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, pre incubation 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. (C) 2000 Elsevier Science Inc. All rights reserved.