O304 is a mitochondrial uncoupler which extends C. elegans lifespan and induces vasorelaxation of rat mesenteric arteries.

O304 was found to be a pan-AMPK activator through inhibiting protein phosphatase 2C and subsequent study indicated it also had mitochondrial uncoupling effect. In the present study, firstly, we confirmed O304 as a mitochondrial uncoupler, then, based on the common pharmacological effects of mitochondrial uncouplers, we examined the effect of O304 on C. elegans lifespan and vasoconstriction and vasorelaxation of rat mesenteric arteries. O304 at 1-20 μmol/L dose-dependently increased mitochondrial oxygen consumption rate (OCR) of vascular smooth muscle cells (A10 cells) and HepG2 cells after mitochondrial ATP synthase was inhibited. O304 at 2-20 μmol/L dose-dependently reduced the mitochondrial membrane potential and activated AMPK in A10 cells and HepG2 cells. These results indicated that O304 was a mitochondrial uncoupler. Based on the role of mitochondrial uncoupling and AMPK activation in extending C. elegans lifespan and vasorelaxation function, we further evaluated the effect of O304 on C. elegans lifespan and vascular tone of rat mesenteric arteries. O304 (100 μmol/L) treatment extended C. elegans lifespan through AMPK-mTOR axis, and autophagy-induced lipid catabolism. O304 (2, 5 μmol/L) also showed an anti-aging effect in mouse embryonic fibroblasts (MEFs). Taken together, the results from nematodes and MEF cells indicated that O304 had anti-aging properties. O304 at 2-10 μmol/L dose-dependently relaxed phenylephrine (PE) and U46619-induced pre-constriction, and pretreatment with O304 at 2-10 μmol/L dose-dependently inhibited PE and U46619-induced constriction of rat mesenteric arteries in an endothelium-independent manner. In conclusion, O304 exerts the common effect of mitochondrial uncouplers on C. elegans lifespan and vascular tone of rat mesenteric arteries.