Martin Fronius

Lungs of air‐breathing vertebrates are constantly exposed to mechanical forces and therefore are suitable for investigation of mechanotransduction processes in nonexcitable cells and tissues. Freshly dissected Xenopus laevis lungs were used for transepithelial short‐circuit current (I _SC ) recordings and were exposed to increased hydrostatic pressure (HP; 5 cm fluid column, modified Ussing chamber). I _SC values obtained under HP ( I _5cm ) were normalized to values before HP ( I _0cm ) application ( I _5cm / I _0cm ). Under control conditions, HP decreased I _SC ( I _5cm / I _0cm =0.84; n =68; P + channel (K _ATP ) inhibitors (HMR1098 and glibenclamide) prevented the decrease in I _SC ( I _5cm / I _0cm : HMR1098=1.19, P P I _5cm / I _0cm : meclofenamic acid=1.09, P P P ATP subunit transcripts in X. laevis lung. These data show an activation of K _ATP in pulmonary epithelial cells in response to HP that is induced by ATP release through mechanosensitive pannexin and connexin hemichannels. These findings represent a novel mechanism of mechanotransduction in nonexcitable cells.—Richter, K., Kiefer, K. P., Grzesik, B. A., Clauss, W. G., Fronius, M. Hydrostatic pressure activates ATP‐sensitive K ⁺ channels in lung epithelium by ATP release through pannexin and connexin hemichannels. FASEB J. 28, 45–55 (2014). www.fasebj.org

Lungs contain a particular amount of fluid that is crucial for proper lung function. This fluid content is tightly controlled within certain limits. Fluid accumulation in the alveolar airspace impairs gas exchange and represents a lifethreatening condition referred to as pulmonary edema. Ion transport processes by pulmonary epithelia represent a mechanism, responsible for fluid absorption from the airspace. Thus, it is obvious to consider ion transport processes as target for therapeutic interventions in pulmonary edema. The principle mechanism responsible for fluid absorption from the airspace is: Na⁺ diffuses through luminal Na⁺ channels into epithelial cells and is extruded by Na⁺/K⁺-ATPases at the basolateral side. This process generates an osmotic gradient that represents the driving force for fluid absorption. The rate of Na⁺ absorption is limited by the number/activity of Na+ channels in the luminal membrane of alveolar epithelial cells. Although different Na+ channels have been identified, the epithelial Na+ channel (ENaC) is a major player that participates in Na⁺-driven fluid absorption and thus a suitable target for the treatment of pulmonary edema. This article reviews cellular mechanisms by which ENaC activity can be increased in alveolar epithelia (lectins, proteases, β-adrenoceptors, mineralo-/glucocorticoid-receptors). These mechanisms are involved in regulating ENaC-dependent fluid absorption under physiological conditions. Additionally, pre-clinical as well as some preliminary clinical studies revealed that “ENaC-activators/stimulators” (β2-adrenoceptor agonists and mineralo-/glucocorticoid-receptor agonists) could be beneficial for therapeutic interventions in patients with pulmonary edema. However, the outcome of subsequently performed multicenter clinical trials with “ENaC-activators/stimulators” for treatment of patients with pulmonary edema was disappointing.

BACKGROUND AND PURPOSERecent studies detected the expression of proteins involved in cholinergic metabolism in airway epithelial cells, although the function of this non‐neuronal cholinergic system is not known in detail. Thus, this study focused on the effect of luminal ACh as a regulator of transepithelial ion transport in epithelial cells.EXPERIMENTAL APPROACHRT‐PCR experiments were performed using mouse tracheal epithelial cells for ChAT and organic cation transporter (OCT) transcripts. Components of tracheal airway lining fluid were analysed with desorption electrospray ionization (DESI) MS. Effects of nicotine on mouse tracheal epithelial ion transport were examined with Ussing‐chamber experiments.KEY RESULTSTranscripts encoding ChAT and OCT1–3 were detected in mouse tracheal epithelial cells. The DESI experiments identified ACh in the airway lining fluid. Luminal ACh induced an immediate, dose‐dependent increase in the transepithelial ion current (EC₅₀: 23.3 µM), characterized by a transient peak and sustained plateau current. This response was not affected by the Na⁺‐channel inhibitor amiloride. The Cl^‐‐channel inhibitor niflumic acid or the K⁺‐channel blocker Ba²⁺attenuated the ACh effect. The calcium ionophore A23187 mimicked the ACh effect. Luminal nicotine or muscarine increased the ion current. Experiments with receptor gene‐deficient animals revealed the participation of muscarinic receptor subtypes M₁and M₃.CONCLUSIONS AND IMPLICATIONSThe presence of luminal ACh and activation of transepithelial ion currents by luminal ACh receptors identifies a novel non‐neuronal cholinergic pathway in the airway lining fluid. This pathway could represent a novel drug target in the airways.

Alveolar macrophages (AM) are crucial for pulmonary host defense, and evidence emerges that ATP-gated P2X receptors are involved in inflammatory processes. This study focuses on the expression and functional characterization of P2X receptors in AM from mouse. In RT-PCR experiments, transcripts encoding the P2X₁, P2X₃, P2X₄, P2X₅, and P2X₇ receptors were detected. In whole-cell patch–clamp recordings, ATP (1 mM) evoked an inward current (mouse and human AM) that was reversible upon washout, and the reversal potential was ~5 mV, indicating the activation of a non-selective conductance—a fingerprint of P2X receptors. Further characterization (mouse AM) revealed that the current was not desensitized by a second ATP application. The ATP-induced current was increased by the removal of extracellular Ca²⁺ (in human and mouse AM), and EC₅₀ in mouse AM were determined with ~1 mM ATP, in the presence as well as in the absence of extracellular Ca²⁺. Pharmacological characterization of mouse AM revealed that the effect was augmented by BzATP and pre-application with ivermectin, but no effect with α,β-meATP was observed. Further, the ATP effect was reduced by PPADS (300 μM), brilliant blue G (5 μM), and about A438079 (10 μM). Although different P2X receptor transcripts were detected in mouse AM, the observed functional and pharmacological characteristics indicate primarily the participation of P2X₄ and P2X₇ receptors as mediators of the ATP-induced ion current in mouse AM. These suggestions were confirmed by experiments with AM from P2X₇-deficient animals, indicating a contribution of P2X₄ and P2X₇ receptors in pulmonary immune function.

Pulmonary epithelia of air-breathing vertebrates are covered by a thin, fluid layer that is essential for immune defense and gas diffusion. The composition of this layer is maintained by ion transport mechanisms, including Cl⁻transport. The present study focuses on the function of basolateral Cl⁻channels in Xenopus pulmonary epithelia, since knowledge concerning this issue is limited. Therefore, Ussing chamber measurements were performed, and transepithelial short-circuit currents ( I_SC) were monitored. Basolateral application of the Cl⁻channel inhibitor N-phenylanthranilic acid (DPC) resulted in an increase of the I_SC, indicating a DPC-sensitive Cl⁻conductance. This observation was confirmed in experiments using an apical-to-basolateral Cl⁻gradient, with and without nystatin (apical side) to permeabilize the epithelia as well as by establishing an iodide gradient. The DPC-sensitive Cl⁻conductance was influenced by procedures interfering with apical Cl⁻secretion. For example, the effect of forskolin was increased when basolateral Cl⁻channels were blocked by the simultaneous application of DPC. Activation of apical Cl⁻secretion by forskolin/IBMX and subsequent DPC application resulted in a significantly reduced DPC effect. Accordingly, DPC led to an increased apical Cl⁻secretion estimated by an increased 5-nitro-2-(3-phenylpropylamino)benzoic acid-sensitive I_SC. Furthermore, inhibition of basolateral anion exchangers responsible for Cl⁻uptake resulted in a decreased DPC-sensitive current. Taken together, we have evidence concerning the function of basolateral Cl⁻channels in Xenopus pulmonary epithelium and that these channels play a significant role in mediating apical Cl⁻secretion involving a novel Cl⁻recycling mechanism across the basolateral membrane.

The lung surface of air‐breathing vertebrates is formed by a continuous epithelium that is covered by a fluid layer. In the airways, this epithelium is largely pseudostratified consisting of diverse cell types such as ciliated cells, goblet cells, and undifferentiated basal cells, whereas the alveolar epithelium consists of alveolar type I and alveolar type II cells. Regulation and maintenance of the volume and viscosity of the fluid layer covering the epithelium is one of the most important functions of the epithelial barrier that forms the outer surface area of the lungs. Therefore, the epithelial cells are equipped with a wide variety of ion transport proteins, among which Na⁺, Cl⁻, and K⁺ channels have been identified to play a role in the regulation of the fluid layer. Malfunctions of pulmonary epithelial ion transport processes and, thus, impairment of the liquid balance in our lungs is associated with severe diseases, such as cystic fibrosis and pulmonary oedema. Due to the important role of pulmonary epithelial ion transport processes for proper lung function, the present paper summarizes the recent findings about composition, function, and ion transport properties of the airway epithelium as well as of the alveolar epithelium.

Nicotinic acetylcholine receptors (nAChR) have been identified on a variety of cells of the immune system and are generally considered to trigger anti-inflammatory events. In the present study, we determine the nAChR inventory of rat alveolar macrophages (AM), and investigate the cellular events evoked by stimulation with nicotine.

Epithelia, in general, and the lung epithelium, in particular, are exposed to mechanical forces, but little is known about their impact on pulmonary ion transport. In our present study, we employed transepithelial ion transport measurements on Xenopus lung preparations using custom-built Ussing chambers. Tissues were exposed to mechanical stress by increasing the water column (5 cm) at one side of the tissues. Apical exposure to hydrostatic pressure significantly decreased the short circuit current (I _SC: 24 ± 1%, n = 152), slightly decreased the transepithelial resistance (R _T: 7 ± 2%, n = 152), but increased the apical membrane capacitance (C _M: 16 ± 6%, n = 9). The pressure-induced effect was sensitive to Na⁺ (amiloride), Cl⁻ (DIDS, NFA, NPPB) and K⁺ channel blockers (Ba²⁺, glibenclamide). Further on, it was accompanied by increased extracellular ATP levels. The results show that mechanical stress leads to an activation of Na⁺, Cl⁻, and K⁺ conductances in a native pulmonary epithelium resulting in a net decrease of ion absorption. This could be of considerable interest, since an altered ion transport may contribute to pathophysiological conditions, e.g., the formation of pulmonary edema during artificial ventilation.