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

Sanda Predescu,Viktor Brovcovych,Cristina Bardita,Dan Predescu,Ivana Knezevic,Radu Florin Neamu,You-Yang Zhao

doi:10.1371/journal.pone.0075846

Sanda Predescu, Viktor Brovcovych + Show 5 more

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https://doi.org/10.1371/journal.pone.0075846

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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.

Highlights

Platelet activating factor (PAF) is one of the most potent phospholipids released by a diversity of cell types [1,2,3,4] upon activation by a variety of agonists [4,5,6]; its release results in a marked increase in vascular permeability at the level of different vascular beds [7,8,9,10]
Because in the initial experiments carried out with isolated pulmonary artery ECs (PAECs) we couldn’t detect modifications in trans monolayers electrical resistance (TER) after PAF, we have to pretreat this monolayers with indometacin (25μM) in order to obtain biological effects; all experiments carried out on isolated PAECs were performed on monolayers treated with indometacin
TER values for mouse PAECs were 14±5 Ω·cm2 and for the MVECs 17± 8 Ω·cm2, comparable to the TER of HUVECs monolayers used as control which showed an average electrical resistance of 12±5 Ω·cm2

Summary

Introduction

Platelet activating factor (PAF) is one of the most potent phospholipids released by a diversity of cell types [1,2,3,4] upon activation by a variety of agonists [4,5,6]; its release results in a marked increase in vascular permeability at the level of different vascular beds [7,8,9,10]. The EC-generated NO, known to control many vital vascular functions [19,20,21,22] is produced by the activation (phosphorylation/dephosphorylation) of endothelial nitric oxide synthase (eNOS). Evidence indicates that eNOS activity is complex and tight regulated; its expression, biochemical activity and subcellular localization controls NO production and NO functional efficacy [23,24,25]. The membrane association of eNOS is required for its phosphorylation and activation by different stimuli [26,27,28], including PAF [29]. PAF-R resides all over plasma membrane outside of any identifiable vesicular carriers, while eNOS is rationalized to be associated with the plasma membrane of caveolae [30]

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