinized ASL pH. As predicted by these results, in vivo data revealed a positive correlation between airway inflammation and CFTR modulator-induced improvement in lung function. These findings suggest that inflammation is a key regulator of HC[O.sub.3]-secretion in CF airways. Thus, they explain earlier observations that ASL pH increases after birth and indicate that, for similar levels of inflammation, the pH of CF ASL is abnormally acidic. These results also suggest that a non-cell-autonomous mechanism, airway inflammation, is an important determinant of the response to CFTR modulators., Introduction Cystic fibrosis (CF) is the most common life-shortening inherited disorder among White individuals (1). CF is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR), [...]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In a newborn pig cystic fibrosis (CF) model, the ability of glandcontaining 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 mm) were nevertheless more acidic (compared with non-CF airways). Therefore, we hypothesized that glandcontaining 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. [ABSTRACT FROM AUTHOR]

Pancreatic ductular epithelial cells comprise the majority of duct cells in pancreas, control cystic fibrosis transmembrane conductance regulator (CFTR)-dependent bicarbonate ([Formula: see text]) secretion, but are difficult to grow as a polarized monolayer. Using NIH-3T3-J2 fibroblast feeder cells and a Rho-associated kinase inhibitor, we produced well-differentiated and polarized porcine pancreatic ductular epithelial cells. Cells grown on semipermeable filters at the air-liquid interface developed typical epithelial cell morphology and stable transepithelial resistance and expressed epithelial cell markers (zona occludens-1 and β-catenin), duct cell markers (SOX-9 and CFTR), but no acinar (amylase) or islet cell (chromogranin) markers. Polarized cells were studied in Ussing chambers bathed in Krebs-Ringer [Formula: see text] solution at 37°C gassed with 5% CO2 to measure short-circuit currents ( Isc). Ratiometric measurement of extracellular pH was performed with fluorescent SNARF-conjugated dextran at 5% CO2. Cells demonstrated a baseline Isc (12.2 ± 3.2 μA/cm2) that increased significantly in response to apical forskolin-IBMX (∆ Isc: 35.4 ± 3.8 μA/cm2, P < 0.001) or basolateral secretin (∆ Isc: 31.4 ± 2.5 μA/cm2, P < 0.001), both of which increase cellular levels of cAMP. Subsequent addition of apical GlyH-101, a CFTR inhibitor, decreased the current (∆ Isc: 20.4 ± 3.8 μA/cm2, P < 0.01). Extracellular pH and [Formula: see text] concentration increased significantly after forskolin-IBMX (pH: 7.18 ± 0.23 vs. 7.53 ± 0.19; [Formula: see text] concentration, 14.5 ± 5.9 vs. 31.8 ± 13.4 mM; P < 0.05 for both). We demonstrate the development of a polarized pancreatic ductular epithelial cell epithelium with CFTR-dependent [Formula: see text] secretion in response to secretin and cAMP. This model is highly relevant, as porcine pancreas physiology is very similar to humans and pancreatic damage in the cystic fibrosis pig model recapitulates that of humans. NEW & NOTEWORTHY Pancreas ductular epithelial cells control cystic fibrosis transmembrane conductance regulator (CFTR)-dependent bicarbonate secretion. Their function is critical because when CFTR is deficient in cystic fibrosis bicarbonate secretion is lost and the pancreas is damaged. Mechanisms that control pancreatic bicarbonate secretion are incompletely understood. We generated well-differentiated and polarized porcine pancreatic ductular epithelial cells and demonstrated feasibility of bicarbonate secretion. This novel method will advance our understanding of pancreas physiology and mechanisms of bicarbonate secretion. [ABSTRACT FROM AUTHOR]

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 cAMPdependent 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. [ABSTRACT FROM AUTHOR]

The airway-surface liquid pH (pHASL) is slightly acidic relative to the plasma and becomes more acidic in airway diseases, leading to impaired host defense. CO2 in the large airways decreases during inspiration (0.04% CO2) and increases during expiration (5% CO2). Thus, we hypothesized that pHASL would fluctuate during the respiratory cycle. We measured pHASL on cultures of airway epithelia while changing apical CO2 concentrations. Changing apical CO2 produced only very slow pHASL changes, occurring in minutes, inconsistent with respiratory phases that occur in a few seconds. We hypothesized that pH changes were slow because airway-surface liquid has little carbonic anhydrase activity. To test this hypothesis, we applied the carbonic anhydrase inhibitor acetazolamide and found minimal effects on CO2-induced pHASL changes. In contrast, adding carbonic anhydrase significantly increased the rate of change in pHASL. Using pH-dependent rates obtained from these experiments, we modeled the pHASL during respiration to further understand how pH changes with physiologic and pathophysiologic respiratory cycles. Modeled pHASL oscillations were small and affected by the respiration rate, but not the inspiratory:expiratory ratio. Modeled equilibrium pHASL was affected by the inspiratory:expiratory ratio, but not the respiration rate. The airway epithelium is the only tissue that is exposed to large and rapid CO2 fluctuations. We speculate that the airways may have evolved minimal carbonic anhydrase activity to mitigate large changes in the pHASL during breathing that could potentially affect pH-sensitive components of ASL. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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/Ca2+., 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/Ca2+ 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)/Ca2+. 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/Ca2+ 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 Ca2+ did not inhibit the NBCe1 current recovery following repolarization from VSP activation, demonstrating that changes in InsP3/Ca2+ 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/Ca2+. [ABSTRACT FROM AUTHOR]