Abstract
The molecular and cellular mechanisms by which long-chain polyunsaturated fatty acids (LCPUFA) exert their beneficial effects on cardiovascular health remain obscure. While both LCPUFA and bradykinin (BK) signaling pathway play a role in the cardiovascular system, any direct link between the two is yet to be established. Using picosecond time-resolved fluorescence microscopy and a genetically engineered bradykinin B2 receptor (B2R) sensor (B2K-CC), we detected LCPUFA-induced conformational responses in the B2R similar to those caused by its cognate ligand, BK. The selective B2R antagonist (HOE-140) blocked the eicosapentaenoic acid (EPA, C20∶5, n-3) induced conformational response of the B2K-CC. Further analysis suggests that LCPUFA are capable of direct, B2R-dependent activation of extracellular ligand-regulated kinases (ERK). From a wide range of fatty acids studied, varying in chain length, saturation, and position of double bonds, EPA, docosahexaenoic (DHA, C22∶6, n-3), docosadienoic (DDA, C22∶2, n-6), and dihomo-gamma linoleic (DGLA, C20∶3, n-6) fatty acids caused the highest ERK phosphorylation. EPA or DHA dependent ERK phosphorylation was inhibited by the selective B2R antagonist. We show that LCPUFA stimulates downstream signaling by B2R such as B2R-dependent phosphorylation and expression regulation of endothelial nitric-oxide synthase (eNOS). Further analysis indicated that LCPUFA also alters levels of the eNOS transcription factor, kruppel-like factor 2 (KLF2). Moreover we show that EPA increases membrane fluidity on the same time scale as B2R conformational response, suggesting that partitioning of LCPUFA into bilayer is a primary step required for receptor activation. In summary our data show that LCPUFA activate B2R receptor at nanomolar concentrations suggesting a novel molecular mechanism by which fatty acids may affect the cardiovascular system.
Highlights
We have recently reported that long-chain polyunsaturated fatty acids (LCPUFA) stimulate conformational changes and activate parathyroid hormone receptor (PTH1R) in a dose dependent manner [1]
B2 receptor (B2R) signaling pathway is involved in activation of endothelial nitric-oxide synthase (eNOS), the enzyme that catalyze the production of nitric oxide (NO) [13], which is essential in maintaining normal vessel homeostasis [14]
GPCR FRET constructs of this type have been shown to have a reduced efficacy of cognate ligands in stimulating conformational changes, we were able to observe significant FRET response to LCPUFA
Summary
We have recently reported that LCPUFAs stimulate conformational changes and activate parathyroid hormone receptor (PTH1R) in a dose dependent manner [1]. A number of earlier studies have demonstrated that omega-3 polyunsaturated fatty acids induce in-vivo vasodilation [15,16,17,18,19,20]. These observations along with established effects of fatty acids on cardiovascular health provide a strong rationale for the study of the signal transduction pathway of LCPUFAs in endothelial cells. Further examination of the fatty acid composition in blood reported that roughly 5.3% of total serum fatty acids are LCPUFA (2.4% docosahexaenoic (DHA, C22:6, n-3), 1.8% eicosapentaenoic (EPA, C20:5, n-3), 1% docosapentaenoic (DPA, C22:5, n-3 ) [25]; similar fatty acid concentration/composition has been reported in mice [26,27]
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