Generation of reactive oxygen species in a human keratinocyte cell line: Role of calcium

In the human keratinocyte cell line HaCaT, reactive oxygen species (ROS) were generated in a dose-and time-dependent manner in response to epidermal growth factor (EGF), bradykinin, thapsigargin, and the Ca2+-ionophore A23187, agonists that interact with different primacy cell targets. ROS formation was assessed by both chemiluminescence-and fluorescence-based methods. The ROS evoked by EGF and bradykinin decayed within 8 and. 4 min, respectively, this transient effect resulting probably from down-regulation of the specific agonist receptors or dissipation of the secondary signals. In contrast, the response to thapsigargin and A23187 was sustained for at least 15 min, Extracellular Ca2+ and a rise in intracellular Ca2+ concentration ([Ca2+](i)) proved essential for ROS production. Chelation by BAPTA suppressed ROS formation. Direct measurement of [Ca2+](i) using fura fluorescence revealed that EGF and bradykinin evoked a modest, transient [Ca2+](i) elevation of less than twofold, whereas with thapsigargin and A23187 there was a sustained two-to fourfold elevation. For each agonist, the kinetics of the rise and decay of [Ca2+](i) were similar to those of ROS. The enzyme(s) involved in ROS formation were inhibited by diphenyleneiodonium, indicating dependence on FAD. Our results suggest a close link between ROS and changes in [Ca2+](i) generated by growth factors and hormones. This is a particularly interesting connection because elevation of ROS and, or [Ca2+](i) has been linked to cell proliferation, differentiation, and apoptosis. (C) 1998 Academic Press.