Generation of Reactive Oxygen Species in a Human Keratinocyte Cell Line: Role of Calcium

Publisher： Elsevier

ISSN： 0003-9861

Source： Archives of Biochemistry and Biophysics, Vol.350, Iss.1, 1998-02, pp. : 10-18

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Abstract

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 primary 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. Copyright 1998 Academic Press.