Role of ERK1/2 activation and nNOS uncoupling on endothelial dysfunction induced by lysophosphatidylcholine.

BACKGROUND AND AIMS: Lysophosphatidylcholine (LPC) - a main component of oxidized LDL - is involved in endothelial dysfunction that precedes atherosclerosis, with an increased superoxide anions and a reduced NO production via endothelial NO synthase (eNOS) uncoupling. However, there is no evidence about the mechanisms involved in neuronal NOS (nNOS) uncoupling. Extracellular signal-regulated kinase (ERK) is related to the control of NO production and inflammatory gene transcription activation in atherosclerosis. Our aim was to investigate the role of nNOS/ERK1/2 pathway on endothelial dysfunction induced by LPC, in mouse aorta and human endothelial cells. METHODS: Thoracic aorta from wild type mice was used to perform vascular reactivity studies in the presence or absence of LPC. Human endothelial cells were used to investigate the effect of LPC on expression of nNOS and his products NO and H2O2. RESULTS: LPC reduced acetylcholine (ACh)-induced vasodilation in mouse aorta (EmaxCT/LPC = âˆ¼95 ± 2/62 ± 3%, p = 0.0004) and increased phenylephrine-induced vasoconstriction (EmaxCT/LPC = âˆ¼4 ± 0,1/6 ± 0,1 mN/mm, p = 0.0002), with a reduction in NO (fluorescence intensityCT/LPC = 91 ± 3/62±2 × 103, p = 0.0002) and H2O2 (fluorescence intensityCT/LPC = âˆ¼16 ± 0,8/10 ± 0,7 × 103, p = 0.0041) production evocated by ACh. An inhibition of nNOS by TRIM (EmaxCT/CT+TRIM = âˆ¼93 ± 1/43 ± 3%, p = 0,0048; EmaxLPC/LPC+TRIM = âˆ¼62 ± 3/65 ± 3%) or H2O2 degradation by catalase (EmaxCT/CT+cat = âˆ¼93 ± 1/46 ± 2%, p < 0,001; EmaxLPC/LPC+cat = âˆ¼62,8 ± 3,2/60,5 ± 4,7%) reduced the relaxation in the control but not in LPC group. PD98059, an ERK1/2 inhibitor, abolished the increase in vasoconstriction in LPC-treated vessels (EmaxLPC/LPC+PD = âˆ¼6 ± 0,1/3 ± 0,1 mN/mm, p = 0.0001). LPC also reduced the dimer/monomer proportion and increased nNOSser852 phosphorylation. CONCLUSIONS: LPC induced nNOS uncoupling and nNOSSer852 phosphorylation, reduced NO and H2O2 production and improved superoxide production by modulating ERK1/2 activity in human and murine endothelial cells.

Vascular Kinin B1 and B2 Receptors Determine Endothelial Dysfunction through Neuronal Nitric Oxide Synthase.

B1- and B2-kinin receptors are G protein-coupled receptors that play an important role in the vascular function. Therefore, the present study was designed to evaluate the participation of kinin receptors in the acetylcholine (ACh)-induced vascular relaxation, focusing on the protein-protein interaction involving kinin receptors with endothelial and neuronal nitric oxide synthases (eNOS and nNOS). Vascular reactivity, nitric oxide (NO·) and reactive oxygen species (ROS) generation, co-immunoprecipitation were assessed in thoracic aorta from male wild-type (WT), B1- (B1R-/-), B2- (B2R-/-) knockout mice. Some vascular reactivity experiments were also performed in a double kinin receptors knockout mice (B1B2R-/-). For pharmacological studies, selective B1- and B2-kinin receptors antagonists, NOS inhibitors and superoxide dismutase (SOD) mimetic were used. First, we show that B1- and B2-kinin receptors form heteromers with nNOS and eNOS in thoracic aorta. To investigate the functionality of these protein-protein interactions, we took advantage of pharmacological tools and knockout mice. Importantly, our results show that kinin receptors regulate ACh-induced relaxation via nNOS signaling in thoracic aorta with no changes in NO· donor-induced relaxation. Interestingly, B1B2R-/- presented similar level of vascular dysfunction as found in B1R-/- or B2R-/- mice. In accordance, aortic rings from B1R-/- or B2R-/- mice exhibit decreased NO· bioavailability and increased superoxide generation compared to WT mice, suggesting the involvement of excessive ROS generation in the endothelial dysfunction of B1R-/- and B2R-/- mice. Alongside, we show that impaired endothelial vasorelaxation induced by ACh in B1R-/- or B2R-/- mice was rescued by the SOD mimetic compound. Taken together, our findings show that B1- and B2-kinin receptors regulate the endothelium-dependent vasodilation of ACh through nNOS activity and indicate that molecular disturbance of short-range interaction between B1- and B2-kinin receptors with nNOS might be involved in the oxidative pathogenesis of endothelial dysfunction.