Nanoscale Viscometry Reveals an Inherent Mucus Defect in Cystic Fibrosis.

The abnormally viscous and thick mucus is a hallmark of cystic fibrosis (CF). How the mutated CF gene causes abnormal mucus remains an unanswered question of paramount interest. Mucus is produced by the hydration of gel-forming mucin macromolecules that are stored in intracellular granules prior to release.

Propidium uptake and ATP release in A549 cells share similar transport mechanisms.

The lipid bilayer of eukaryotic cells' plasma membrane is almost impermeable to small ions and large polar molecules, but its miniscule basal permeability in intact cells is poorly characterized. This report describes the intrinsic membrane permeability of A549 cells toward the charged molecules propidium (Pr) and ATP. Under isotonic conditions, we detected with quantitative fluorescence microscopy, a continuous low-rate uptake of Pr (∼150 × 10 moles (zmol)/h/cell, [Pr] = 150 μM, 32°C).

Lytic Release of Cellular ATP: Physiological Relevance and Therapeutic Applications.

The lytic release of ATP due to cell and tissue injury constitutes an important source of extracellular nucleotides and may have physiological and pathophysiological roles by triggering purinergic signalling pathways. In the lungs, extracellular ATP can have protective effects by stimulating surfactant and mucus secretion. However, excessive extracellular ATP levels, such as observed in ventilator-induced lung injury, act as a danger-associated signal that activates NLRP3 inflammasome contributing to lung damage.

Type 2 secretory cells are primary source of ATP release in mechanically stretched lung alveolar cells.

Extracellular ATP and its metabolites are potent paracrine modulators of lung alveolar cell function, including surfactant secretion and fluid transport, but the sources and mechanism of intra-alveolar ATP release remain unclear. To determine the contribution of gas-exchanging alveolar type 1 (AT1) and surfactant-secreting type 2 (AT2) cells to stretch-induced ATP release, we used quantitative real-time luminescence ATP imaging and rat primary alveolar cells cultured on silicon substrate for 2-7 days. When cultured on solid support, primary AT2 cells progressively transdifferentiated into AT1-like cells with ~20% of cells showing AT1 phenotype by day 2-3 (AT2:AT1 ≈ 4:1), while on day 7, the AT2:AT1 cell ratio was reversed with up to 80% of the cells displaying characteristics of AT1 cells.

Wide field of view quantitative imaging of cellular ATP release.

Although several mechanical stressors promote ATP secretion from eukaryotic cells, few mechanosensitive pathways for ATP release have been precisely characterized and none have been clearly identified. To facilitate progress, we report here a wide field of view (∼20 × 20 mm sample area) imaging technique paired with a quantitative image analysis to accurately map the dynamics of ATP release from a cell population. The approach has been tested on A549 cells stretched at high initial strain rate (2-5 s) or swelled by hypotonic shock.

Mechanosensitive ATP release in the lungs: New insights from real-time luminescence imaging studies.

Extracellular ATP and other nucleotides are important autocrine/paracrine mediators that stimulate purinergic receptors and regulate diverse processes in the normal lungs. They are also associated with pathogenesis of a number of respiratory diseases and clinical complications including acute respiratory distress syndrome and ventilator induced lung injury. Mechanical forces are major stimuli for cellular ATP release but precise mechanisms responsible for this release are still debated.

Search for Upstream Cell Volume Sensors: The Role of Plasma Membrane and Cytoplasmic Hydrogel.

The plasma membrane plays a prominent role in the regulation of cell volume by mediating selective transport of extra- and intracellular osmolytes. Recent studies show that upstream sensors of cell volume changes are mainly located within the cytoplasm that displays properties of a hydrogel and not in the plasma membrane. Cell volume changes occurring in anisosmotic medium as well as in isosmotic environment affect properties of cytoplasmic hydrogel that, in turn, trigger rapid regulatory volume increase and decrease (RVI and RVD).

Zinc deficiency primes the lung for ventilator-induced injury.

Mechanical ventilation is necessary to support patients with acute lung injury, but also exacerbates injury through mechanical stress-activated signaling pathways. We show that stretch applied to cultured human cells, and to mouse lungs in vivo, induces robust expression of metallothionein, a potent antioxidant and cytoprotective molecule critical for cellular zinc homeostasis. Furthermore, genetic deficiency of murine metallothionein genes exacerbated lung injury caused by high tidal volume mechanical ventilation, identifying an adaptive role for these genes in limiting lung injury.

Real-time imaging of inflation-induced ATP release in the ex vivo rat lung.

Extracellular ATP and other nucleotides are important autocrine/paracrine mediators that regulate diverse processes critical for lung function, including mucociliary clearance, surfactant secretion, and local blood flow. Cellular ATP release is mechanosensitive; however, the impact of physical stimuli on ATP release during breathing has never been tested in intact lungs in real time and remains elusive. In this pilot study, we investigated inflation-induced ATP release in rat lungs ex vivo by real-time luciferin-luciferase (LL) bioluminescence imaging coupled with simultaneous infrared tissue imaging to identify ATP-releasing sites.

Calcium is not required for triggering volume restoration in hypotonically challenged A549 epithelial cells.

Maintenance of cell volume is a fundamental housekeeping function in eukaryotic cells. Acute cell swelling activates a regulatory volume decrease (RVD) process with poorly defined volume sensing and intermediate signaling mechanisms. Here, we analyzed the putative role of Ca signaling in RVD in single substrate-adherent human lung epithelial A549 cells.

Role of cytoskeleton network in anisosmotic volume changes of intact and permeabilized A549 cells.

Recently we found that cytoplasm of permeabilized mammalian cells behaves as a hydrogel displaying intrinsic osmosensitivity. This study examined the role of microfilaments and microtubules in the regulation of hydrogel osmosensitivity, volume-sensitive ion transporters, and their contribution to volume modulation of intact cells. We found that intact and digitonin-permeabilized A549 cells displayed similar rate of shrinkage triggered by hyperosmotic medium.

Salt and osmosensing: role of cytoplasmic hydrogel.

Osmotic perturbations, occurring frequently under physiological and pathological conditions, alter cell size/volume and function. To protect cellular homeostasis, cell osmo- and volume-sensing mechanisms activate volume compensatory processes. The plasma membrane plays a prominent role in cell volume regulation by mediating the selective transport of extra- and intracellular osmolytes.

Temperature-induced inactivation of cytoplasmic biogel osmosensing properties is associated with suppression of regulatory volume decrease in A549 cells.

Upstream intermediates of intracellular signaling involved in cell volume regulation remain poorly explored. Recently, we demonstrated that osmolarity-induced volume changes in permeabilized cells were several-fold higher than those observed with intact cells, indicating the osmosensing properties of cytoplasmic gel. To further examine the role of cytoplasmic biogel in cell volume regulation, we compared the action of short-term heat treatment on volume changes in intact and permeabilized A549 cells.

Identification of dedifferentiation and redevelopment phases during postpneumonectomy lung growth.

Surgical resection of pulmonary tissue exerts a proregenerative stretch stimulus in the remaining lung units. Whether this regeneration process reenacts part or whole of lung morphogenesis developmental program remains unclear. To address this question, we analyzed the stretch-induced regenerating lung transcriptome in mice after left pneumonectomy (PNX) in its developmental context.

Lysophosphatidic acid stimulates epidermal growth factor-family ectodomain shedding and paracrine signaling from human lung fibroblasts.

Lysophospatidic acid (LPA) is a bioactive lipid mediator implicated in tissue repair and wound healing. It mediates diverse functional effects in fibroblasts, including proliferation, migration and contraction, but less is known about its ability to evoke paracrine signaling to other cell types involved in wound healing. We hypothesized that human pulmonary fibroblasts stimulated by LPA would exhibit ectodomain shedding of epidermal growth factor receptor (EGFR) ligands that signal to lung epithelial cells.

Recent advances and new opportunities in lung mechanobiology.

Lung function is inextricably linked to mechanics. On short timescales every breath generates dynamic cycles of cell and matrix stretch, along with convection of fluids in the airways and vasculature. Perturbations such airway smooth muscle shortening or surfactant dysfunction rapidly alter respiratory mechanics, with profound influence on lung function.

Stretch-induced mitogen-activated protein kinase activation in lung fibroblasts is independent of receptor tyrosine kinases.

Lung growth and remodeling are modulated by mechanical stress, with fibroblasts thought to play a leading role. Little mechanistic information is available about how lung fibroblasts respond to mechanical stress. We exposed cultured lung fibroblasts to tonic stretch and measured changes in phosphorylation status of mitogen-activated protein kinases (MAPKs), selected receptor tyrosine kinases (RTKs), and phospholipase Cgamma1 (PLCgamma1) and activation of the small G-protein Ras.

Membrane reserves and hypotonic cell swelling.

To accommodate expanding volume (V) during hyposmotic swelling, animal cells change their shape and increase surface area (SA) by drawing extra membrane from surface and intracellular reserves. The relative contributions of these processes, sources and extent of membrane reserves are not well defined. In this study, the SA and V of single substrate-attached A549, 16HBE14o(-), CHO and NIH 3T3 cells were evaluated by reconstructing cell three-dimensional topology based on conventional light microscopic images acquired simultaneously from two perpendicular directions.

Cell swelling-induced ATP release is tightly dependent on intracellular calcium elevations.

Mechanical stresses release ATP from a variety of cells by a poorly defined mechanism(s). Using custom-designed flow-through chambers, we investigated the kinetics of cell swelling-induced ATP secretion, cell volume and intracellular calcium changes in epithelial A549 and 16HBE14o- cells, and NIH/3T3 fibroblasts. Fifty per cent hypotonic shock triggered transient ATP release from cell confluent monolayers, which consistently peaked at around 1 min 45 s for A549 and NIH/3T3, and at 3 min for 16HBE14o- cells, then declined to baseline within the next 15 min.

Comparison of multistage versus one-stage destabilization of a type II external fixator used to stabilize an oblique tibial osteotomy in dogs.

Objective: To compare the biomechanical effects of multistage versus one-stage destabilization of a type II external skeletal fixator (ESF) used to stabilize an oblique unstable tibial osteotomy in dogs.

Study Design: In vitro, in vivo, and ex vivo experimental study.

Animal Population: Twelve healthy adult dogs.

Cell swelling-induced ATP release and gadolinium-sensitive channels.

ATP release induced by hypotonic swelling is an ubiquitous phenomenon in eukaryotic cells, but its underlying mechanisms are poorly defined. A mechanosensitive (MS) ATP channel has been implicated because gadolinium (Gd(3+)), an inhibitor of stretch-activated channels, suppressed ATP efflux monitored by luciferase bioluminescence. We examined the effect of Gd(3+) on luciferase bioluminescence and on ATP efflux from hypotonically swollen cells.