No abstract

Nitric oxide (NO) is known to have various biologic and pathophysiologic effects on organisms. The molecular mechanisms by which NO exerts harmful effects are unknown, although various O 2 radicals and ions that result from reactivity of NO are presumed to be involved. Here we report that adaptive cellular response controlled by the transcription factor hypoxia-inducible factor 1 (HIF-1) in hypoxia is suppressed by NO. Induction of erythropoietin and glycolytic aldolase A mRNAs in hypoxically cultured Hep3B cells, a human hepatoma cell line, was completely and partially inhibited, respectively, by the addition of sodium nitroprusside (SNP), which spontaneously releases NO. A reporter plasmid carrying four hypoxia-response element sequences connected to the luciferase structural gene was constructed and transfected into Hep3B cells. Inducibly expressed luciferase activity in hypoxia was inhibited by the addition of SNP and two other structurally different NO donors, S-nitroso-Lglutathione and 3-morpholinosydnonimine, giving IC 50 values of 7.8, 211, and 490 M, respectively. Inhibition by SNP was also observed in Neuro 2A and HeLa cells, indicating that the inhibition was not cell-type-specific. The vascular endothelial growth factor promoter activity that is controlled by HIF-1 was also inhibited by SNP (IC 50 ‫؍‬ 6.6 M). Induction generated by the addition of cobalt ion (this treatment mimics hypoxia) was also inhibited by SNP (IC 50 ‫؍‬ 2.5 M). Increased luciferase activity expressed by cotransfection of effector plasmids for HIF-1␣ or HIF-1␣-like factor in hypoxia was also inhibited by the NO donor. We also showed that the inhibition was performed by blocking an activation step of HIF-1␣ to a DNA-binding form.

Photodenitrogenation of the diazenes 4 affords exclusively the housanes 5 through intramolecular cyclization of the spectrally detected and characterized singlet diradicals 3. The lifetime of singlet diradical 3, determined by transient absorption measurements, depends on the Y and Z substituents at the para position of the phenyl ring and has the following order: Y, Z = OMe, OMe > OMe, CN > CN, CN > OMe, H > Cl, Cl approximately CN, H approximately Me, Me > H, H. This unprecedented substituent effect reveals stabilization of the singlet 2,2-dimethoxycyclopentane-1,3-diyl diradicals 3 through radical, zwitterionic, pi-bonding, and hyperconjugative structures.