Contribution Of TRPV4 Channels To Calcium Signaling Profiles Of The Lung Microvascular Endothelium Under Acidic pH

Abstract

Maintenance of lung endothelial barrier integrity is achieved to a great extent through effcient homeostatic endothelial calcium signaling in the lung microvasculature. The transient receptor potential vanilloid 4 (TRPV4), a non-selective calcium permeable channel has been implicated in several pulmonary pathologies involving lung barrier disruption. These channels are activated by various physical forces and chemical stimuli, but their underlying contribution to inherent and distinct microvascular endothelial Ca2+ signaling profiles are unknown. We previously assessed the role of TRPV4 channels in basal Ca2+ signaling dynamics of the pulmonary microcirculation under differing conditions of stretch and temperature. In the current study, we evaluate the impact of acidic pH (7.1, resembling acidosis) on the distinct endothelial Ca2+ signaling events in the mouse lung microcirculation and examine the participation of TRPV4 channels. Lung slices isolated from the Cdh5-GCaMP8 mice (transgenic mice with Ca2+-dependent fluorescent indicator expressed specifically within vascular endothelial cells) were mounted on Sylgard inserts with HEPES buffer (pH 7.45) and imaged for their basal signaling events using spinning disk confocal microscopy. After an initial baseline recording, the bathing solution was replaced with acidic HEPES buffer (pH 7.1) and recordings were made for approximately two minutes at 5 frames per second. These slices were then treated with the TRPV4 channel inhibitor GSK2193874 (3μM) or vehicle. The recorded image sequences were analyzed by using ImageJ and our custom signal tracking software S8, for quantification of the Ca2+ signaling profiles through event frequency, amplitude, duration and spread. Subjecting the lung slices to pH 7.1 increased the amplitude and spatial spread of the signaling events but the duration and frequency of the events remained relatively constant when compared to baseline recorded events. Addition of the channel blocker diminished the signaling event frequency, amplitude and spatial spread of the lung tissues. The normal pH and vehicle control for the above experiments did not show any relative change from the baseline signaling event profiles. To summarize, treating the lung slices to acidic pH increased the event amplitude and event spatial area distributions, indicating widespread activation of Ca2+ entry events. These increased event parameters were reduced upon addition of the antagonist, suggesting higher contribution of TRPV4 channel induced Ca2+ signaling events under acidic pH. We conclude that the channels contribute minimally to basal dynamics under homeostatic conditions but are activated to a greater extent under stimulations resembling pathological conditions like acidosis or acid aspirations. NIH P01 HL066299, R01 HL155288, NIH S10 0D020149. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.