5‐Hydroxytryptamine Evokes Endothelial Nitric Oxide Synthase Activation in Bovine Aortic Endothelial Cell Cultures

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Abstract
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 μM) 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 μM): L‐Nω ‐monomethyl‐L‐arginine (L‐NMMA) and L‐N ω‐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.

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