Antisense oligodeoxynucleotides to human inducible nitric oxide synthase selectively inhibit induced nitric oxide production by human vascular endothelial cells: an experimental study.
OBJECTIVE
To find out whether the antisense oligodeoxynucleotide designed against the complementary DNA to human inducible NO synthase (iNOS) would block the translation from iNOS mRNA to NO in human vascular endothelial cells.


METHODS
Prospective controlled study.


METHODS
Research laboratory, Japan.


METHODS
Cultured human vascular endothelial cells.


METHODS
Human vascular endothelial cells were cultured for 24 hours, and divided into two groups, one of which was exposed to antisense oligodeoxynucleotides and the other to sense oligodeoxynucleotides at 100, 200, 400 and 800 micromol/L, respectively, for 18 hours. They were then exposed to interferon-gamma (1000 units/ml) and recombinant human tumour necrosis factor alpha (500 units/ml) to stimulate NO production. Each experiment was repeated ten times.


RESULTS
Clear bands expressing cDNA of iNOS mRNA were identified in agarose gels in all cultured cells in both groups. The mean nitrite concentration in the supernatants of cultured cells in the group with the addition of antisense oligodeoxynucleotides was significantly lower than that in the group with sense oligonucleotides added at 200, 400, and 800 micromol/L.


CONCLUSIONS
These findings indicate that the antisense oligodeoxynucleotides inhibited the NO production dependently, by preventing only their translation but not the processing, before their transcription from DNA into iNOS mRNA.
Tanjoh
K
K
Department of Emergency and Critical Care Medicine, Nihon University School of Medicine, Tokyo, Japan.
Tomita
R
R
Hayashi
N
N
D002478:Cells, Cultured; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D003627:Data Interpretation, Statistical; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D009102:Multiple Organ Failure / Q000235:genetics / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000235:genetics* / Q000378:metabolism; D009573:Nitrites / Q000032:analysis; D016376:Oligonucleotides, Antisense / Q000235:genetics*; D011446:Prospective Studies; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018805:Sepsis / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
D017931:DNA Primers; D018076:DNA, Complementary; D009573:Nitrites; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase