Antisense oligodeoxynucleotides to the inducible no synthase rescue
epithelial cells from oxidative stress injury.
Peresleni, Tatyana, Eisei Noiri, Wadie F. Bahou, Michael S.
Goligorsky.
Departments of Medicine, Physiology and Biophysics, State
University of New York, Stony Brook, NY 11794-8152
APStracts 3:0005F, 1996.
Until recently, the lack of specific inhibitors of various forms of
nitric oxide synthase (NOS) hampered a stringent evaluation of the
role played by iNOS in cell damage. Phosphorothioate derivatives of
iNOS antisense and control sense or scrambled oligodeoxynucleotides
(S-ODNs) were synthesized and their effect on epithelial cell
viability was examined under oxidant stress. Exposure of BSC-1 kidney
tubular epithelial cells to H2O2 resulted in elevation of NO release,
accompanied by a significant decrease in the population of viable
cells (from 97.4+/-1.7% to 72.4+/-2.4.% population). Nitrite
production by BSC-1 cells exposed to H2O2 increased almost ten-fold
compared to control. Pretreatment of the cells with 10 uM antisense
ODNs significantly blunted this response, whereas sense or scrambled
ODNs did not modify it. Pretreatment of BSC-1 cells with 10 uM
antisense ODNs virtually prevented lethal cell damage in response to
H2O2, whereas sense ODNs were ineffective. Lipopolysaccharide
induction of iNOS, also preventable by the antisense construct,
resulted in a lesser compromise to cell viability.
Immunocytochemistry of iNOS in cells pretreated with antisense ODNs
showed minimal cytoplasmic staining, as opposed to the untreated or
sense ODNs-treated positively stained cells. Staining with antibodies
to nitrotyrosine was conspicuous in stressed, but undetectable in
antisense ODNs-treated cells. In conclusion, oxidant stress is
accompanied by the induction of iNOS, increased production of NO, and
impaired cell viability; selective inhibition of iNOS using the
designed antisense ODNs dramatically improved BSC-1 cell viability
after oxidant stress.
Received 18 August 1995; accepted in final form 14 December 1995.
APS Manuscript Number F277-5.
Article publication pending Am. J. Physiol. (Renal Fluid Electrolyte
Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 22 January 96