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起重要作用。然而，ho依赖的生物学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，以解决同工酶的特异性作用，寻找内源性CO的新靶标，并在体内研究CO和其他气体分子如一氧化氮（NO）对同一靶标分子的影响。为了避免HO-1基因失活导致的胚胎致死性和不育，我们构建了一个新的靶向载体，该载体含有该基因的外显子2，两侧是loxP序列，可以通过Cre重组酶以组织特异性的方式在体内删除，以产生条件HO-1敲除小鼠。到目前为止，已经获得了两个不同的胚胎干细胞克隆，其中发生了适当的同源重组，并且嵌合小鼠已经产生了更多。为了寻找新的CO靶向分子，我们使用CE-MS检测了对乙酰氨基酚（AAP）处理小鼠肝脏代谢物的变化，通过AAP，肝内HO活性和随之而来的CO生成增加。我们发现AAP治疗显著降低了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，蛋氨酸的大量积累发生在受影响的肝脏中。这些结果表明，CO可能通过结合和抑制胱硫氨酸β-合成酶（CBS）来抑制进入转硫途径的通量，CBS是该途径的限速酶。接下来，我们在体外研究了CO如何使用部分纯化的酶抑制CBS活性。我们的研究结果表明，毫摩尔水平的一氧化碳可以切割血红素-半胱氨酸键的铁，形成羧基-血红素复合物，导致酶活性抑制。为了明确CO和NO在体内调节可溶性鸟苷酸环化酶（sGC）中的作用，我们进行了免疫组织化学分析，分别通过抑制内源性这些气体分子来观察气体介导的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活化的增强。另一方面，在no抑制条件下，消除CO以层特异性的方式消除了sGC的基础激活。这些结果表明，内源性CO通过抑制视网膜特定层的最大反应和适度提高最小反应来微调no依赖性sGC功能的激活。少

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