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Correspondence  |   August 2007
Heme as a Playmaker in the Regulation of the Nitric Oxide System
Author Affiliations & Notes
  • Chun-Jen Huang, M.D., Ph.D.
    *
  • *Mackay Memorial Hospital, Taipei, Taiwan.
Article Information
Correspondence
Correspondence   |   August 2007
Heme as a Playmaker in the Regulation of the Nitric Oxide System
Anesthesiology 8 2007, Vol.107, 356. doi:10.1097/01.anes.0000271925.56781.d3
Anesthesiology 8 2007, Vol.107, 356. doi:10.1097/01.anes.0000271925.56781.d3
In Reply:—
We appreciate the interest of Li Volti et al.  in our recently published article.1 In their letter, Li Volti et al.  pointed out that our conclusions could have been strengthened if there had been data indicating the effect of hemin on nitric oxide. Incidentally, we did perform these experiments, but we decided not to present these data in the article because of page limitations. We appreciate these comments and are glad to present these data in this response letter. Briefly, production of nitric oxide and expression of inducible nitric oxide synthase, i.e.  , the main enzyme for nitric oxide production during sepsis,2 were evaluated by chemiluminescence and immunoblotting assays, as we previously reported.3 The data revealed that hemin significantly attenuated inducible nitric oxide synthase expression and nitric oxide production in lipopolysaccharide-stimulated macrophages (fig. 1). In addition, the effects of hemin on inducible nitric oxide synthase expression could be attenuated by tin protoporphyrin, the potent heme oxygenase-1 inhibitor (fig. 1). Along with our previous reports,4,5 these data provide strong evidence to support the crucial role of heme oxygenase 1 on regulating inducible nitric oxide synthase expression and nitric oxide production during sepsis.
Fig. 1. Representative gel photography illustrated the effects of hemin on inducible nitric oxide synthase (iNOS) expression and nitric oxide production in lipopolysaccharide-stimulated murine macrophages. The effects of tin protoporphyrin (SnPP) on iNOS expression and nitric oxide production in lipopolysaccharide (LPS) plus hemin–stimulated murine macrophages were also illustrated. The iNOS protein concentrations were normalized by β-actin. The nitric oxide concentrations were determined using chemiluminescence assay. Data are expressed as mean ± SD. H(5) and Hemin(5) = 5 μm hemin; H(50) and Hemin(50) = 50 μm hemin; H(500) and Hemin(500) = 500 μm hemin; PBS = phosphate-buffered saline; S = tin protoporphyrin. *  P  < 0.05 compared with the PBS group. #  P  < 0.05 compared with the LPS group. †  P  < 0.05, the LPS + Hemin(500) group  versus  the LPS + Hemin(5) group. ‡  P  < 0.05, the LPS + Hemin(500) group  versus  the LPS + Hemin(50) group. ¶  P  < 0.05, the LPS + Hemin(5) group  versus  the LPS + Hemin(5) + SnPP group or the LPS + Hemin(50) group  versus  the LPS + Hemin(50) + SnPP group. 
Fig. 1. Representative gel photography illustrated the effects of hemin on inducible nitric oxide synthase (iNOS) expression and nitric oxide production in lipopolysaccharide-stimulated murine macrophages. The effects of tin protoporphyrin (SnPP) on iNOS expression and nitric oxide production in lipopolysaccharide (LPS) plus hemin–stimulated murine macrophages were also illustrated. The iNOS protein concentrations were normalized by β-actin. The nitric oxide concentrations were determined using chemiluminescence assay. Data are expressed as mean ± SD. H(5) and Hemin(5) = 5 μm hemin; H(50) and Hemin(50) = 50 μm hemin; H(500) and Hemin(500) = 500 μm hemin; PBS = phosphate-buffered saline; S = tin protoporphyrin. *  P  < 0.05 compared with the PBS group. #  P  < 0.05 compared with the LPS group. †  P  < 0.05, the LPS + Hemin(500) group  versus  the LPS + Hemin(5) group. ‡  P  < 0.05, the LPS + Hemin(500) group  versus  the LPS + Hemin(50) group. ¶  P  < 0.05, the LPS + Hemin(5) group  versus  the LPS + Hemin(5) + SnPP group or the LPS + Hemin(50) group  versus  the LPS + Hemin(50) + SnPP group. 
Fig. 1. Representative gel photography illustrated the effects of hemin on inducible nitric oxide synthase (iNOS) expression and nitric oxide production in lipopolysaccharide-stimulated murine macrophages. The effects of tin protoporphyrin (SnPP) on iNOS expression and nitric oxide production in lipopolysaccharide (LPS) plus hemin–stimulated murine macrophages were also illustrated. The iNOS protein concentrations were normalized by β-actin. The nitric oxide concentrations were determined using chemiluminescence assay. Data are expressed as mean ± SD. H(5) and Hemin(5) = 5 μm hemin; H(50) and Hemin(50) = 50 μm hemin; H(500) and Hemin(500) = 500 μm hemin; PBS = phosphate-buffered saline; S = tin protoporphyrin. *  P  < 0.05 compared with the PBS group. #  P  < 0.05 compared with the LPS group. †  P  < 0.05, the LPS + Hemin(500) group  versus  the LPS + Hemin(5) group. ‡  P  < 0.05, the LPS + Hemin(500) group  versus  the LPS + Hemin(50) group. ¶  P  < 0.05, the LPS + Hemin(5) group  versus  the LPS + Hemin(5) + SnPP group or the LPS + Hemin(50) group  versus  the LPS + Hemin(50) + SnPP group. 
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*Mackay Memorial Hospital, Taipei, Taiwan.
References
Tsai PS, Chen CC, Tsai PS, Yang LC, Huang WY, Huang CJ: Heme oxygenase 1, nuclear factor E2–related factor 2, and nuclear factor κB are involved in hemin inhibition of type 2 cationic amino acid transporter expression and l-arginine transport in stimulated macrophages. Anesthesiology 2006; 105:1201–10Tsai, PS Chen, CC Tsai, PS Yang, LC Huang, WY Huang, CJ
Xie QW, Cho HJ, Calaycay J, Mumford RA, Swiderek KM, Lee TD, Ding A, Troso T, Nathan C: Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science 1992; 256:225–8Xie, QW Cho, HJ Calaycay, J Mumford, RA Swiderek, KM Lee, TD Ding, A Troso, T Nathan, C
Huang CJ, Haque IU, Slovin PN, Nielsen RB, Fang X, Skimming JW: Environmental pH regulates LPS-induced nitric oxide formation in murine macrophages. Nitric Oxide 2002; 6:73–8Huang, CJ Haque, IU Slovin, PN Nielsen, RB Fang, X Skimming, JW
Huang TY, Tsai PS, Wang TY, Huang CL, Huang CJ: Hyperbaric oxygen attenuation of lipopolysaccharide-induced acute lung injury involves heme oxygenase-1. Acta Anaesthesiol Scand 2005; 49:1293–301Huang, TY Tsai, PS Wang, TY Huang, CL Huang, CJ
Chang KY, Tsai PS, Huang TY, Wang TY, Yang S, Huang CJ: HO-1 mediates the effects of HBO pretreatment against sepsis. J Surg Res 2006; 136:143–53Chang, KY Tsai, PS Huang, TY Wang, TY Yang, S Huang, CJ
Fig. 1. Representative gel photography illustrated the effects of hemin on inducible nitric oxide synthase (iNOS) expression and nitric oxide production in lipopolysaccharide-stimulated murine macrophages. The effects of tin protoporphyrin (SnPP) on iNOS expression and nitric oxide production in lipopolysaccharide (LPS) plus hemin–stimulated murine macrophages were also illustrated. The iNOS protein concentrations were normalized by β-actin. The nitric oxide concentrations were determined using chemiluminescence assay. Data are expressed as mean ± SD. H(5) and Hemin(5) = 5 μm hemin; H(50) and Hemin(50) = 50 μm hemin; H(500) and Hemin(500) = 500 μm hemin; PBS = phosphate-buffered saline; S = tin protoporphyrin. *  P  < 0.05 compared with the PBS group. #  P  < 0.05 compared with the LPS group. †  P  < 0.05, the LPS + Hemin(500) group  versus  the LPS + Hemin(5) group. ‡  P  < 0.05, the LPS + Hemin(500) group  versus  the LPS + Hemin(50) group. ¶  P  < 0.05, the LPS + Hemin(5) group  versus  the LPS + Hemin(5) + SnPP group or the LPS + Hemin(50) group  versus  the LPS + Hemin(50) + SnPP group. 
Fig. 1. Representative gel photography illustrated the effects of hemin on inducible nitric oxide synthase (iNOS) expression and nitric oxide production in lipopolysaccharide-stimulated murine macrophages. The effects of tin protoporphyrin (SnPP) on iNOS expression and nitric oxide production in lipopolysaccharide (LPS) plus hemin–stimulated murine macrophages were also illustrated. The iNOS protein concentrations were normalized by β-actin. The nitric oxide concentrations were determined using chemiluminescence assay. Data are expressed as mean ± SD. H(5) and Hemin(5) = 5 μm hemin; H(50) and Hemin(50) = 50 μm hemin; H(500) and Hemin(500) = 500 μm hemin; PBS = phosphate-buffered saline; S = tin protoporphyrin. *  P  < 0.05 compared with the PBS group. #  P  < 0.05 compared with the LPS group. †  P  < 0.05, the LPS + Hemin(500) group  versus  the LPS + Hemin(5) group. ‡  P  < 0.05, the LPS + Hemin(500) group  versus  the LPS + Hemin(50) group. ¶  P  < 0.05, the LPS + Hemin(5) group  versus  the LPS + Hemin(5) + SnPP group or the LPS + Hemin(50) group  versus  the LPS + Hemin(50) + SnPP group. 
Fig. 1. Representative gel photography illustrated the effects of hemin on inducible nitric oxide synthase (iNOS) expression and nitric oxide production in lipopolysaccharide-stimulated murine macrophages. The effects of tin protoporphyrin (SnPP) on iNOS expression and nitric oxide production in lipopolysaccharide (LPS) plus hemin–stimulated murine macrophages were also illustrated. The iNOS protein concentrations were normalized by β-actin. The nitric oxide concentrations were determined using chemiluminescence assay. Data are expressed as mean ± SD. H(5) and Hemin(5) = 5 μm hemin; H(50) and Hemin(50) = 50 μm hemin; H(500) and Hemin(500) = 500 μm hemin; PBS = phosphate-buffered saline; S = tin protoporphyrin. *  P  < 0.05 compared with the PBS group. #  P  < 0.05 compared with the LPS group. †  P  < 0.05, the LPS + Hemin(500) group  versus  the LPS + Hemin(5) group. ‡  P  < 0.05, the LPS + Hemin(500) group  versus  the LPS + Hemin(50) group. ¶  P  < 0.05, the LPS + Hemin(5) group  versus  the LPS + Hemin(5) + SnPP group or the LPS + Hemin(50) group  versus  the LPS + Hemin(50) + SnPP group. 
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