Enhancement of Receptor-mediated Calcium Responses by Phenytoin Through the Suppression of Calcium Excretion in Human Gingival Fibroblasts

Background And Objectives: Gingival overgrowth caused by phenytoin is proposed to be associated with Ca signaling; however, the mechanisms that increase the intracellular Ca concentration ([Ca ] ) are controversial. The current study aimed to elucidate the mechanism underlying the phenytoin-induced increase in [Ca ] in human gingival fibroblasts (HGFs).

Methods: Effects of 100 μM phenytoin on [Ca ] 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 [Ca ] in the absence of extracellular Ca , indicating that the phenytoin-induced increase in [Ca ] does not require an influx of extracellular Ca . In addition, phenytoin increased [Ca ] in HGFs depleted of intracellular Ca stores by thapsigargin, indicating that neither Ca release from stores nor inhibition of Ca uptake is involved. Furthermore, the phenytoin-induced [Ca ] elevation was reduced to 18.8% in the absence of extracellular Na , and [Ca ] elevation upon removal of extracellular Na was reduced to 25.9% in the presence of phenytoin. These results imply that phenytoin increases [Ca ] of HGFs by suppressing the Na /Ca exchanger. Suppression of intracellular Ca excretion is thought to enhance the Ca responses induced by various stimuli. Analysis at the single-cell level showed that stimulation with 1 μM ATP or 3 μM histamine increased [Ca ] in 20-50% of cells, and [Ca ] increased in many unresponsive cells in the presence of phenytoin.

Conclusion: Our findings demonstrate that phenytoin induced increase in [Ca ] by the inhibition of Ca efflux in HGFs. It was also found that phenytoin strongly enhanced small Ca responses induced by stimulation with a low concentration of ATP or histamine by inhibiting Ca efflux. These findings suggest a possibility that phenytoin causes drug-induced gingival overgrowth by interacting with inflammatory bioactive substances in the gingiva.