Constitutive Nitric Oxide Synthase Is Present in Normal Human Keratinocytes

Abstract

Normal human keratinocytes in culture exhibit a nitric oxide synthase (NOS) activity ranging from 50 to 150 pmol/min/mg of protein. The enzyme is cytosolic and requires the presence of calcium, nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), and flavin adenine dinucleotide. Calmodulin antagonists (trifluoperazine and calmidazolium) inhibit the enzyme activity. We show that NG-nitro-L-arginine inhibits NOS more potently than NG-monomethyl-L-arginine and that L-canavanine is a weak inhibitor. NOS was partially purified using a 2',5'-ADP Sepharose affinity column eluted with NADPH. A partially purified fraction was analyzed by sodium dodecyl sulfate-polyacrylamide gel electro-phoresis and Western blotting. A protein with an apparent molecular weight of 152 kDa cross-reacted with monoclonal antibodies raised against the neuronal constitutive isoform of NOS. The enzyme had a Vmax of 7.3 nmol/min/mg of protein and a Km for L-arginine of 22.3 microM. These results indicate that normal human keratinocytes contain a constitutive nitric oxide synthase related to NOS I.

Protective effects of an extract from Citrus bergamia against inflammatory injury in interferon-gamma and histamine exposed human keratinocytes

“…NO produced by the constitutive isoform of nitric oxide synthase (NOS) is a key regulator of homeostasis, whereas NO produced by inducible NOS plays an important role in inflammation and tissue repair. Skin keratinocytes express all three NOS isoforms (Baudouin and Tachon, 1996;RomeroGraillet et al, 1996;Shimizu et al, 1997): constitutive (cNOS), endothelial (eNOS) and inducible (iNOS). In response to inflammatory cytokines due to cutaneous injury, NO formation is increased during inflammation (Kolb and Kolb-Bachofen, 1992), and several classic inflammatory symptoms are reversed by NOS inhibitors (Frank et al, 2000;Stallmeyer et al, 1999).…”

Nitric Oxide Stimulates a Large-Conductance Ca²⁺-Activated K⁺ Channel in Human Skin Fibroblasts through Protein Kinase G Pathway

“…Not only is NO normally involved in maintaining skin homeostasis, but it has also been demonstrated to directly modify several skin reactions [7] . NO synthase (NOS), which catalyses NO synthesis, manifests itself in three different isoforms (neuronal NOS, endothelial NOS, and inducible NOS), all of which are expressed in skin tissues [8][9][10] . In particular, human skin fi broblasts constitutively possess the capacity to produce NO, which is either linked to Ca 2+ -dependent endothelial NOS or to the inducible NOS isoform [9] .…”

Ni2+, a Double-Acting Inhibitor of Neuronal Nitric Oxide Synthase Interfering with -Arginine Binding and Ca2+/Calmodulin-Dependent Enzyme Activation

“…Mechanisms controlling NOS activity are of particular interest because of the involvement of NO in a broad variety of physiological processes, including vasodilation, neurotransmission and nonspecific immunity (22). In the skin, both constitutive and inducible isoforms of NOS have been identified in a variety of cells, including macrophages, Langerhans cells, dermal fibroblasts, endothelial cells, melanocytes and keratinocytes (23)(24)(25)(26)(27), and can be implicated in the control of tissue homeostasis as well as in the modulation of inflammatory processes. Since Ni 2ϩ -induced allergic contact dermatitis is considered to be an inflammatory response caused by antigen-specific T cells, it can be speculated that inhibition of cutaneous NOS by the metal plays at least a contributory role in amplifying inflammatory tissue damage by suppression of NO-dependent defense mechanisms.…”