The Protective Effects of Eicosapentaenoic Acid are Triggered Unusal Mechanisms of Ca2+ Release and Ca2+ Influx in Mouse Cerebral Cortical Endothelial Cells

Abstract

Aims: Eicosapentaenoic acid (EPA), an omega-3 fatty acid abundant in fish oil, protects endothelial cells (EC) from lipotoxicity and triggers EC nitric oxide release, has been demonstrated to have beneficial effects in cardiovascular system and brain protection, but the detail molecular mechanism is unclear and how EPC affects EC Ca2+-signaling and other functions remain largely unexplored. In this work, we investigated the effects of EPA on mouse cerebral cortical endothelial cells (bEND.3 cells) as well as the influence of Ca2+ channel and cytosolic Ca2+ level. Methods: Brain microvascular bEND.3 cells cultured in Dulbecco¡¦s modified Eagle¡¦s medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cytosolic Ca2+ in bEND was measured with Fura-2 method. Mitochondria membrane potential (MMP) measured by MMP-Assay Kit. Cell viability was measured By MTT-assay. The p < 0.05 were considered significant (ANOVA). Results: EPA was shown to cause intracellular Ca2+ release in mouse cerebral cortex endothelial bEND.3 cells; the EPA-sensitive intracellular Ca2+ pool(s) appeared to encompass and was larger than the Ca2+ pool mobilized by sarcoplasmic-endoplasmic reticulum Ca2+-ATPase inhibition by cyclopiazonic acid. EPA also opened a Ca2+ influx pathway pharmacologically distinct from store-operated Ca2+ influx. Additionally, EPA-triggered Ca2+ influx was Ni2+-insensitive; and EPA did not trigger Mn2+ influx. Further, EPA-triggered Ca2+ influx did not involve Na+-Ca2+ exchangers. Conclusion: EPA caused Ca2+ release and Ca2+ Influx via unusual mechanisms in bEND.3 cells. Given the versatile health effects of EPA, it is interesting and important to study the Ca2+ signaling triggered by this fatty acid in EC and other cell types, whose Ca2+ responses to EPA are very different from those in EC. (*Acknowledgement: Funded by: Ministry of Science & Technology, Taiwan MOST 105-2320- B039-028 & China Medical University Grant DMR-105-069 to KL Wong)