Analysis of molecular mechanisms by which heme oxygenase-carbon monoxide pathway regulates cellular functions under physiological and pathophysiological conditions using newly developed gene targeting mice.

血红素加氧酶 (HO) 是一种能够将原血红素 IX 降解为一氧化碳 (CO)、游离二价铁和胆绿素-IXα 的酶，在许多细胞功能的调节中发挥着重要作用。然而，H2O2依赖性生物功能的详细分子机制仍然难以捉摸。在本研究中，我们旨在建立新的HO基因靶向小鼠来解决同工酶特异性作用，寻找内源性CO的新靶点，并检查CO和其他气态分子如一氧化氮（NO）对体内相同靶向分子的影响。为了避免HO-1基因失活引起的胚胎致死和不育，我们构建了一种新的靶向载体，该载体包含侧翼为loxP序列的基因外显子2，该外显子可以被Cre删除在体内以组织特异性方式重组酶，以产生条件性 HO-1 基因敲除小鼠。迄今为止，已经获得了两个不同的 ES 细胞克隆，其中发生了适当的同源重组，并且已经生成了嵌合小鼠。为了寻找新的 CO 靶向分子，我们使用 CE-MS 检查了用对乙酰氨基酚 (AAP) 处理的小鼠肝脏中代谢物的变化，通过这种变化，肝内 H2O 活性和随之而来的 CO 产生增加。我们发现 AAP 处理显着降低了 S-腺苷甲硫氨酸、半胱氨酸、谷胱甘肽和牛磺酸的水平。另一方面，甲硫氨酸在受影响的肝脏中大量积聚。这些结果表明，CO 可能通过结合并抑制胱硫醚 β-合酶 (CBS)（该途径的限速酶）来抑制进入转硫途径的通量。接下来我们在体外使用部分纯化的酶研究了 CO 如何抑制 CBS 活性。我们的研究结果表明，毫摩尔水平的CO可以裂解血红素-半胱氨酸键的Fe并形成羧基-血红素复合物，从而抑制酶活性。为了阐明CO和NO在体内调节可溶性鸟苷酸环化酶（sGC）的作用，我们进行了免疫组织化学分析，通过分别抑制内源性这些气体分子来可视化气体介导的sGC激活。在 ZnPP 抑制 CO 的条件下，所有视网膜细胞层均表现出 NO 依赖性 sGC 激活的增强。另一方面，在 NO 抑制条件下，消除 CO 以层特异性方式消除了基础 sGC 激活。这些结果表明，内源性 CO 通过抑制最大反应以及适度提高视网膜特定层的最小反应，在微调 sGC 功能的 NO 依赖性激活中发挥作用。较少的

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 … More 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