Rudi Samapati, Yang Yang, Jun Yin, Christof Stoerger, Christoph Arenz, Alexander Dietrich, Thomas Gudermann, Dieter Adam, Songwei Wu, Marc Freichel, Veit Flockerzi, Stefan Uhlig, and Wolfgang M. Kuebler Am. J. Respir. Crit. Care Med. January 15, 2012 vol. 185 no. 2 160-170
Abstract
Rationale: Platelet-activating factor (PAF) increases lung vascular permeability within minutes by activation of acid sphingomyelinase (ASM) and a subsequent nitric oxide (NO)-inhibitable and Ca2+-dependent loss in barrier function.
Objectives: To elucidate the molecular mechanisms underlying this response.
Methods: In isolated perfused rat and mouse lungs, endothelial Ca2+ concentration ([Ca2+]i) was quantified by real-time fluorescence imaging, and caveolae of endothelial cells were isolated and probed for Ca2+ entry channels. Regulation of transient receptor potential classical (TRPC) 6–mediated currents in lung endothelial cells was assessed by patch clamp technique.
Measurements and Main Results: PAF increased lung weight gain and endothelial [Ca2+]i. This response was abrogated by inhibitors of ASM or in ASM-deficient mice, and replicated by lung perfusion with exogenous ASM or C2-ceramide. PAF increased the caveolar abundance of TRPC6 channels, which was similarly blocked by ASM inhibition. PAF-induced increases in lung endothelial [Ca2+]i, vascular filtration coefficient, and edema formation were attenuated by the TRPC inhibitor SKF96365 and in TRPC6-deficient mice, whereas direct activation of TRPC6 replicated the [Ca2+]i and edema response to PAF. The exogenous NO donor PapaNONOate or the cyclic guanosine 3′,5′-monophosphate analog 8Br-cGMP blocked the endothelial [Ca2+]i and permeability response to PAF, in that they directly blocked TRPC6 channels without interfering with their PAF-induced recruitment to caveolae.
Conclusions: The present findings outline a new signaling cascade in the induction of PAF-induced lung edema, in that stimulation of ASM causes recruitment of TRPC6 channels to caveolae, thus allowing for Ca2+ influx and subsequent increases in endothelial permeability that are amplified in the absence of endothelial NO synthesis.
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