Comparison Of Dynamic Calcium Signaling Profiles On Activation Of TRPV4 Channel And T‐Type Calcium Channel In Pulmonary Microvasculature

Abstract

BackgroundAcute Lung Injury (ALI) is characterized by inflammation, barrier disruption and edema within lung microvasculature. A dysregulation in intercellular signaling of calcium (Ca2+) within pulmonary microvascular endothelium potentially damages lung endothelial barrier integrity. Recently, two plasma membrane ion channels, the vanilloid transient receptor potential IV (TRPV4) and the T‐type voltage gated calcium channels (TTCC) have been implicated as potential conduits for spatially and temporally targeted Ca2+ signals within lung endothelium. Ca2+ entry via TRPV4 causes barrier disruption and Ca2+ entry via TTCC regulates P‐selectin expression, but their underlying patterns of specific Ca2+ signaling events are unknown. Therefore, in this study we aim to determine whether these channels elicit distinct Ca2+ signaling events that might contribute to their different functions in lung endothelium.MethodPulmonary microvascular endothelial cells (PMVECs) grown in 35mm glass‐bottomed dishes were loaded with fluorescent Ca2+ indicator Cal‐520 AM and recorded for signals via spinning disk confocal microscopy. After baseline recording, cells were subjected to GSK1016790 (TRPV4 activator) and then to GSK2798754 (TRPV4 inhibitor). Another set of cells were treated with high K+ solution (TTCC activator) and then with mibefradil (TTCC inhibitor). Lung slices were mounted on Sylgard inserts, and loaded with Cal‐520 AM. Two different slices were recorded for baseline activities and were respectively subjected to same drug treatments as PMVECs. Time‐lapse image sequences recorded after each addition were analyzed for amplitude and frequency of events using ImageJ and customized signal detection software LC_Pro.ResultsIn PMVECs with TRPV4 channel activation, the amplitude and frequency of events increased 1.5‐fold when compared to baseline, whereas the TTCC activation showed an increase by 1.3‐fold. In lung slices, the TRPV4 channel activation exhibited an increase by 1.4‐fold compared to baseline, as opposed to an increase of only 1.1‐fold with TTCC activation. The addition of respective inhibitors brought the signals back to near baseline levels.ConclusionOur findings suggest both TRPV4 channel and TTCC activation caused an increase in Ca2+ dynamics, with the TRPV4 channel activation showing a greater increase in event amplitude and frequency than TTCCs, thereby indicating they both contribute to distinct signaling events in PMVECs and lung slices. Further studies will focus on how these different signatures contribute to the differential functional roles of the channels.