Insulin and lysophosphatidylcholine synergistically stimulate NO-dependent cGMP production in human endothelial cells.

Abstract

Nitric oxide (NO) is an important regulator of cardiovascular homeostasis. Lysophosphatidylcholine (lyso-PC), a major constituent of oxidized low density lipoproteins (oxLDL), has been reported to impair nitric oxide-dependent vasodilatation. This study investigated the possible mechanism of the lyso-PC effect on insulin-stimulated NO-dependent of cyclic guanosine 3',5'-monophosphate (cGMP) generation in human endothelial cells. The intracellular concentration of cGMP in cultured human umbilical vein endothelial cells (HUVECs) was used to estimate NO production. The levels of endothelial nitric oxide synthase (eNOS) protein expression were assessed by Western blotting analyses. Both insulin, at physiological concentration, and lyso-PC stimulated rapid and prolonged intracellular of cGMP production, and together induced a marked synergistic response (for short-term stimulation: 1185 +/- 285.9% over control level (100%) compared with insulin and lyso-PC alone (384.8 +/- 67.4% and 357 +/- 205%, respectively; P < 0.001), for long-term stimulation: 3495 +/- 1377%, compared with insulin and lyso-PC alone (663 +/- 131% and 487 +/- 250%, P = 0.002)). Stimulated levels of cGMP accumulation were completely abrogated by NOS inhibitor, indicating NO involvement in the effects of insulin and lyso-PC. Stimulated NO synthesis was not associated with altered eNOS protein expression. Cell subfractionation studies demonstrate that insulin and lyso-PC each alone induced translocation of eNOS from the membrane to the cytosolic compartment and together caused a synergistic translocation. The presented data suggest that insulin and lyso-PC synergistically upregulate endothelial NO production via eNOS translocation from the membrane fraction to the cytosol. This study raises the possibility that an interplay between various factors accompanying diabetes can lead to endothelial NO overproduction or desensitization of NO-dependent responses. Appropriate rather than necessarily high levels of nitric oxide is the determinant of vascular health.