Calcium-dependent PAF-stimulated generation of reactive oxygen species in a human keratinocyte cell line

During inflammation and other pathological states, the lipid mediator plate let-activating factor (PAF) and reactive oxygen species (ROS) are both gene rated. We have been investigating the effect of exogenous PAF on ROS format ion in the human keratinocyte cell line (HaCaT). ROS production, measured u sing luminol-enhanced chemiluminescence (CL), proved to be rapid, transient , PAF receptor-mediated, and totally dependent on an increase in intracellu lar Ca2+ ([Ca2+](i)) and on the presence of extracellular Ca2+. Repeated ad ministration of PAF resulted in refractoriness to the agonist in terms of b oth capacities to increase [Ca2+](i) and generate ROS. The cells, however, continued to respond fully to other stimulants (bradykinin, epidermal growt h factor, thapsigargin). The PAF-induced increases in [Ca2+](i) (monitored using the fluorescent probe Fluo-3) were also rapid and transient and paral leled those of ROS generation. Relatively specific inhibitors of potential ROS-producing systems were administered in an attempt to characterize the R OS producing system(s). Inhibitors of xanthine oxidase, phospholipase A(2), lipoxygenase, cyclooxygenase and NO synthase did not interfere with PAF ev oked ROS. The flavoprotein inhibitor diphenyleneiodonium and the mitochondr ial cytochrome oxidase inhibitor KCN, prevented generation of ROS, making N AD(P)H a candidate for the electron source of the ROS and the mitochondria a potential major site of formation. (C) 1999 Elsevier Science B.V. All rig hts reserved.