PDF herunterladen
Thromb Haemost 1997; 77(01): 163-167
DOI: 10.1055/s-0038-1655925
Platelets
Schattauer GmbH Stuttgart

Identification of the NO Synthase isoforms Expressed in Human Neutrophil Granulocytes, Megakaryocytes and Platelets

Thomas Wallerath
1   The Department of Pharmacology, Vascular Biology Center, Medical College of Georgia, Augusta, GA, USA
,
Ingolf Gath
1   The Department of Pharmacology, Vascular Biology Center, Medical College of Georgia, Augusta, GA, USA
,
Walter E Aulitzky
2   The Department of Hematology, Johannes Gutenberg University, Mainz, Germany
,
Jennifer S Pollock
3   The Department of Pharmacology, Vascular Biology Center, Medical College of Georgia, Augusta, GA, USA
,
Hartmut Kleinert
1   The Department of Pharmacology, Vascular Biology Center, Medical College of Georgia, Augusta, GA, USA
,
Ulrich Förstermann
1   The Department of Pharmacology, Vascular Biology Center, Medical College of Georgia, Augusta, GA, USA
› Institutsangaben
Weitere Informationen

Publikationsverlauf

Received 26. Juni 1996

Accepted after revision 09. September 1996

Publikationsdatum:
11. Juli 2018 (online)

Auch verfügbar auf
  als PDF herunterladen  Lizenzen und Reprints

Summary

Using Western blot and fluorescent immunocytochemistry, NOS III (or ecNOS) and NOS II (or iNOS), but no NOS I (or ncNOS), were identified in preparations of human platelets. Reverse-transcription polymerase chain reactions (RT-PCR) demonstrated NOS III mRNA, but no NOS II mRNA (which is short-lived) and no NOS I mRNA in platelets. Immunofluorescent staining of human bone marrow smears showed the presence of NOS III, but not NOS I in megakaryocytes. A subpopulation of megakaryocytes also expressed NOS II. In preparations of human neutrophils, immunocytochemistry demonstrated NOS I in all cells, whereas no NOS III was detected. The few NOS II positive cells were characterized as contaminating eosinophils. Similarly, in RT-PCR, transcripts for NOS I and NOS II, but not for NOS III, were identified. Thus, the constitutive NOS isoform in megakaryocytes and platelets is NOS III, whereas neutrophils express NOS I. Some megakaryocytes and eosinophils also express NOS II.

 

  • References

  • 1 Radomski MW, Palmer RM, Moncada S. An L-arginine/nitric oxide pathway present in human platelets regulates aggregation. Proc Natl Acad Sci USA 1990; 87: 5193-5197
    CrossrefPubMedGoogle Scholar
  • 2 Schmidt HHHW, Seifert R, Bohme E. Formation and release of nitric oxide from human neutrophils and HL-60 cells induced by a chemotactic peptide platelet activating factor and leukotriene B4. FEBS Lett 1989; 244: 357-360
    CrossrefPubMedGoogle Scholar
  • 3 De Beider A, Radomski M, Hancock V, Brown A, Moncada S, Martin J. Megakaryocytes from patients with coronary atherosclerosis express the inducible nitric oxide synthase. Arterioscler Thromb Vase Biol 1995; 15: 637-641
    CrossrefPubMedGoogle Scholar
  • 4 Bandaletova T, Brouet I, Bartsch H, Sugimura T, Esumi H, Ohshima H. Immunohistochemical localization of an inducible form of nitric oxide synthase in various organs of rats treated with propionibacterium-acnes and lipopolysaccharide. APMIS 1993; 101: 330-336
    CrossrefPubMedGoogle Scholar
  • 5 Lelchuk R, Radomski MW, Martin JF, Moncada S. Constitutive and inducible nitric oxide synthases in human megakaryoblastic cells. J Pharmacol Exp Ther 1992; 262: 1220-1224
    PubMedGoogle Scholar
  • 6 Sato K, Miyakawa K, Takeya M, Hattori R, Yui Y, Sunamoto M, Ichimori Y, Ushio Y, Takahashi K. Immunohistochemical expression of inducible nitric oxide synthase (iNOS) in reversible endotoxic shock studied by a novel monoclonal antibody against rat iNOS. J Leukocyte Biol 1995; 57: 36-44
    CrossrefPubMedGoogle Scholar
  • 7 Alheid U, Frӧlich JC, Fӧrstermann U. Endothelium-derived relaxing factor from cultured human endothelial cells inhibits aggregation of human platelets. Thromb Res 1987; 47: 561-571
    CrossrefPubMedGoogle Scholar
  • 8 Radomski MW, Palmer RM, Moncada S. The role of nitric oxide and cGMP in platelet adhesion to vascular endothelium. Biochem Biophys Res Commun 1987; 148: 1482-1489
    CrossrefPubMedGoogle Scholar
  • 9 Kubes P, Suzuki M, Granger DN. Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc Natl Acad Sci USA 1991; 88: 4651-4655
    CrossrefPubMedGoogle Scholar
  • 10 Salvemini D, de Nucci G, Gryglewski RJ, Vane JR. Human neutrophils and mononuclear cells inhibit platelet aggregation by releasing a nitric oxidelike factor. Proc Natl Acad Sci USA 1989; 86: 6328-6332
    CrossrefPubMedGoogle Scholar
  • 11 Spitzer JA, Zhang P, Mayer AMS. Functional characterization of peripheral circulating and liver recruited neutrophils in endotoxic rats. J Leukocyte Biol 1994; 56: 166-173
    CrossrefPubMedGoogle Scholar
  • 12 Smith JA. Neutrophils, host defense and inflammation: a double-edged sword. J Leukocyte Biol 1994; 56: 672-686
    CrossrefPubMedGoogle Scholar
  • 13 Yan L, Vandivier RW, Suffredini AF, Danner RL. Human polymorphonuclear leukocytes lack detectable nitric oxide synthase activity. J Immunol 1994; 153: 1825-1834
    PubMedGoogle Scholar
  • 14 Förstermann U, Gath I, Schwarz P, Closs El, Kleinert H. Isoforms of nitric oxide synthase – properties, cellular distribution and expressional control. Biochem Pharmacol 1995; 50: 1321-1332
    CrossrefPubMedGoogle Scholar
  • 15 Muruganandam A, Mutus B. Isolation of nitric oxide synthase from human platelets. Biochem Biophys Acta 1994; 1200: 1-6
    CrossrefPubMedGoogle Scholar
  • 16 Yui Y, Hattori R, Kosuga K, Eizawa H, Hiki K, Kawai C. Purification of nitric oxide synthase from rat macrophages. J Biol Chem 1991; 266: 12544-12547
    PubMedGoogle Scholar
  • 17 Förstermann U, Gath I. Purification of isoforms of nitric oxide synthase. In: Methods in Enzymology – Nitric Oxide. Vol Abelson JN, Melvin IS. eds 268. Academic Press Orlando: FL; 1996. pp 334-339
    Google Scholar
  • 18 Cutts HJ. Cell separation Methods in Hematology. Orlando FL: Academic Press; 1970
    Google Scholar
  • 19 Pertoft H, Hirtenstein M, Kadegal L. Cell separation in a new density medium Percoll. Biochemistry 1979; 09: 67-80
    PubMedGoogle Scholar
  • 20 Haurand M, Flohe L. Leukotriene formation by human polymorphonuclear leukocytes from endogenous arachidonate. Biochem Pharmacol 1989; 38: 2129-2137
    CrossrefPubMedGoogle Scholar
  • 21 Schmidt HHHW, Gagne GD, Nakane M, Pollock JS, Miller MF, Murad F. Mapping of neural nitric oxide synthase in the rat suggests frequent co-localization with NADPH diaphorase but not with soluble guanylyl cyclase and novel paraneural functions for nitrinergic signal transduction. J Histo-chem Cytochem 1992; 40: 1439-1456
    CrossrefPubMedGoogle Scholar
  • 22 Pollock JS, Nakane M, Buttery LK, Martinez A, Springall D, Polak JM, Förstermann U, Murad F. Characterization and localization of endothelial nitric oxide synthase using specific monoclonal antibodies. Am J Physiol 1993; 265C: 1379-1387
    PubMedGoogle Scholar
  • 23 Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162: 156-159
    PubMedGoogle Scholar
  • 24 Nakane M, Schmidt HHHW, Pollock JS, Forstermann U, Murad F. Cloned human brain nitric oxide synthase is highly expressed in skeletal muscle. FEBS Lett 1993; 316: 175-180
    CrossrefPubMedGoogle Scholar
  • 25 Geller DA, Lowenstein CJ, Shapiro RA, Nussler AK, Di SM, Wang SC, Nakayama DK, Simmons RL, Snyder SH, Billiar TR. Molecular cloning and expression of inducible nitric oxide synthase from human hepatocytes. Proc Natl Acad Sci USA 1993; 90: 3491-3495
    CrossrefPubMedGoogle Scholar
  • 26 Marsden PA, Schappert KT, Chen HS, Flowers M, Sundell CL, Wilcox JN, Lamas S, Michel T. Molecular cloning and characterization of human endothelial nitric oxide synthase. FEBS Lett 1992; 307: 287-293
    CrossrefPubMedGoogle Scholar
  • 27 Ponte P, Ng SY, Engel J, Gunning P, Kedes L. Evolutionary conservation in the untranslated regions of actin mRNAs: DNA sequence of a human beta-actin cDNA. Nucleic Acids Res 1984; 12: 1687-1696
    CrossrefPubMedGoogle Scholar
  • 28 Förstermann U, Closs El, Pollock JS, Nakane M, Schwarz P, Gath I, Kleinert H. Nitric oxide synthase isozymes: characterization, molecular cloning and functions. Hypertension 1994; 23: 1121-1131
    CrossrefPubMedGoogle Scholar
  • 29 Chen LY, Mehta P, Mehta JL. Oxidized LDL decreases L-arginine uptake and nitric oxide synthase protein expression In human platelets: relevance of the effect of oxidized LDL on platelet function. Circulation 1996; 93: 1740-1746
    CrossrefPubMedGoogle Scholar
  • 30 Chen LY, Metha JL. Further evidence of the presence of constitutive and inducible nitric oxide synthase isoforms in human platelets. J Cardiovasc Pharmacol 1996; 27: 154-158
    CrossrefPubMedGoogle Scholar
  • 31 Pollock JS, Förstermann U, Mitchell JA, Warner TD, Schmidt HHHW, Nakane M, Murad F. Purification and characterization of particulate endothelium-derived relaxing factor synthase from cultured and native bovine aortic endothelial cells. Proc Natl Acad Sci USA 1991; 88: 10480-10484
    CrossrefPubMedGoogle Scholar
  • 32 Tracey WR, Pollock JS, Murad F, Nakane M, Förstermann U. Identification of a type III (endothelial-like) particulate nitric oxide synthase in LLC-PK1 kidney tubular epithelial cells. Am J Physiol 1994; 266C: 22-26
    PubMedGoogle Scholar
  • 33 Förstermann U, Pollock JS, Schmidt HHHW, Heller M, Murad F. Calmodulin-dependent endothelium-derived relaxing factor/nitric oxide synthase activity is present in the particulate and cytosolic fractions of bovine aortic endothelial cells. Proc Natl Acad Sci USA 1991; 88: 1788-1792
    CrossrefPubMedGoogle Scholar
  • 34 Gilbert RS, Herschman HR. “Macrophage” nitric oxide synthase is a glucocorticoid-inhibitable primary response gene in 3T3 cells. J Cell Physiol 1993; 157: 128-132
    CrossrefPubMedGoogle Scholar
  • 35 Kleinert H, Euchenhofer C, Ihrig-Biedert I, Fӧrstermann U. In murine 3T3 fibroblasts, different second messenger pathways resulting in the induction of NO synthase II (iNOS) converge in the activation of transcription factorNF-kB . J Biol Chem 1996; 271: 6039-6244
    CrossrefPubMedGoogle Scholar
  • 36 Chu SC, Wu HP, Banks TC, Eissa NT, Moss J. Structural diversity in the 5'-untranslated region of cytokine-stimulated human inducible nitric oxide synthase mRNA. J Biol Chem 1995; 270: 10625-10630
    CrossrefPubMedGoogle Scholar
  • 37 Sase K, Michel T. Expression of constitutive endothelial nitric oxide synthase in human blood platelets. Life Sci 1995; 57: 2049-2055
    CrossrefPubMedGoogle Scholar
  • 38 Mehta JL, Chen LY, Kone BC, Mehta P, Turner P. Identification of constitutive and inducible forms of nitric oxide synthase in human platelets. J Lab Clin Med 1995; 125: 370-377
    PubMedGoogle Scholar
  • 39 Geng Y, Lotz M. Increased intracellular Ca2+selectively suppresses IL-1-induced NO production by reducing iNOS mRNA stability. J Cell Biol 1995; 129: 1651-1657
    CrossrefPubMedGoogle Scholar
  • 40 Yoshizumi M, Perrella MA, Burnett JC, Lee ME. Tumor necrosis factor downregulates an endothelial nitric oxide synthase messenger RNA by shortening its half-life. Circ Res 1993; 73: 205-209
    CrossrefPubMedGoogle Scholar
  • 41 Ohshima H, Bandaletova TY, Brouet I, Bartsch H, Kirby G, Ogunbiyi F, Vatanasapt V, Pipitgool V. Increased nitrosamine and nitrate biosynthesis mediated by nitric oxide synthase induced in hamsters infected with liver fluke (Opisthorchis viverrini). Carcinogenesis 1994; 15: 271-275
    CrossrefPubMedGoogle Scholar
  • 42 Bryant JJ, Mehta P, von derPorten A, Mehta JL. Co-purification of 130 kD nitric oxide synthase and a 22 kD link protein from human neutrophils. Biochem Biophys Res Commun 1992; 189: 558-564
    CrossrefPubMedGoogle Scholar
  • 43 Bredt DS, Snyder SH. Isolation of nitric oxide synthetase a calmodulin-requiring enzyme. Proc Natl Acad Sci USA 1990; 87: 682-685
    CrossrefPubMedGoogle Scholar
  • 44 Schmidt HHHW, Pollock JS, Nakane M, Gorsky LD, Förstermann U, Murad F. Purification of a soluble isoform of guanylyl cyclase-activating-factor synthase. Proc Natl Acad Sci USA 1991; 88: 365-369
    CrossrefPubMedGoogle Scholar
  • 45 Kleinert H, Euchenhofer C, Ihrig-Biedert I, Förstermann U. Glucocorticoids inhibit the induction of nitric oxide synthase II by downregulating cytokine-induced activity of transcription factorNF-kB . Mol Pharmacol 1996; 49: 15-21
    PubMedGoogle Scholar
  • 46 Edgell CJS, McDonald CC, Graham JB. Permanent cell line expressing factor VIII antigen established by hybridization. Proc Natl Acad Sci USA 1983; 80: 3734-3737
    CrossrefPubMedGoogle Scholar