Thrombin induces a temporal biphasic vascular response through the differential phosphorylation of endothelial nitric oxide synthase via protease-activated receptor-1 and protein kinase C

Endothelial nitric oxide synthase (eNOS) is a critical regulatory enzyme that controls vascular tone via the production of nitric oxide. Although thrombin also modulates vascular tone predominantly via the activation of protease-activated receptors (PARs), the time course and mechanisms involved in how thrombin controls eNOS enzymatic activity are unknown. eNOS enzymatic activity is enhanced by the phosphorylation of eNOS-Ser1177 and reduced by the phosphorylation of eNOS-Thr495. In this study, we hypothesized that thrombin regulates vascular tone through the differential phosphorylation of eNOS. Using rat descending aorta, we show that thrombin modulates vascular tone in an eNOS-dependent manner via activated PAR-1. We also show that thrombin causes a temporal biphasic response. Protein kinase C (PKC) is associated with second phase of thrombin-induced response. Western blot analysis demonstrated thrombin phosphorylated eNOS-Ser1177 and eNOS-Thr495 in human umbilical vein endothelial cells. A PKC inhibitor suppressed the thrombin-induced phosphorylation of eNOS-Thr495, but not that of eNOS-Ser1177. Our results suggest that thrombin induces a temporal biphasic vascular response through the differential phosphorylation of eNOS via activated PAR-1. Thrombin causes transient vasorelaxation by the phosphorylation of eNOS-Ser1177, and subsequent attenuation of vasorelaxation by the phosphorylation of eNOS-Thr495 via PKC, leading to the modulation of vascular tone. (c) 2022 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/ 4.0/).

Automated summary

This study reveals that thrombin regulates vascular tone through the differential phosphorylation of eNOS via activated PAR-1, causing a temporal biphasic vascular response. Thrombin induces transient vasorelaxation by phosphorylating eNOS-Ser1177, followed by attenuation of vasorelaxation through PKC-mediated phosphorylation of eNOS-Thr495, ultimately modulating vascular tone.