Phosphatidic acid-induced elevation of intracellular Ca²⁺ is mediated by RhoA and H₂O₂ in rat-2 fibroblasts

We have investigated possible roles of RhoA and H₂O₂ in the elevation of intracellular Ca²⁺ ([Ca²⁺](i)) by phosphatidic acid (PA) in Rat-2 fibroblasts. PA induced a transient elevation of [Ca²⁺](i) in the presence or absence of EGTA. Lysophosphatidic acid (LPA) also increased [Ca²⁺](i), but the sustained Ca²⁺ response was inhibited by EGTA. LPA stimulated the production of inositol phosphates, but PA did not. In the presence of EGTA, preincubation with LPA completely blocked the subsequent elevation of [Ca²⁺](i) by PA, but not vice versa. PA stimulated the translocation of RhoA to the particulate fraction as did LPA. Scrape loading of C3 transferase inhibited the transient Ca²⁺ response to PA, but not to LPA, suggesting an essential role of RhoA in the elevation of [Ca²⁺](i) by PA. H₂O₂ also induced a transient increase of [Ca²⁺](i) as did PA. H₂O₂ scavengers, catalase and N-acetyl-L-cysteine, completely blocked the rise of [Ca²⁺](i) stimulated by PA, but not by LPA. Furthermore, preincubation with PA blocked the subsequent Ca²⁺ response to H₂O₂, and the incubation with H₂O₂ also blocked the PA-induced rise of [Ca²⁺](i). Thus, it was suggested that PA stimulated Ca²⁺ release from PA-sensitive, but not inositol 1,4,5- trisphosphate-sensitive, Ca²⁺ stores by the activation of RhoA and intracellular H₂O₂.