Kanji Nakatsu

Does carbon monoxide have a physiological function

Recently endothelium-derived relaxing factor (EDRF) has been identified as nitric oxide. The source of the nitric oxide is L-arginine, and the L-arginine-nitric oxide pathway has been proposed to function as a widespread transduction mechanism for the regulation of cell function and communication. Gerald Marks and colleagues suggest that carbon monoxide, which is formed endogenously from heme catabolism and which shares some of the chemical and biological properties of nitric oxide, may play a similar role. This would be achieved by carbon monoxide binding to the iron atom of the heme moiety of soluble guanylyl cyclase and to the iron-sulfur centers of macrophage enzymes.

Selectivity of imidazole–dioxolane compounds for in vitro inhibition of microsomal haem oxygenase isoforms

Haem oxygenases (HO) are involved in the catalytic breakdown of haem to generate carbon monoxide (CO), iron and biliverdin. It is widely accepted that products of haem catabolism are involved in biological signaling in many physiological processes. Conclusions to most studies in this field have gained support from the judicious use of synthetic metalloporphyrins such as chromium mesoporphyrin (CrMP) to selectively inhibit HO. However, metalloporphyrins have also been found to inhibit other haem‐dependent enzymes, such as nitric oxide synthase (NOS), cytochromes P‐450 (CYPs) and soluble guanylyl cyclase (sGC), induce the expression of HO‐1 or exhibit varied toxic effects. To obviate some of these problems, we have been examining non‐porphyrin HO inhibitors and the present study describes imidazole–dioxolane compounds with high selectivity for inhibition of HO‐1 (rat spleen microsomes) compared to HO‐2 (rat brain microsomes) in vitro. (2R,4R)‐2‐[2‐(4‐chlorophenyl)ethyl]‐2‐[(1H‐imidazol‐1‐yl)methyl]‐4‐methyl‐1,3‐dioxolane hydrochloride) was identified as the most selective inhibitor with a concentration of 0.6 μM inhibiting HO‐1(inducible) by 50% compared with 394 μM for HO‐2 (constitutive). These compounds were found to have no effects on the catalytic activities of rat brain NOS and lung sGC, but were potent inhibitors of microsomal CYP2E1 and CYP3A1/3A2 activities. In conclusion, we have identified imidazole–dioxolanes that are able to inhibit microsomal HO in vitro with high selectivity for HO‐1 compared to HO‐2, and little or no effect on the activities of neuronal NOS and sGC. These molecules could be used to facilitate studies on the elucidation of physiological roles of HO/CO in biological systems.

Quantitation of nitric oxide formation from nitrovasodilator drugs by chemiluminescence analysis of headspace gas

A rapid and reliable method has been developed for the quantitation of nitric oxide by chemiluminescence analysis of headspace gas. Aqueous nitric oxide standards are used to calibrate the method. There is a linear relationship between the amount of nitric oxide and the redox chemiluminescence detector response over the range of 52-2445 pmol of nitric oxide in 2.0 mL of deionized water contained in a sealed 6.2-mL flask. The intra-day and inter-day coefficient of variation values of the method do not exceed 4% and 9%, respectively. The lower limit of quantitative sensitivity and the lower limit of qualitative detection are 52 pmol and 26 pmol of nitric oxide, respectively. This method has been used to measure nitric oxide formation during the incubation of glyceryl trinitrate or sodium nitroprusside in the presence of cysteine. This method has two major advantages over the currently available procedures for the quantitation of nitric oxide, viz., no artifactual formation of nitric oxide during sample preparation and decreased instrumental contamination.

Measurement of endogenous carbon monoxide formation in biological systems.

Endogenous carbon monoxide (CO) formation has been measured in different biological systems using a variety of analytical procedures. The methods include gas chromatography-reduction gas detection, gas chromatography-mass spectroscopic detection, laser sensor-infrared absorption, UV-visible spectrophotometric measurement of CO-hemoglobin or CO-myoglobin complex, and formation of (14)CO from (14)C-heme formed following [2-(14)C]glycine administration. CO formation ranged from a low of 0.029 nmol/mg of protein/h in chorionic villi of term human placenta to a high of 0.28 nmol/mg of protein/h in rat olfactory receptor neurons in culture and rat liver perfusate.

Heme oxygenase and nitric oxide synthase in the placenta of the guinea-pig during gestation

Nitric oxide (NO) and carbon monoxide (CO) are novel gaseous chemical messengers that play key roles in cell function and cell-cell communication in many organ systems, including the cardiovascular system. Although the presence of NO synthase (NOS) in the placenta and its role in the regulation of fetoplacental and uteroplacental blood flow are well established, little is known about placental expression and activity of heme oxygenase (HO), the enzyme that catalyses the oxidation of heme to CO, biliverdin and iron, during gestation. The objectives of this study were to elucidate the localization of HO-1 and HO-2 isoforms relative to NOS III protein, and to determine the enzymatic activity of HO in the placenta of the guinea-pig during gestation. Placentae were obtained from pregnant guinea-pigs at gestational day (GD) 34, 50, 62 and full term (term, about GD 68). Immunohistochemical localization of HO-1, HO-2 and NOS III protein was conducted using selective polyclonal antibodies. HO activity was determined by using a gas chromatographic method to measure the rate of formation of CO from heme. Faint staining for HO-1 was observed in the adventitial layer of larger fetal blood vessels of the placenta at GD 34. The intensity of this staining was higher at GD 50 and GD 62, and decreased at full term. Similar areas in serial sections of placentae obtained at these selected times during gestation exhibited lower staining intensity when incubated with anti-HO-2 antiserum. Placental HO activity was significantly increased (P<0.05) at GD 62 compared with GD 34, GD 50 and full term. NOS III (endothelial constitutive NOS) staining was highest at GD 34, decreasing thereafter, and was localized mostly to trophoblast lining maternal channels. The data demonstrate that, in the guinea-pig, placental HO and NOS differ in tissue localization during the second half of gestation, with expression of HO protein and its catalytic activity being higher during near-term pregnancy. In a preliminary immunohistochemical investigation of the full-term human placenta, HO-1 protein was localized primarily in the adventitial region of fetal blood vessels of stem chorionic villi. In view of the vasodilator action of CO and NO, the HO and NOS systems may play key roles in the regulation of placental haemodynamics.

Heme oxygenase activity and immunohistochemical localization in bovine pulmonary artery and vein.

Recent studies suggest that carbon monoxide (CO) derived from heme oxygenase (HO)-catalyzed metabolism of heme plays a role in the regulation of cell function and communication. In blood vessels, CO may regulate vascular smooth-muscle tone through the activation of soluble guanylyl cyclase, in a manner similar to that of nitric oxide. The objective of this study was to determine the relation between HO enzymatic activity and localization of HO protein in bovine pulmonary blood vessels. HO enzymatic activity was determined by quantitating the rate of CO formation in the microsomal fraction of homogenates of bovine pulmonary artery (BPA) and vein (BPV). HO protein was localized by immunohistochemical analysis of paraformaldehyde-fixed tissue by using polyclonal antibodies to inducible HO (HO-1) and noninducible HO (HO-2). HO enzymatic activity was measured in BPA and BPV, which correlated with the presence of HO protein. In BPA, HO enzymatic activity was found in the adventitia and medial layer; HO protein was localized in the nerves and vasa vasorum of the adventitia and was found throughout the smooth-muscle cells in the medial layer. The data clearly demonstrate the presence of HO enzymatic activity for the formation of CO in blood vessels that contain HO protein.

X-ray crystal structure of human heme oxygenase-1 in complex with 1-(adamantan-1-yl)-2-(1H-imidazol-1-yl)ethanone: a common binding mode for imidazole-based heme oxygenase-1 inhibitors.

Development of inhibitors specific for heme oxygenases (HOs) should aid our understanding of the HO system and facilitate future therapeutic applications. The crystal structure of human HO-1 complexed with 1-(adamantan-1-yl)-2-(1H-imidazol-1-yl)ethanone (3) was determined. This inhibitor binds to the HO-1 distal pocket such that the imidazolyl moiety coordinates with heme iron while the adamantyl group is stabilized by a hydrophobic binding pocket. Distal helix flexibility, coupled with shifts in proximal residues and heme, acts to expand the distal pocket, thus accommodating the bulky inhibitor without displacing heme. Inhibitor binding effectively displaces the catalytically critical distal water ligand. Comparison with the binding of 2-[2-(4-chlorophenyl)ethyl]-2-[1H-imidazol-1-yl)methyl]-1,3-dioxolane (2) revealed a common binding mode, despite differing chemical structures beyond the imidazolyl moiety. The inhibitor binding pocket is flexible, yet contains well-defined subpockets to accommodate appropriate functional groups. On the basis of these structural insights, we rationalize binding features to optimize inhibitor design.

Peroxynitrite-mediated inactivation of heme oxygenases

BackgroundEndogenous nitric oxide (NO) and carbon monoxide (CO) are generated by nitric oxide synthase and heme oxygenase, respectively. Like NO, CO has been accepted as an important cellular signaling molecule in biological systems. An up-regulation in both gene and protein expression of heme oxygenase-1 (HO-1) under oxidative/nitrosative stress has been well documented, and the protective role of HO-1 and HO-2 against oxidative damage is proposed. However, data on the direct effect of reactive oxygen/nitrogen species (ROS/RNS) on HO function is incomplete. Using gas chromatography to quantify carbon monoxide (CO) formation from heme oxidation, we investigated the effects of peroxynitrite (ONOO-) on the in vitro catalytic activity of rat spleen (HO-1) and brain (HO-2) microsomal heme oxygenases.ResultsExposure to ONOO- led to concentration-dependent but reversible decreases in the activity of microsomal rat spleen and brain HO activity. Spleen HO activity was 100-fold more sensitive to ONOO--dependent inactivation compared to that of the brain, with IC50 values of 0.015 ± 0.005 mM and 1.25 ± 0.25 mM respectively. Inhibition of both rat spleen and brain microsomal HO activity was also observed with tetra-nitromethane, a tyrosine nitrating agent, as well as two NO donors, S-nitrosoglutathione (GSNO) and diethylamine NONOate (DEA-NONOate). However, no additive effect was found following the application of NO donors and ONOO- together.ConclusionThese results indicate that ONOO- may regulate HO-1 and HO-2 activities by mechanisms that involve different interactions with these proteins. It is suggested that while nitration of tyrosine residues and oxidation of sulfhydryl groups may be involved, consideration should be given to other facets of ONOO- chemistry. This inhibition of HO activity offers a mechanism for cross talk between the nitric oxide synthase and HO systems.

Heme Oxygenase Expression in Selected Regions of Term Human Placenta

Carbon monoxide (CO), formed during heme oxygenase (HO)-catalyzed oxidation of heme, has been proposed to play a complementary role with nitric oxide in the regulation of placental hemodynamics. The objective of this study was to elucidate HO enzymatic activity and HO-1 (inducible) and HO-2 (constitutive) protein content in the microsomal subcellular fraction of homogenate of selected regions of placenta from normotensive and mild pre-eclamptic pregnancies. HO enzymatic activity was measured under optimized conditions by gas chromatography using CO formation as an index of activity, and HO-1 and HO-2 protein content were determined by Western immunoblot analysis. Microsomal HO activity in each of the four placental regions was not different between normotensive and mild pre-eclamptic pregnancies. Microsomal HO-2 protein content was not different between normotensive and mild pre-eclamptic pregnancies, whereas there was increased expression of microsomal HO-1 protein in chorionic villi and fetal membranes from pre-eclamptic pregnancy compared with normotensive pregnancy. Microsomal HO enzymatic activity correlated with HO-2, but not HO-1, protein content.

Long-Term Circulatory Consequences of Perinatal Iron Deficiency in Male Wistar Rats

Perinatal iron deficiency (PID) has been reported to induce developmental abnormalities, including cardiovascular complications in rats. These complications are believed to be “programmed” by an aberrant perinatal environment because the changes persist long after the insult is corrected (ie, despite subsequent iron replenishment). Little is known about the mechanisms by which PID affects blood pressure in the offspring, although the kidney is likely to play a central role. The objective of this study was to investigate the circulatory complications of PID and the putative role of the kidney involved therein. Before and throughout gestation, female Wistar rats were fed either a low-iron diet (3 ppm/10 ppm Fe) or an iron-enriched diet (225 ppm Fe). After giving birth, all of the dams were placed on a standard grain-based diet. At 24 hours postpartum, hematocrits and hemoglobin levels from offspring of iron-deficient mothers were 60% and 59% of control values, respectively. Adult PID animals had greater mean arterial pressures (110 versus 106 mm Hg) and systolic blood pressures (129 versus124 mm Hg) than controls, as assessed by radiotelemetry. The relationship between renal arterial pressure and renal interstitial hydrostatic pressure, assessed in anesthetized rats, was blunted by 41% in the PID group compared with controls. In addition, arterial pressure changes were significantly greater in response to altered sodium in the PID animals compared with controls. These data confirm that PID adversely affects blood pressure control, which seems to be mediated, at least in part, by altered intrarenal hemodynamic properties.