Effects of oxidized low density lipoprotein on nitric oxide synthetase and protein kinase C activities in bovine endothelial cells.

Oxidized low-density lipoprotein (Ox-LDL) is an atherogenic lipoprotein. It has been suggested that Ox-LDL causes endothelial dysfunction by decreasing the release of endothelium-derived factors (EDRF-NO) or increasing the inactivation of EDRF-NO. The mechanism by which Ox-LDL causes dysfunctional NO during early stages of atherosclerosis is not clear. The purpose of this study was to examine the role of Ox-LDL on nitric oxide synthetase (eNOS), protein kinase C (PKC) activities and cAMP production in bovine aortic endothelial cells (BAEC). Ox-LDL stimulated PKC activity of BAEC but it inhibited both eNOS activity and cAMP production. Ox-LDL partially inhibited the forskolin stimulated cAMP production. Furthermore, we observed that 8Br-cAMP treatment decreased the activity of eNOS in a concentration dependent manner. Serotonin which has a profound inhibitory effect on cAMP production also stimulated eNOS activity. Pertusis toxin treatment blocked the stimulatory action of serotonin on the stimulation of eNOS activity. Our results thus suggest that Ox-LDL inhibit the endothelium-dependent relaxation. One possible mechanism is that Ox-LDL stimulates PKC activity, which in turn increases the phosphorylation of the Gi-protein. Inhibition of Gi-protein then leads to reduced release of NO from endothelial cells and thus causes endothelial dysfunction.

5-hydroxytryptamine evokes endothelial nitric oxide synthase activation in bovine aortic endothelial cell cultures.

Activation of endothelial nitric oxide synthase (eNOS) results in the production of nitric oxide (NO) that mediates the vasorelaxing properties of endothelial cells. The goal of this project was to address the possibility that 5-hydroxytryptamine (5-HT) stimulates eNOS activity in bovine aortic endothelial cell (BAEC) cultures. Here, we tested the hypothesis that 5-HT receptors mediate eNOS activation by measuring agonist-stimulated [3H]L-citrulline ([3H]L-Cit) formation in BAEC cultures. We found that 5-HT stimulated the conversion of [3H]L-arginine ([3H]L-Arg) to [3H]L-Cit, indicating eNOS activation. The high affinity 5-HT1B receptor agonist, 5-nonyloxytryptamine (5-NOT)-stimulated [3H]L-Cit turnover responses were concentration-(0.01 nM to 100 microM) and time-dependent. Maximal responses were observed within 10 min following agonist exposures. These responses were effectively blocked by the 5-HT1B receptor antagonist, isamoltane, the 5-HT1B/5-HT2 receptor antagonist, methiothepin, and the eNOS selective antagonists (0.01-10 microM): L-Nomega -monomethyl-L-arginine (L-NMMA) and L-N omega-iminoethyl-L-ornithine (L-NIO). Pretreatment of BAEC cultures with pertussis toxin (PTX; 1-100 ng/ml) for 16 hr resulted in significant inhibition of the agonist-stimulated eNOS activity, indicating the involvement of Gi proteins. These findings lend evidence of a 5-HT1B receptor/eNOS pathway, accounting in part for the activation of eNOS by 5-HT. Further investigation is needed to determine the role of other vascular 5-HT receptors in the stimulation of eNOS activity.