Cyclooxygenase products stimulate carbon monoxide production by piglet cerebral microvessels

Products of arachidonic acid (AA) metabolism by cyclooxygenase (Cox) are important in regulation of neonatal cerebral circulation. The brain and cerebral microvessels also express heme oxygenase (HO) that metabolizes heme to carbon monoxide (CO), biliverdin, and iron. The purpose of this study in newborn pig cerebral microvessels was to address the hypothesis that Cox products affect HO activity and HO products affect Cox activity. AA (2.0-20 microM) increased prostaglandin E2 (PGE2) measured by radioimmunoassay (RIA) and also CO measured by gas chromatography/mass spectrometry (GC/MS). Further, 10(-4) M indomethacin, which inhibited Cox, reduced both AA and heme-induced CO production. Conversely, neither exogenous 2 x 10(-6) M heme, which markedly increased CO production, nor the inhibitor of HO, chromium mesoporphyrin, altered PGE2 synthesis. Because AA metabolism by Cox generates both prostanoids and superoxides, we determined the effects of the predominant prostanoid and superoxide on CO production. Although PGE2 caused a small increase in CO production, xanthine oxidase plus hypoxanthine, which produces superoxide, strongly stimulated the production of CO by cerebral microvessels. This increase was mildly attenuated by catalase. These data suggest that Cox-catalyzed AA metabolites, most likely superoxide and/or a subsequent reactive oxygen species, increase cerebrovascular CO production. This increase seems to be caused, at least in part, by the elevation of HO-2 catalytic activity. Conversely, Cox activity is not affected by HO-catalyzed heme metabolites. These data suggest that some cerebrovascular functions attributable to Cox activity could be mediated by CO.     

Nitric oxide increases carbon monoxide production by piglet cerebral microvessels

Carbon monoxide (CO) and nitric oxide (NO) can be involved in the regulation of cerebral circulation. Inhibition of production of either one of these gaseous intercellular messengers inhibits newborn pig cerebral arteriolar dilation to the excitatory amino acid glutamate. Glutamate can increase NO production. Therefore, the present study tests the hypothesis that NO, which is increased by glutamate, stimulates the production of CO by cerebral microvessels. Experiments used freshly isolated cerebral microvessels from piglets that express only heme oxygenase-2 (HO-2). CO production was measured by gas chromatography-mass spectrometry. Although inhibition of nitric oxide synthase (NOS) with N(omega)-nitro-l-arginine (l-NNA) did not alter basal HO-2 catalytic activity or CO production, l-NNA blocked glutamate stimulation of HO-2 activity and CO production. Furthermore, the NO donor sodium nitroprusside mimicked the actions of glutamate on HO-2 and CO production. The action of NO appears to be via cGMP because 8-bromo-cGMP mimics and 1H-[1,2,4]oxadiazole-[4,3-a]quinoxalin-1-one (ODQ) blocks glutamate stimulation of CO production and HO-2 catalytic activity. Inhibitors of neither casein kinase nor phosphotidylinositol 3-kinase altered HO-2 catalytic activity. Conversely, inhibition of calmodulin with calmidazolium chloride blocked glutamate stimulation of CO production and reduced HO-2 catalytic activity. These data suggest that glutamate may activate NOS producing NO that leads to CO synthesis via a cGMP-dependent elevation of HO-2 catalytic activity. These results are consistent with the findings in vivo that either HO or NOS inhibition blocks cerebrovascular dilation to glutamate in piglets.     

Inhaled carbon monoxide provides cerebral cytoprotection in pigs

Carbon monoxide (CO) at low concentrations imparts protective effects in numerous preclinical small animal models of brain injury. Evidence of protection in large animal models of cerebral injury, however, has not been tested. Neurologic deficits following open heart surgery are likely related in part to ischemia reperfusion injury that occurs during cardiopulmonary bypass surgery. Using a model of deep hypothermic circulatory arrest (DHCA) in piglets, we evaluated the effects of CO to reduce cerebral injury. DHCA and cardiopulmonary bypass (CPB) induced significant alterations in metabolic demands, including a decrease in the oxygen/glucose index (OGI), an increase in lactate/glucose index (LGI) and a rise in cerebral blood pressure that ultimately resulted in increased cell death in the neocortex and hippocampus that was completely abrogated in piglets preconditioned with a low, safe dose of CO. Moreover CO-treated animals maintained normal, pre-CPB OGI and LGI and corresponding cerebral sinus pressures with no change in systemic hemodynamics or metabolic intermediates. Collectively, our data demonstrate that inhaled CO may be beneficial in preventing cerebral injury resulting from DHCA and offer important therapeutic options in newborns undergoing DHCA for open heart surgery.     

Exercise restores beta-adrenergic vasorelaxation in aged rat carotid arteries

Aging is associated with alterations in beta-adrenergic receptor (beta-AR) signaling and reduction in cardiovascular responses to beta-AR stimulation. Because exercise can attenuate age-related impairment in myocardial beta-AR signaling and function, we tested whether training could also exert favorable effects on vascular beta-AR responses. We evaluated common carotid artery responsiveness in isolated vessel ring preparations from 8 aged male Wistar-Kyoto (WKY) rats trained for 6 wk in a 5 days/wk swimming protocol, 10 untrained age-matched rats, and 10 young WKY rats. Vessels were preconstricted with phenylephrine (10-6 M), and vasodilation was assessed in response to the beta-AR agonist isoproterenol (10-10-3 x 10-8 M), the alpha2-AR agonist UK-14304 (10-9-10-6 M), the muscarinic receptor agonist ACh (10-9-10-6 M), and nitroprusside (10-8-10-5 M). beta-AR density and cytoplasmic beta-AR kinase (beta-ARK) activity were tested on pooled carotid arteries. beta-ARK expression was assessed in two endothelial cell lines from bovine aorta and aorta isolated from a 12-wk WKY rat. beta-AR, alpha2-AR, and muscarinic responses, but not that to nitroprusside, were depressed in untrained aged vs. young animals. Exercise training restored beta-AR and muscarinic responses but did not affect vasodilation induced by UK-14304 and nitroprusside. Aged carotid arteries showed reduced beta-AR number and increased beta-ARK activity. Training counterbalanced these phenomena and restored beta-AR density and beta-ARK activity to levels observed in young rat carotids. Our data indicate that age impairs beta-AR vasorelaxation in rat carotid arteries through beta-AR downregulation and desensitization. Exercise restores this response and reverts age-related modification in beta-ARs and beta-ARK. Our data support an important role for beta-ARK in vascular beta-AR vasorelaxation.     

Alterations in heme oxygenase/carbon monoxide system in pulmonary arteries in hypertension

Enhancement of the heme oxygenase/carbon monoxide (HO/CO) system has been shown to lower blood pressure (BP) in young (8 weeks), but not in adult (20 weeks) spontaneously hypertensive (SHR) rats. The reasons for this selective effect still remain puzzling. We investigated the effects of hemin on the HO/CO system of the pulmonary artery (PA) in SHR and Wistar-Kyoto (WKY) rats at different ages and evaluated the hemin-dependent changes in sGC and cGMP pathways. Hemin administration resulted in an evident reduction of BP (from 148.6 +/- 3.2 to 125.8 +/- 2.6 mmHg, P < 0.01) in young, but not in prehypertensive (4 weeks) or adult SHR or WKY rats at all ages. Coadministration of the HO inhibitor, chromium mesoporphyrin, with hemin, cancelled the BP-lowering effect of hemin. Remarkably, lower expression levels of HO-1, HO-2, and sGC paralleled with reduced HO activity and cGMP content were observed in PA from 8-week SHR rats, but not from adult SHR or WKY rats of all ages. Interestingly, hemin treatment restored these deficiencies, although the expression level of non-inducible HO-2 protein remained unchanged. We conclude that in young and prehypertensive SHR rats, an impaired HO/CO-sGC/cGMP system in the PA might be indicative of the pathogenesis and development of hypertension. In contrast, the HO/CO system in the PA of adult SHR rats was upregulated as a compensatory reaction to elevated BP and desensitization of the downstream targets of the sGC/cGMP pathway occurred.     

Characterization of the vasorelaxation to methanandamide in rat gastric arteries

In the present study, the relaxant effect of the cannabinoid methanandamide was explored in rat gastric arteries. Since in some vessels cannabinoids have been shown to release calcitonin gene-related peptide (CGRP) from perivascular nerves, the influence of methanandamide was compared with that of exogenous CGRP. Methanandamide and CGRP elicited concentration-dependent, endothelium-independent relaxations. Methanandamide-induced relaxations were unaffected by the CB1 receptor antagonist AM251, the CB2 receptor antagonists AM630 and SR144528, and combined pre-exposure to AM251 and SR144528. Pre-exposure to O-1918, an antagonist of a novel nonCB1/nonCB2 cannabinoid receptor, did not influence the relaxations to methanandamide. Capsaicin or capsazepine treatment slightly inhibited methanandamide-induced relaxations. Preincubation with 30 mmol/L extracellular K+ or 3 mmol/L TEA had no significant effect on the responses elicited by methanandamide, but reduced CGRP-induced relaxations. Relaxation to 10(-5) mol/L methanandamide was significantly blunted by Bay K8644 and by preincubation with nifedipine. Furthermore, 10(-5) mol/L methanandamide significantly inhibited CaCl2-induced contractions in norepinephrine-stimulated vessels previously depleted of intra- and extracellular Ca2+. Finally, preincubation with 10(-5) mol/L methanandamide almost completely abolished high K+-induced contractions. These findings suggest that the vasorelaxant action of methanandamide in rat gastric arteries is not mediated by stimulation of known cannabinoid receptors and only partly related to stimulation of TRPV1 receptors on perivascular nerves. At high concentrations, methanandamide might induce relaxation by reducing calcium entry into the smooth muscle cells.     

The effects of acute carbon monoxide intoxication on the cerebral energy metabolism of the rat.

The cerebal metabolic effects of 60 min exposure to 0.5, 1.0, 1.5, and 2.0% carbon monoxide (CO) and 60 min exposure to 1.0% CO were studied in lightly anesthetized rats by measurement of brain tissue contents of glycolytic and citric acid cycle intermediates, as well as tissue energy phosphates. The results indicate that cerebral energy homeostasis is maintained until advanced levels of CO intoxication (2.0%) are reached. Animals exposed to 2.0% CO developed significant decreases in systemic blood pressure, with attendent decreases in cerebral ATP, increases in ADP and AMP, plus early depletions of tissue citrate and alpha-oxyglutarate. The similarity of this pattern to that previously documented for various cerebral oligemic states suggests a possible modifying role for altered cerebral production in its production. A correlation between conscious behavior and cerebral energy state was suggested by the observation that unanesthetized animals exposed to 1.0% CO for 30 and 60 min retained consciousness, whereas animals exposed to 2.0% CO for 30 min became unresponsive late on in the exposure. A comparison of CO induced changes in intermediary metabolites, energy phosphates, intracellular pH, and cytoplasmic redox state with those seen in hypoxemia indicate no basic qualitative or quantiative differences in the metabolic response of brain tissue to the two conditions.     

The measurement of endogenous carbon monoxide production

Recent findings that heme oxygenase-1 can be induced by oxidative stress and inflammation in many different cellular systems, and that carbon monoxide (CO) produced as a by-product of this enzyme is a signaling molecule, have generated a major research area with hundreds of studies published over the last few years. The measurement of expired CO concentration has been used in humans as a biomarker of induced heme oxygenase resulting from inflammation or oxidative stress, but a precise method of measuring endogenous CO production that can be easily used to study patients is needed. The present study describes such a method. The described method allows calculation of the rate of heme catabolism with a precision of ±2 μmol/h, ～10% of the mean normal rate in subjects used in this investigation. This method, which is subject-patient friendly, precise, and inexpensive to perform, should be applicable to studies performed on humans with induced heme oxygenase and studies of effects of therapy for inflammatory and hemolytic diseases.     

Mechanism of oxidation of carbon monoxide by bacteria

Double label experiments were performed employing ¹³CO and either H₂¹⁸O or ¹⁸O₂ in the presence of a CO utilizing bacterium. CO₂ generated was trapped and $\text{m}\text{e}$ ratios $\text{47}\text{45}$ showed that the second oxygen atom in the oxidation of CO to CO₂ by this bacterium comes neither from O₂ nor H₂O.     

Photooxidative production of carbon monoxide by phototrophic microorganisms

Net photosynthesis and CO production were measured in cell suspensions of Chlorella fusca. The rate of net photosynthesis showed saturation curves with increasing radiation intensities and CO₂-mixing ratios. Maximum rates were found at 35° C with a sharp decrease at higher temperatures. By contrast, the rate of CO production was proportional to the radiation intensity and did not show any saturation up to 1.5 kW · m^?2 white light. The CO-production rate was higher in blue than in red light and was independent of the CO₂-mixing ratio of the carrier gas within a range of 0–1000 ppmv. We found that the CO-production rate was constant within the physiological temperature range of 10–35° C, but increased considerably at higher temperatures and that CO production by the chlorophyll-deficient mutant of C. fusca was 5 times that of the wild type. In addition, we measured CO production in cell suspensions of Chromatium vinosum, Rhodopseudomonas sphaeroides and Rhodopseudomonas acidophila, which were grown either anaerobically in the light or aerobically in the dark. CO production could only be observed when the cells were incubated in the presence of oxygen and light. Under these conditions more CO was produced by aerobically grown cells than by phototrophically grown cells of R. sphaeroides and R. acidophila. The results obtained indicate that CO was produced by photosensitized oxidations and not by metabolic processes.     

Regional cerebral blood flow of the rat in acute carbon monoxide intoxication.

The effect of carbon monoxide (CO) on the regional cerebral blood flow was studied by exposing lightly anesthetized rats for 30 min to 0.5, 1.0, 1.5, and 2.0% CO gas mixtures. Cortical cerebral blood flow (CBF) increases of near 200%, 300%, and 400% control were observed at 0.5, 1.0, and 1.5% CO, respectively; whereas at 2.0% CO a reversal of the CBF increase was observed with values declining to near 300% control. The CBF response of subcortical, cerebellar, and brain stem areas was quantitatively similar to that of cortex, indicating that the CBF changes in CO intoxication are general. The decrease in CBF at 2.0% CO was related to significant decreases in arterial CO2 tension. Comparison of the CBF data to previous metabolic results in CO poisoning suggests that the CBF increases are a principal factor in the maintenance of an intact energy state in CO poisoning.