Thrombin-induced Ca²⁺ mobilization in human gingival fibroblasts is mediated by protease-activated receptor-1 (PAR-1)

By reverse transcription (RT)-PCR analyses, human gingival fibroblasts (HGFs) were demonstrated to express mRNAs for protease-activated receptor-1 (PAR-1) and PAR-3, although the expression of PAR-3 was much weaker than that of PAR-1. The mRNAs for PAR-2 and PAR-4 were not found by RT-PCR. Furthermore, PAR activation was studied by monitoring cytosolic Ca²⁺ concentration ([Ca²⁺]i) in cultured HGFs loaded with fura-2. At concentrations > 0.1 nM, α-thrombin caused a transient increase in [Ca²⁺]i in a concentration-dependent manner, and the maximum response was obtained with 10 nM α-thrombin. After the [Ca²⁺]i response, the HGFs were completely desensitized to the second stimulation with α-thrombin. The PAR-1 agonist peptide SFLLRN produced approximately the same transient [Ca²⁺]i response as α-thrombin. After stimulation with SFLLRN, the HGFs did not respond to α-thrombin, indicating that treatment with SFLLRN results in complete desensitization to α-thrombin. The PAR-2 and PAR-4 agonist peptides had no effect on [Ca²⁺]i in HGFs. These results suggest that α-thrombin-induced Ca²⁺ mobilization in HGFs is solely mediated by PAR-1.