The essential role of H₂O₂ in the regulation of intracellular Ca²⁺ by epidermal growth factor in Rat-2 fibroblasts

We have investigated a new mechanism by which epidermal growth factor (EGF) increases intracellular Ca²⁺ ([Ca²⁺](i)) in Rat-2 fibroblasts. EGF induced a transient increase of [Ca²⁺](i), and sustained Ca²⁺ increase disappeared in the absence of extracellular Ca²⁺. However, EGF had no effect on the formation of inositol phosphates. Expression of N17Rac or scrape-loading of C3 transferase blocked the elevation of [Ca²⁺](i) by EGF, but not by lysophosphatidic acid (LPA). EGF increased intracellular H₂O₂, with a maximal increase at 5 min, which was blocked by catalase, scrape-loading of C3 transferase, or expression of N17Rac. H₂O₂ scavengers, catalase and N-acetyl-L-cysteine, also blocked the Ca²⁺ response to EGF, but not to LPA. In the presence of EGTA, preincubation with EGF completely inhibited subsequent Ca²⁺ response to extracellular H₂O₂ and vice versa. Incubation with EGF or phosphatidic acid abolished subsequent elevation of [Ca²⁺](i) by phosphatidic acid or EGF, respectively. Furthermore, preincubation with LPA inhibited the subsequent Ca²⁺ response to EGF, but not vice versa. These results suggested that intracellular H₂O₂ regulated by Rac and RhoA, but not inositol phosphates, was responsible for the EGF-stimulated elevation of [Ca²⁺](i). It was also suggested that EGF cross talked with LPA in the regulation of [Ca²⁺](i) by producing intracellular H₂O₂. Copyright (C) 2000 Elsevier Science Inc.