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. IntroductionMast cells (MCs) are tissue-resident effector cells that arise from bone marrow precursors. 1 They produce and secrete numerous bioactive agents and have been implicated in diverse homeostatic functions such as angiogenesis, wound healing, and tissue remodeling. 2 Due to their plethora of mediators and strategic localization, MCs also play central roles in various disease states, including multiple sclerosis and T-helper type 2 (T H 2)-driven inflammatory conditions such as asthma. 3,4 Nitric oxide (NO) is a potent radical with diverse roles in regulating cellular activation. 5 NO is derived from L-arginine by the nitric oxide synthase (NOS) family of enzymes. The Ca 2ϩ -dependent members include endothelial (eNOS) and neuronal (nNOS), characterized by constitutive expression and low NO production. Inducible NOS (iNOS) is up-regulated by a variety of inflammatory mediators and functions independently of cellular Ca 2ϩ levels and releases large amounts of NO. 6 Numerous investigators have shown that rodent MCs are regulated by endogenous NO from both constitutive and inducible sources. 7,8 However, little is known about the production of NO by human MCs or the potential involvement of NO as modulator of human MC function.The aim of this study was to investigate the expression of NOS and production of NO in human MCs. Furthermore, we studied the regulation of NOS and the resulting functional effects on the release of leukotrienes. Our results indicate that NO may be a novel modulator that determines the functional phenotype of human MCs. Materials and methods ReagentsThe NOS inhibitor N G -nitro-L-arginine methyl ester (L-NAME), inactivate enantiomer N G -nitro-D-arginine methyl ester (D-NAME), and NO donor S-n...

Mast cells (MC)

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Percutaneous coronary intervention has resulted in a paradigm shift in the treatment of coronary artery disease and myocardial infarction. However, neither bare-metal stents nor polymer-coated drug-eluting stents represent ideal therapies at this time due to the undesired in-stent stenosis or delayed thrombosis. Hence there is pressing clinical need for greater understanding of the cellular mechanisms involved. It is hoped that this in turn will provide insight into designing and developing the next generation of stents. Although immunohistochemistry and immunofluorescence are appropriate tools in understanding the molecular histology, performing these techniques on stented blood vessels is technically challenging because of poor permeability of antibodies into the stented blood vessels which are embedded in methacrylate-based resins and inadequate image resolution due to autofluorescence. Hence there is a need to develop techniques which can facilitate immunohistochemistry/immunofluorescence procedures on stented blood vessel cross-sections. In this study we describe an improved protocol for processing stented porcine coronary arteries for immunostaining with smooth muscle cell, endothelial cell, monocyte and macrophage markers. We first identified the optimal conditions for resin embedding of stented artery and cross sectioned the vessels using high speed precision wafering diamond blade. The sections were then ground using two levels of water sandpaper on a Metaserve 2000 grinder to achieve the desired thickness. For immunostaining, we developed a novel deplasticization protocol which favors optimal antibody permeabilization. Our protocol not only provides feasibility of improved immunostaining of stented artery sections but also results in high quality images.