Effect of chronic hypoxia on inducible nitric oxide synthase expression in rat myocardial tissue.
The purpose of our study was to evaluate the effect of chronic exposure to low cellular oxygen tension (90% N2 and 10% O2 for 14 days) in inducing apoptosis and activation of transcription and translation of inducible nitric oxide (NO) synthase (iNOS) in rat hearts tissue. Rats were divided into four groups: normoxic, hypoxic, rats maintained in normoxic condition for 7 days and subjected to hypoxic conditions for another 7 days, and rats maintained in hypoxic condition for 7 days and subjected to normoxic conditions for another 7 days. At the 7th and 14th days, five rats from each group were sacrificed. Immunohistochemical and Western blot analysis were performed on myocardial tissue to reveal the presence of iNOS. Expression of iNOS was determined by RT-PCR. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and by detection of internucleosomal DNA fragmentation by electrophoresis. Electrophoretic analysis of DNA showed oligonucleosomal fragmentation in the hypoxic groups, but no ladder was observed in the other groups. This data was confirmed through end labeling with streptavidin-biotin (biotin d-UTP). iNOS expression was evaluated through immunohistochemical techniques (Ab anti-iNOS) and Western blotting, and the results were quantified with a computerized imaging analysis. The expression of iNOS protein was greater in the hypoxic groups; in the normoxic groups, only a nonspecific background was detected. This data was supported with results obtained through RT-PCR, which showed the specific transcription of mRNA for iNOS in the same experimental conditions. In addition, the iNOS activity was also evaluated and was found to be more active in the hypoxic groups (0.1 +/- 0.01 vs 0.02 +/- 0.003). The present study shows that exposure to low oxygen tension is capable of inducing programmed cell death and activating iNOS.
Grilli
Alfredo
A
Department of Biomorphology, University G. D'Annunzio, 66013 Chieti, Italy.
De Lutiis
Maria Anna
MA
Patruno
Antonia
A
Speranza
Lorenza
L
Cataldi
Amelia
A
Centurione
Lucia
L
Taccardi
Alfonso A
AA
Di Napoli
Pericle
P
De Caterina
Raffaele
R
Barbacane
Renato
R
Conti
Pio
P
Felaco
Mario
M
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D053938:DNA Fragmentation; D004586:Electrophoresis; D015971:Gene Expression Regulation, Enzymologic; D000860:Hypoxia / Q000201:enzymology*; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D008297:Male; D009206:Myocardium / Q000201:enzymology* / Q000473:pathology; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic
D012333:RNA, Messenger; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat