gms | German Medical Science

Asymmetric-dimethyl-L-arginine (ADMA) produces endothelial dysfunction via endogenous inhibition of endothelial nitric oxide synthase (eNOS). Recently we have shown, that ADMA levels in the CSF were strongly correlated with arteriographic vasospasm after subarachnoid hemorrhage (SAH). We sought to determine the cellular mechanism of ADMA elevation, using the putative spasmogen oxidized bilirubin products (BOXes), and Probucol, an ADMA inhibitor.

Human umbilical vein endothelial cells (HUVECs) in EGM were used as control and HUVECs exposed to oxidized low density protein (oxLDL;10mg/dl), known to induce ADMA production, were used as positive controls. HUVECs were exposed to BOXes (1:100) for 24 hours. HUVECs exposed to oxLDL or BOXes were also co-incubated with Probucol (50μM), known to decrease ADMA levels. Cell-media and lysates were harvested to assess ADMA levels using HPLC and nitrite levels.

In the media the control ADMA levels were 7± 3.6pmol/μg protein. OxLDL (positive control) (p<0.001) and BOXes (p<0.01) increased ADMA levels. Probucol decreased ADMA levels evoked by either oxLDL (p<0.001) or BOXes (p<0.01) and significantly attenuated the decrease of nitrite levels produced by BOXes and oxLDL. In cell-lysates the same effects were detected in all experimental groups.

BOXes increased ADMA production and release in endothelial cells. Probucol significantly decreased ADMA levels in the media and cell-lysate. These results suggest that BOXes may be responsible for endothelial dysfunction via endogenous inhibition of eNOS. ADMA-dependent endothelial dysfunction may be prevented by Probucol.