Acetylcholine-induced Calcium Signaling and Contraction of Airway Smooth Muscle Cells in Lung Slices

The Ca2+ signaling and contractility of airway smooth muscle cells (SMCs) were investigated with confocal microscopy in murine lung slices (∼75-μm thick) that maintained the in situ organization of the airways and the contractility of the SMCs for at least 5 d. 10–500 nM acetylcholine (ACH) induced a contraction of the airway lumen and a transient increase in [Ca2+]i in individual SMCs that subsequently declined to initiate multiple intracellular Ca2+ oscillations. These Ca2+ oscillations spread as Ca2+ waves through the SMCs at ∼48 μm/s. The magnitude of the airway contraction, the initial Ca2+ transient, and the frequency of the subsequent Ca2+ oscillations were all concentration-dependent. In a Ca2+-free solution, ACH induced a similar Ca2+ response, except that the Ca2+ oscillations ceased after 1–1.5 min. Incubation with thapsigargin, xestospongin, or ryanodine inhibited the ACH-induced Ca2+ signaling. A comparison of airway contraction with the ACH-induced Ca2+ response of the SMCs revealed that the onset of airway contraction correlated with the initial Ca2+ transient, and that sustained airway contraction correlated with the occurrence of the Ca2+ oscillations. Buffering intracellular Ca2+ with BAPTA prohibited Ca2+ signaling and airway contraction, indicating a Ca2+-dependent pathway. Cessation of the Ca2+ oscillations, induced by ACH-esterase, halothane, or the absence of extracellular Ca2+ resulted in a relaxation of the airway. The concentration dependence of the airway contraction matched the concentration dependence of the increased frequency of the Ca2+ oscillations. These results indicate that Ca2+ oscillations, induced by ACH in murine bronchial SMCs, are generated by Ca2+ release from the SR involving IP3- and ryanodine receptors, and are required to maintain airway contraction

IL-13R alpha 2 reverses the effects of IL-13 and IL-4 on bronchial reactivity and acetylcholine-induced Ca2+ signaling

Background: The interleukins IL-4 and IL-13 play a key role in the pathophysiology of asthma. The interleukin receptor IL-13R alpha 2 is believed to act as a decoy receptor, but until now, the functional significance of IL-13R alpha 2 remains vague. Methods: Bronchial reactivity was quantified in murine lung slices by digital video microscopy and acetylcholine (ACH)-induced Ca2+ signaling was measured in human airway smooth muscle cells (ASMC) using fluorescence microscopy. Results: IL-4 or IL-13 up to 50 ng/ml induced bronchial hyperreactivity. But after incubation with 100 ng/ml this effect was lost and bronchial responsiveness was again comparable to the control level. The effects of IL-4 and IL-13 on bronchial reactivity were paralleled by the effects on ASMC proliferation. Fifty nanograms per milliliter of IL-4 and IL-13 increased the Ca2+ response of human ASMC to ACH. At 100 ng/ml, however, the effects of the cytokines on the Ca2+ response were no longer evident. The expression of IL-13R alpha 2 increased with increasing concentrations of IL-4 or IL-13, reaching its maximum at 100 ng/ml. Blocking IL-13R alpha 2, the loss of the effect of IL-4 and IL-13 at 100 ng/ml on human ASMC proliferation and the ACH-induced Ca2+ response were no longer present. Conclusions: IL-4 and IL-13 induce bronchial hyperreactivity by changing the Ca2+ homeostasis of ASMC. These effects are counteracted by IL-13R alpha 2. The biological significance of IL-13R alpha 2 might be a protective function by regulating IL-13- and IL-4-mediated signal transduction and thereby limiting pathological alterations in Th2-mediated inflammatory diseases. Copyright (c) 2007 S. Karger AG, Basel

Mechanisms altering airway smooth muscle cell Ca(2+) homeostasis in two asthma models

Background: Asthma is characterized by airway remodeling, altered mucus production and airway smooth muscle cell (ASMC) contraction causing extensive airway narrowing. In particular, alterations of ASMC contractility seem to be of crucial importance. The elevation of the cytoplasmic Ca(2+) concentration is a key event leading to ASMC contraction and changes in the agonist- induced Ca(2+) increase in ASMC have been reported in asthma. Objective: The aim of this study was to investigate mechanisms underlying these changes. Methods: Murine tracheal smooth muscle cells (MTSMC) from T- bet KO mice and human bronchial smooth muscle cells (HBSMC) incubated with IL-13 and IL-4 served as asthma models. Acetylcholine- induced changes in the cytoplasmic Ca(2+) concentration were recorded using fluorescence microscopy and the expression of Ca(2+) homeostasis regulating proteins was investigated with Western blot analysis. Results: Acetylcholine- induced Ca(2+) transients were elevated in both asthma models. This correlated with an increased Ca(2+) content of the sarcoplasmic reticulum (SR). In MTSMC from T-bet KO mice, the expression of the SR Ca(2+) buffers calreticulin and calsequestrin was higher compared to wild- type mice. In HBSMC incubated with IL-13 or IL-4, the expression of ryanodine receptors, inositol-3-phosphate receptors and sarcoplasmic/ endoplasmic reticulum Ca 2+ ATPases 2 was increased compared to HBSMC without incubation with interleukins. The enlarged acetylcholine- induced Ca(2+) transients could be reversed by blocking inositol-3- phosphate receptors. Conclusions: We conclude that in the murine asthma model the SR Ca(2+) buffer capacity is increased, while in the human asthma model the expression of SR Ca(2+) channels is altered. The investigation of the Ca(2+) homeostasis of ASMC has the potential to provide new therapeutical options in asthma. Copyright (C) 2008 S. Karger AG, Basel

Nucleosomes indicate the in vitro radiosensitivity of irradiated bronchoepithelial and lung cancer cells

Nucleosomes, which are typical cell death products, are elevated in the serum of cancer patients and are known to rapidly increase during radiotherapy. As both normal and malignant cells are damaged by irradiation, we investigated to which extent both cell types contribute to the release of nucleosomes. We cultured monolayers of normal bronchoepithelial lung cells (BEAS-2B, n = 18) and epithelial lung cancer cells (EPLC, n = 18), exposed them to various radiation doses (0, 10 and 30 Gy) and observed them for 5 days. Culture medium was changed every 24 h. Subsequently, nucleosomes were determined in the supernatant by the Cell Death Detection-ELISA(plus) ( Roche Diagnostics). Additionally, the cell number was estimated after harvesting the cells in a second preparation. After 5 days, the cell number of BEAS-2B cultures in the irradiated groups (10 Gy: median 0.03 x 10(6) cells/culture, range 0.02-0.08 x 10(6) cells/culture; 30 Gy: median 0.08 x 10(6) cells/culture, range 0.02-0.14 x 10(6) cells/culture) decreased significantly (10 Gy: p = 0.005; 30 Gy p = 0.005; Wilcoxon test) compared to the non-irradiated control group (median 4.81 x 10(6) cells/culture, range 1.50-9.54 x 10(6) cells/culture). Consistently, nucleosomes remained low in the supernatant of nonirradiated BEAS-2B. However, at 10 Gy, BEAS-2B showed a considerably increasing release of nucleosomes, with a maximum at 72 h ( before irradiation: 0.24 x 10(3) arbitrary units, AU, range 0.13-4.09 x 10(3) AU, and after 72 h: 1.94 x 10(3) AU, range 0.11-5.70 x 10(3) AU). At 30 Gy, the release was even stronger, reaching the maximum earlier (at 48 h, 11.09 x 10(3) AU, range 6.89-18.28 x 10(3) AU). In non-irradiated EPLC, nucleosomes constantly increased slightly. At 10 Gy, we observed a considerably higher release of nucleosomes in EPLC, with a maximum at 72 h (before irradiation: 2.79 x 10(3) AU, range 2.42-3.80 x 10(3) AU, and after 72 h: 7.16 x 10(3) AU, range 4.30-16.20 x 10(3) AU), which was more than 3.5 times higher than in BEAS-2B. At 30 Gy, the maximum (6.22 x 10(3) AU, range 5.13-9.71 x 10(3) AU) was observed already after 24 h. These results indicate that normal bronchoepithelial and malignant lung cancer cells contribute to the release of nucleosomes during irradiation in a dose-and time-dependent manner with cancer cells having a stronger impact at low doses. Copyright (C) 2004 S. Karger AG, Basel

Endoplasmic reticulum Ca2+-homeostasis is altered in small and non-small cell lung cancer cell lines

<p>Abstract</p> <p>Background</p> <p>Knowledge of differences in the cellular physiology of malignant and non-malignant cells is a prerequisite for the development of cancer treatments that effectively kill cancer without damaging normal cells. Calcium is a ubiquitous signal molecule that is involved in the control of proliferation and apoptosis. We aimed to investigate if the endoplasmic reticulum (ER) Ca²⁺-homeostasis is different in lung cancer and normal human bronchial epithelial (NHBE) cells.</p> <p>Methods</p> <p>The intracellular Ca²⁺-signaling was investigated using fluorescence microscopy and the expression of Ca²⁺-regulating proteins was assessed using Western Blot analysis.</p> <p>Results</p> <p>In a Small Cell Lung Cancer (H1339) and an Adeno Carcinoma Lung Cancer (HCC) cell line but not in a Squamous Cell Lung Cancer (EPLC) and a Large Cell Lung Cancer (LCLC) cell line the ER Ca²⁺-content was reduced compared to NHBE. The reduced Ca²⁺-content correlated with a reduced expression of SERCA 2 pumping calcium into the ER, an increased expression of IP₃R releasing calcium from the ER, and a reduced expression of calreticulin buffering calcium within the ER. Lowering the ER Ca²⁺-content with CPA led to increased proliferation NHBE and lung cancer cells.</p> <p>Conclusion</p> <p>The significant differences in Ca²⁺-homeostasis between lung cancer and NHBE cells could represent a new target for cancer treatments.</p

Scientific drilling projects in ancient lakes: integrating geological and biological histories

Sedimentary sequences in ancient or long-lived lakes can reach several thousands of meters in thickness and often provide an unrivalled perspective of the lake's regional climatic, environmental, and biological history. Over the last few years, deep drilling projects in ancient lakes became increasingly multi- and interdisciplinary, as, among others, seismological, sedimentological, biogeochemical, climatic, environmental, paleontological, and evolutionary information can be obtained from sediment cores. However, these multi- and interdisciplinary projects pose several challenges. The scientists involved typically approach problems from different scientific perspectives and backgrounds, and setting up the program requires clear communication and the alignment of interests. One of the most challenging tasks, besides the actual drilling operation, is to link diverse datasets with varying resolution, data quality, and age uncertainties to answer interdisciplinary questions synthetically and coherently. These problems are especially relevant when secondary data, i.e., datasets obtained independently of the drilling operation, are incorporated in analyses. Nonetheless, the inclusion of secondary information, such as isotopic data from fossils found in outcrops or genetic data from extant species, may help to achieve synthetic answers. Recent technological and methodological advances in paleolimnology are likely to increase the possibilities of integrating secondary information, e.g., through molecular dating of molecular phylogenies. Some of the new approaches have started to revolutionize scientific drilling in ancient lakes, but at the same time, they also add a new layer of complexity to the generation and analysis of sediment core data. The enhanced opportunities presented by new scientific approaches to study the paleolimnological history of these lakes, therefore, come at the expense of higher logistic, communication, and analytical efforts. Here we review types of data that can be obtained in ancient lake drilling projects and the analytical approaches that can be applied to empirically and statistically link diverse datasets for creating an integrative perspective on geological and biological data. In doing so, we highlight strengths and potential weaknesses of new methods and analyses, and provide recommendations for future interdisciplinary deep drilling projects

ATP stimulates Ca2+ oscillations and contraction in airway smooth muscle cells of mouse lung slices

In airway smooth muscle cells (SMCs) from mouse lung slices, \u3e or =10 microM ATP induced Ca2+ oscillations that were accompanied by airway contraction. After approximately 1 min, the Ca2+ oscillations subsided and the airway relaxed. By contrast, \u3e or =0.5 microM adenosine 5\u27-O-(3-thiotriphosphate) (nonhydrolyzable) induced Ca2+ oscillations in the SMCs and an associated airway contraction that persisted for \u3e2 min. Adenosine 5\u27-O-(3-thiotriphosphate)-induced Ca2+ oscillations occurred in the absence of external Ca2+ but were abolished by the phospholipase C inhibitor U-73122 and the inositol 1,4,5-trisphosphate receptor inhibitor xestospongin. Adenosine, AMP, and alpha,beta-methylene ATP had no effect on airway caliber, and the magnitude of the contractile response induced by a variety of nucleotides could be ranked in the following order: ATP = UTP \u3e ADP. These results suggest that the SMC response to ATP is impaired by ATP hydrolysis and mediated via P2Y(2) or P2Y(4) receptors, activating phospholipase C to release Ca2+ via the inositol 1,4,5-trisphosphate receptor. We conclude that ATP can serve as a spasmogen of airway SMCs and that Ca2+ oscillations in SMCs are required to sustain airway contraction