Abstract:
The vascular endothelium is a single cell layer that lines the lumen of the entire
vasculature. It is the site of synthesis of nitric oxide (NO), a vasodilatory compound
synthesized by endothelial nitric oxide synthase (eNOS). NO causes intracellular
calcium sequestration of the vascular smooth muscle cells, relaxing and dilating the
arteries. Age profoundly affects endothelium-dependent vasodilation, leading to
specific losses of NO. We sought to determine what causes the age-specific loss of
endothelial NO. This was accomplished by investigating whether there are differences
in markers of eNOS post-translational regulation elements in the aortic endothelium of
young (2-4 months; corresponding to an adolescent human adult) and old (32-34
months; corresponding to a 65-75 year-old human). F 344 x Brown Norway hybrid
rats.
Results show that maximal eNOS activity significantly declines with age
(n=4;p≤0.05) though there was no change in eNOS protein levels in the aortic
endothelium. Endothelial NOS exists in two distinct subcellular fractions. No alterations were detected in the soluble, inactive fraction while significantly less eNOS
protein is detected in the active, plasma membrane fraction of the endothelium
(n=4;p≤0.02). Endothelial NOS activation is also controlled by its phosphorylation
state. In this work we demonstrate that free ceramides and ceramide-activated
phosphatase (PP2A) activity are significantly elevated with age in the endothelium and
correlate with specific alterations in eNOS phosphorylation status consistent with its
inactivation. These changes were concomittent with an age-associated decline in
endothelial glutathione (GSH) and increased sphingomyelinase activity which
liberates ceramides from membrane sphingolipids.
In previously published reports we demonstrated that the dithiol compound R-α-lipoic acid (LA) increased maximal NO synthesis in cultured endothelial cells and
that LA improved age-associated loss of eNOS stimulatory phosphorylation in rats.
Therefore, we administered pharmacologic doses of LA (40 mg/kg, i.p. over 24 h) to
old rats to determine whether it restored NO-dependent vasomotor function. Results
show that LA significantly increased endothelial GSH (p≤0.05 compared to saline
controls), decreased sphingomyelinase activity and reversed the age-related increase in
ceramide (p≤0.01) in old animals. Finally, LA significantly improved endothelium-dependent
vasodilation, suggesting that it might be a good therapeutic agent for age-related
vascular endothelial dysfunction.
