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Substance P Induce Nitric Oxide Production in Human Microvascular Endothelial Cells
Author(s) -
Muangman P,
Muffley L.A.,
Gibran N.S.
Publication year - 2008
Publication title -
wound repair and regeneration
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.847
H-Index - 109
eISSN - 1524-475X
pISSN - 1067-1927
DOI - 10.1111/j.1067-1927.2005.130216z.x
Subject(s) - enos , nitric oxide , nitrite , chemistry , nitric oxide synthase , proinflammatory cytokine , endothelial nos , cytokine , endothelial stem cell , endothelium , downregulation and upregulation , arginase , microbiology and biotechnology , biochemistry , pharmacology , arginine , nitrate , medicine , in vitro , biology , inflammation , amino acid , organic chemistry , gene
Substance P(SP) modulates cytokine synthesis and adhesion molecule expression by inflammatory and endothelial cells in response to injury. A major proinflammatory effect includes nitric oxide(NO) production. Three nitric oxide synthase(NOS) isoforms include neuronal(nNOS), inducible(iNOS), and endothelial(eNOS). In this study, we determined which NOS enzymes mediate SP‐induced NO upregulation in endothelial cells. Confluent human microvascular endothelial cells (HMECs) were treated with SP(10 −8 M) for 48 h with and without the nonspecific NOS inhibitor N‐nitro l‐arginine methyl ester (L‐NAME; 4 uM) or specific inhibitors against nNOS (L‐thiocitrulline;60 nM), iNOS (n‐3‐aminomethyl benzyl acetamidine;7 nM), eNOS (N‐5‐1‐iminoethyl‐L‐ornithine; 0.5 uM). Inhibitors were added for 3 h prior to and continued throughout SP stimulation. Nitrate/nitrite levels were measured in cell extracts using Greiss reagents and standardized with protein concentration for each sample. Results are expressed as uM nitrate/mg protein. Statistical analysis was determined using ANOVA. Result are expressed as mean ± sd; p < 0.05 was considered statistically significant. SP effectively elevated nitrate levels (2.96 uM/mg protein;p ≤ 0.05) compared to control treatment (2.1 uM/mg protein). All NOS inhibitors showed some inhibitory effect on substance P induced NO production. However, only L‐NAME (1.9 uM/mg protein) and the specific eNOS inhibitor (2.32 uM/mg protein) demonstrated a significant decrease (p < 0.05.) Our data suggest that SP up‐regulation of NO in HMECs is primarily mediated by eNOS, but that other NOS enzymes may also contribute. Further studies are warranted to better understand the role of NOS in SP signaling.