Abstract : Exposure to diesel exhaust particles (DEPs) affects endothelial function and may contribute to the development of atherosclerosis and vasomotor dysfunction. As intracellular calcium concentration [Ca] is considered important in myoendothelial signalling, we explored the effects of extractable organic matter from DEPs (DEP-EOM) on [Ca] and membrane microstructure in endothelial cells. DEP-EOM of increasing polarity was obtained by pressurized sequential extraction of DEPs with -hexane (-Hex-EOM), dichloromethane (DCM-EOM), methanol, and water. Chemical analysis revealed that the majority of organic matter was extracted by the -Hex- and DCM-EOM, with polycyclic aromatic hydrocarbons primarily occurring in -Hex-EOM. The concentration of calcium was measured in human microvascular endothelial cells (HMEC-1) using micro-spectrofluorometry. The lipophilic -Hex-EOM and DCM-EOM, but not the more polar methanol- and water-soluble extracts, induced rapid [Ca] increases in HMEC-1. -Hex-EOM triggered [Ca] increase from intracellular stores, followed by extracellular calcium influx consistent with store operated calcium entry (SOCE). By contrast, the less lipophilic DCM-EOM triggered [Ca] increase via extracellular influx alone, resembling receptor operated calcium entry (ROCE). Both extracts increased [Ca] via aryl hydrocarbon receptor (AhR) non-genomic signalling, verified by pharmacological inhibition and RNA-interference. Moreover, DCM-EOM appeared to induce an AhR-dependent reduction in the global plasma membrane order, as visualized by confocal fluorescence microscopy. DCM-EOM-triggered [Ca] increase and membrane alterations were attenuated by the membrane stabilizing lipid cholesterol. In conclusion, lipophilic constituents of DEPs extracted by -hexane and DCM seem to induce rapid AhR-dependent [Ca] increase in HMEC-1 endothelial cells, possibly involving both ROCE and SOCE-mediated mechanisms. The semi-lipophilic fraction extracted by DCM also caused an AhR-dependent reduction in global membrane order, which appeared to be connected to the [Ca] increase.
