Sulfated and Glucuronidated Conjugates of 3-(4-Hydroxy-3-methoxyphenyl) Propionic Acid Can Promote NO Production by Elevated Ca²⁺ Release from the Endoplasmic Reticulum in HUVECs

We aimed to clarify whether metabolic conjugates of sulfated and glucuronidated forms have the physiological potential to produce the vasorelaxant nitric oxide (NO) in human umbilical vein endothelial cells (HUVECs), using 3-(4-hydroxy-3-methoxyphenyl) propionic acid (HMPA), a metabolite of dietary flavonoids in the gut. Treatment of HUVECs with sulfated and glucuronidated HMPAs significantly increased NO production and eNOS phosphorylation. A transporter-inhibitor-aided cellular uptake experiment of HMPAs revealed that both conjugates were incorporated into cells via MCT, OATP1A2, and GLUT transporters, whereas intact HMPA was transported via the MCT and OATP1A2 routes. A Fluo-4-probe Ca²⁺ assay demonstrated that the incorporated HMPAs significantly increased intracellular Ca²⁺ concentration by stimulating the IP₃R of the endoplasmic reticulum in the CaMKII/eNOS signaling cascade. In conclusion, to our knowledge, this study provides the first evidence that sulfated and glucuronidated forms of HMPAs may stimulate NO production in HUVECs.