Non-heme iron protein: a potential target of nitric oxide in acute cardiac allograft rejection.
We examined iron nitrosylation of non-heme protein and enzymatic activity of the Fe-S cluster protein, aconitase, in acute cardiac allograft rejection. Heterotopic transplantation of donor hearts was performed in histocompatibility matched (isografts: Lewis --> Lewis) and mismatched (allografts: Wistar-Furth --> Lewis) rats. On postoperative days (POD) 4-6, Western blot analysis and immunohistochemistry revealed inducible nitric-oxide synthase (iNOS) protein in allografts but not isografts. EPR spectroscopy revealed background signals at g = 2.003 (for semiquinone) and g = 2.02 and g = 1.94 (for Fe-S cluster protein) in isografts and normal hearts. In contrast, in allografts on POD4, a new axial signal at g = 2.04 and g = 2.02 appeared that was attributed to the dinitrosyl-iron complex formed by nitrosylation of non-heme protein. Appearance of this signal occurred at or before significant nitrosylation of heme protein. Iron nitrosylation of non-heme protein was coincidental with decreases in the nonnitrosylated Fe-S cluster signal at g = 1.94. Aconitase enzyme activity was decreased to approximately 50% of that observed in isograft controls by POD4. Treatment with cyclosporine blocked the (i) elevation of plasma nitrate + nitrite, (ii) up-regulation of iNOS protein, (iii) decrease in Fe-S cluster EPR signal, (iv) formation of dinitrosyl-iron complexes, and (v) loss of aconitase enzyme activity. Formation of dinitrosyl-iron complexes and loss of aconitase activity within allografts also was inhibited by treatment of recipients with a selective iNOS inhibitor, l-N(6)-(1-iminoethyl)lysine. This report shows targeting of an important non-heme Fe-S cluster protein in acute solid organ transplant rejection.
Pieper
Galen M
GM
Department of Surgery, Division of Transplant Surgery, Medical College of Wisconsin, 9200 West Wisconsin Avenue, Milwaukee, WI 53226, USA. gmpieper@mcw.edu
Halligan
Nadine L N
NL
Hilton
Gail
G
Konorev
Eugene A
EA
Felix
Christopher C
CC
Roza
Allan M
AM
Adams
Mark B
MB
Griffith
Owen W
OW
D000154:Aconitate Hydratase / Q000378:metabolism; D000818:Animals; D016572:Cyclosporine / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006084:Graft Rejection / Q000188:drug therapy / Q000209:etiology / Q000378:metabolism*; D016027:Heart Transplantation / Q000009:adverse effects / Q000502:physiology*; D006420:Hemeproteins / Q000737:chemistry / Q000378:metabolism; D007166:Immunosuppressive Agents / Q000494:pharmacology; D008239:Lysine / Q000031:analogs & derivatives* / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D019159:Nonheme Iron Proteins / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D011917:Rats, Inbred Lew; D011920:Rats, Inbred WF; D017207:Rats, Sprague-Dawley; D014184:Transplantation, Homologous; D014185:Transplantation, Isogeneic
D004791:Enzyme Inhibitors; D006420:Hemeproteins; D007166:Immunosuppressive Agents; C090663:N(6)-(1-iminoethyl)lysine; D019159:Nonheme Iron Proteins; D009569:Nitric Oxide; D016572:Cyclosporine; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D000154:Aconitate Hydratase; D008239:Lysine