The Antibacterial Peptide PR39 Alleviates Liver Tissue
Hypoxia During LPS-Induced Septic Shock
1Madhani, M., 2Barchowsky, A.,,2Klei, L., 3Ross, C., 4Jackson, S.K.,
5Swartz, H.M. and 1James, P.E.
1Department of Cardiology, Wales Research Heart Institute, University of
Wales College of Medicine,Wales, U.K
2Department of Pharmacology and Toxicology, Dartmouth Medical School,
Hanover, NH, USA
3Department of Anatomy and Physiology, College of Veterinary Medicine,
Kansas State, USA
4Department of Medical Microbiology, University of Wales College of
Medicine, Wales,
5EPR Center, Department of Radiology, Dartmouth Medical School, Hanover, NH,
USA
INTRODUCTION: We investigated the effects of lipopolysaccharide
(LPS)-induced NO on
liver pO2 and tissue damage in mice. We previously had found NO plays a
major role in both
circulatory and local effects of LPS on liver oxygenation. An NADPH oxidase
inhibitor, PR39,
was utilized to inhibit superoxide production in vivo. We hypothesized this
would prevent local
peroxynitrite formation and alleviate oxidative damage to the liver. In vivo
EPR was used to make
direct measurements of liver NO and pO2.
METHODS: Mice were challenged with LPS, PR39, PR39 plus LPS, or
saline. In order to
distinguish between effects of LPS on blood supply (and hence oxygen supply)
and oxygen
utilization, pO2 measurements were made at two different anatomical
locations within the hepatic
tissue; either as an average pO2 across the tissue bed (measured from
implantation of particles of
oxygen-sensitive gloxy directly into liver tissue), or selectively from the
liver sinusoids (by
injecting a slurry of fine particles of gloxy i.v. so as to be taken up by
Kupffer cells lining the
liver sinusoids). RT-PCR was used to assess levels of iNOS in liver tissue
samples. To assess NO
in blood, frozen EPR analysis of nitrosylated hemoglobin in red blood cell
samples was used. We
also assessed the effect of PR39 on activation of Nf-kB, a marker protein
for oxidative stress that
mediates the cascade of cytokines and induction of iNOS, as a result of LPS
stimulation.
RESULTS: We found PR39 (1) increased levels of NO locally in liver
(compared to LPS alone)
(2) had no effect on circulatory NO, (3) had little effect on Nf-kB activity
and (4) reduced iNOS
expression in the liver. LPS-induced hypoxia was alleviated by PR39. Our
selective
measurements of pO2 showed that sinusoidal perfusion was improved by PR39,
and parenchymal
pO2 was increased to a level that could not be accounted for by increased
perfusion alone. We
also observed increased local NO in the hepatic parenchyma. PR39 is known to
inhibit superoxide
production, and this may also have reduced oxygen consumption by
NADPH-oxidase.
CONCLUSIONS: These studies demonstrate the relative contribution of
NO-induced defects in
oxygen supply versus oxygen utilization in liver, and also provide direct in
vivo evidence for the
indirect role of superoxide during sepsis. PR39 preserves sinusoidal
perfusion and alleviates
parenchymal hypoxia, and our results indicate this maybe the result of
decreased local superoxide
and increased local NO.