Platelet-activating factor reduces endothelial nitric oxide production: role of acid sphingomyelinase

Abstract

Platelet-activating factor (PAF) is a mediator of pulmonary oedema in acute lung injury that increases vascular permeability within minutes, partly through activation of acid sphingomyelinase (ASM). Since caveolae are rich in sphingomyelin and caveolin-1, which block endothelial nitric oxide (NO) synthase (eNOS) by direct binding, we examined the relationship between ASM, caveolin-1 and eNOS activity in the regulation of vascular permeability by PAF.In caveolar fractions from pulmonary vascular endothelial cells (isolated from perfused rat lungs) the abundance of caveolin-1 and eNOS increased rapidly after PAF perfusion. PAF treatment decreased endothelial NO (eNO) formation as assessed by in situ fluorescence microscopy. Restoration of eNO levels with PAPA-NONOate ((Z)-1-[N-(3-ammoniopropyl)-N-(npropyl)amino]diazen-1-ium-1,2-diolate) mitigated the PAF-induced oedema.PAF treatment increased the ASM activity in caveolar fractions and perfusion with ASM decreased eNO production. Pharmacological inhibition of the ASM pathway with imipramine, D609 or dexamethasone blocked the PAF-induced increase of caveolin-1 and eNOS in caveolae, and the decrease in eNO production and oedema formation.We conclude that PAF causes ASM-dependent enrichment of caveolin-1 in caveolae of endothelial cells, leading to decreased eNO production which contributes to pulmonary oedema formation. These findings suggest rapid reduction in eNO production as a novel mechanism in the regulation of vascular permeability.

Differential Regulation of Lung Endothelial Permeability <b><i>in Vitro</i></b> and <b><i>in Situ</i></b>

“…In isolated lungs PAF increases vascular permeability within minutes by a complex mechanism that starts with activation of the acid sphingomyelinase with subsequent ceramide formation [30] that leads to the recruitment of caveolin-1 into the caveolae of pulmonary endothelial cells. The recruited caveolin-1 engages TRPC6 channels that become disinhibited in part by the dramatic decrease in endothelial NO levels through the silencing of eNOS by its interaction with caveolin-1 [31,32]. We have recently reviewed this mechanism in detail [11,33].…”

“…The reasons why PAF is active in terms of increasing the permeability of cultured endothelial cells in some labs but not in others are not clear. Importantly, however, the mechanisms between the PAF-induced edema formation in isolated lungs and in endothelial cells in culture show principal differences, as recently reviewed by us in detail [11]: in isolated lungs PAF causes edema by a decrease in NO-levels [31], whereas in cultured cells PAF causes edema by an increase in NO-levels [37]. This is an important difference, because the induction of high NO levels by PAF is a response typical for non-pulmonary vascular beds [11].…”

“…Weight gain in isolated rat lungs that were perfused and treated with a bolus injection of 5 nMol PAF into the pulmonary artery as described [31,32,110]. One µM S1P was added 10 min prior to administration of PAF.…”

“…5 Role of Rho-kinase in bronchoconstriction and edema formation. Isolated perfused rat lungs were perfused with Krebs-Henseleit buffer supplemented with albumin as described [31,32,110]. Airway resistance and lung weight were measured continuously.…”

Microparticles and acute lung injury

“…Likewise, EMPs attenuate vasorelaxation in rat aortas in a dose-dependent fashion involving impaired NO signaling and endothelial function (29). These findings gain relevance in the context of ALI in view of the frequently barrier-protective (85,179), anti-inflammatory, and anticoagulatory effects of basal endothelial NO production in the lung. Endothelial dysfunction and impaired aortic vasorelaxation were likewise inducible in mice by LMPs, which caused overexpression of caveolin-1 and concomitant inhibition of eNOS (106).…”