Direct Effect Of Carbon Monoxide Research Articles

Direct Effect of Carbon Monoxide on Relaxation Induced by Electrical Field Stimulation in Rat Corpus Cavernosum

PurposeCarbon monoxide (CO) may mediate smooth muscle relaxation in the rat corpus cavernosum smooth muscle (CCSM). We hypothesized that CO plays a role in neurally derived, frequency-dependent relaxation of rat CCSM.Materials and MethodsTo study the effect of CO on CCSM relaxation induced by electrical field stimulation (EFS), a CCSM bundle was mounted on a force transducer and perfused with Hanks' balanced salt solution at 37℃ with 95% O2 and 5% CO2. After 1 hour equilibration with -500 mg of passive tension, contraction of the CCSM bundle was elicited by 10-5 M phenylephrine, which was continuously added with different concentrations of CO (1%, 2%, and 5%). Frequency-dependent relaxation was induced by EFS trains (0.2 ms at 0.5-32 Hz, for 10 s) repeated at 2 min intervals over 15 min in the presence of adrenergic and muscarinic receptor blocking agents (guanethidine and atropine, respectively). To study the distribution of heme oxygenase-2 (HO-2) in the rat CCSM, we performed immunohistochemical evaluation.ResultsCO produced a dose-dependent enhancement of EFS-induced relaxation. Pretreatment with NG-nitro-L-arginine (a nitric oxide synthase blocker) greatly reduced the EFS-induced relaxation in the presence of CO (-45%). Pretreatment with zinc protoporphyrin-IX (ZnPP-9, a heme oxygenase inhibitor) had no significant effect on EFS-induced relaxation in the absence or the presence of CO. We found immunoreactivity for HO-2 in CCSM and immunoreactivity for protein gene product 9.5 (PGP 9.5) in nerve fibers.ConclusionsWe conclude that CO produced a dose-dependent enhancement of EFS-induced relaxation in rat CCSM bundles, but neurally derived, frequency-dependent relaxation in the rat CCSM depended mostly on nitric oxide in response to nonadrenergic noncholinergic neurotransmission. Immunoreactivity for HO-2 was found in rat CCSM but not nerve fibers.

Protective Effect of Carbon Monoxide Donor Compounds in Endotoxin-Induced Acute Renal Failure

Background: Sepsis is a common cause of acute renal failure (ARF) in clinical practice. However, the precise mechanism of endotoxin-induced ARF is not fully understood. There have been several reports that inhalation of carbon monoxide (CO) gas could be protective against acute rejection in intestine, lung, and kidney transplantation. Thus, we investigated the direct effect of CO in an experimental ARF model of septic shock induced by lipopolysaccharide (LPS). Method: Mice were pretreated with [Ru(CO)₃Cl₂]₂ (CO donor compounds) at various concentrations (0.5, 1.0 and 2.0 µg) which were intravenously injected 24 h before intraperitoneal LPS injection. Biomarkers including myeloperoxidase activity and histochemical staining were evaluated. Results: The elevation of plasma creatinine was suppressed in CO donor-pretreated mice compared with vehicle-treated mice (creatinine 0.35 vs. 0.25; p < 0.05) 24 h after LPS injection. Renal myeloperoxidase activity slightly decreased in CO donor-pretreated mice. In the histological examination, neutrophil infiltration was significantly diminished in CO donor-treated mice. Real-time polymerase chain reaction revealed significant improvements in inflammatory related genes, such as TNFα, MCP-1, RANTES and IL4. Conclusion: Our results suggest the protective effect of the CO donor against endotoxin-induced renal injury; however, further study is needed to elucidate the mechanism.

Nitric oxide inhibits, and carbon monoxide activates, islet acid α-glucoside hydrolase activities in parallel with glucose-stimulated insulin secretion

We have studied the influence of nitric oxide (NO) and carbon monoxide (CO), putative messenger molecules in the brain as well as in the islets of Langerhans, on glucose-stimulated insulin secretion and on the activities of the acid alpha-glucoside hydrolases, enzymes which we previously have shown to be implicated in the insulin release process. We have shown here that exogenous NO gas inhibits, while CO gas amplifies glucose-stimulated insulin secretion in intact mouse islets concomitant with a marked inhibition (NO) and a marked activation (CO) of the activities of the lysosomal/vacuolar enzymes acid glucan-1,4-alpha-glucosidase and acid alpha-glucosidase (acid alpha-glucoside hydrolases). Furthermore, CO dose-dependently potentiated glucose-stimulated insulin secretion in the range 0.1-1000 microM. In intact islets, the heme oxygenase substrate hemin markedly amplified glucose-stimulated insulin release, an effect which was accompanied by an increased activity of the acid alpha-glucoside hydrolases. These effects were partially suppressed by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one. Hemin also inhibited inducible NO synthase (iNOS)-derived NO production probably through a direct effect of CO on the NOS enzyme. Further, exogenous CO raised the content of both cGMP and cAMP in parallel with a marked amplification of glucose-stimulated insulin release, while exogenous NO suppressed insulin release and cAMP, leaving cGMP unaffected. Emiglitate, a selective inhibitor of alpha-glucoside hydrolase activities, was able to markedly inhibit the stimulatory effect of exogenous CO on both glucose-stimulated insulin secretion and the activityof acid glucan-1,4-alpha-glucosidase and acid alpha-glucosidase, while no appreciable effect on the activities of other lysosomal enzyme activities measured was found. We propose that CO and NO, both produced in significant quantities in the islets of Langerhans, have interacting regulatory roles on glucose-stimulated insulin secretion. This regulation is, at least in part, transduced through the activity of cGMP and the lysosomal/vacuolar system and the associated acid alpha-glucoside hydrolases, but probably also through a direct effect on the cAMP system.

Hypoxia-independent apoptosis in neural cells exposed to carbon monoxide in vitro

The neurotoxic effects of carbon monoxide (CO) are well known. Brain hypoxia due to the binding of CO to hemoglobin is a recognized cause of CO neurotoxicity, while the direct effect of CO on intracellular targets remains poorly understood. In the present study, we have investigated the pathways leading to neural cell death induced by in vitro exposure to CO using a gas exposure chamber that we have developed. Mouse hippocampal neurons (HT22) and human glial cells (D384) were exposed to concentrations of CO ranging from 300 to 1000 ppm in the presence of 20% oxygen. Cytotoxicity was observed after 48 h exposure to 1000 ppm, corresponding to approximately 1 μM CO in the cultured medium, as measured by gas chromatography. CO induced cell death with characteristic features of apoptosis. Exposed cells exhibited loss of mitochondrial membrane potential, release of cytochrome c into the cytosol, nuclei with chromatin condensation, and exposure of phosphatidyl serine on the external leaflet of the plasma membrane. CO also triggered activation of caspase and calpain proteases. Pre-incubation with either the pancaspase inhibitor Z-VAD-fmk (20μM) or the calpain inhibitor E64d (25 μM) reduced by 50% the occurrence of apoptosis. When pre-incubating the cells with the two inhibitors together there was an additional reduction in the number of cells with apoptotic nuclei. These data suggest that CO causes apoptosis via activation of parallel proteolytic pathways involving both caspases and calpains. Furthermore, pre-treatment with the antioxidant MnTBAP (100 μM) significantly reduced the number of apoptotic nuclei, pointing to a critical role of oxidative stress in CO toxicity.

Carbon monoxide inhibits capacitative calcium entry in human platelets

The cytosolic calcium concentration in human platelets is elevated by several agonists via receptor-operated mechanisms involving both Ca 2+ release from intracellular stores and Ca 2+ entry. In order to get a mechanistic insight in the effect of carbon monoxide (CO)-containing solutions, this work examines the changes in [Ca 2+] i induced by 100 μM adenosine 5′diphosphate (ADP), 0.1 IU/ ml thrombin, 0.5 μM thapsigargin or 0.5 μM ionomycin in human platelets. In a saline solution bubbled with CO, the increase of [Ca 2+] i produced by thrombin was 72±4% of the response evoked in the control solution (CO-free) and the response elicited by ADP was 64±8% of the control. When a mixture of 5% CO/95% N 2 was used, the responses were 70±7% of control for thrombin and 79±6% of control for ADP. The mobilization of stored calcium produced by thrombin in a calcium-free solution and the increase of [Ca 2+] i produced by subsequent introduction of 1 mM extracellular calcium were both reduced in the presence of CO (82±6% and 78±5% of control, respectively). Similar reductions in the presence of CO were found when platelets were stimulated by ADP (62±8% and 60±8% for mobilization in calcium-free media and calcium entry, respectively). Although the change in [Ca 2+] i induced by ionomycin in the presence of extracellular calcium was almost the same in the absence or presence of CO (97±5% of control), the entry induced by depletion of reservoirs with the ionophore undergoes a significant reduction in a solution bubbled with CO (84±5% of control). In agreement with the concept that CO has a direct inhibitory effect on capacitative calcium entry, a reduction to 47±6% of control was obtained when sarco/endoplasmic reticulum ATPase was blocked by thapsigargin. Diverse mechanisms could be responsible for the effect of CO on calcium entry. On the one hand, a decrease in the calcium release from intracellular stores or an increase in the rate of its back-sequestration could occur, being the reduction of capacitative calcium entry an indirect consequence of a diminished emptying of reservoirs. On the other hand, CO could have a direct inhibitory effect on the pathway that produces the calcium entry. The decrease in the Ca 2+ signal in the presence of CO evoked by receptor-independent emptying of reservoirs indicates that a direct effect of CO on capacitative calcium entry participates in the antiaggregatory properties of CO. The proposal that CO inhibits directly store-operated calcium influx widens the potential mechanisms by which heme oxygenase regulates cell functions.

Carbon monoxide specifically inhibits cytochrome c oxidase of human mitochondrial respiratory chain.

Carbon monoxide (CO) toxicity is the result of a combination of tissue hypoxia and direct CO-mediated damage at a cellular level, since not all the signs and symptoms presented can be explained only by the formation of carboxyhaemoglobin. Mitochondria, specially the electron transport chain, seem to be the target for CO at a subcellular level. However, the direct effect of CO in individual complexes of the human mitochondrial respiratory chain has not been completely elucidated. We here studied the in vitro effect of CO on individual complexes of the mitochondrial respiratory chain of human mitochondria. We obtained muscle tissue from 10 healthy people who underwent orthopaedic surgery for hip replacement. Isolated mitochondria were incubated for 5 min. under CO concentrations of 50, 100 and 500 ppm. Afterwards, enzymatic activities of individual complexes of the mitochondrial respiratory chain were assessed in vitro and compared with those obtained in basal (synthetic air without CO) conditions. Cytochrome c oxidase (complex IV of the mitochondrial respiratory chain) activity showed a decrease from 836+/-439 nmol/min./mg of mitochondrial protein after air incubation to 670+/-401, 483+/-182, and 379+/-131 nmol/min./mg after 50, 100 and 500 ppm of CO incubation, respectively (20%, 42% and 55% decrease in cytochrome c oxidase activity). This gradual decrease in cytochrome c oxidase was found to be statistically significant (P<0.001). Other complex activities showed no any significant variation. Carbon monoxide is toxic for mitochondria in man, altering the mitochondrial respiratory chain at the cytochrome c oxidase level. This inhibition in cytochrome c oxidase may play a role in the development of the symptoms observed in acute CO poisoning, and in some diseases related to smoking.

A possible dual site of action for carbon monoxide-mediated chemoexcitation in the rat carotid body.

High tensions of carbon monoxide (CO), relative to oxygen, were used as a tool to investigate the mechanism of chemotransduction. In an in vitro whole organ, rat carotid body preparation, CO increased sinus nerve chemoafferent discharge in the dark, an effect that was significantly reduced (by ca 70 %) by bright white light and by the removal of extracellular Ca(2+) from the superfusate or by the addition of either Ni(2+) (2 mM) or methoxyverapamil (100 microM). Addition of the P(2) purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (50 microM) also significantly reduced the neural response to CO. In perforated patch, whole-cell recordings of isolated rat type I cells, CO induced a depolarisation of ca 11 mV and a decrease in the amplitude of an outward current around and above the resting membrane potential. Membrane conductance between -50 and -60 mV was significantly reduced by ca 40 % by CO. These effects were not photolabile and were present also when a 'blocking solution' containing TEA, 4-AP, Ni(2+) and zero extracellular Ca(2+) was used. In conventional whole-cell recordings, CO only decreased current amplitudes above +10 mV and was without effect around the resting membrane potential. These data demonstrate a direct effect of CO upon type I cell K(+) conductances and strongly suggest an effect upon a background, leak conductance that requires an intracellular mediator. The photolabile effect of CO only upon afferent neural discharge adds further evidence to a dual site of action of CO with a separate action at the afferent nerve terminal that, additionally, requires the permissive action of the neurotransmitter ATP.

The direct effect of carbon monoxide on KCa channels in vascular smooth muscle cells.

The vasorelaxation induced by carbon monoxide (CO) has been demonstrated previously. Both a guanosine cyclic monophosphate (cGMP) signalling pathway and cGMP-independent mechanisms have been proposed to be responsible for the vascular action of CO. A direct effect of CO on the activity of calcium-activated K (KCa) channels in vascular smooth muscle cells (SMCs) and the underlying mechanisms were investigated in the present study. It was found that CO hyperpolarized single SMCs isolated from rat tail arteries. The whole-cell outward K+ channel currents in vascular SMCs, but not in neuroblastoma cells, were enhanced by CO. Extracellularly or intracellularly applied CO increased the open probability of single high-conductance KCa channels concentration-dependently without affecting the single channel conductance. Although it did not increase the resting level of intracellular free calcium concentration, CO significantly enhanced the calcium sensitivity of single KCa channels in SMCs. Furthermore, the effect of CO on KCa channels was not mediated by cGMP or guanine nucleotide-binding proteins (G proteins, Gi/Go or Gs) in excised membrane patches. Our results suggest that the direct modulation of high-conductance KCa channels in vascular SMCs by CO may constitute a novel mechanism for the vascular effect of CO.

The effect of carbon monoxide on oxygen metabolism in the brains of awake sheep

Eight conscious chronically instrumented sheep were exposed to 1% inspired carbon monoxide (CO) for 35 min. In all sheep, carboxyhaemoglobin (COHb) levels at the end of the exposure were approximately 65%. Mean arterial blood pressure was unchanged with the exception of 2 sheep in which administration was stopped at 25 min following the sudden onset of hypotension. Oxygen delivery to the brain was sustained throughout the administration of CO due to a significant increase in cerebral blood flow (CBF). There was no evidence of either a metabolic acidosis or of lactate production by the brain suggesting the brain did not become hypoxic during the time-course of this study. Despite the apparent lack of hypoxia, oxygen consumption by the brain fell progressively and the sheep showed behavioural changes which varied from agitation to sedation and narcosis. The mechanism of these changes was therefore probably unrelated to hypoxia, but may have been due to raised intracranial pressure or a direct effect of CO on brain function. It is proposed that the time-course of progressive CO poisoning includes a phase in which CBF is elevated, blood pressure is unchanged and the brain is normoxic despite high COHb levels, but that this situation can rapidly evolve into a phase of haemodynamic collapse and severe hypoxia.

Regulation of cyclic nucleotide-gated channels and membrane excitability in olfactory receptor cells by carbon monoxide.

1. The effect of the putative neural messenger carbon monoxide (CO) and the role of the cGMP second-messenger system for olfactory signal generation was examined in isolated olfactory receptor neurons (ORNs) of the tiger salamander. 2. With the use of whole cell voltage-clamp recordings in combination with a series of ionic and pharmological tests, it is demonstrated that exogenously applied CO is a potent activator (K1/2 = 2.9 microM) of cyclic nucleotide-gated (CNG) channels previously described to mediate odor transduction. 3. Several lines of evidence suggest that CO mediates its effect through stimulation of a soluble guanylyl cyclase (sGC) leading to formation of the second-messenger cGMP. This conclusion is based on the findings that CO responses show an absolute requirement for guanosine 5'-triphosphate (GTP) in the internal solution, that no direct effect of CO on CNG currents in the absence of GTP is detectable, and that a blocker of sGC activation, LY85383 (10 microM), completely inhibits the CO response. 4. The dose-response curve for cGMP at CNG channels is used as a calibration to provide a quantitative estimate of the CO-stimulated cGMP formation. This analysis implies that CO is a potent activator of olfactory sGC. 5. Perforated patch recordings using amphotericin B demonstrate that low micromolar doses of CO effectively depolarize the membrane potential of ORNs through tonic activation of CNG channels. This effect in turn regulates excitable and adaptive properties of ORNs and modulates neuronal responsiveness. 6. These data argue for an important role of the cGMP pathway in olfactory signaling and support the idea that CO may function as a diffusible messenger in the olfactory system.

Direct effect of carbon monoxide on hexobarbital metabolism in the isolated perfused liver in the absence of hemoglobin.

The interaction of carbon monoxide (CO) with cytochrome P-450 associated with hexobarbital metabolism was observed in hemoglobin-free perfused rat liver by using a scanning reflectance spectrophotometer. The evidence obtained showed that CO bound to the substrate complexed cytochrome P-450 and, at a CO/O2 ratio of over 0.1 in the perfusate, inhibited the hexobarbital metabolism estimated from the hexobarbital uptake, and oxygen consumption. Although the oxygen supply to the liver cell was one of the major limiting factors during CO hypoxia, CO binding to cytochrome P-450 significantly enhanced the suppression of hexobarbital oxidation caused by hypoxic hypoxia.

Direct effects of carbon monoxide on cardiac function.

The direct effects of carbon monoxide (CO) on cardiac function were investigated in hemoglobin-free living rabbits treated with perfluorochemical blood substitutes. After exchange transfusion with a perfluorochemical emulsion, the erythrocyte count was below 10 X 10(4)/mm3 in each animal. Gas mixtures of oxygen with 5%, 10%, and 20% of CO or nitrogen were administered via a respirator. The results showed that the cardiac effects of CO and nitrogen were significantly different as regards changes in arterial pressure, pulse pressure, heart rate, and the product of heart rate and systolic arterial pressure in spite of the same oxygen tension in the inhaled gases. It was concluded that there was a direct effect of CO not mediated by hemoglobin.

Inhibition of DNA replication in Escherichia coli by cyanide and carbon monoxide.

The inhibition of DNA replication in aerobically growing Escherichia coli by cyanide or carbon monoxide occurs within about 20 s at 15 degrees, as previously reported by Cairns and Denhardt (Cairns, J., and Denhardt, D.T. (1968) J. Mol. Biol. 36, 335-342). This rapid inhibition can be explained by the nearly complete depletion of both intracellular ATP and deoxynucleoside triphosphates which occurs during the time that replication stops. There is probably no direct effect of carbon monoxide on any of the enzymes involved in replication because this reagent has no effect on replication rate or ATP level in anaerobic cells. These cells produce ATP by glycolysis. The inhibition of replication by cyanide, a highly reactive compound, appears to be more complex since anaerobically growing cells can still be completely inhibited, although higher concentrations are required than for aerobically growing cells. The sensitivity of anaerobic cells to cyanide is probably due to the ability of this highly reactive compound to react nonspecifically with many proteins and other molecules.