Expression, localization, and regulation of NOS in human mast cell lines: effects on leukotriene production.
Nitric oxide (NO) is a potent radical produced by nitric oxide synthase (NOS) and has pleiotrophic activities in health and disease. As mast cells (MCs) play a central role in both homeostasis and pathology, we investigated NOS expression and NO production in human MC populations. Endothelial NOS (eNOS) was ubiquitously expressed in both human MC lines and skin-derived MCs, while neuronal NOS (nNOS) was variably expressed in the MC populations studied. The inducible (iNOS) isoform was not detected in human MCs. Both growth factor-independent (HMC-1) and -dependent (LAD 2) MC lines showed predominant nuclear eNOS protein localization, with weaker cytoplasmic expression. nNOS showed exclusive cytoplasmic localization in HMC-1. Activation with Ca(2+) ionophore (A23187) or IgE-anti-IgE induced eNOS phosphorylation and translocation to the nucleus and nuclear and cytoplasmic NO formation. eNOS colocalizes with the leukotriene (LT)-initiating enzyme 5-lipoxygenase (5-LO) in the MC nucleus. The NO donor, S-nitrosoglutathione (SNOG), inhibited, whereas the NOS inhibitor, N(G)-nitro-l-arginine methyl ester (L-NAME), potentiated LT release in a dose-dependent manner. Thus, human MC lines produce NO in both cytoplasmic and nuclear compartments, and endogenously produced NO can regulate LT production by MCs.
Gilchrist
Mark
M
Institute for Systems Biology, Seattle, WA 98103, USA. mgilchrist@systemsbiology.org
McCauley
Scott D
SD
Befus
A Dean
AD
D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology; D003593:Cytoplasm / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D015289:Leukotrienes / Q000096:biosynthesis*; D008407:Mast Cells / Q000166:cytology / Q000201:enzymology* / Q000276:immunology*; D018613:Microscopy, Confocal; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D052250:Nitric Oxide Synthase Type III; D010641:Phenotype; D012333:RNA, Messenger / Q000032:analysis; D012867:Skin / Q000166:cytology
D004791:Enzyme Inhibitors; D015289:Leukotrienes; D012333:RNA, Messenger; D009569:Nitric Oxide; D001094:Arachidonate 5-Lipoxygenase; C496320:NOS1 protein, human; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052250:Nitric Oxide Synthase Type III; D019331:NG-Nitroarginine Methyl Ester