Recent interest has focused on the role of Ca²⁺ in regulating health problems, including cardiovascular disease and colorectal cancer. The inverse correlation between colon cancer and serum Ca²⁺ underlines the importance of understanding intracellular Ca²⁺ dynamics. Studies are also evaluating the contributions of abnormalities in Ca²⁺ homeostasis and intracellular dysfunction to the pathogenesis of metabolic syndrome as a precursor of cardiovascular disease. In this study, we investigated the changes of Ca²⁺ dynamics and ginsenoside Rb₁ (Rb₁)-induced recovery in two vascular cell lines exposed to palmitic acid (PA), the most abundant active ingredient in palm oil. The mechanism underlying the Rb₁-induced recovery was examined in a store operated Ca²⁺ entry model by Ca²⁺ store depletion. PA reduced the Ca²⁺ influx in both EA.hy926 (EA) and MOVAS cells, and this change was restored by Rb₁. In EA cells, the Rb₁-induced restoration was abolished by U73122 or 2-APB. In MOVAS cells, meanwhile, the effect of Rb₁ was abolished by FIPI, U73122 and U73343. Under normal conditions, Rb₁ itself altered phospholipid signaling (PLC in EA cells and PLD in MOVAS cells), but did not affect Ca²⁺ homeostasis. These differences resulted in differences in downstream actions, as KB-R7943 and nifedipine inhibited Rb₁-mediated Ca²⁺ influx recovery only in MOVAS cells. In conclusion, Rb₁ rescues the PA-induced Ca²⁺ influx by appropriately activating PLC in EA cells and PLD in MOVAS cells. This demonstrates that Ca²⁺ dynamics are elaborately regulated via intracellular Ca²⁺ signaling networks, suggesting a potential strategy for maintaining vascular Ca²⁺ homeostasis in hyperlipidemic environments.

Inflammation and loss of cholinergic transmission are involved in neurodegenerative diseases, but possible interactions between them within neurons, astrocytes, and microglia have not yet been investigated. We aimed to compare store-operated Ca²⁺ entry (SOCE) in neurons, astrocytes, and microglia following cholinergic dysfunction in combination with (or without) an inflammatory stimulus and to investigate the effects of linalyl acetate (LA) on this process. We used the SH-SY5Y, U373, and BV2 cell lines related to neurons, astrocytes, and microglia, respectively. Scopolamine or lipopolysaccharide (LPS) was used to antagonize the muscarinic receptors or induce inflammatory responses, respectively. The concentration of intracellular Ca²⁺ was measured using Fura-2 AM. Treatment with scopolamine and LPS significantly increased SOCE in the neuron-like cells and microglia but not in the scopolamine-pretreated astrocytes. LA significantly reduced SOCE in the scopolamine-pretreated neuron-like cells and microglia exposed to LPS, which was partially inhibited by the Na⁺-K⁺ ATPase inhibitor ouabain and the Na⁺/Ca²⁺ exchanger (NCX) inhibitor Ni²⁺. Notably, SOCE was significantly reduced in the LPS plus scopolamine-pretreated cells mixed with astrocytes and microglia, with a two-fold increase in the applied number of astrocytes. LA may be useful in protecting neurons and microglia by reducing elevated SOCE that is induced by inflammatory responses and inhibiting the muscarinic receptors via Na⁺-K⁺ ATPase and the forward mode of NCX. Astrocytes may protect microglia by reducing increased SOCE under the conditions of inflammation and a muscarinic receptor blockade.

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.

Parkinson’s disease is one of the most common neurodegenerative disorders, and the pesticide rotenone is widely used to model Parkinson’s disease in experimental studies. Rotenone-induced mitochondrial dysfunction is associated with oxidative stress and intracellular Ca ²⁺ ([Ca ²⁺ ]ᵢ) dysregulation in neuronal systems. L-type Ca ²⁺ channels (LTCCs) contribute to Ca² ⁺ influx under oxidative stress conditions, but pharmacological inhibition of LTCCs is limited by cardiovascular side effects. Phospholipase D (PLD) has also been implicated in cellular stress-associated signaling pathways. Ginsenosides are known to influence Ca² ⁺ -related signaling; however, differences among ginsenosides in neuronal and microglial Ca² ⁺ regulation have not been fully characterized. Therefore, this study aimed to investigate the effects of ginsenosides on intracellular Ca² ⁺ homeostasis and oxidative stress of rotenone-exposed SH-SY5Y neuronal cells and BV2 microglial cells. Cell viability, superoxide dismutase (SOD) activity, interleukin-6 (IL-6) levels, intracellular Ca² ⁺ influx, and malondialdehyde (MDA) levels were evaluated. Intracellular Ca² ⁺ was measured using Fura-2 AM ratiometric fluorescence analysis. The effects of ginsenosides Rg1, Rg2, and Rd were examined using pharmacological inhibitors targeting PLD, LTCCs, and protein kinase A (PKA). Rotenone reduced cell viability and SOD activity, while increasing IL-6 levels, [Ca ²⁺ ]ᵢ influx, and MDA levels in both cell types. Treatment with ginsenosides immediately before rotenone exposure attenuated rotenone-induced increases in [Ca ²⁺ ]ᵢ and MDA levels. Ca² ⁺ responses in both SH-SY5Y and BV2 cells were sensitive to PLD inhibition. In SH-SY5Y cells, responses were additionally sensitive to LTCC inhibition, whereas BV2 cells showed weaker LTCC-associated pharmacological responses. Rd showed broader pharmacological sensitivity involving PLD-, LTCC-, and PKA-associated components, whereas Rg1 and Rg2 showed predominantly PLD-associated response patterns. These findings suggest that ginsenosides differentially modulate rotenone-associated Ca² ⁺ dysregulation through PLD-associated pharmacological pathways in a cell type-dependent manner. Overall, the present findings demonstrate distinct pharmacological response profiles among structurally different ginsenosides under rotenone-induced oxidative stress conditions.

Background: Intracellular Ca²⁺ dysregulation and neurovascular dysfunction are increasingly recognized as important mechanisms underlying neurodegenerative disorders, including Alzheimer’s disease (AD). Restoration of pathological Ca²⁺ imbalance has therefore emerged as a potential therapeutic strategy. Although ginsenoside compound K (CK) has demonstrated neuroprotective and anti-inflammatory properties, its role in Ca²⁺ homeostasis remains unclear. Methods: SH-SY5Y neuronal cells, U373 astrocytes, BV2 microglia, bEND brain endothelial cells, and MOVAS vascular smooth muscle cells were exposed to trimethyltin chloride (TMT, 5 μM, 24 h). Cell viability, real-time cell confluence, intracellular Ca²⁺ influx, and endoplasmic reticulum (ER) Ca²⁺ store release were evaluated. Ca²⁺ dynamics were analyzed using Fura-2 fluorescence, and signaling associated with CK-mediated Ca²⁺ regulation was investigated using pharmacological inhibitors. Orai1 protein expression was evaluated by Western blot analysis in SH-SY5Y and U373 cells. Results: TMT increased store-operated Ca²⁺ entry (SOCE)-mediated Ca²⁺ influx in SH-SY5Y neuronal cells without significant cytotoxicity. In contrast, TMT reduced both Ca²⁺ influx and cell viability in U373 astrocytes, BV2 microglia, and MOVAS cells, whereas bEND cells showed minimal changes. CK restored abnormal Ca²⁺ responses in a cell type-specific manner, reducing elevated Ca²⁺ influx in neuronal cells while restoring suppressed Ca²⁺ signaling in astrocytes. Consistent with these functional findings, Orai1 protein expression was increased by TMT in SH-SY5Y cells and attenuated by CK, whereas TMT reduced Orai1 expression in U373 cells, which was restored by CK. Pharmacological analyses suggested involvement of PKA- and LTCC-associated signaling in neuronal cells and PLC- and PLD-associated signaling in astrocytes. In both cell types, CK-associated Ca²⁺ responses were sensitive to NAC and the SOCE inhibitor BTP2, supporting ROS- and SOCE-associated mechanisms. Additional controls showed no significant inhibitor effects under control or CK-only conditions, strengthening the pharmacological interpretation of responses under TMT-containing conditions. Conclusions: TMT-induced Ca²⁺ dysregulation differed markedly by cell type. CK restored disrupted Ca²⁺ homeostasis in association with distinct pharmacological signaling profiles in neuronal and astrocytic cells. These findings suggest that CK may function as a cell type-specific Ca²⁺ homeostatic regulator under neurotoxic stress conditions and highlight the importance of differential Ca²⁺ regulation in neurodegenerative disease models.