Enhancement of receptor-mediated calcium responses by phenytoin through the suppression of calcium excretion in human gingival fibroblasts

Background and ObjectivesGingival 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). MethodsEffects of 100 mu M 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. ResultsExposure of HGFs to 100 mu M 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 mu M ATP or 3 mu M histamine increased [Ca2+](i) in 20-50% of cells, and [Ca2+](i) increased in many unresponsive cells in the presence of phenytoin. ConclusionOur 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.