Mechanical Forces Impair Alveolar Ion Transport Processes – A Putative Mechanism Contributing to the Formation of Pulmonary Edema

The aim of this chapter is to highlight the importance of transepithelial ion transport processes for lung function in general and to focus on the impact of mechanical forces on pulmonary ion transport in particular. Linking mechanical forces with pulmonary ion transport derives from the fact that the lung is a dynamic organ as well as from several studies providing evidence that the amount of mechanical forces as used during artificial ventilation correlates with mortality rates in patients with respiratory failure such as ALI (acute lung injury) and ARDS (acute respiratory distress syndrome) (ARDS Network Investigators, 2000). In these patients the formation of pulmonary edema is a characteristic symptom (Frank and Matthay, 2003; Ricard et al., 2003) and the basic rationale behind this is, that mechanical perturbations cause epithelial leakage in response to mechanically induced damage of the epithelial layer. This damage is suggested to be a major cause for the formation of pulmonary edema as well as the inability to reabsorb the edema fluid. However, little is known whether or not mechanical forces may directly interfere with pulmonary ion transport processes and this represents a putative mechanism that facilitates the formation of pulmonary edema – in addition to damages of the epithelial layer

Elektrophysiologische Untersuchungen zur Elektrolyt-Resorption am Lungenepithel von Xenopus laevis

In der vorliegenden Arbeit wurden Untersuchungen am Alveolarepithel von Xenopus laevis durchgeführt. Im Mittelpunkt der Untersuchungen stand der transepitheliale Ionentransport über das pulmonale Epithel, wobei ausschließlich elektrophysiologische Messmethoden zum Einsatz kamen. Die einfache Anatomie der Xenopus-Lunge ermöglichte es, Messungen in einer Ussingkammer durchzuführen; mit der Möglichkeit, verschiedene Regulations-mechanismen des transepithelialen Ionentransports zu untersuchen und zu charakterisieren. Haupteigenschaft des Studienobjekts ist die elektrogene Aufnahme von Na+-Ionen durch hochselektive, epitheliale Kanäle (ENaCs). Die Injektion von Tieren mit ACTH vor der Entnahme der Lungen resultierte in einer deutlichen Steigerung der Na+-Resorption durch ENaCs. Diese Beobachtung unterstreicht zum einen die Relevanz von ACTH bei der Regulation des Salz-/Wasserhaushalts in Xenopus, sowie dass die Xenopus-Lunge ein potentielles Zielorgan einer ACTH-induzierten Freisetzung von Steroiden der Nebennieren ist – wichtige Parallelen zu bekannten, hormonellen Regulationsmechanismen in Säugetieren. Untersuchungen mit bekannten auto- bzw. parakrin wirksamen Substanzen ermöglichten die Identifikation unterschiedlicher Membranrezeptoren, sowie den Nachweis einer Beteiligung an der Elektrolytregulation. Die Applikation von Histamin z. B. führte zu einer Erhöhung des amilorid-sensitiven Stroms. Diese Stimulation konnte durch einen bekannten Antagonisten von H 1 –Rezeptoren unterbunden werden, was auf die Anwesenheit von H1-Rezeptoren im Alveolarepithel von Xenopus deutet. Ebenso konnten durch den Einsatz von ATP, UTP und Adenosin verschiedene purinerge Rezeptoren nachgewiesen werden. Alle drei Moleküle vermochten den transepithelialen Ionenstrom zu erhöhen, wobei sich die Wirkung von ATP und Adenosin nicht nur auf den amilorid-sensitiven Ionenstrom beschränkte. Zusätzlich Experimente deuten auf eine parallele Stimulation einer Cl–-Sekretion durch ATP und Adenosin hin. Eine abschließende Charakterisierung bzw. Identifikation der purinergen Rezeptoren anhand der durchgeführten funktionellen Messungen ist nicht möglich. Dies bedarf zusätzlicher Experimente sowie des Einsatzes anderer Methoden. Die vorliegenden Ergebnisse deuten auf die Anwesenheit von mindestens je einem Typ eines P2Y-, eines P2X- und eines P1-Rezeptors hin. Der Nachweis einer nicht-hormonellen Kontrolle der epithelialen Na+-Kanäle im Lungenepithel des Krallenfrosches ist eine wichtige Gemeinsamkeit mit anderen, gut charakterisierten und akzeptierten Na+-resorbierenden Epithelien. Überraschend war die Entdeckung, dass das Erdmetall Gadolinium als Aktivator dieser Kanäle in Erscheinung treten kann. Versuche mit Substanzen, die bekanntermaßen die self-/feedback inhibition der Na+-Kanäle aufheben, sowie zusätzlich durchgeführte Einzelkanalmessungen, ergaben Hinweise, dass Gadolinium mit extrazellulären Komponenten der Kanäle wechselwirkt und das gating der Kanäle beeinflusst – Folge dieser Wechselwirkung ist eine gesteigerte Aktivität der Kanäle und damit eine erhöhte transepitheliale Na+-Resorption, wie sie in Versuchen an der Froschlunge beobachtet werden konnte. Die morphologisch/anatomischen Vorteile der Froschlunge waren die Grundlage für die nach-folgend durchgeführten Messungen. Zum einen konnten mit einfachsten Mitteln Potentiale an ganzen, intakten Organen abgeleitet werden. Vorbild waren Messungen, wie sie an isolierten, perfundierten Säugerlungen gemacht werden. Zum anderen konnten mit einer modifizierten Ussingkammer Messungen durchgeführt werden, in denen Effekte mechanischer Kräfte, die in Form von hydrostatischen Drücken appliziert wurden, untersucht werden. Dabei zeigte sich, dass mechanische Kräfte den Ionentransport signifikant beeinflussen. Die zweistufige Erhöhung des Druckes auf der apikalen Seite führte dabei zu einem entsprechenden zwei-stufigen Abfall des transepithelialen Ionenstroms. Die Charakterisierung der beobachteten Wirkung mechanischer Kräfte bedarf allerdings weiterer Untersuchungen. Die Ergebnisse dieser Arbeit zeigen einige Parallelen zu bekannten Mechanismen von alveolarem Elektrolyttransport in Säugetieren und unterstreichen eine gewisse Allgemeingültigkeit, die Regulation pulmonalen Ionentransports betreffend – artenunabhängig. Die Lunge von Xenopus laevis bietet die Möglichkeit, grundlegende Fragestellungen der alveolaren Elektrolytregulation unter weitgehend physiologischen Bedingungen zu untersuchen

Why Do We have to Move Fluid to be Able to Breathe?

The ability to breathe air represents a fundamental step in vertebrate evolution that was accompanied by several anatomical and physiological adaptations. The morphology of the air-blood barrier is highly conserved within air-breathing vertebrates. It is formed by three different plies, which are represented by the alveolar epithelium, the basal lamina, and the endothelial layer. Besides these conserved morphological elements, another common feature of vertebrate lungs is that they contain a certain amount of fluid that covers the alveolar epithelium. The volume and composition of the alveolar fluid is regulated by transepithelial ion transport mechanisms expressed in alveolar epithelial cells. These transport mechanisms have been reviewed extensively. Therefore, the present review focuses on the properties and functional significance of the alveolar fluid. How does the fluid enter the alveoli? What is the fate of the fluid in the alveoli? What is the function of the alveolar fluid in the lungs? The review highlights the importance of the alveolar fluid, its volume and its composition. Maintenance of the fluid volume and composition within certain limits is critical to facilitate gas exchange. We propose that the alveolar fluid is an essential element of the air-blood barrier. Therefore, it is appropriate to refer to this barrier as being formed by four plies, namely (1) the thin fluid layer covering the apical membrane of the epithelial cells, (2) the epithelial cell layer, (3) the basal membrane, and (4) the endothelial cells

Amiloride-Sensitive Sodium Channels and Pulmonary Edema

The development of pulmonary edema can be considered as a combination of alveolar flooding via increased fluid filtration, impaired alveolar-capillary barrier integrity, and disturbed resolution due to decreased alveolar fluid clearance. An important mechanism regulating alveolar fluid clearance is sodium transport across the alveolar epithelium. Transepithelial sodium transport is largely dependent on the activity of sodium channels in alveolar epithelial cells. This paper describes how sodium channels contribute to alveolar fluid clearance under physiological conditions and how deregulation of sodium channel activity might contribute to the pathogenesis of lung diseases associated with pulmonary edema. Furthermore, sodium channels as putative molecular targets for the treatment of pulmonary edema are discussed

Nicotine stimulates ion transport via metabotropic β4 subunit containing nicotinic ACh receptors

Background and Purpose Mucociliary clearance is an innate immune process of the airways, essential for removal of respiratory pathogens. It depends on ciliary beat and ion and fluid homeostasis of the epithelium. We have shown that nicotinic ACh receptors (nAChRs) activate ion transport in mouse tracheal epithelium. Yet the receptor subtypes and signalling pathways involved remained unknown. Experimental Approach Transepithelial short circuit currents (ISC) of freshly isolated mouse tracheae were recorded using the Ussing chamber technique. Changes in [Ca2+]i were studied on freshly dissociated mouse tracheal epithelial cells. Key Results Apical application of the nAChR agonist nicotine transiently increased ISC. The nicotine effect was abolished by the nAChR antagonist mecamylamine. α‐Bungarotoxin (α7 antagonist) had no effect. The agonists epibatidine (α3β2, α4β2, α4β4 and α3β4) and A‐85380 (α4β2 and α3β4) increased ISC. The antagonists dihydro‐β‐erythroidine (α4β2, α3β2, α4β4 and α3β4), α‐conotoxin MII (α3β2) and α‐conotoxin PnIA (α3β2) reduced the nicotine effect. Nicotine‐ and epibatidine‐induced currents were unaltered in β2−/−mice, but in β4−/− mice no increase was observed. In the presence of thapsigargin (endoplasmatic reticulum Ca2+‐ATPase inhibitor) or the ryanodine receptor antagonists JTV‐519 and dantrolene there was a reduction in the nicotine‐effect, indicating involvement of Ca2+ release from intracellular stores. Additionally, the PKA inhibitor H‐89 and the TMEM16A (Ca2+‐activated chloride channel) inhibitor T16Ainh‐A01 significantly reduced the nicotine‐effect. Conclusion and Implications α3β4 nAChRs are responsible for the nicotine‐induced current changes via Ca2+ release from intracellular stores, PKA and ryanodine receptor activation. These nAChRs might be possible targets to stimulate chloride transport via TMEM16A

Nicotinic receptors on rat alveolar macrophages dampen ATP-induced increase in cytosolic calcium concentration

Background: 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. Methods: Rat AM were isolated freshly by bronchoalveolar lavage. The expression of nAChR subunits was analyzed by RT-PCR, immunohistochemistry, and Western blotting. To evaluate function of nAChR subunits, electrophysiological recordings and measurements of intracellular calcium concentration ([Ca2+]i) were conducted. Results: Positive RT-PCR results were obtained for nAChR subunits α3, α5, α9, α10, β1, and β2, with most stable expression being noted for subunits α9, α10, β1, and β2. Notably, mRNA coding for subunit α7 which is proposed to convey the nicotinic anti-inflammatory response of macrophages from other sources than the lung was not detected. RT-PCR data were supported by immunohistochemistry on AM isolated by lavage, as well as in lung tissue sections and by Western blotting. Neither whole-cell patch clamp recordings nor measurements of [Ca2+]i revealed changes in membrane current in response to ACh and in [Ca2+]i in response to nicotine, respectively. However, nicotine (100 μM), given 2 min prior to ATP, significantly reduced the ATP-induced rise in [Ca2+]i by 30%. This effect was blocked by α-bungarotoxin and did not depend on the presence of extracellular calcium. Conclusions: Rat AM are equipped with modulatory nAChR with properties distinct from ionotropic nAChR mediating synaptic transmission in the nervous system. Their stimulation with nicotine dampens ATP-induced Ca2+-release from intracellular stores. Thus, the present study identifies the first acute receptor-mediated nicotinic effect on AM with anti-inflammatory potential

COVID-19 and Liquid Homeostasis in the Lung—A Perspective through the Epithelial Sodium Channel (ENaC) Lens

Infections with a new corona virus in 2019 lead to the definition of a new disease known as Corona Virus Disease 2019 (COVID-19). The sever cases of COVID-19 and the main cause of death due to virus infection are attributed to respiratory distress. This is associated with the formation of pulmonary oedema that impairs blood oxygenation and hypoxemia as main symptoms of respiratory distress. An important player for the maintenance of a defined liquid environment in lungs needed for normal lung function is the epithelial sodium channel (ENaC). The present article reviews the implications of SARS-CoV-2 infections from the perspective of impaired function of ENaC. The rationale for this perspective is derived from the recognition that viral spike protein and ENaC share a common proteolytic cleavage site. This cleavage site is utilized by the protease furin, that is essential for ENaC activity. Furin cleavage of spike ‘activates’ the virus protein to enable binding to host cell membrane receptors and initiate cell infection. Based on the importance of proteolytic cleavage for ENaC function and activation of spike, it seems feasible to assume that virus infections are associated with impaired ENaC activity. This is further supported by symptoms of COVID-19 that are reminiscent of impaired ENaC function in the respiratory tract