Establishment of the deuterium oxide dilution method as a new possibility for determining the transendothelial water permeability.

Increase in transendothelial water permeability is an essential etiological factor in a variety of diseases like edema and shock. Despite the high clinical relevance, there has been no precise method to detect transendothelial water flow until now. The deuterium oxide (D2O) dilution method, already established for measuring transepithelial water transport, was used to precisely determine the transendothelial water permeability. It detected appropriate transendothelial water flow induced by different hydrostatic forces. This was shown in four different endothelial cell types. The general experimental setup was verified by gravimetry and absorbance spectroscopy. Determination of transendothelial electrical resistance (TEER) and immunocytochemical staining for proteins of the cell-cell contacts were performed to ensure that no damage to the endothelium occurred because of the measurements. Furthermore, endothelial barrier function was modulated. Measurement of transendothelial water flux was verified by measuring the TEER, the apparent permeability coefficient and the electrical capacity. The barrier-promoting substances cyclic adenosine monophosphate and iloprost reduced TEER and electrical capacity and increased permeability. This was accompanied by a reduced transendothelial water flux. In contrast, the barrier-damaging substances thrombin, histamine and bradykinin reduced TEER and electrical capacity, but increased permeability. Here, an increased water flow was shown. This newly established in vitro method for direct measurement of transendothelial water permeability was verified as a highly precise technique in various assays. The use of patient-specific endothelial cells enables individualized precision medicine in the context of basic edema research, for example regarding the development of barrier-protective pharmaceuticals.

Serially passaged, conditionally reprogrammed nasal epithelial cells as a model to study epithelial functions and SARS-CoV-2 infection.

Primary airway epithelial cells (pAECs) cultivated at air-liquid interface (ALI) conditions are widely used as surrogates for human in vivo epithelia. To extend the proliferative capacity and to enable serially passaging of pAECs, conditional reprogramming (cr) has been employed in recent years. However, ALI epithelia derived from cr cells often display functional changes with increasing passages. This highlights the need for thorough validation of the ALI cultures for the respective application. In our study, we evaluated the use of serially passaged cr nasal epithelial cells (crNECs) as a model to study SARS-CoV-2 infection and effects on ion and water transport. NECs were obtained from healthy individuals and cultivated as ALI epithelia derived from passages 1, 2, 3, and 5. We compared epithelial differentiation, ion and water transport, and infection with SARS-CoV-2 between passages. Our results show that epithelia maintained major differentiation characteristics and physiological ion and water transport properties through all passages. However, the frequency of ciliated cells, short circuit currents reflecting epithelial Na+ channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR) activity and expression of aquaporin 3 and 5 decreased gradually over passages. crNECs also expressed SARS-CoV-2 receptors angiotensin converting enzyme 2 (ACE2) and transmembrane serin2 protease 2 (TMPRSS2) across all passages and allowed SARS-CoV-2 replication in all passages. In summary, we provide evidence that passaged crNECs provide an appropriate model to study SARS-CoV-2 infection and also epithelial transport function when considering some limitations that we defined herein.

TGF-ß1 increases permeability of ciliated airway epithelia via redistribution of claudin 3 from tight junction into cell nuclei.

TGF-ß1 is a major mediator of airway tissue remodelling during atopic asthma and affects tight junctions (TJs) of airway epithelia. However, its impact on TJs of ciliated epithelia is sparsely investigated. Herein we elaborated effects of TGF-ß1 on TJs of primary human bronchial epithelial cells. We demonstrate that TGF-ß1 activates TGF-ß1 receptors TGFBR1 and TGFBR2 resulting in ALK5-mediated phosphorylation of SMAD2. We observed that TGFBR1 and -R2 localize specifically on motile cilia. TGF-ß1 activated accumulation of phosphorylated SMAD2 (pSMAD2-C) at centrioles of motile cilia and at cell nuclei. This triggered an increase in paracellular permeability via cellular redistribution of claudin 3 (CLDN3) from TJs into cell nuclei followed by disruption of epithelial integrity and formation of epithelial lesions. Only ciliated cells express TGF-ß1 receptors; however, nuclear accumulations of pSMAD2-C and CLDN3 redistribution were observed with similar time course in ciliated and non-ciliated cells. In summary, we demonstrate a role of motile cilia in TGF-ß1 sensing and showed that TGF-ß1 disturbs TJ permeability of conductive airway epithelia by redistributing CLDN3 from TJs into cell nuclei. We conclude that the observed effects contribute to loss of epithelial integrity during atopic asthma.

Retinoic acid signalling adjusts tight junction permeability in response to air-liquid interface conditions.

The pulmonary epithelium separates the gaseous intraluminal space of the airways and the aqueous interstitium. This compartimentalization is required for appropriate lung function, it is established during perinatal periods and can be disturbed in lung edema. Herein we elaborated the impact of the air-liquid interface (ALI) on the function of the pulmonary epithelium. We used NCI-H441 epithelia as a well-established and characterized model of distal airway epithelia, which were cultivated either at ALI or (at submerged conditions) at liquid-liquid interface conditions (LLI). Our study revealed that paracellular permeability was increased and claudin 1 (CLDN1) expression levels were reduced under LLI conditions. This was accompanied by elevated c-FOS, c-JUN and retinoic acid receptor α (RARA) expression, as well as cellular retinoic acid (RA) content. Exposure of epithelia to RA derivatives of ALI cultivated epithelia mimicked effects of LLI. The increase in RA content was in line with the identified upregulation of retinoic acid anabolizing enzymes ALDH1A3 and DHRS3. CLDN1 promoter analysis revealed c-FOS and c-JUN as activating transcription factors, whereas activation of RARA reduced CLDN1 promoter activity. We then concluded that ALI/LLI dependent modulation of CLDN1 expression and TJ permeability is under the control of RA synthesis. Activation of RARA results in an inhibition of c-FOS/c-JUN dependent CLDN1 promoter activation and increased TJ permeability. Our results underscore RA signalling as a pivotal mechanism in adjusting TJ properties, which could play a role during birth when the lung changes from LLI to ALI conditions.

IL-13 Impairs Tight Junctions in Airway Epithelia.

Interleukin-13 (IL-13) drives symptoms in asthma with high levels of T-helper type 2 cells (Th2-cells). Since tight junctions (TJ) constitute the epithelial diffusion barrier, we investigated the effect of IL-13 on TJ in human tracheal epithelial cells. We observed that IL-13 increases paracellular permeability, changes claudin expression pattern and induces intracellular aggregation of the TJ proteins zonlua occludens protein 1, as well as claudins. Furthermore, IL-13 treatment increases expression of ubiquitin conjugating E2 enzyme UBE2Z. Co-localization and proximity ligation assays further showed that ubiquitin and the proteasomal marker PSMA5 co-localize with TJ proteins in IL-13 treated cells, showing that TJ proteins are ubiquitinated following IL-13 exposure. UBE2Z upregulation occurs within the first day after IL-13 exposure. Proteasomal aggregation of ubiquitinated TJ proteins starts three days after IL-13 exposure and transepithelial electrical resistance (TEER) decrease follows the time course of TJ-protein aggregation. Inhibition of JAK/STAT signaling abolishes IL-13 induced effects. Our data suggest that that IL-13 induces ubiquitination and proteasomal aggregation of TJ proteins via JAK/STAT dependent expression of UBE2Z, resulting in opening of TJs. This may contribute to barrier disturbances in pulmonary epithelia and lung damage of patients with inflammatory lung diseases.

Pharmacological cholesterol depletion disturbs ciliogenesis and ciliary function in developing zebrafish.

Patients with an inherited inability to synthesize sufficient amounts of cholesterol develop congenital malformations of the skull, toes, kidney and heart. As development of these structures depends on functional cilia we investigated whether cholesterol regulates ciliogenesis through inhibition of hydroxymethylglutaryl-Coenzyme A reductase (HMG-CoA-R), the rate-limiting enzyme in cholesterol synthesis. HMG-CoA-R is efficiently inhibited by statins, a standard medication for hyperlipidemia. When zebrafish embryos are treated with statins cilia dysfunction phenotypes including heart defects, left-right asymmetry defects and malformation of ciliated organs develop, which are ameliorated by cholesterol replenishment. HMG-CoA-R inhibition and other means of cholesterol reduction lowered ciliation frequency and cilia length in zebrafish as well as several mammalian cell types. Cholesterol depletion further triggers an inability for ciliary signalling. Because of a reduction of the transition zone component Pi(4,5)P2 we propose that cholesterol governs crucial steps of cilium extension. Taken together, we report that cholesterol abrogation provokes cilia defects.

Aquaporins in the lung.

The lung is the interface between air and blood where the exchange of oxygen and carbon dioxide occurs. The surface liquid that is directly exposed to the gaseous compartment covers both conducting airways and respiratory zone and forms the air-liquid interface. The barrier that separates this lining fluid of the airways and alveoli from the extracellular compartment is the pulmonary epithelium. The volume of the lining fluid must be kept in a range that guarantees an appropriate gas exchange and other functions, such as mucociliary clearance. It is generally accepted that this is maintained by balancing resorptive and secretory fluid transport across the pulmonary epithelium. Whereas osmosis is considered as the exclusive principle of fluid transport in the airways, filtration may contribute to alveolar fluid accumulation under pathologic conditions. Aquaporins (AQP) facilitate water flux across cell membranes, and as such, they provide a transcellular route for water transport across epithelia. However, their contribution to near-isosmolar fluid conditions in the lung still remains elusive. Herein, we discuss the role of AQPs in the lung with regard to fluid homeostasis across the respiratory epithelium.

Inflammation-induced upregulation of P2X4 expression augments mucin secretion in airway epithelia.

Mucus clearance provides an essential innate defense mechanism to keep the airways and lungs free of particles and pathogens. Baseline and stimulated mucin secretion from secretory airway epithelial cells need to be tightly regulated to prevent mucus hypersecretion and mucus plugging of the airways. It is well established that extracellular ATP is a potent stimulus for regulated mucus secretion. Previous studies revealed that ATP acts via metabotropic P2Y2 purinoreceptors on goblet cells. Extracellular ATP, however, is also a potent agonist for ionotropic P2X purinoreceptors. Expression of several P2X isoforms has been reported in airways, but cell type-specific expression and the function thereof remained elusive. With this study, we now provide evidence that P2X4 is the predominant P2X isoform expressed in secretory airway epithelial cells. After IL-13 treatment of either human primary tracheal epithelial cells or mice, P2X4 expression is upregulated in vitro and in vivo under conditions of chronic inflammation, mucous metaplasia, and hyperplasia. Upregulation of P2X4 is strongest in MUC5AC-positive goblet cells. Moreover, activation of P2X4 by extracellular ATP augments intracellular Ca2+ signals and mucin secretion, whereas Ca2+ signals and mucin secretion are dampened by inhibition of P2X4 receptors. These data provide new insights into the purinergic regulation of mucin secretion and add to the emerging picture that P2X receptors modulate exocytosis of large secretory organelles and secretion of macromolecular vesicle cargo.

P2X4 receptor re-sensitization depends on a protonation/deprotonation cycle mediated by receptor internalization and recycling.

KEY POINTS: Re-sensitization of P2X4 receptors depends on a protonation/de-protonation cycle Protonation and de-protonation of the receptors is achieved by internalization and recycling of P2X4 receptors via acidic compartments Protonation and de-protonation occurs at critical histidine residues within the extracellular loop of P2X4 receptors Re-sensitization is blocked in the presence of the receptor agonist ATP ABSTRACT: P2X4 receptors are members of the P2X receptor family of cation-permeable, ligand-gated ion channels that open in response to the binding of extracellular ATP. P2X4 receptors are implicated in a variety of biological processes, including cardiac function, cell death, pain sensation and immune responses. These physiological functions depend on receptor activation on the cell surface. Receptor activation is followed by receptor desensitization and deactivation upon removal of ATP. Subsequent re-sensitization is required to return the receptor into its resting state. Desensitization and re-sensitization are therefore crucial determinants of P2X receptor signal transduction and responsiveness to ATP. However, the molecular mechanisms controlling desensitization and re-sensitization are not fully understood. In the present study, we provide evidence that internalization and recycling via acidic compartments is essential for P2X4 receptor re-sensitization. Re-sensitization depends on a protonation/de-protonation cycle of critical histidine residues within the extracellular loop of P2X4 receptors that is mediated by receptor internalization and recycling. Interestingly, re-sensitization under acidic conditions is completely revoked by receptor agonist ATP. Our data support the physiological importance of the unique subcellular distribution of P2X4 receptors that is predominantly found within acidic compartments. Based on these findings, we suggest that recycling of P2X4 receptors regulates the cellular responsiveness in the sustained presence of ATP.

Tight junctions in pulmonary epithelia during lung inflammation.

Inflammatory lung diseases like asthma bronchiale, chronic obstructive pulmonary disease and allergic airway inflammation are widespread public diseases that constitute an enormous burden to the health systems. Mainly classified as inflammatory diseases, the treatment focuses on strategies interfering with local inflammatory responses by the immune system. Inflammatory lung diseases predispose patients to severe lung failures like alveolar oedema, respiratory distress syndrome and acute lung injury. These life-threatening syndromes are caused by increased permeability of the alveolar and airway epithelium and exudate formation. However, the mechanism underlying epithelium barrier breakdown in the lung during inflammation is elusive. This review emphasises the role of the tight junction of the airway epithelium as the predominating structure conferring epithelial tightness and preventing exudate formation and the impact of inflammatory perturbations on their function.

Water Permeability Adjusts Resorption in Lung Epithelia to Increased Apical Surface Liquid Volumes.

The apical surface liquid (ASL) layer covers the airways and forms a first line of defense against pathogens. Maintenance of ASL volume by airway epithelia is essential for maintaining lung function. The proteolytic activation of epithelial Na+ channels is believed to be the dominating mechanism to cope with increases in ASL volumes. Alternative mechanisms, in particular increases in epithelial osmotic water permeability (Posm), have so far been regarded as rather less important. However, most studies mainly addressed immediate effects upon apical volume expansion (AVE) and increases in ASL. This study addresses the response of lung epithelia to long-term AVE. NCI-H441 cells and primary human tracheal epithelial cells, both cultivated in air-liquid interface conditions, were used as models for the lung epithelium. AVE was established by adding isotonic solution to the apical surface of differentiated lung epithelia, and time course of ASL volume restoration was assessed by the deuterium oxide dilution method. Concomitant ion transport was investigated in Ussing chambers. We identified a low resorptive state immediately after AVE, which coincided with proteolytic ion transport activation within 10-15 minutes after AVE. The main clearance of excess ASL occurred during a delayed (hours after AVE) high resorptive state, which did not correlate with ion transport activation. Instead, high resorptive state onset coincided with an increase in Posm, which depended on aquaporin up-regulation. In summary, our data demonstrate that, aside from ion transport activation, modulation of Posm is a major mechanism to compensate for long-term AVE in lung epithelia.

Glucocorticoids Regulate Tight Junction Permeability of Lung Epithelia by Modulating Claudin 8.

The lung epithelium constitutes a selective barrier that separates the airways from the aqueous interstitial compartment. Regulated barrier function controls water and ion transport across the epithelium and is essential for maintaining lung function. Tight junctions (TJs) seal the epithelial barrier and determine the paracellular transport. The properties of TJs depend especially on their claudin composition. Steroids are potent drugs used to treat a variety of airway diseases. Therefore, we addressed whether steroid hormones directly act on TJ properties in lung epithelia. Primary human tracheal epithelial cells and NCI-H441 cells, both cultivated under air-liquid interface conditions, were used as epithelial cell models. Our results demonstrate that glucocorticoids, but not mineralocorticoids, decreased paracellular permeability and shifted the ion permselectivity of TJs toward Cl(-). Glucocorticoids up-regulated claudin 8 (cldn8) expression via glucocorticoid receptors. Silencing experiments revealed that cldn8 is necessary to recruit occludin at the TJs. Immunohistochemistry on human lung tissue showed that cldn8 is specifically expressed in resorptive epithelia of the conducting and respiratory airways but not in the alveolar epithelium. We conclude that glucocorticoids enhance lung epithelia barrier function and increase paracellular Cl(-) selectivity via modulation of cldn8-dependent recruitment of occludin at the TJs. This mode of glucocorticoid action on lung epithelia might be beneficial to patients who suffer from impaired lung barrier function in various diseased conditions.

Amiloride-sensitive fluid resorption in NCI-H441 lung epithelia depends on an apical Cl(-) conductance.

Proper apical airway surface hydration is essential to maintain lung function. This hydration depends on well-balanced water resorption and secretion. The mechanisms involved in resorption are still a matter of debate, especially as the measurement of transepithelial water transport remains challenging. In this study, we combined classical short circuit current (I SC) measurements with a novel D2O dilution method to correlate ion and water transport in order to reveal basic transport mechanisms in lung epithelia. D2O dilution method enabled precise analysis of water resorption with an unprecedented resolution. NCI-H441 cells cultured at an air-liquid interface resorbed water at a rate of 1.5 ± 0.4 µL/(h cm(2)). Water resorption and I SC were reduced by almost 80% in the presence of the bulk Cl(-) channel inhibitor 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) or amiloride, a specific inhibitor of epithelial sodium channel (ENaC). However, water resorption and I SC were only moderately affected by forskolin or cystic fibrosis transmembrane regulator (CFTR) channel inhibitors (CFTRinh-172 and glybenclamide). In line with previous studies, we demonstrate that water resorption depends on ENaC, and CFTR channels have only a minor but probably modulating effect on water resorption. However, the major ENaC-mediated water resorption depends on an apical non-CFTR Cl(-) conductance.

Deuterium oxide dilution: a novel method to study apical water layers and transepithelial water transport.

Lung epithelia regulate the water flux between gas filled airways and the interstitial compartment in order to maintain organ function. Current methodology to assess transepithelial water transport is limited. We present a D2O dilution method to quantify submicroliter volumes of aqueous solutions on epithelial cell layers. Evaluating D2O/H2O mixtures using mid-infrared (2-25 µm) attenuated total reflection (ATR) spectroscopy, with a resolution of 0.06% vol/vol change, corresponding to 24 nL, was achieved. Using this method, we demonstrate that water transport across NCI-H441 lung epithelial cell layers and apical surface liquid (ASL) volumes are coupled to dexamethasone dependent amiloride-sensitive ion transport. However, contrary to current dogma, electrogenic transport is not rate-limiting for water transport. This clearly indicates the need to directly assess net water rather than ion transport across epithelial cell layers. The presented D2O dilution method enables such direct and quick quantification of transepithelial water transport with high resolution.

Fusion-activated cation entry (FACE) via P2X4 couples surfactant secretion and alveolar fluid transport.

Two fundamental mechanisms within alveoli are essential for lung function: regulated fluid transport and secretion of surfactant. Surfactant is secreted via exocytosis of lamellar bodies (LBs) in alveolar type II (ATII) cells. We recently reported that LB exocytosis results in fusion-activated cation entry (FACE) via P2X4 receptors on LBs. We propose that FACE, in addition to facilitating surfactant secretion, modulates alveolar fluid transport. Correlative fluorescence and atomic force microscopy revealed that FACE-dependent water influx correlated with individual fusion events in rat primary ATII cells. Moreover, ATII cell monolayers grown at air-liquid interface exhibited increases in short-circuit current (Isc) on stimulation with ATP or UTP. Both are potent agonists for LB exocytosis, but only ATP activates FACE. ATP, not UTP, elicited additional fusion-dependent increases in Isc. Overexpressing dominant-negative P2X4 abrogated this effect by ∼50%, whereas potentiating P2X4 lead to ∼80% increase in Isc. Finally, we monitored changes in alveolar surface liquid (ASL) on ATII monolayers by confocal microscopy. Only stimulation with ATP, not UTP, led to a significant, fusion-dependent, 20% decrease in ASL, indicating apical-to-basolateral fluid transport across ATII monolayers. Our data support the first direct link between LB exocytosis, regulation of surfactant secretion, and transalveolar fluid resorption via FACE.

Combined atomic force microscopy-fluorescence microscopy: analyzing exocytosis in alveolar type II cells.

Hybrid atomic force microscopy (AFM)-fluorescence microscopy (FM) investigation of exocytosis in lung epithelial cells (ATII cells) allows the detection of individual exocytic events by FM, which can be simultaneously correlated to structural changes in individual cells by AFM. Exocytosis of lamellar bodies (LBs) represents a slow form of exocytosis found in many non-neuronal cells. Exocytosis of LBs, following stimulation with adenosine-5'-triphosphate (ATP) and phorbol 12-myristate 13-acetate (PMA), results in a cation influx via P2X(4) receptors at the site of LB fusion with the plasma membrane (PM), which should induce a temporary increase in cell height/volume. AFM measurements were performed in single-line scans across the cell surface. Five minutes after stimulation, ATII cells revealed a cell height and volume increase of 13.7% ± 4.1% and 15.9 ± 4.8% (N = 9), respectively. These transient changes depend on exocytic LB-PM fusion. Nonstimulated cells and cells lacking LB fusions did not show a significant change in cell height/volume (N = 8). In addition, a cell height decrease was observed in ATII cells stimulated by uridine-5'-triphosphate (UTP) and PMA, agonists inducing LB fusion with the PM, but not activation of P2X(4) receptors. The cell height and volume decreased by -8.6 ± 3.6% and -11.2 ± 3.9% (N = 5), respectively. Additionally, low force contact and dynamic mode AFM imaging of cell areas around the nucleus after stimulation with ATP/PMA was performed. Fused LBs are more pronounced in AFM topography images compared to nonfused LBs, concluding that different "dynamic states" of LBs or locations from the PM are captured during imaging.

Actin coating and compression of fused secretory vesicles are essential for surfactant secretion--a role for Rho, formins and myosin II.

Secretion of vesicular contents by exocytosis is a fundamental cellular process. Increasing evidence suggests that post-fusion events play an important role in determining the composition and quantity of the secretory output. In particular, regulation of fusion pore dilation and closure is considered a key regulator of the post-fusion phase. However, depending on the nature of the cargo, additional mechanisms might be essential to facilitate effective release. We have recently described that in alveolar type II (ATII) cells, lamellar bodies (LBs), which are secretory vesicles that store lung surfactant, are coated with actin following fusion with the plasma membrane. Surfactant, a lipoprotein complex, does not readily diffuse out of fused LBs following opening and dilation of the fusion pore. Using fluorescence microscopy, atomic force microscopy and biochemical assays, we present evidence that actin coating and subsequent contraction of the actin coat is essential to facilitate surfactant secretion. Latrunculin B prevents actin coating of fused LBs and inhibits surfactant secretion almost completely. Simultaneous imaging of the vesicle membrane and the actin coat revealed that contraction of the actin coat compresses the vesicle following fusion. This leads to active extrusion of vesicle contents. Initial actin coating of fused vesicles is dependent on activation of Rho and formin-dependent actin nucleation. Actin coat contraction is facilitated by myosin II. In summary, our data suggest that fusion pore opening and dilation itself is not sufficient for release of bulky vesicle cargos and that active extrusion mechanisms are required.

Molecular basis of early epithelial response to streptococcal exotoxin: role of STIM1 and Orai1 proteins.

Streptolysin O (SLO) is a cholesterol-dependent cytolysin (CDC) from Streptococcus pyogenes. SLO induces diverse types of Ca(2+) signalling in host cells which play a key role in membrane repair and cell fate determination. The mechanisms behind SLO-induced Ca(2+) signalling remain poorly understood. Here, we show that in NCI-H441 cells, wild-type SLO as well as non-pore-forming mutant induces long-lasting intracellular Ca(2+) oscillations via IP(3) -mediated depletion of intracellular stores and activation of store-operated Ca(2+) (SOC) entry. SLO-induced activation of SOC entry was confirmed by Ca(2+) add-back experiments, pharmacologically and by overexpression as well as silencing of STIM1 and Orai1 expression. SLO also activated SOC entry in primary cultivated alveolar type II (ATII) cells but Ca(2+) oscillations were comparatively short-lived in nature. Comparison of STIM1 and Orai1 revealed a differential expression pattern in H441 and ATII cells. Overexpression of STIM1 and Orai1 proteins in ATII cells changed the short-lived oscillatory response into a long-lived one. Thus, we conclude that SLO-mediated Ca(2+) signalling involves Ca(2+) release from intracellular stores and STIM1/Orai1-dependent SOC entry. The phenotype of Ca(2+) signalling depends on STIM1 and Orai1 expression levels. Our findings suggest a new role for SOC entry-associated proteins in S. pyogenes-induced lung infection and pneumonia.

An ultra fast detection method reveals strain-induced Ca(2+) entry via TRPV2 in alveolar type II cells.

A commonly used technique to investigate strain-induced responses of adherent cells is culturing them on an elastic membrane and globally stretching the membrane. However, it is virtually impossible to acquire microscopic images immediately after the stretch with this method. Using a newly developed technique, we recorded the strain-induced increase of the cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) in rat primary alveolar type II (ATII) cells at an acquisition rate of 30ms and without any temporal delay. We can show that the onset of the mechanically induced rise in [Ca(2+)](c) was very fast (<30 ms), and Ca(2+) entry was immediately abrogated when the stimulus was withdrawn. This points at a direct mechanical activation of an ion channel. RT-PCR revealed high expression of TRPV2 in ATII cells, and silencing TRPV2, as well as blocking TRPV channels with ruthenium red, significantly reduced the strain-induced Ca(2+) response. Moreover, the usually homogenous pattern of the strain-induced [Ca(2+)](c) increase was converted into a point-like response after both treatments. Also interfering with actin/myosin and integrin binding inhibited the strain-induced increase of [Ca(2)](c). We conclude that TRPV2 participates in strain-induced Ca(2+) entry in ATII cells and suggest a direct mechanical activation of the channel that depends on FAs and actin/myosin. Furthermore, our results underline the importance of cell strain systems that allow high temporal resolution.