Calcium-dependent PAF-stimulated generation of reactive oxygen species in a human keratinocyte cell line.
During inflammation and other pathological states, the lipid mediator platelet-activating factor (PAF) and reactive oxygen species (ROS) are both generated. We have been investigating the effect of exogenous PAF on ROS formation in the human keratinocyte cell line (HaCaT). ROS production, measured using luminol-enhanced chemiluminescence (CL), proved to be rapid, transient, PAF receptor-mediated, and totally dependent on an increase in intracellular Ca2+ ([Ca2+]i) and on the presence of extracellular Ca2+. Repeated administration of PAF resulted in refractoriness to the agonist in terms of both capacities to increase [Ca2+]i and generate ROS. The cells, however, continued to respond fully to other stimulants (bradykinin, epidermal growth factor, thapsigargin). The PAF-induced increases in [Ca2+]i (monitored using the fluorescent probe Fluo-3) were also rapid and transient and paralleled those of ROS generation. Relatively specific inhibitors of potential ROS-producing systems were administered in an attempt to characterize the ROS producing system(s). Inhibitors of xanthine oxidase, phospholipase A2, lipoxygenase, cyclooxygenase and NO synthase did not interfere with PAF evoked ROS. The flavoprotein inhibitor diphenyleneiodonium and the mitochondrial cytochrome oxidase inhibitor KCN, prevented generation of ROS, making NAD(P)H a candidate for the electron source of the ROS and the mitochondria a potential major site of formation.
Goldman
R
R
Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel. bmgold@weizmann.weizmann.ac.il
Moshonov
S
S
Zor
U
U
D000814:Aniline Compounds; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D005453:Fluorescence; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism*; D008163:Luminescent Measurements; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D009861:Onium Compounds / Q000494:pharmacology; D010972:Platelet Activating Factor / Q000494:pharmacology*; D010980:Platelet Membrane Glycoproteins / Q000819:agonists / Q000037:antagonists & inhibitors; D011190:Potassium Cyanide / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D013696:Temperature; D014966:Xanthenes
D000814:Aniline Compounds; D008244:Lysophosphatidylcholines; D009861:Onium Compounds; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D017382:Reactive Oxygen Species; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D014966:Xanthenes; C039351:platelet activating factor receptor; C059715:Fluo-3; C007517:diphenyleneiodonium; D011190:Potassium Cyanide; D002118:Calcium