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Despite its fundamental role in providing an extensive surface for gas exchange, the alveolar epithelium (AE) serves as an immunological barrier through, e.g., the release of proinflammatory cytokines and secretion of surfactant to prevent alveolar collapse. Thus, AE is important for sustaining lung homeostasis. Extracellular ATP secreted by alveolar epithelial cells (AECs) is involved in physiological and pathological conditions and acts mainly through the activation of purine receptors (P2Rs). When studying P2R-mediated processes, primary isolated type II AECs (piAECs) still represent the gold standard in in vitro research, although their preparation is time-consuming and requires the sacrifice of many animals. Hence, cultivated immortalized and tumor-derived AEC lines may constitute a valuable alternative. In this work, we examined P2R expression and functionality in piAECs, in immortalized and tumor-derived AEC lines with the purpose of gaining a better understanding of purinergic signaling in different cell systems and assisting researchers in the choice of a suitable cell line with a certain P2R in demand. We combined mRNA and protein analysis to evaluate the expression of P2R. For pharmacological testing, we conducted calcium ([Ca 2+ ]) measurements and siRNA receptor knockdown. Interestingly, the mRNA and protein levels of P2Y 2 , P2Y 6, and P2X 4 were detected on all cell lines. Concerning functionality, P2XR could be narrowed to L2 and piAECs while P2YR were active in all cell lines.

Bacterial pneumonia is a global health challenge that causes up to 2 million deaths each year. Purinergic signaling plays a pivotal role in healthy alveolar epithelium. Here, we used fluorophore-based analysis and live-cell calcium imaging to address the question of whether the bacterial pathogen Streptococcus pneumoniae directly interferes with purinergic signaling in alveolar epithelial cells. Disturbed purinergic signaling might result in pathophysiologic changes like edema formation and atelectasis, which are commonly seen in bacterial pneumonia. Purine receptors are mainly activated by ATP, mediating a cytosolic calcium response. We found that this purinergic receptor P2Y 2 -mediated response is suppressed in the presence of S. pneumoniae in A549 and isolated primary alveolar cells in a temperature-dependent manner. Downstream inositol 3-phosphate (IP 3 ) signaling appeared to be unaffected, as calcium signaling via protease-activated receptor 2 remained unaltered. S. pneumoniae -induced suppression of the P2Y 2 -mediated calcium response depended on the P2Y 2 phosphorylation sites Ser-243, Thr-344, and Ser-356, which are involved in receptor desensitization and internalization. Spinning-disk live-cell imaging revealed that S. pneumoniae induces P2Y 2 translocation into the cytosol. In conclusion, our results show that S. pneumoniae directly inhibits purinergic signaling by inducing P2Y 2 phosphorylation and internalization, resulting in the suppression of the calcium response of alveolar epithelial cells to ATP, thereby affecting cellular integrity and function., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2019 Olotu et al.)

In adult respiratory distress syndrome (ARDS) pulmonary perfusion failure increases physiologic dead-space (V D /V T ) correlating with mortality. High V D /V T results in alveolar hypocapnia, which has been demonstrated to cause edema formation, atelectasis, and surfactant depletion, evoked, at least in part, by apoptosis of alveolar epithelial cells (AEC). However, the mechanism underlying the hypocapnia-induced AEC apoptosis is unknown. Here, using fluorescent live-cell imaging of cultured AEC type 2 we could show that in terms of CO 2 sensing the tricarboxylic acid cycle enzyme isocitrate dehydrogenase (IDH) 3 seems to be an important player because hypocapnia resulted independently from pH in an elevation of IDH3 activity and subsequently in an increase of NADH, the substrate of the respiratory chain. As a consequence, the mitochondrial transmembrane potential (ΔΨ) rose causing a Ca 2+ shift from cytosol into mitochondria, whereas the IDH3 knockdown inhibited these responses. Furthermore, the hypocapnia-induced mitochondrial Ca 2+ uptake resulted in reactive oxygen species (ROS) production, and both the mitochondrial Ca 2+ uptake and ROS production induced apoptosis. Accordingly, we provide evidence that in AEC type 2 hypocapnia induces elevation of IDH3 activity leading to apoptosis. This finding might give new insight into the pathogenesis of ARDS and may help to develop novel strategies to reduce tissue injury in ARDS.