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(Received for publication, February 21, 1996, and in revised form, September 4, 1996)
From the Molecular and Cellular Biophysics Laboratories, Department
of Medicine, Division of Cardiology, and the Electron Paramagnetic
Resonance Center, The Johns Hopkins Medical Institutions, Johns
Hopkins Bayview Medical Center, Baltimore, Maryland 21224
Altered nitric oxide (NO
Volume 271, Number 46,
Issue of November 15, 1996
pp. 29223-29230
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
EVIDENCE FOR PEROXYNITRITE-MEDIATED REPERFUSION INJURY
) production is a
critical factor in tissue reperfusion injury; however, controversy
remains regarding these alterations and how they cause injury. Since
superoxide (O
2) generation is triggered during the early
period of reperfusion the cytotoxic oxidant peroxynitrite
(ONOO
) could be formed, but it is not known if this
occurs. Therefore electron paramagnetic resonance and chemiluminescence
studies were performed of the magnitude and time course of NO,
O2, and ONOO formation in the postischemic
heart. Isolated rat hearts were subjected either to normal perfusion or
to reperfusion after 30 min of ischemia in the presence of the
NO trap
Fe2+-N-methyl-D-glucamine
dithiocarbamate with electron paramagnetic resonance measurements
performed on the effluent. Although only trace signals were present
prior to ischemia, prominent NO adduct signals were seen during
the first 2 min of reflow which were abolished by nitric oxide synthase
(NOS) inhibition. Similar studies with the O2 trap
5,5-dimethyl-1-pyrroline N-oxide demonstrated a burst of
O2 generation over the first 2 min of reflow.
Chemiluminescence measurements using
5-amino-2,3-dihydro-1,4-phthalazinedione (luminol) demonstrated a
similar marked increase in ONOO which was blocked by NOS
inhibitors or superoxide dismutase. NOS inhibition or superoxide
dismutase greatly enhanced the recovery of contractile function in
postischemic hearts. Immunohistology demonstrated that the
ONOO-mediated nitration product nitrotyrosine was formed
in postischemic hearts but not in normally perfused controls. Thus,
NO formation is increased during the early period of reflow and
reacts with O2 to form ONOO, which results in
amino acid nitration and cellular injury.
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