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

We have investigated a novel function of calpeptin, a commonly used inhibitor of calpain, in the production of intracellular reactive oxygen species (ROS) in Swiss 3T3 fibroblasts. Calpeptin induced a rapid increase of intracellular ROS by a dose-dependent manner, with a maximal increase at 10 min, which was inhibited by ROS scavengers, catalase and 2-MPG. However, other calpain inhibitors, E64d and N-acetyl-Leu-Leu-Nle-CHO (ALLN), had no effect on the level of intracellular ROS, indicating that calpain was not involved in the ROS production by calpeptin. The role of Rho in the ROS production by calpain was studied by scrape-loading of C3 transferase. C3 transferase, which inhibited stress fiber formation by calpeptin, had no effect on the ROS production in response to calpeptin, suggesting that Rho was not involved in the ROS production by calpeptin. But the elevation of intracellular ROS was inhibited by mepacrine, a phospholipase A2 inhibitor. In addition, scavenging intracellular ROS by the incubation with catalase and 2-MPG had no effect on the stress fiber formation by calpeptin. These results suggested that calpeptin stimulated the production of intracellular ROS and stress fiber formation by independent mechanisms.