Heme oxygenase-1 inhibits cigarette smoke-induced increase in the tracheal mucosal permeability in guinea pigs in vivo

Objective: To examine whether heme oxygenase (HO)-1 inhibits cigarette smoke (CS)-induced increase in the airway mucosal permeability.Methods: Mucosal permeability was quantified by monitoring the rate of appearance in the circulation of horseradish peroxidase that had been instilled into the isolated tracheal segment in guinea pigs in vivo, after exposure to CS or room air.Results: Exposure to 10 puffs of CS did not increase the tracheal mucosal permeability but did increase the permeability after pretreatment with zinc protoporphyrin, a competitive inhibitor of HO-1. Moreover, pretreatment with hemin, a potent inducer of HO-1, inhibited the increase in the permeability of the tracheal mucosa induced by 20 puffs of CS exposure.Conclusion: It is concluded that HO-1 has an important role in suppressing the increase in the mucosal permeability induced by CS in guinea pig trachea.Received 10 October 2004; returned for revision 17 December 2004; returned for final revision 28 January 2005; accepted by N. Boughton-Smith 4 February 2005     

A structural motif in the C-terminal tail of slo1 confers carbon monoxide sensitivity to human BKCa channels

Carbon monoxide (CO) is a potent activator of large conductance, calcium-dependent potassium (BK_Ca) channels of vascular myocytes and carotid body glomus cells or when heterologously expressed. Using the human BK_Ca channel α₁-subunit (hSlo1; KCNMA1) stably and transiently expressed in human embryonic kidney 293 cells, the mechanism and structural basis of channel activation by CO was investigated in inside–out, excised membrane patches. Activation by CO was concentration dependent (EC₅₀ ∼20 μM), rapid, reversible, and evoked a shift in the V_0.5 of −20 mV. CO evoked no changes in either single channel conductance or in deactivation rate but augmented channel activation rate. Activation was independent of the redox state of the channel, or associated compounds/protein partners, and was partially dependent on [Ca²⁺]_i in the physiological range (100–1,000 nM). Importantly, CO “super-stimulated” BK_Ca activity even in saturating [Ca²⁺]_i. Single or double mutation of two histidine residues previously implicated in CO sensing did not suppress CO activation but replacing the S9–S10 module of the C-terminal of Slo1 with that of Slo3 completely prevented the action of CO. These findings show that a motif in the S9–S10 part of the C-terminal is essential for CO activation and suggest that this gas transmitter activates the BK_Ca channel by redox-independent changes in gating.     

Heme oxgenase-1 is expressed in viable astrocytes and microglia but in degenerating pyramidal neurons in the kainate-lesioned rat hippocampus

The present study aimed to elucidate the distribution of heme oxygenase-1 (HO-1) in the hippocampus after intracerebroventricular injections of kainate. Very little or no staining of HO-1 was observed in the normal CA1, whilst moderate staining of dentate hilar neurons was observed in the dentate gyrus, in the normal hippocampus. At postinjection day 1, a slight increase in immunoreactivity in the neuropil of the lesioned CA fields and a marked increase in HO-1 immunoreactivity in glial cells of the stratum lacunosum moleculare of CA fields and the stratum moleculare of the dentate gyrus was observed. Electron microscopy showed that the glial cells had features of viable astrocytes. At postinjection day 3, glial cells in the dentate gyrus continued to express HO-1, whilst pyramidal neurons in the degenerating CA fields started to express intense HO-1 immunoreactivity in their cell bodies. At postinjection weeks 1–3, HO-1 was observed in glial cells in the center of the lesion, but also in neurons at the perifocal region of the glial scar. The glial cells were found to have features of viable astrocytes and microglia, whilst the neurons contained discontinuous cell membranes and nuclear outlines, and had features of degenerating neurons. Intense immunoreactivity was observed in the cytoplasm of the degenerating neurons. The density of staining was greater than that observed in astrocytes or microglia. Recent in vitro results on fibroblasts transfected with HO-1 cDNA showed that, despite cytoprotection with low (less than fivefold compared with untransfected cells) HO-1 activity, high levels of HO-1 expression (more than 15-fold) were associated with significant oxygen toxicity. These and the present observations suggest a destructive effect of increased expression of HO-1 in neurons, and possible novel therapeutic approaches involving overexpression of HO-1 must therefore be approached with caution. Electronic Publication     

Reactive oxygen species regulated mitochondria-mediated apoptosis in PC12 cells exposed to chlorpyrifos

Reactive oxidative species (ROS) generated by environmental toxicants including pesticides could be one of the factors underlying the neuronal cell damage in neurodegenerative diseases. In this study we found that chlorpyrifos (CPF) induced apoptosis in dopaminergic neuronal components of PC12 cells as demonstrated by the activation of caspases and nuclear condensation. Furthermore, CPF also reduced the tyrosine hydroxylase-positive immunoreactivity in substantia nigra of the rat. In addition, CPF induced inhibition of mitochondrial complex I activity. Importantly, N-acetyl cysteine (NAC) treatment effectively blocked apoptosis via the caspase-9 and caspase-3 pathways while NAC attenuated the inhibition of mitochondrial complex I activity as well as the oxidative metabolism of dopamine (DA). These results demonstrated that CPF-induced apoptosis was involved in mitochondrial dysfunction through the production of ROS. In the response of cellular antioxidant systems to CPF, we found that CPF treatment increased HO-1 expression while the expression of CuZnSOD and MnSOD was reduced. In addition, we found that CPF treatment activated MAPK pathways, including ERK 1/2, the JNK, and the p38 MAP kinase in a time-dependent manner. NAC treatment abolished MAPK phosphorylation caused by CPF, indicating that ROS are upstream signals of MAPK. Interestingly, MAPK inhibitors abolished cytotoxicity and reduced ROS generation by CPF treatment. Our results demonstrate that CPF induced neuronal cell death in part through MAPK activation via ROS generation, suggesting its potential to generate oxidative stress via mitochondrial damage and its involvement in oxidative stress-related neurodegenerative disease.     

丙泊酚对急性肺损伤大鼠血红素氧合酶-1表达的影响

目的探讨丙泊酚对肺的保护作用及其可能作用机制。方法通过尾静脉注射内毒素建立急性肺损伤模型,丙泊酚和锌原卟啉同样经过尾静脉给药。将Wistar大鼠随机分入实验对照组（C组）、脂多糖组（L组）、脂多糖＋丙泊酚组（LP组）和锌原卟啉组（LZ组）。给药前及开始后第3、6和12h测定动脉血氧分压（PaO2）,实验结束后观察肺湿/干重（W/D）比值、肺组织病理学检查并进行肺损伤轻重程度评分,同时观察各组大鼠肺组织血红素氧合酶-1（HO-1）含量。结果实验前各组PaO2无明显差异,注射LPS后L组PaO2持续下降,和C组相比其PaO2显著降低（P〈0.05）。实验结束后L组和C组相比其W/D比值、肺组织病理学检查评分及HO-1含量显著增加（P〈0.05）。而LP组和L组相比W/D比值、肺组织病理学检查评分含量显著降低（P〈0.05）,而PaO2和HO-1含量显著升高（P〈0.05）。而LZ组加入锌原卟啉后明显抑制丙泊酚以上保护作用。结论丙泊酚具有肺保护作用,且该作用可能与其通过增加HO-1表达作用相关。     

State-of-the-art Tools for Computational Site of Metabolism Predictions: Comparative Analysis,Mechanistical Insights,and Future Applications

In drug design, it is crucial to have reliable information on how a chemical entity behaves in the presence of metabolizing enzymes. This requires substantial experimental efforts. Consequently, being able to predict the likely site/s of metabolism in any compound, synthesized or virtual, would be highly beneficial and time efficient.

In this work, six different methodologies for predictions of the site of metabolism (SOM) have been compared and validated using structurally diverse data sets of drug-like molecules with well-established metabolic pattern in CYP3A4, CYP2C9, or both. Three of the methods predict the SOM based on the ligand's chemical structure, two additional methods use structural information of the enzymes, and the sixth method combines structure and ligand similarity and reactivity. The SOM is correctly predicted in 50 to 90% of the cases, depending on method and enzyme, which is an encouraging rate. We also discuss the underlying mechanisms of cytochrome P450 metabolism in the light of the results from this comparison.     

内源性一氧化碳在生殖系统中作用的研究

内源性一氧化碳(endogenous CO)主要由血红素加氧酶(hemeoxygenase,HO)催化分解血红素而产生,是继一氧化氮之后发现的另一种具有重要生物学效应的气体分子,广泛参与体内的各种生理和病理过程,在心血管系统、神经系统、消化系统中起非常重要的作用。近年来,其在生殖系统的研究也日益受到重视。在女性生殖系统,HO/CO不仅会影响动情周期、分娩和哺乳行为,而且会抑制受孕子宫肌层的收缩,参与保护作用。而在男性生殖系统,HO/CO能够通过抗炎、抗氧化以及抗凋亡机制保护睾丸免受应激的损伤。这种可诱导的保护性作用将成为研究的新热点。