Linalyl acetate prevents D-galactose and nicotine-induced increases in intracellular Ca2+ by inhibiting phospholipase D hyperactivation in MOVAS and L6 cells

Abstract

Linalyl acetate (LA) is known for its protective effects on vascular and skeletal muscle and for regulating intracellular Ca²⁺ ([Ca²⁺]_i). In this study, we investigated the mechanisms by which nicotine and D-galactose (D-gal) increase [Ca²⁺]_i in vascular smooth muscle cells (MOVAS) and skeletal muscle cells (L6), and examined the protective role of LA. Using a Fura-2 AM–based assay, we found that D-gal+nicotine significantly elevated [Ca²⁺]_i in both cell types, with a greater effect in MOVAS cells. Mechanistically, this abnormal [Ca²⁺]_i increase was mediated through phospholipase D (PLD) hyperactivation in MOVAS cells, while both PLD and phospholipase C (PLC) hyperactivation were involved in L6 cells. In D-gal–treated L6 cells, nicotine further increased [Ca²⁺]_i via additional PLD hyperactivation, suggesting that nicotine exacerbates D-gal–induced Ca²⁺ dysregulation. Treatment with LA effectively rescued these abnormalities by suppressing PLD hyperactivation, thereby restoring [Ca²⁺]_i to normal levels in both cell types. Moreover, our findings indicate that VSMCs are more vulnerable than skeletal muscle cells to D-gal+nicotine–induced [Ca²⁺]_i elevation, likely due to the stronger NCX activation associated with PLD hyperactivation compared to PLC. Taken together, these results demonstrate that D-gal+nicotine induces abnormal Ca²⁺ signaling through PLD hyperactivation, contributing to vascular and skeletal muscle cell stress, and that LA effectively prevents this pathological process. Thus, LA may serve as a promising plant-derived natural compound for preventing cellular aging and Ca²⁺-mediated pathological changes induced by nicotine and D-gal.