Lung endothelial Ca2+ and permeability response to platelet-activating factor is mediated by acid sphingomyelinase and transient receptor potential classical 6.

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 Ca(2+)-dependent loss in barrier function.

OBJECTIVES

To elucidate the molecular mechanisms underlying this response.

METHODS

In isolated perfused rat and mouse lungs, endothelial Ca(2+) concentration (Ca(2+)) was quantified by real-time fluorescence imaging, and caveolae of endothelial cells were isolated and probed for Ca(2+) 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 Ca(2+). 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 Ca(2+), 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 Ca(2+) and edema response to PAF. The exogenous NO donor PapaNONOate or the cyclic guanosine 3',5'-monophosphate analog 8Br-cGMP blocked the endothelial Ca(2+) 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 Ca(2+) influx and subsequent increases in endothelial permeability that are amplified in the absence of endothelial NO synthesis.