HDL-associated lysophospholipids induce vasorelaxation in isolated arteries and NO production and eNOS phosphorylation in endothelial cells. (a) Following precontraction of thoracic aortic rings from WKY rats with PE (1 0 10^–6 mol/l), measurements were taken of direct relaxation responses to HDL (0.5 mg/ml), the HDL-lipid fraction (Lipid, equivalent to 0.5 mg/ml HDL), the HDL protein fraction (Protein, equivalent to 0.5 mg/ml HDL), apoAI (0.1 mg/ml), cholesterol (Chol, 10 μmol/l), phosphatidylcholine (PC, 10 μmol/l), and sphingomyelin (Sm, 10 μmol/l). Cumulative findings (mean ± SEM) for maximal relaxation in eight experiments are shown (*P < 0.01 vs. HDL). (b) HPLC profile of S1P and dihydro-S1P separated on a reverse-phase C18 column after a two-step lipid extraction and derivatization with o-phthaldialdehyde (upper left panel) is shown beside a representative HPLC chromatogram of HDL after addition of dihdro-S1P (50 pmol) before extraction procedures (upper right panel). HPLC chromatogram of o-phthaldialdehyde derivatives of SPC: 0.5 μmol SPC standard (lower left panel) and HDL (lower right panel). arb U, arbitrary units. (c) Following precontraction of thoracic aortic rings from WT 129/C57BL/6 mice (WT) or eNOS–/– mice with PE (1 × 10^–6 mol/l), direct relaxation responses to HDL (0.5 mg/ml), SPC (10 μmol/l), LSF (10 μmol/l), and S1P (10 μmol/l) were tested. Cumulative findings (mean ± SEM) for maximal relaxation in eight experiments are shown (*P < 0.01 vs. WT). (d) Following precontraction of thoracic aortic rings from WKY rats with PE, direct relaxation responses to different doses of SPC, S1P, and LSF were measured. Cumulative findings (mean ± SEM) for maximal relaxation in eight experiments are shown.