GODA Nobuhito Keio University, School of Medicine, Associate Professor, 医学部, 講師 (00245549)
ONISHI Yasuyuki Central Institute for Experimental Animals, Research Project Center, Senior Research Investigator, プロジェクト推進センター, 副部長 (70201382)
江川 毅 慶應義塾大学, 医学部, 助手 (10232935)
|Budget Amount *help
¥11,200,000 (Direct Cost: ¥11,200,000)
Fiscal Year 2005: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 2004: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 2003: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 2002: ¥4,900,000 (Direct Cost: ¥4,900,000)
Heme oxygenase (HO), an enzyme capable to degrade protoheme IX into carbon monoxide (CO), free divalent iron, and biliverdin-IXα, plays important roles in the regulation of many cellular functions. However, the detailed molecular mechanisms of HO-dependent biological functions remain elusive. In this study, we aimed to establish new HO gene-targeting mice to address isozyme-specific roles, to find new targets of endogenous CO, and to examine effects of CO and other gaseous molecules such as nitric oxide (NO) on the same targeting molecules in vivo.
To circumvent embryonic lethality and infertility caused by an inactivation of the HO-1 gene, we constructed a new targeting vector containing exon2 of the gene flanked by loxP sequences, which can be deleted by Cre recombinase in a tissue-specific manner in vivo, in order to generate conditional HO-1 knockout mice. Two distinct ES cell clones, in which an appropriate homologous recombination occurred, were obtained so far, and chimeric mice
have been generated.
To find novel targeting molecules of CO, we examined alterations of metabolites in mouse liver treated with acetaminophen (AAP), by which intrahepatic HO activity and consequent CO production were increased, using CE-MS. We found that the levels of S-adenosylmethionine, cysteine, glutathione, and taurine were significantly reduced by AAP treatment. On the other hand, great accumulation of methionine occurred in the affected liver. These results suggest that CO might suppress the flux into a transsulfuration pathway by binding to and inhibiting cystathionine β-synthase (CBS), a rate-limiting enzyme of the pathway. We next examined how CO inhibits CBS activity using the partially purified enzyme in vitro. Our findings showed that mili molar levels of CO could cleave the Fe of heme-cysteine bond and form a carboxy-heme complex, resulting in an inhibition of the enzyme activity.
To clarify the roles of CO and NO to regulate soluble guanylate cyclase (sGC) in vivo, we carried out immunohistochemical analyses to visualize the gas-mediated sGC activation by suppressing endogenous these gaseous molecules separately. Under conditions where CO was suppressed by ZnPP, all retinal cell layers homogenously exhibited enhancement of NO-dependent activation of sGC. On the other hand, under NO-suppressing conditions, eliminating CO abrogated the basal sGC activation in a layer-specific manner. These results suggest that endogenous CO plays a role in fine tuning the NO-dependent activation of sGC function by suppressing the maximum response as well as by modestly elevating the minimum response in particular layer of retina. Less