Platelet Activating Factor-Induced Ceramide Micro-Domains Drive Endothelial NOS Activation and Contribute to Barrier Dysfunction

Abstract

The spatial and functional relationship between platelet activating factor-receptor (PAF-R) and nitric oxide synthase (eNOS) in the lateral plane of the endothelial plasma membrane is poorly characterized. In this study, we used intact mouse pulmonary endothelial cells (ECs) as well as endothelial plasma membrane patches and subcellular fractions to define a new microdomain of plasmalemma proper where the two proteins colocalize and to demonstrate how PAF-mediated nitric oxide (NO) production fine-tunes ECs function as gatekeepers of vascular permeability. Using fluorescence microscopy and immunogold labeling electron microscopy (EM) on membrane patches we demonstrate that PAF-R is organized as clusters and colocalizes with a subcellular pool of eNOS, outside recognizable vesicular profiles. Moreover, PAF-induced acid sphingomyelinase activation generates a ceramide-based microdomain on the external leaflet of plasma membrane, inside of which a signalosome containing eNOS shapes PAF-stimulated NO production. Real-time measurements of NO after PAF-R ligation indicated a rapid (5 to 15 min) increase in NO production followed by a > 45 min period of reduction to basal levels. Moreover, at the level of this new microdomain, PAF induces a dynamic phosphorylation/dephosphorylation of Ser, Thr and Tyr residues of eNOS that correlates with NO production. Altogether, our findings establish the existence of a functional partnership PAF-R/eNOS on EC plasma membrane, at the level of PAF-induced ceramide plasma membrane microdomains, outside recognized vesicular profiles.

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

“…2) and functional as was shown by the PAF-dependent production of prostacyclin [35]. Some groups were successful in increasing vascular permeability in endothelial cell monolayers by PAF in HUVEC cells, in murine pulmonary artery and in murine pulmonary microvascular endothelial cells [29,36,37]. 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.…”

“…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].…”

“…To some extent, it appears as though with respect to endothelial cell permeability all endothelial cells - irrespective of their origin - develop a similar phenotype in culture. This phenotype seems to be more representative of systemic than of pulmonary endothelial cells, because these cells respond to PAF (if they respond at all) with increased NO production [37] which is typical for extrapulmonary endothelial cells rather than with decreased NO-production which is characteristic of pulmonary venous endothelial cells in situ [11,31]. However, these cells do also show properties that are not observed by their counterparts in vivo , in particular the increased permeability in response to LPS, TNF-α and thrombin and the preponderance of Rho-kinase-dependent mechanisms.…”

Lipid Biomarkers for Risk Assessment in Acute Coronary Syndromes

“…The inflammatory cytokines interferon-γ, tumor necrosis factor-α (TNFα), and interleukin-1ß all stimulate ceramide synthesis [39]. Finally, ceramides are implicated in platelet activation and endothelial dysfunction via uncoupling of nitric oxide signaling pathways [40]. In addition to the biochemical role in atherosclerosis progression, elevated plasma concentrations of ceramides are associated with multiple CVD risk factors including hypertension [41], heart failure [42], and type 2 diabetes mellitus [43].…”

Insights into the Possible Mechanisms By Which Platelet-Activating Factor and PAF-receptors Function in Vascular Smooth Muscle in Magnesium Deficiency and Vascular Remodeling: Possible Links to Atherogenesis, Hypertension and Cardiac Failure

“…Although these initiated membrane receptor phenomena are mostly established for several tissuecell systems [1][2][3][4][5][6][7][8], exactly how PAF and PAF-Rs cause vascular remodeling in hypertension and atherosclerosis is not clear. A little more than 15 years ago, PAF was identified as a molecule that stimulated activation of nuclear factor-kappaB (NF-kB) [1,2,8].…”