Low-dose carbon monoxide reduces airway hyperresponsiveness in mice

Carbon monoxide (CO) in expired gas has been shown to be elevated with asthma; however, its function is not known, and there is some potential that it may serve a bronchoprotective role to decrease airway hyperresponsiveness (AHR). Thus the ability of CO to reverse methacholine (MCh)-induced bronchoconstriction was evaluated in C57BL/6 (C57) and A/J mice with and without airway inflammation produced by ovalbumin (OVA). Acutely administered CO (1% in air, 10 min) reduced MCh-driven increases in lung resistance in OVA-challenged C57 mice by an average of 50% (from 14.5 to 7.1 cmH2O.ml-1.s-1), whereas no effect was observed in na?ve C57 mice or OVA-challenged C57 mice inhaling air alone. Acutely inhaled CO (500 ppm = 0.05%, for 10 min) reduced MCh-induced airway reactivity (AR) by 20-60% in airway hyperresponsive na?ve A/J mice, whereas repeated 10-min administrations of 500 ppm CO over a 5-day period decreased AR by 50%. Repeated administration of low-dose CO [250 (0.025%) and (0.05%) 500 ppm, 1 h/day, 5 days] to A/J mice with airway inflammation likewise resulted in a drop of AR by 50%, compared with those not receiving CO. Inhibition of guanylyl cyclase/guanosine 3',5'-cyclic monophosphothioate (cGMP) using 1H-[1,2,4] oxydiazolo[4,3-a]quinoxalin-1-one or a competitive inhibitor, Rp diastereomers of 8-bromo-cGMP, resulted in inhibition of the effect of CO on AHR, suggesting that the effects of CO were mediated through this mechanism. These results indicate that low-dose CO can effectively reverse AHR in the presence and absence of airway inflammation in mice and suggest a potential role for CO in the modulation of AHR.     

Ventricular function following acute carbon monoxide exposure.

Cardiac output function curves were used to investigate the effects of carbon monoxide on the heart in the conscious dog. Each dog was briefly exposed to 1,500 ppm carbon monoxide through a permanent tracheostomy. Immediately upon attaining either 10%, 20%, or 30% HbCO a rapid infusion of Ringer's lactate was given to test cardiac capabilities. The combined effects of carbon monoxide and infusion produced significant increases in cardiac output, heart rate, mean left ventricular pressure, dP/dt and (dP/dt)/IP. Cardiac output was sufficient to prevent peripheral hypoxia at all HbCO levels; however, there was evidence of impending cardiac depression beginning at 20% HbCO.     

Role of carbon monoxide in glutamate receptor-induced dilation of newborn pig pial arterioles

The hypothesis that glutamate dilates pial arterioles of newborn pigs through the production of carbon monoxide (CO) was addressed. Anesthesized newborn pigs were equipped with cranial windows to measure pial arteriolar responses to stimuli. Heme oxygenase (HO) inhibitors added topically inhibited dilation to glutamate and to specific glutamate receptor agonists. The initial dilation to glutamate (10(-5) M) was 22% from baseline without an inhibitor and decreased to 9% with the HO inhibitor chromium mesoporphyrin (CrMP). Inhibition of dilation upon HO inhibition was similar when specific glutamate receptor agonists were employed. RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid caused 24% dilation from the baseline without an inhibitor, and the dilation was decreased to 1% with tin protoporphyrin (SnPP). (RS)-2-amino-3-(3-hydroxy-5-t-butylisoxazol-4-yl)propionic acid (kainate receptors) caused dilation of 18% from baseline without an inhibitor, but only 2% when tin mesoporphyrin was applied. 1-Aminocyclopropanecarboxylic acid (N-methyl-D-aspartate receptors) dilated pial arterioles 33% from baseline in control, but only to 2% in the presence of SnPP. Neither copper mesoporphyrin, which does not inhibit HO, nor light-inactivated CrMP affected the dilations. Furthermore, cerebral microvessels removed from the brain produced CO (stable isotope dilution gas chromatography-mass spectrometry), and this production was dose dependently increased by glutamate and inhibited by metal porphyrin HO inhibitors. These data suggest that dilation of newborn pig pial arterioles to glutamate and specific glutamate receptor agonists involves vascular production of CO. Additional cerebral sources of CO also could be stimulated by glutamate and contribute to the dilation.     

Myocardial cytochrome oxidase activity is decreased following carbon monoxide exposure

Carbon monoxide (CO) inhalation often leads to cardiac dysfunction, dysrhythmias, ischemia, infarction, and death. However, the underlying mechanism of CO toxicity is poorly understood. We hypothesize that inhaled CO interrupts myocardial oxidative phosphorylation by decreasing the activity of myocardial cytochrome oxidase (CcOX), the terminal oxidase of the electron transport chain. Male C57Bl6 mice were exposed to either 1000 ppm (0.1%) CO or air for 3 h. Cardiac ventricles were harvested and mitochondria were isolated. CcOX kinetics and heme aa(3) content were measured. V(max), K(m), and turnover number were determined. Levels of CcOX subunit I message and protein were evaluated. Carboxyhemoglobin (COHb) levels were measured and tissue hypoxia was assessed with immunohistochemistry for pimonidazole hydrochloride. CO significantly decreased myocardial CcOX activity and V(max) without altering K(m). Heme aa(3) content and CcOX I protein levels significantly decreased following CO exposure while enzyme turnover number and CcOX I mRNA levels remained unchanged. CO exposure increased COHb levels without evidence of tissue hypoxia as compared to sham and hypoxic controls. Decreased CcOX activity following CO inhalation was likely due to decreased heme aa(3) and CcOX subunit I content. Importantly, myocardial CcOX impairment could underlie CO induced cardiac dysfunction.     

Effects of inhaled carbon monoxide on acute lung injury in mice

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are major causes of morbidity and mortality in the intensive care unit, but despite continuing research few effective therapies have been identified. In recent years, inhaled carbon monoxide (CO) has been reported to have cytoprotective effects in several animal models of tissue injury. We therefore evaluated the effects of inhaled CO in three different in vivo mouse models of ALI. Anesthetized C57BL/6 mice were ventilated with oxygen in the presence or absence of CO (500 parts per million) for 1 h before lung injury was induced by lipopolysaccharide (LPS) or oleic acid (OA) administration. Ventilation was then continued with the same gases for a further 2-3 h, with hemodynamic and respiratory parameters monitored throughout. Intratracheal LPS administration induced lung injury with alveolar inflammation (increased lavage fluid neutrophils, total protein, and cytokines). In contrast, intravenous LPS induced a predominantly vascular lung injury, with increased plasma TNF and increased neutrophil activation (surface Mac-1 upregulation and L-selectin shedding) and sequestration within the pulmonary vasculature. Intravenous OA produced deteriorations in lung function, reflected by changes in respiratory mechanics and blood gases and lavage fluid neutrophil accumulation. However, addition of CO to the inspired gas did not produce significant changes in the measured physiological or immunological parameters in the mouse models used in this study. Thus the results do not support the hypothesis that use of inhaled CO is beneficial in the treatment of ALI and ARDS.     

Teratogenic potential of inhaled carbon monoxide in mice and rabbits.

Pregnant CF-1 mice and New Zealand rabbits were exposed to carbon monoxide at a concentration of 250 ppm for 7 or 24 hours daily during the period of major organogenesis, days 6 through 15 of gestation in mice and 6 through 18 of gestation in rabbits. Carboxyhemoglobin levels in the range of 10--15% were observed in both species (control animals had 0.7% or less). Carbon monoxide was not found to be teratogenic in either species. In mice, a significant increase in the incidence of some minor skeletal variants was observed. One litter in each of the carbon monoxide-exposed groups of mice was completely resorbed; none of the litters of control mice or of control or exposed rabbits were completely resorbed. The fetuses of mice exposed to carbon monoxide for seven hours daily were heavier than control fetuses, and those exposed for 24 hours daily were lighter than control fetuses. The reason for this result is not known.     

Brief exposure to carbon monoxide preconditions cardiomyogenic cells against apoptosis in ischemia-reperfusion

We examined whether and how pretreatment with carbon monoxide (CO) prevents apoptosis of cardioblastic H9c2 cells in ischemia-reperfusion. Reperfusion (6 h) following brief ischemia (10 min) induced cytochrome c release, activation of caspase-9 and caspase-3, and apoptotic nuclear condensation. Brief CO pretreatment (10 min) or a caspase-9 inhibitor (Z-LEHD-FMK) attenuated these apoptotic changes. Ischemia-reperfusion increased phosphorylation of Akt at Ser472/473/474, and this was enhanced by CO pretreatment. A specific Akt inhibitor (API-2) blunted the anti-apoptotic effects of CO in reperfusion. In normoxic cells, CO enhanced generation, which was inhibited by a mitochondrial complex III inhibitor (antimycin A) but not by a NADH oxidase inhibitor (apocynin). The CO-enhanced Akt phosphorylation was suppressed by an scavenger (Tiron), catalase or a superoxide dismutase (SOD) inhibitor (DETC). These results suggest that CO pretreatment induces mitochondrial generation of , which is then converted by SOD to H₂O₂, and subsequent Akt activation by H₂O₂ attenuates apoptosis in ischemia-reperfusion.     

Effect of chronic carbon monoxide exposure on experimental alcoholic liver injury in rats

Two groups of experimental animals with pair-fed controls were studied to evaluate the effect of chronic carbon monoxide (CO) exposure on progression of experimental alcoholic liver injury. Eight pairs of male Wistar rats were continuously infused liquid diet and ethanol or isocaloric dextrose for four months. Four pairs were also exposed to CO. Liver damage was followed monthly by serum ALT and morphologic assessment of liver biopsy. Serum levels of ALT were significantly higher in the CO-ethanol group compared to other groups. Electron microscopy revealed a greater degree of cell necrosis in the CO exposed group which explained the significantly higher ALT activity in these animals. Both experimental groups (CO-ethanol and air-ethanol) had significantly greater liver damage than controls. Carboxyhemoglobin levels were not different in the ethanol-fed and control group. Our results show that chronic CO exposure enhances liver cell necrosis in ethanol-fed rats thereby lending support to the hypothesis that ethanol and hypoxia enhance cellular disruption in the liver which could be important in the pathogenesis of alcoholic liver disease in rats.