Tyrosine phosphorylation is involved in Ca²⁺entry in human gingival fibroblasts

Bradykinin (1 μM) and histamine (100 μM) evoked an initial transient increase and a subsequent sustained increase in intracellular Ca²⁺concentration ([Ca²⁺]_i) in fura-2-loaded human gingival fibroblasts, which may be attributed to Ca²⁺release from intracellular stores and Ca²⁺entry from extracellular sites, respectively. In fibroblasts pretreated with tyrosine kinase inhibitors such as herbimycin A (1 μM) and tyrphostin 47 (20 μM)_,the sustained level of [Ca²⁺]_iinduced by bradykinin and histamine increased, but not the initial peak level. In the absence of external Ca²⁺, bradykinin and histamine induced only the transient increase in [Ca²⁺]_i, but a subsequent addition of Ca²⁺to the medium resulted in a sustained increase in [Ca²⁺]_icaused by Ca²⁺entry. Thapsigargin, an inhibitor of Ca²⁺-ATPase in inositol 1,4,5-trisphosphate-sensitive Ca²⁺stores, mimicked the effect of bradykinin and histamine. In the fibroblasts pretreated with tyrosine kinase inhibitors, the bradykinin-, histamine- and thapsigargin-induced Ca²⁺entry was clearly enhanced, but not the transient [Ca²⁺]_iincrease. Tyrosine phosphatase inhibitor benzylphosphonic acid (200 μM) had no effect on Ca²⁺entry or transient [Ca²⁺]_iincrease. These results suggest that tyrosine phosphorylation is involved in Ca²⁺entry in human gingival fibroblasts.