Occurrence of cGMP/nitric oxide-sensitive store-operated calcium entry in fibroblasts and its effect on matrix metalloproteinase secretion

Abstract

AIM: To examine the existence of Nitric oxide/cGMP sensitive store-operated Ca²⁺ entry in mouse fibroblast NIH/3T3 cells and its influence on matrix metalloproteinase (MMP) production and adhesion ability of fibroblasts.

METHODS: NIH/3T3 cells were cultured. Confocal laser scanning microscopy was used to examine the existence of thapsigargin-induced store-operated Ca²⁺ entry in fibroblasts. Gelatin zymography and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) were employed to detect the involvement of [Ca²⁺]_i and NO/cGMP in MMP secretion. The involvement of NO/cGMP-sensitive Ca²⁺ entry in adhesion was determined using matrigel-coated culture plates.

RESULTS: 8-bromo-cGMP inhibited the thapsigargin-induced Ca²⁺ entry in 3T3 cells. The cGMP-induced inhibition was abolished by an inhibitor of protein kinase G, KT5823 (1μmol/L). A similar effect on the Ca²⁺ entry was observed in 3T3 cells in response to a NO donor, (±)-S-nitroso-N-acetylpenicillamine (SNAP). The inhibitory effect of SNAP on the thapsigargin-induced Ca²⁺ entry was also observed, indicating NO/cGMP-regulated Ca²⁺ entry in 3T3 cells. Results of gelatin zymography assay showed that addition of extracellular Ca²⁺ concentration induced MMP release and activation in a dose-dependent manner. RT-PCR also showed that cGMP and SNAP reduced the production of MMP mRNA in 3T3 cells. Experiments investigating adhesion potentials demonstrated that cGMP and SNAP could upgrade 3T3 cell attachment rate to the matrigel-coated culture plates.

CONCLUSION: NO/cGMP sensitive store-operated Ca²⁺ entry occurs in fibroblasts, and attenuates their adhesion potentials through its influence on MMP secretion.

Keywords: cGMP, Nitric oxide, Protein kinase G, Store-operated Ca²⁺ entry, Matrix metalloproteinase, Fibroblast