Distinguishing Dynamic Ca2+ Signals Originating from Different Entry Pathways in Microvascular Endothelial Cells

Abstract

Background Pseudomonas aeruginosa ExoU infection causes formation of an infectious prion like tau proteinopathy in lung microvasculature, leading to respiratory endothelial cell injury. ExoU, a soluble phospholipase A2, disrupts the host cell membrane phospholipid chains, producing arachidonic acid (AA), and lysophosphatidic acid. A synthetic AA molecule has been shown to potently inhibit –cellular calcium channel transients through AA metabolites. Calcium transients have been linked to autophagy whereby the host endothelial cell entraps misfolded proteins. Although the mechanisms inherent to the cytotoxicity of ExoU infection are not clear, recent findings support an important role for AA metabolites. It is suspected that specific Ca2+ dynamics drive autophagy and inhibition of these transients by AA metabolites contributes to the pathologic signaling of Pseudomonas aeruginosa ExoU infection. The current project aims to evaluate whether our automated detection and analysis algorithm can effectively discern dynamic Ca2+ signals originating from distinct dynamic membrane ion channels. Method Microvascular endothelial cells (MVC's) were seeded and grown in 35mm glass-bottomed dishes. Cells were loaded with the fluorescent calcium indicator Cal-520 and assessed via confocal microscopy for calcium signals. Signals were recorded before and after treatment with either the 4α-PDD, which solicits Ca2+ entry through membrane TRPV4 channels, or the SERCA inhibitor thapsigargin (TG), which solicits Ca2+ entry through membrane store-operated Ca2+ channels. All image sequences were analyzed for frequency, duration, and amplitude of events using ImageJ and customized calcium signal detection software LC Pro. Data were expressed as mean ± SEM. Group data were subjected to Mann-Whitney U statistical method. Results We observed that TG treatments elicited Ca2+ signals with significant increase in mean amplitude of 7 ± 0.17 (F/F0) from mean amplitude of 4α-PDD treatment which was 5 ± 0.43 (F/F0) (p<0.05, n=3). The mean frequency of TG treatment was 1.46 ± 0.12 events/second, whereas mean frequency of 4α-PDD treatment was found to be 0.1 ± 0.04 events/second (p<0.05, n=3). Also, the mean event duration was 150 ± 17.56 seconds when subjected to TG as opposed to 4α-PDD treatment which elicited a mean event duration of 30 ± 7.12 seconds (p<0.05, n=3). Conclusion In these experiments, we were able to discern specific Ca2+ signaling profiles resulting from stimulation of two distinct Ca2+ entry pathways. Thus, application of automated dynamic signal tracking may provide a useful approach for distinguishing distinct Ca2+ entry mechanisms in future studies.