Background and Objectives Gingival overgrowth caused by phenytoin is proposed to be associated with Ca2+ signaling; however, the mechanisms that increase the intracellular Ca2+ concentration ([Ca2+]i) are controversial. The current study aimed to elucidate the mechanism underlying the phenytoin‐induced increase in [Ca2+]i in human gingival fibroblasts (HGFs). Methods Effects of 100 μM phenytoin on [Ca2+]i in HGFs were examined at the single‐cell level using fluorescence images of fura‐2 captured by an imaging system consisting of an EM‐CCD camera coupled to an inverted fluorescence microscope at room temperature. Results Exposure of HGFs to 100 μM phenytoin induced a transient increase in [Ca2+]i in the absence of extracellular Ca2+, indicating that the phenytoin‐induced increase in [Ca2+]i does not require an influx of extracellular Ca2+. In addition, phenytoin increased [Ca2+]i in HGFs depleted of intracellular Ca2+ stores by thapsigargin, indicating that neither Ca2+ release from stores nor inhibition of Ca2+ uptake is involved. Furthermore, the phenytoin‐induced [Ca2+]i elevation was reduced to 18.8% in the absence of extracellular Na+, and [Ca2+]i elevation upon removal of extracellular Na+ was reduced to 25.9% in the presence of phenytoin. These results imply that phenytoin increases [Ca2+]i of HGFs by suppressing the Na+/Ca2+ exchanger. Suppression of intracellular Ca2+ excretion is thought to enhance the Ca2+ responses induced by various stimuli. Analysis at the single‐cell level showed that stimulation with 1 μM ATP or 3 μM histamine increased [Ca2+]i in 20–50% of cells, and [Ca2+]i increased in many unresponsive cells in the presence of phenytoin. Conclusion Our findings demonstrate that phenytoin induced increase in [Ca2+]i by the inhibition of Ca2+ efflux in HGFs. It was also found that phenytoin strongly enhanced small Ca2+ responses induced by stimulation with a low concentration of ATP or histamine by inhibiting Ca2+ efflux. These findings suggest a possibility that phenytoin causes drug‐induced gingival overgrowth by interacting with inflammatory bioactive substances in the gingiva.

Genetically-encoded calcium indicators such as G-GECO are useful for studying Ca 2+ responses during long-term processes. In this study, we employed a lentiviral vector and established a rat dental epithelial cell line that stably expressed G-GECO (SF2-G-GECO). Ca 2+ imaging analysis under cell culture conditions revealed that SF2-G-GECO cells exhibited spontaneous Ca 2+ responses, which could be classified into the following three major patterns depending on the cell density: localized Ca 2+ responses at cell protrusions at a low density, a cell-wide spread of Ca 2+ responses at a medium density, and Ca 2+ responses in clusters of 3-20 cells at a high density. The P2Y receptor inhibitor suramin (10 μM), the ATP-degrading enzyme apyrase (5 units/mL), and the fibroblast growth factor (FGF) receptor inhibitor FIIN-2 (1 μM) decreased the frequency of spontaneous Ca 2+ responses. These results indicate that ATP and FGF are involved in the spontaneous Ca 2+ responses. SF2 cells differentiate into ameloblasts via interactions with mesenchymal cells. Therefore, SF2-G-GECO cells are expected to be a useful tool for studying the functions of Ca 2+ responses in regulating gene expression during tooth development.