Non-heme iron protein: A potential target of nitric oxide in acute cardiac allograft rejection

doi:10.1073/pnas.0636938100

Non-heme iron protein: A potential target of nitric oxide in acute cardiac allograft rejection

^*Department of Surgery, Division of Transplant Surgery, ^†Cardiovascular Research Center, ^‡Free Radical Research Center, ^¶Biophysics Research Institute, and ^‖Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226

Edited by Louis J. Ignarro, University of California School of Medicine, Los Angeles, CA, and approved January 16, 2003 (received for review November 18, 2002)

Abstract

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 ≈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.

graft rejection‖aconitase‖dinitrosyl–iron complexes

Footnotes

↵ § To whom correspondence should be addressed at: Division of Transplant Surgery, Medical College of Wisconsin, 9200 West Wisconsin Avenue, Milwaukee, WI 53226. E-mail: gmpieper{at}mcw.edu.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:

iNOS,

inducible NO synthase;

CsA,

cyclosporine;

L-NIL,

l-N6-(1-iminoethyl)lysine;

POD,

postoperative day