Heme Oxygenase Substrate Research Articles

Hemin-primed dendritic cells suppress allergic airway inflammation through releasing extracellular vesicles.

Hemin, a substrate of heme oxygenase (HO)‐1, induces HO‐1 expression on a variety of cells to exert anti‐oxidant and anti‐inflammatory roles. However, the role of HO‐1 in allergic diseases for dendritic cells (DCs) is not fully understood. Here, we report that HO‐1 modulates asthmatic airway inflammation by hemin‐treated DC‐released extracellular vesicles (DCEVs). Following induction of bone marrow‐derived DCs by hemin and then by house dust mite (HDM) in vitro, mouse CD4+ naïve T cells were cocultured with DCEVs to determine T helper (h) cell differentiation. C57BL/6 mice were sensitized by different stimuli‐induced DCEVs and challenged with HDM to analyze the changes of inflammatory cells and cytokines in the lung and bronchoalveolar lavage fluid. The results showed that hemin‐treated DCEVs (hemin‐DCEVs) express phosphatidylserine (PS), CD81, heat shock protein 70, and HO‐1, which facilitates regulatory T (Treg) cells differentiation in vitro and in vivo. In HDM‐induced asthmatic mouse model, hemin‐DCEVs inhalation reduced eosinophils infiltration and mucus secretion in the airway, decreased the levels of IL‐4, IL‐5, and IL‐13 in the lung and the number of Th2 cells in mediastinal lymph nodes (MLNs), and increased the number of Treg cells in MLNs. Thus, our study demonstrated, for the first time, that EVs from HO‐1‐overexpressing DCs alleviate allergic airway inflammation of eosinophilic asthma by potentiating Treg cells differentiation and limiting proinflammatory cytokine secretion, which expands our understanding of HO‐1 function, opening the door for HO‐1 inducer‐like hemin as a novel therapeutic strategy for asthma or other allergic diseases.

Endogenous Carbon Monoxide Signaling Modulates Mitochondrial Function and Intracellular Glucose Utilization: Impact of the Heme Oxygenase Substrate Hemin.

Stress-inducible heme oxygenase-1 (HO-1) catalyzes the oxidative cleavage of heme yielding biliverdin, ferrous iron, and carbon monoxide (CO). Heme oxygenase activity has been attributed to antioxidant defense via the redox cycling system of biliverdin and bilirubin. There is increasing evidence that CO is a gaseous signaling molecule and plays a role in the regulation of energy metabolism. Inhibitory effects of CO on the respiratory chain are well established, but the implication of such a process on the cellular stress response is not well understood. By means of extracellular flux analyses and isotopic tracing, we studied the effects of CO, either released from the CO donor CORM-401 or endogenously produced by heme oxygenases, on the respiratory chain and glucose metabolism. CORM-401 was thereby used as a tool to mimic endogenous CO production by heme oxygenases. In the long term (>60 min), CORM-401-derived CO exposure inhibited mitochondrial respiration, which was compensated by increased glycolysis accompanied by a loss of the ATP production rate and an increase in proton leakage. This effect pattern was likewise observed after endogenous CO production by heme oxygenases. However, in the present setting, these effects were only observed when sufficient substrate for heme oxygenases (hemin) was provided. Modulation of the HO-1 protein level was less important. The long-term influence of CO on glucose metabolism via glycolysis was preceded by a short-term response (<30 min) of the cells to CO. Stable isotope-labeling experiments and metabolic flux analysis revealed a short-term shift of glucose consumption from glycolysis to the pentose phosphate pathway (PPP) along with an increase in reactive oxygen species (ROS) generation. Overall, we suggest that signaling by endogenous CO stimulates the rapid formation of reduction equivalents (NADPH) via the PPP, and plays an additional role in antioxidant defense, e.g., via feed-forward stimulation of the bilirubin/biliverdin redox cycling system.

Heme Oxygenase Activity Correlates with Serum Indices of Iron Homeostasis in Healthy Nonsmokers.

Heme oxygenase (HO) catalyzes the breakdown of heme to carbon monoxide, iron, and biliverdin. While the use of genetically altered animal models in investigation has established distinct associations between HO activity and systemic iron availability, studies have not yet confirmed such participation of HO in iron homeostasis of humans. Carbon monoxide produced through HO activity will bind to hemoglobin in circulating erythrocytes, and therefore, blood carboxyhemoglobin (COHb) can be used as an index of HO activity. Using the second National Health and Nutrition Examination Survey, we tested the postulate that HO activity correlates with serum indices of iron homeostasis in healthy nonsmokers. The investigation included 844 lifetime nonsmokers (586 females) 18 years of age and older in the study population. Significant correlations were demonstrated between COHb and several indices of iron homeostasis including serum levels of both ferritin and iron and percentage iron saturation of transferrin. There was no significant association between COHb and hemoglobin, the largest repository of heme in the human body, which functions as the substrate for HO. We conclude that HO activity contributes to human iron homeostasis with significant correlations between COHb and serum ferritin and iron levels and percentage iron saturation of transferrin.

The peripheral antinociceptive effect induced by the heme oxygenase/carbon monoxide pathway is associated with ATP-sensitive K+ channels

Carbon monoxide (CO), a product of the enzyme heme oxygenase (HO), has been recognized to act as an atypical neurotransmitter or neuromodulator in the nervous system, and several lines of evidence suggest that CO may play a role through multiple mechanisms in nociceptive processing. Therefore, the aim of our study was to assess the interaction between the HO/CO pathway and ATP-sensitive K+ channels in hypernociception in response to carrageenan. The electronic von Frey and Randall Selitto tests were applied before and after intraplantar carrageenan administration. The intraplantar hemin carrageenan administration (HO substrate) into the right hindpaw elicited an antinociceptive effect, which was determined to be local because it produced no effect when injected into the contralateral paw. The administration of a HO pathway inhibitor was capable of potentiating the hypersensitivity evoked by carrageenan. Among the three different HO products, CO appears to be the one that attenuated the nociceptive response, whereas biliverdin and iron (II) sulfate failed to cause any significant changes. This blockade of the carrageenan mechanical hyperalgesia induced by the hemin was antagonized by the administration of glybenclamide, and a combination of hemin and diazoxide decreased the hyperalgesic action of carrageenan. These results also suggest that an endogenous opioid system may not be involved because naloxone did not affect the hemin-induced antinociception in the carrageenan model. Our study provides evidence that the peripheral antinociceptive effect of the HO/CO pathway may result from the activation of ATP-sensitive K+ channels.

Carbon Monoxide Involved in Modulating HCO3− Secretion in Rat Duodenum

We examined the effect of the tricarbonyl-dichlororuthenium (II) dimer (CORM-2), a carbon monoxide (CO) donor, on duodenal HCO(3)(-) secretion in rats and investigated whether endogenous CO produced by heme oxygenase (HO) is involved in the regulation of this secretion. Under urethane anesthesia, a duodenal loop was perfused with saline, and HCO(3)(-) secretion was measured at pH 7.0 using a pH stat method. CORM-2, biliverdin, FeCl(2), or ruthenium (III) chloride hydrate (RuCl(3)) was applied to the loop for 5 min. The mucosal application of CORM-2 dose-dependently increased HCO(3)(-) secretion, whereas neither RuCl(3), FeCl(2), nor biliverdin had an effect. The stimulatory effect was significantly attenuated by indomethacin but not N(G)-nitro-L-arginine methyl ester. The application of CORM-2 increased the mucosal prostaglandin (PG) E(2) content of the duodenum. The acid-induced HCO(3)(-) response was markedly inhibited by indomethacin and Sn(IV) protoporphyrin IX dichloride (SnPP; an inhibitor of HO) but not Cu(II) protoporphyrin dichloride, and the inhibitory effect of SnPP was significantly reversed by pretreatment with hemin, a substrate of HO. Perfusion of the duodenal loop with 100 mM HCl for 2 h caused a few hemorrhagic lesions in the mucosa, and this response was significantly worsened by the prior administration of SnPP and indomethacin. The expression of HO-1 but not HO-2 protein was up-regulated in the duodenum after the acid treatment. These results suggest that CO, generated endogenously or exogenously, stimulates HCO(3)(-) secretion in the duodenum, and this effect is mediated by endogenous PGs. It is assumed that HO/CO plays a role in maintaining the integrity of the duodenal mucosa.

Glutamate‐stimulated, astrocyte‐derived carbon monoxide production is intracellular calcium concentration‐dependent in piglets

Glutamate‐stimulated, astrocyte‐derived carbon monoxide (CO) induces vasodilation by activating big conductance Ca2+‐activated potassium (BKCa) channels in arterial myocytes. Here, we examined the hypotheses that glutamate stimulates astrocyte‐derived CO production via heme oxygenase (HO)‐2 activation by elevating [Ca2+]i and Ca2+‐calmodulin complexes in piglets. The major findings are: (1) Exogenous glutamate stimulates Ca2+ oscillations and increases [Ca2+]i in primary‐cultured astrocytes. (2) A [Ca2+]i elevation increases CO production in astrocytes with an EC50 of 310 nM. (3) If [Ca2+]i is held constant, glutamate does not affect CO production. (4) Depleting intracellular Ca2+ stores in astrocytes with a sarco/endoplasmic reticulum Ca2+‐ATPases blocker, thapsigargin, decreases CO production, and blocks the glutamate‐induced increase in CO in astrocytes caused by glutamate and the exogenous HO substrate, hemin. (5) The calmodulin inhibitor, calmidazolium, blocks glutamate‐induced CO production in freshly isolated piglet astrocytes. Taken together, our data are consistent with the hypothesis that glutamate elevates [Ca2+]i in astrocytes, which triggers Ca2+‐calmodulin complex formation, leading to HO‐2 activation and CO production. (NIH 5R37HL042851 and 5R01HL034059).

Enhanced Hemeoxygenase‐1 Signaling in the Rostral Ventrolateral Medulla Mediates a Critical Protective Role Against Genetically Elevated Blood Pressure in SHR Rats

Heme oxygenase (HO)‐derived carbon monoxide (CO) contributes to central regulation of blood pressure (BP). However, the few reported studies demonstrating reductions in BP following microinjection into the nucleus tractus solitarius of the HO substrate hemin, were carried out in anesthetized normotensive rats. We hypothesized that HO‐CO signaling is enhanced in the RVLM of conscious SHRs to prevent exacerbation of hypertension in this genetic model of hypertension. To test this hypothesis, we investigated the effects of intra‐RVLM hemin on BP, HR and RVLM neuronal norepinephrine (index of sympathetic activity; measured in real‐time by in vivo electrochemistry) in SHRs and their normotensive control WKY rats. Furthermore, the type of the specific HO isoenzyme implicated in the hypotensive response was investigated by administering the selective HO‐1 inhibitor zinc protoporphyrin IX (ZNPPIX) 30 minutes prior to hemin. Intra‐RVLM hemin elicited greater and longer‐lasting reductions in BP and RVLM NE in SHRs. These hemodynamic and electrochemical response, which suggest enhanced HO‐CO signaling in the RVLM of SHRs, were confirmed by the greater enzymatic activity of HO in SHRs than in WKY rats, Moreover, ZNPPIX, not only abolished the hemin‐evoked responses in either strain, albeit to a greater extent in SHRs, but also caused a marked increase in baseline pressure in SHRs. The findings suggest an exaggerated HO‐CO signaling, due to higher HO‐1 activity, in the RVLM of SHR, a brainstem area that controls sympathetic outflow. Such enhancement of HO‐CO signaling seems to serve as a protective mechanism that opposes further increases in BP in the SHR.

Heme oxygenase-derived carbon monoxide modulates gonadotropin-releasing hormone release in immortalized hypothalamic neurons

Heme oxygenase (HO), the main enzyme deputed to heme metabolism, has been identified as two main isoforms called HO-1 and HO-2 both present in the central nervous system. Heme oxygenase has been shown to regulate the hypothalamic release of neuropeptides such as corticotrophin-releasing hormone and arginin-vasopressin. The aim of this study was to investigate and further characterize the presence of HO in gonadotropin-releasing hormone (GnRH) secreting hypothalamic neurons, GT1-7 and the role of HO by-products on GnRH secretion. The pulsatile release of GnRH from scattered hypothalamic neurons is the key regulator of mammalian fertility in the central nervous system. GT1-7 cells are immortalized hypothalamic neurons, characterized by spontaneous electrical activity and pulsatile GnRH release, resembling the central control pathway of the hypothalamic pituitary gonadal axis (HPG) in mammals. Hemin, the substrate of HO, significantly stimulated HO activity in static cultures, causing a rapid increase in GnRH release. Neither biliverdin nor bilirubin were able to mimic this rapid stimulatory effect, which was instead caused by carbon monoxide. Evidence of a possible involvement of prostaglandin E 2 in the HO by-product modulated GnRH secretion was reported. The hemin-evoked effect on GT1-7 neurons suggests a direct activity of HO by-products on the hypothalamic neuropeptide secretion, and claims for a possible role of CO in both the modulation of gonadotropin secretion and crosstalk among HPG and stress axis within the mammalian hypothalamus.

Heme Oxygenase Induced Endothelial BKCa Channel Activity Following Chronic Hypoxia

Exposure to chronic hypoxia (CH) results in blunted myogenic and agonist‐induced tone in systemic arteries that persists upon return to normoxia. This impaired vasoconstrictor reactivity is reversed by either removal of the endothelium or by blockade of the CO producing enzyme heme oxygenase (HO). Interestingly, endothelial cell (EC) specific blockade of large conductance Ca2+‐activated K+ (BKCa) channels also restores vasoreactivity in arteries from CH rats. Patch clamp studies demonstrate EC BKCa activity following CH, but not in ECs from control rats, although channels are observed by immunofluorescence (IF) in cells from both groups. Since HO‐derived CO is an endogenous activator of BKCa channels, we hypothesized that HO and BKCa form a functional unit that results in enhanced channel activity following CH. In support of the hypothesis, the HO inhibitor zinc protoporphyrin IX (ZnPPIX) significantly decreased K+ currents in ECs from CH rats, but was without effect in EC from control rats. In addition, the HO substrate hemin increased K+ currents only in the CH group. HO‐2, but not HO‐1, was found to co‐localize with BKCa in cells from each group. These results suggest that although HO and BKCa channels are expressed in EC from each group, HO‐derived CO only stimulates channel activity following CH.

Gene Targeting and Heme Oxygenase-1 Expression in Prevention of Hypertension Induced by Angiotensin II

The report by Vera et al1 that appears in this issue demonstrates that the kidney-specific induction of heme oxygenase (HO)-1 prevents angiotensin (Ang) II hypertension in CL57BL/6J mice. Kidney-specific induction of HO-1 was achieved by implanting intrarenal medullary interstitial catheters in the left kidney of uninephrectomized mice. Infusion of cobalt protoporphyrin, a known inducer of HO-1, produced a significant induction of HO-1 protein levels and increased HO activity largely in the renal medulla. Ang II–dependent hypertension was examined by measuring mean arterial pressure and was found to be attenuated in cobalt protoporphyrin–treated animals. The levels of heme, the substrate of HO, and superoxide were increased in Ang II–treated animal. These increases were blocked in cobalt protoporphyrin–treated animals. The authors conclude that the specific induction of renal HO-1 is responsible for the prevention of Ang II–dependent hypertension and the lowering of blood pressure in this animal model of hypertension. This article raises important findings related to gene targeting in renal injury and HO-1. As shown previously, the degradation of heme is regarded as pivotal in cellular defense for 2 reasons. First, the pro-oxidant heme is removed and, second, increased production of bilirubin/biliverdin and CO, heme degradation products, is now considered beneficial to cellular cytoprotection. Iron, the third heme degradation product, which can stimulate free radical formation, is immediately bound by ferritin. Thus, CO and bilirubin are central to the defense mechanisms that occur in times of stress, a result of elevated levels of HO-1 protein and HO activity. The HO-1/HO-2 system has been long implicated in the regulation of renal function and hypertension (reviewed in Reference 2). Sacerdoti et al3 first reported the benefits of an acute effect, describing that treatment with stannous chloride prevented the development of high blood pressure in the spontaneously hypertensive rats. Subsequently, …

Heme oxygenase–carbon monoxide pathway is involved in regulation of respiration in medullary slice of neonatal rats

Carbon monoxide (CO) is a novel biological messenger molecule. It is well known that CO can be synthesized in mammalian cells. In addition, CO is also demonstrated to participate in many physiological processes, such as vasomotion, thermoregulation and respiratory regulation. The purpose of our present study was to investigate the role of heme oxygenase-carbon monoxide (HO-CO) pathway in central regulation of respiration. The experiments were carried out on the medullary slices of neonatal Sprague-Dawley rats. The discharge activity of the hypoglossal rootlets was recorded to indicate the central rhythmic respiratory activity and its duration (DD), interval (DI), frequency (DF) and integrated amplitude (IA) were analyzed. The slices were perfused with ZnPP-9 (a potent inhibitor of heme oxygenase), CO and hemin (substrate of heme oxygenase), respectively, to observe their effects on respiratory activity. The results obtained were as follows: ZnPP-9 could decrease DD, DI and IA, and increase DF (P<0.05); exogenous CO caused a decrease in DD and DF, and an increase in DI and IA (P<0.05); in response to hemin, DI and IA decreased, DF increased (P<0.05), and DD did not change significantly (P>0.05); administration of both ZnPP-9 and hemin could decrease DI, and increase DF (P<0.05), but did not affect DD and IA significantly (P>0.05). It can be concluded from the results above that the HO-CO pathway may be involved in the regulation of rhythmic respiration at the level of medulla oblongata.

Carbon Monoxide Stimulates the Ca 2+ –Activated Big Conductance K Channels in Cultured Human Endothelial Cells

We used the whole-cell patch-clamp technique to study K channels in the human umbilical vein endothelial cells and identified a 201 pS K channel, which was blocked by tetraethylammonium and iberiotoxin but not by TRAM34 and apamin. This suggests that the Ca(2+)-activated big-conductance K channel (BK) is expressed in endothelial cells. Application of carbon monoxide (CO) or tricarbonylchloro(glycinato)ruthenium(II), a water soluble CO donor, stimulated the BK channels. Moreover, application of hemin, a substrate of heme oxygenase, mimicked the effect of CO and increased the BK channel activity. The stimulatory effect of hemin was significantly diminished by tin mesoporphyrin, an inhibitor of heme oxygenase. To determine whether the stimulatory effect of CO on the BK channel was mediated by NO and the cGMP-dependent pathway, we examined the effect of CO on BK channels in cells treated with, N(G)-nitro-l-arginine methyl ester, 1H(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one, an inhibitor of soluble guanylate cyclase, or KT5823, an inhibitor of protein kinase G. Addition of either diethylamine NONOate or sodium nitroprusside significantly increased BK channel activity. Inhibition of endogenous NO synthesis with N(G)-nitro-l-arginine methyl ester, blocking soluble guanylate cyclase or protein kinase G, delayed but did not prevent the CO-induced activation of BK channels. Finally, application of an antioxidant agent, ebselen, had no effect on CO-mediated stimulation of BK channels in human umbilical vein endothelial cells. We conclude that BK channels are expressed in human umbilical vein endothelial cells and that they are activated by both CO and NO. CO activates BK channels directly, as well as via a mechanism involving NO or the cGMP-dependent pathway.

Non-heme Induction of Heme Oxygenase-1 Does Not Alter Cellular Iron Metabolism

The catabolism of heme is carried out by members of the heme oxygenase (HO) family. The products of heme catabolism by HO-1 are ferrous iron, biliverdin (subsequently converted to bilirubin), and carbon monoxide. In addition to its function in the recycling of hemoglobin iron, this microsomal enzyme has been shown to protect cells in various stress models. Implicit in the reports of HO-1 cytoprotection to date are its effects on the cellular handling of heme/iron. However, the limited amount of uncommitted heme in non-erythroid cells brings to question the source of substrate for this enzyme in non-hemolytic circumstances. In the present study, HO-1 was induced by either sodium arsenite (reactive oxygen species producer) or hemin or overexpressed in the murine macrophage-like cell line, RAW 264.7. Both of the inducers elicited an increase in active HO-1; however, only hemin exposure caused an increase in the synthesis rate of the iron storage protein, ferritin. This effect of hemin was the direct result of the liberation of iron from heme by HO. Cells stably overexpressing HO-1, although protected from oxidative stress, did not display elevated basal ferritin synthesis. However, these cells did exhibit an increase in ferritin synthesis, compared with untransfected controls, in response to hemin treatment, suggesting that heme levels, and not HO-1, limit cellular heme catabolism. Our results suggest that the protection of cells from oxidative insult afforded by HO-1 is not due to the catabolism of significant amounts of cellular heme as thought previously.

Heme oxygenase/carbon monoxide‐biliverdin pathway down regulates neutrophil rolling, adhesion and migration in acute inflammation

Heme oxygenase (HO) activity is known to down-regulate inflammatory events. Here, we address the role of HO and its metabolites, carbon monoxide (CO) and biliverdin (BVD), in leukocyte rolling, adhesion and neutrophil migration during inflammatory processes. Intravital microscopy was used to evaluate leukocyte rolling and adhesion in the mesenteric microcirculation of mice. TNFalpha and IL-1beta were determined by ELISA and HO-1 protein expression by Western blot. Intraperitoneal challenge with carrageenan enhanced HO-1 protein expression in mesentery and bilirubin concentration in peritoneal exudates. Pretreatment of mice with a non-specific inhibitor of HO (ZnDPBG) or with a HO-1 specific inhibitor (ZnPP IX) enhanced neutrophil migration, rolling and adhesion on endothelium induced by carrageenan. In contrast, HO substrate (hemin), CO donor (DMDC) or BVD reduced these parameters. The reduction of neutrophil recruitment promoted by HO metabolites was independent of the production of chemotactic cytokines. Inhibitory effects of CO, but not of BVD, were counteracted by treatment with a soluble guanylate cyclase (sGC) inhibitor, ODQ. Furthermore, inhibition of HO prevented the inhibitory effect of a nitric oxide (NO) donor (SNAP) upon neutrophil migration, while the blockade of NO synthase (NOS) activity by aminoguanidine did not affect the CO or BVD effects. Metabolites of HO decreased leukocyte rolling, adhesion and neutrophil migration to the inflammatory site by a mechanism partially dependent on sGC. Moreover, inhibition by NO of neutrophil migration was dependent on HO activity.

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.

Determination of in vivo carbon monoxide production in laboratory animals via exhaled air

?>Introduction In vitro assays play an important role in the understanding of the heme oxygenase (HO)/carbon monoxide (CO) pathway. However, because physiological roles for the products of this pathway are hypothesized, it is becoming increasingly important to perform in vivo studies. Since CO production is primarily mediated by HO and is excreted mainly by the lungs, measurements of total body CO excretion (VeCO) via the breath allow continuous, noninvasive monitoring of heme degradation and CO and bilirubin production. Here, we describe a modified flow-through method for the collection and quantitation of CO from small laboratory animals. ?>Methods Mice and rats were studied in gas-tight chambers supplied with a continuous flow of CO-free air. CO in the exhaust air was measured by gas chromatography with a reduction gas analyzer. After establishing baseline VeCO levels, animals were administered various xenobiotics known to alter HO activity and further monitored for changes in CO production for up to 12 h without observable distress. ?>Results Administration of heme (substrate for HO) resulted in reproducible increases in CO production; whereas, prior administration of zinc protoporphyrin (ZnPP, HO inhibitor) or cobalt protoporphyrin (CoPP, HO inducer) resulted in respective dose-dependent decreases and increases in the heme-induced CO production. ?>Discussion We have demonstrated that this noninvasive method of CO quantitation reliably estimates heme degradation with sensitivity to distinguish between different types of HO-manipulating xenobiotics in a dose-dependant manner in both mouse and rat models. Furthermore, VeCO measurements allow nearly real-time determinations of CO and bilirubin formation, which helps to illustrate the time course of drug action.

CARBON MONOXIDE ACTUATES O 2-LIMITED HEME DEGRADATION IN THE RAT BRAIN

The biochemical paradigm for carbon monoxide (CO) is driven by the century-old Warburg hypothesis: CO alters O 2-dependent functions by binding heme proteins in competitive relation to 1/oxygen partial pressure ( PO 2). High PO 2 thus hastens CO elimination and toxicity resolution, but with more O 2, CO-exposed tissues paradoxically experience less oxidative stress. To help resolve this paradox we tested the Warburg hypothesis using a highly sensitive gas-reduction method to track CO uptake and elimination in brain, heart, and skeletal muscle in situ during and after exogenous CO administration. We found that CO administration does increase tissue CO concentration, but not in strict relation to 1/ PO 2. Tissue gas uptake and elimination lag behind blood CO as predicted, but 1/ PO 2 vs. [CO] fails even at hyperbaric PO 2. Mechanistically, we established in the brain that cytosol heme concentration increases 10-fold after CO exposure, which sustains intracellular CO content by providing substrate for heme oxygenase (HO) activated after hypoxia when O 2 is resupplied to cells rich in reduced pyridine nucleotides. We further demonstrate by analysis of CO production rates that this heme stress is not due to HO inhibition and that heme accumulation is facilitated by low brain PO 2. The latter becomes rate limiting for HO activity even at physiological PO 2, and the heme stress leads to doubling of brain HO-1 protein. We thus reveal novel biochemical actions of both CO and O 2 that must be accounted for when evaluating oxidative stress and biological signaling by these gases.

Mechanism of internal anal sphincter relaxation by CORM-1, authentic CO, and NANC nerve stimulation.

The present studies compared the effects of CO-releasing molecule (CORM-1), authentic CO, and nonadrenergic noncholinergic (NANC) nerve stimulation in the internal anal sphincter (IAS). Functional in vitro experiments and Western blot studies were conducted in rat IAS smooth muscle. We examined the effects of CORM-1 (50-600 microM) and authentic CO (5-100 microM) and NANC nerve stimulation by electrical field stimulation (EFS; 0.5-20 Hz, 0.5-ms pulse, 12 V, 4-s train). The experiments were repeated after preincubation of the tissues with the neurotoxin TTX, the guanylate cyclase inhibitor 1H-(1,2,4)oxadiazolo-(4,3-a)quinoxalin-1-one (ODQ), the selective heme oxygenase (HO) inhibitor tin protoporphyrin IX (SnPP-IX), the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine (L-NNA), and SnPP-IX + L-NNA. We also investigated the effects of the HO substrate hematin (100 microM). CORM-1, as well as CO, produced concentration-dependent IAS relaxation, whereas hematin had no effect. TTX abolished and L-NNA significantly blocked IAS relaxation by EFS without any effect on CORM-1 and CO. ODQ blocked IAS relaxation by CORM-1, authentic CO, and EFS. SnPP-IX had no significant effect on IAS relaxation by CORM-1, CO, or EFS. The presence of neuronal nitric oxide synthase, HO-1, and HO-2 in IAS smooth muscle was confirmed by Western blot studies. CORM-1 and CO, as well as NANC nerve stimulation, produced IAS relaxation via guanylate cyclase/cGMP-dependent protein kinase activation. The advent of CORM-1 with potent effects in the IAS has significant implications in anorectal motility disorders with regard to pathophysiology and therapeutic potentials.

In vivo role of heme oxygenase in ischemic coronary vasodilation.

The heart constitutively expresses heme oxygenase (HO)-2, which catabolizes heme-containing proteins to produce biliverdin and carbon monoxide (CO). The heart also contains many possible substrates for HO-2 such as heme groups of myoglobin and cytochrome P-450s, which potentially could be metabolized into CO. As a result of observations that CO activates guanylyl cyclase and induces vascular relaxation and that HO appears to confer protection from ischemic injury, we hypothesized that the HO-CO pathway is involved in ischemic vasodilation in the coronary microcirculation. Responses of epicardial coronary arterioles to ischemia (perfusion pressure approximately 40 mmHg; flow velocity decreased by approximately 50%; dL/dt reduced by approximately 60%) were measured using stroboscopic fluorescence microangiography in 34 open-chest anesthetized dogs. Ischemia caused vasodilation of coronary arterioles by 36 +/- 6%. Administration of N(G)-monomethyl-L-arginine (L-NMMA, 3 micromol.kg(-1).min(-1) intracoronary), indomethacin (10 mg/kg iv), and K(+) (60 mM, epicardial suffusion) to prevent the actions of nitric oxide, prostaglandins, and hyperpolarizing factors, respectively, partially inhibited dilation during ischemia (36 +/- 6 vs. 15 +/- 4%; P < 0.05). The residual vasodilation during ischemia after antagonist administration was inhibited by tin mesoporphyrin IX (SnMP, 10 mg/kg iv), which is an inhibitor of HO (15 +/- 4 vs. 7 +/- 2%; P < 0.05 vs. before SnMP). The guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (10(-5) M, epicardial suffusion) also inhibited vasodilation during ischemia in the presence of L-NMMA with indomethacin and KCl. Moreover, administration of heme-L-arginate, which is a substrate for HO, produced dilation after ischemia but not after control conditions. We conclude that during myocardial ischemia, HO-2 activation can produce cGMP-mediated vasodilation presumably via the production of CO. This vasodilatory pathway appears to play a backup role and is activated only when other mechanisms of vasodilation during ischemia are exhausted.

The source of heme for vascular heme oxygenase I: heme uptake in rat aorta

During the last decade, heme oxygenase (HO) and carbon monoxide (CO) have garnered substantial research interest in terms of cell and organ regulation, especially as they bear on the central nervous system, organ transplantation, and the cardiovascular system. While the enzymatic mechanism, substrates, and products of HO are well known, it is not clear whether the cardiovascular system derives its supply of the heme substrate through de novo synthesis or uptake from the extracellular milieu. The objective of the present study was to test the latter possibility in rat aorta and to determine the influence of plasma proteins that bind heme in vivo, viz. hemopexin and albumin. Aortic tissue was exposed to [14C]heme in vitro, and the concentration and time dependence of heme uptake was assessed. The presence of hemopexin or albumin in the incubation medium dramatically decreased heme uptake by the aorta. Heme uptake by aortic tissue was not altered after induction of HO-1, which would be expected to increase tissue heme demand. In summary, the rat, isolated aorta was capable of obtaining heme from its external milieu, but this was obtunded in the presence of the plasma proteins hemopexin or albumin. For normal physiological situations, heme uptake may not be a usual source of substrate for vascular HO and hemoenzymes such as nitric oxide synthase, soluble guanylyl cyclase, and cyclooxygenase.