Chromium Mesoporphyrin Research Articles

Heme oxygenase-mediated endothelial dysfunction in DOCA-salt, but not in spontaneously hypertensive, rat arterioles.

Vascular heme oxygenase (HO) metabolizes heme to form carbon monoxide. Carbon monoxide inhibits nitric oxide synthase and promotes endothelium-dependent vasoconstriction. We reported HO-1-mediated endothelial dysfunction in Dahl salt-sensitive hypertension. Previous studies suggested that salt-sensitive hypertensive rats, but not spontaneously hypertensive rats (SHR), display endothelial dysfunction. This study examines the hypothesis that HO-1-mediated arteriolar endothelial dysfunction develops in deoxycorticosterone acetate (DOCA)-salt hypertensive (DOCA) rats, but not in SHR. Uninephrectomized (isoflurane anesthesia) male Sprague-Dawley rats received DOCA injections and saline drinking solution for 4 wk. Rats subjected to sham surgery received vehicle injections and tap water. Blood pressure was elevated in DOCA rats and SHR compared with sham and Wistar-Kyoto (WKY) groups. Aortic HO-1 expression and blood carboxyhemoglobin levels were elevated in the DOCA group, but not in SHR. In isolated gracilis muscle arterioles, ACh caused concentration-related vasodilation in all groups, with attenuated maximum responses in DOCA, but not in SHR, arterioles. Acute pretreatment with an inhibitor of HO, chromium mesoporphyrin, restored ACh-induced responses in DOCA arterioles to sham levels. ACh responses remained the same in SHR and WKY arterioles after chromium mesoporphyrin treatment. These data show that HO-1 levels and activity are increased and arteriolar responses to ACh are decreased in DOCA rats, but not in SHR. Furthermore, in DOCA arterioles, an inhibitor of HO restores ACh-induced vasodilation to sham levels. These results suggest that elevated HO-1 levels and activity, not resulting from hypertension per se, contribute to endothelial dysfunction in DOCA rats.

Protective mechanisms during ischemic tolerance in skeletal muscle

The purpose of this study was to test specific mechanisms of protection afforded the rat extensor digitorum longus (EDL) muscle during ischemic tolerance. Two days following five cycles of 10 min ischemia and 10 min reperfusion, heme oxygenase (HO) and calcium-dependent nitric oxide synthase (cNOS) activities were increased 2- and 2.5-fold ( p < .05), respectively. Interestingly, calcium-independent NOS (iNOS) activity was completely downregulated ( p < .05). The levels of superoxide dismutase (SOD) and catalase were increased 2-fold ( p < .05), while glutathione peroxidase activity remained unchanged from non-preconditioned controls. Using intravital microscopy combined with chromium mesoporphyrin (CrMP), a selective HO inhibitor, and l-NAME, a NOS inhibitor, the roles of HO and cNOS were evaluated. Ischemic tolerance in the EDL muscle, 48 h after the preconditioning stimulus, was characterized by complete protection from both microvascular perfusion deficits and tissue injury after a 2-h period of ischemia. Removal of NOS activity completely removed the benefit afforded microvascular perfusion, while inhibition of HO activity prevented the parenchymal protection. These data suggest that ischemic tolerance within skeletal muscle is associated with the upregulation of specific cytoprotective proteins and that the benefits afforded by cNOS and HO activity are spatially discrete to the microvasculature and parenchyma, respectively.

Heme oxygenase-1 protects HepG2 cells against cytochrome P450 2E1-dependent toxicity

The inducible form of heme oxygenase (HO-1) is increased during oxidative injury and HO-1 is believed to be an important defense mechanism against such injury. Arachidonic acid (AA) and l-buthionine-( S,R)-sulfoximine (BSO), which lowers GSH levels, cause cytochrome P450 2E1 (CYP2E1)-dependent oxidative injuries in HepG2 cells (E47 cells). Treatment of E47 cells with 50 μM AA or 100 μM BSO for 48 h was recently shown to increase HO-1 mRNA, protein, and activity. The possible functional significance of this increase in protecting against CYP2E1-dependent toxicity was evaluated in the current study. The treatment with AA and BSO caused loss of cell viability (40 and 50%, respectively) in E47 cells. Chromium mesoporphyrin (CrMP), an inhibitor of HO activity, significantly potentiated this cytotoxicity. ROS production, lipid peroxidation, and the decline in mitochondrial membrane potential produced by AA and BSO were also enhanced in the presence of CrMP in E47 cells. Infection with an adenovirus expressing rat HO-1 protected E47 cells from AA toxicity, increasing cell viability and reducing LDH release. HO catalyzes formation of CO, bilirubin, and iron from the oxidation of heme. Bilirubin was not protective whereas iron catalyzed the AA toxicity. The carbon monoxide (CO) scavenger hemoglobin enhanced AA toxicity in E47 cells analogous to CrMP, whereas exposure to exogenous CO partially reduced AA toxicity and the enhanced AA toxicity by CrMP. Addition of exogenous CO to the cells inhibited CYP2E1 catalytic activity, as did overexpression of the rat HO-1 adenovirus. These results suggest that induction of HO-1 protects against CYP2E1-dependent toxicity and this protection may be mediated in part via production of CO and CO inhibition of CYP2E1 activity.

Hyperosmolar solution effects in guinea pig airways. III. Studies on the identity of epithelium-derived relaxing factor in isolated perfused trachea using pharmacological agents.

Hyperosmolar challenge of airway epithelium stimulates the release of epithelium-derived relaxing factor (EpDRF), but the identity of EpDRF is not known. We examined the effects of pharmacological agents on relaxant responses of methacholine (3 x 10(-7) M)-contracted guinea pig perfused trachea to mucosal hyperosmolar challenge using D-mannitol. Responses were inhibited by gossypol (5 x 10(-6) M), an agent with diverse actions, by the carbon monoxide (CO) scavenger hemoglobin (10(-6) M), and by the heme oxygenase (HO) inhibitor zinc (II) protoporphyrin IX (10(-4) M). The HO inhibitor chromium (III) mesoporphyrin IX (10(-4) M) was not inhibitory, and the HO activator heme-L-lysinate (3 x 10(-4) M) did not evoke relaxant responses. The CO donor tricarbonyldichlororuthenium (II) dimer (2.2 x 10(-4) M) elicited small relaxation responses. Other agents without an effect on responses included: apyrase, adenosine, 6-anilino-5,8-quinolinequinone (LY83583), proadifen, (E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl][[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid (MK 571), diphenhydramine, glibenclamide, HgCl2, tetrodotoxin, nystatin, alpha-hemolysin, 8-bromoguanosine 3',5'-cyclic monophosphothioate, Rp-isomer, 12-O-tetradecanoylphorbol-13-acetate, cholera toxin, pertussis toxin, thapsigargin, nifedipine, Ca(2+)-free mucosal solution, hydrocortisone, and epidermal growth factor. Cytoskeleton inhibitors, includingerythro-9-(2-hydroxyl-3-nonyl)adenine, colchicine, nocodazole, latrunculin B, and cytochalasins B and D, had no effect on relaxation responses. The results suggest provisionally that a portion of EpDRF activity may be due to CO and that the release of EpDRF does not involve cytoskeletal reorganization.

CO modulates pulmonary vascular response to acute hypoxia: relation to endothelin.

Pulmonary intralobar arteries express heme oxygenase (HO)-1 and -2 and release carbon monoxide (CO) during incubation in Krebs buffer. Acute hypoxia elicits isometric tension development (0.77 +/- 0.06 mN/mm) in pulmonary vascular rings treated with 15 micromol/l chromium mesoporphyrin (CrMP), an inhibitor of HO-dependent CO synthesis, but has no effect in untreated vessels. Acute hypoxia also induces contraction of pulmonary vessels taken from rats injected with HO-2 antisense oligodeoxynucleotides (ODN), which decrease pulmonary HO-2 vascular expression and CO release. Hypoxia-induced contraction of vessels treated with CrMP is attenuated (P < 0.05) by endothelium removal, by CO (1-100 micromol/l) in the bathing buffer, and by endothelin-1 (ET-1) receptor blockade with L-754142 (10 micromol/l). CrMP increases ET-1 levels in pulmonary intralobar arteries, particularly during incubation in hypooxygenated media. CrMP also causes a leftward shift in the concentration-response curve to ET-1, which is offset by exogenous CO. In anesthetized rats, pretreatment with CrMP (40 micromol/kg iv) intensifies the elevation of pulmonary artery pressure elicited by breathing a hypoxic gas mixture. However, acute hypoxia does not elicit augmentation of pulmonary arterial pressure in rats pretreated concurrently with CrMP and the ET-1 receptor antagonist L-745142 (15 mg/kg iv). These data suggest that a product of HO activity, most likely CO, inhibits hypoxia-induced pulmonary vasoconstriction by reducing ET-1 vascular levels and sensitivity.

Inhibition of heme oxygenase decreases sodium and fluid absorption in the loop of Henle.

We previously demonstrated that carbon monoxide (CO) stimulates the apical 70-pS K+ channel in the thick ascending limb (TAL) of the rat kidney (Liu HJ, Mount DB, Nasjletti A, and Wang WH. J Clin Invest 103: 963-970, 1999). Because the apical K+ channel plays a key role in K+ recycling, we tested the hypothesis that heme oxygenase (HO)-dependent metabolites of heme may affect Na+ transport in the TAL. We used in vivo microperfusion to study the effect of chromium mesoporphyrin (CrMP), an inhibitor of HO, on fluid absorption (Jv) and Na+ absorption (JNa) in the loop of Henle and renal clearance methods to examine the effect of CrMP on renal sodium excretion. Microperfusion experiments demonstrated that addition of CrMP to the loop of Henle decreased Jv by 13% and JNa by 20% in animals on normal rat chow and caused a decrease in Jv (39%) and JNa (40%) in rats on a high-K+ (HK) diet. The effect of CrMP is the result of inhibition of HO because addition of MgPP, an analog of CrMP that does not inhibit HO, had no effect on Jv. Western blot analysis showed that HO-2 is expressed in the kidney and that the level of HO-2 was significantly elevated in animals on a HK diet. Renal clearance studies demonstrated that the infusion of CrMP increased the excretion of urinary Na+ (ENa) and volume (UV) without changes in glomerular filtration rate. The effect of CrMP on ENa and UV was larger in HK rats than those kept on normal chow. We conclude that HK intake increases HO-2 expression in the kidney and that HO-dependent metabolites of heme, presumably CO, play a significant role in the regulation of Na+ transport in the loop of Henle.

Age-related alterations in soluble guanylyl cyclase and cGMP pathway in spontaneously hypertensive rats.

Vascular contractility and blood pressure (BP) are regulated by soluble guanylyl cyclase (sGC) and cyclic guanosine monophosphate (cGMP) pathway, which can be influenced by heme oxygenase (HO)-derived carbon monoxide (CO). The age-related changes in sGC/cGMP pathway in tail artery smooth muscle cells (SMCs) in hypertension have not been systematically investigated. In the present study, spontaneously hypertensive rats (SHR) of 4, 8, and 20 weeks old were used. The basal and hemin-modulated levels of sGC and cGMP in tail artery tissues were examined. Although BP of 20-week SHR was significantly elevated, sGC and cGMP levels were unaltered compared with age-matched Wistar-Kyoto rats (WKY). The levels of sGC and cGMP were significantly lower in 4- and 8-week SHR compared with age-matched WKY although BP of 4-week SHR was normotensive. Hemin administration resulted in a significant decrease in BP in 8-week (158.7 +/- 2.4 versus 123.5 +/- 1.3 mmHg, P < 0.01), but not in pre-hypertensive (4 weeks) or 20-week SHR or WKY at all ages. Coincidently, sGC and cGMP levels in 8-week SHRs were significantly elevated and so did the expression levels of HO-1. Hemin treatment did not increase the cyclic adenosine monophosphate (cAMP) content of tail artery from 8-week SHR. The constitutive HO-2 levels remained unchanged in 8- and 20-week SHR and age-matched WKY. The HO-activity inhibitor, chromium mesoporphyrin, abolished the BP-lowering and HO- stimulating effects of hemin in young SHR. Our results suggest that alteration in sGC/cGMP pathway in vascular SMCs precedes the occurrence of hypertension but returns to normal once hypertension is fully manifested.

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.

Carbon monoxide mediates vasodilator effects of glutamate in isolated pressurized cerebral arterioles of newborn pigs.

The excitatory neurotransmitter glutamate causes dilation of newborn pig cerebral arterioles in vivo that is blocked by inhibition of carbon monoxide (CO) production. CO, a potent dilator in cerebral circulation in vivo, is produced endogenously in cerebral microvessels via heme oxygenase (HO). In isolated pressurized cerebral arterioles (approximately 200 microm) from newborn pigs, we investigated the involvement of CO and the endothelium in response to glutamate. A CO-releasing molecule, dimanganese decacarbonyl (10(-8)-10(-6) M), dilated cerebral arterioles. Glutamate (10(-6)-10(-4) M) and 1-aminocyclopentane-cis-1,3-dicarboxylic acid (cis-ACPD; 10(-6)-10(-5) M), a N-methyl-D-aspartate (NMDA) receptor agonist, caused cerebral vascular dilation. Dilation of cerebral arterioles to glutamate and cis-ACPD was abolished by chromium mesoporphyrin (CrMP; 10(-6) M), a HO inhibitor. In contrast, CrMP did not alter dilation to isoproterenol, a -adrenergic receptor agonist. Endothelium-denuded cerebral arterioles did not dilate to glutamate or bradykinin (endothelium-dependent dilator), whereas responses to isoproterenol were preserved. These data indicate that cerebral arterioles from newborn pigs may directly respond to glutamate and the NMDA receptor agonists by endothelium-dependent dilation that involves stimulation of CO production via the HO pathway in the endothelium.

Heme oxygenase in the rat anterior pituitary: immunohistochemical localization and possible role in gonadotropin and prolactin secretion.

The objectives of this study were to determine if heme oxygenase (HO), which catalyzes the degradation of heme and the formation of carbon monoxide (CO), is localized in the rat anterior pituitary and, if so, to determine if hemin (a substrate for HO) or chromium mesoporphyrin (CrMP) (an inhibitor of HO), alter pituitary gonadotropin and prolactin secretion. For localization of HO, sections of anterior pituitaries obtained from mature Holtzman Sprague-Dawley rats in different stages of the estrous cycle were immunostained for two of the HO isoforms, HO-1 and HO-2. The immunostaining for the inducible HO isoform (HO-1) was limited to discrete populations of pituitary cells, whereas the constitutive isoform (HO-2) had a more widespread distribution. The afternoon surge of leutinizing hormone (LH) in the plasma of ovariectomized, estradiol-treated rats was advanced by 2 hr after 7 days of treatment with CrMP (4 micro M/kg), and this effect was reversed when hemin (30 micro M/kg) was co-administered with CrMP. The afternoon follicle-stimulating hormone (FSH) surge was not affected by either treatment. In contrast, the afternoon prolactin (PRL) surge was completely blocked or delayed by CrMP treatment, and this effect was not reversed by hemin. In vitro perifusion of pituitary explants with CrMP also significantly reduced PRL release compared with secretion from untreated explants. In vitro gonadotropin-releasing hormone (GnRH)-stimulated FSH secretion was significantly increased from pituitary explants of ovariectomized, estradiol-treated rats treated in vivo with hemin but was unaffected by CrMP treatment, whereas GnRH-stimulated LH release was not affected by hemin but was increased by CrMP treatment. In conclusion, this study demonstrates that HO exists in the rat anterior pituitary gland, and that a substrate and an inhibitor of this enzyme alter the secretion of gonadotropins and PRL.

Heme oxygenase in the rat ovary: immunohistochemical localization and possible role in steroidogenesis.

The objectives of this study were to determine if heme oxygenase (HO), which catalyzes the degradation of heme and the formation of carbon monoxide (CO), is localized in the rat ovary and, if so, to determine if hemin (a substrate for HO) or chromium mesoporphyrin (CrMP, an inhibitor of HO), alter basal or gonadotropin-induced steroidogenesis. The hypothesis was that CO produced endogenously by HO suppresses steroid hormone production by the ovary similar to the action of nitric oxide. For the histological localization of HO, sections of ovaries obtained from mature Holtzman Sprague-Dawley rats were immunostained for two of the HO isoforms, HO-1 and HO-2. Theca cells and granulosa cells of follicles and luteal cells stained for HO-1, whereas the ovarian stroma showed a low intensity of staining. Theca, granulosa cells, and corpora lutea as well as the ovarian stroma exhibited HO-2 staining. HO-2 immunostaining appeared more intense for theca cells than granulosa cells. In the study of steroidogenesis, three daily injections of hemin stimulated basal- and gonadotropin-induced androstenedione and estradiol secretion from ovaries of pregnant mare serum gonadotropin-treated immature rats in vitro, but had no effect on progesterone production. A similar treatment with CrMP suppressed basal- and gonadotropin-induced secretion of progesterone and androstenedione, but had no effect on estradiol production. These data, taken together, show the existence of HO in the rat ovary and suggest a possible stimulatory role of endogenous CO in the production of ovarian steroids.

Carbon monoxide as an attenuator of vasoconstriction in piglet cerebral arterioles.

Carbon monoxide (CO) is an endogenous dilator in the newborn cerebral circulation. The present study addressed the hypothesis that endogenous CO attenuates pial arteriolar vasoconstriction caused by hypocapnia, platelet activating factor, and elevated blood pressure. Experiments used anesthetized piglets with implanted, closed cranial windows. Topical application of a metal porphyrin inhibitor of heme oxygenase was used to inhibit production of CO. Chromium mesopophyrin increased vasoconstriction in response to hypocapnia. The constrictor response to a topical stimulus, platelet activating factor, was also increased by application of chromium mesoporphyrin. Inhibition of heme oxygenase did not constrict pial arterioles in normotensive newborn pigs (mean arterial pressure of about 70 mmHg), but did constrict pial arterioles of piglets with experimentally induced increases in arterial pressure (mean arterial pressure greater than 90 mmHg). In fact, pial arterioles of normotensive piglets transiently dilated to chromium mesoporphyrin, whereas those of hypertensive piglets progressively constricted during 10 min of chromium mesoporphyrin treatment. Therefore, inhibition of heme oxygenase augments cerebral vasoconstriction in response to several very different constrictor stimuli. These data suggest endogenous CO attenuates vasoconstrictor responses in the newborn cerebral circulation.

Heme oxygenase inhibitor restores arteriolar nitric oxide function in dahl rats.

Vascular tissues express heme oxygenase (HO), which metabolizes heme to form carbon monoxide (CO). CO relaxes vascular smooth muscle but inhibits nitric oxide (NO) formation. Decreased NO synthesis may contribute to salt-induced hypertension in Dahl salt-sensitive (DS) rats. The current study examines the hypothesis that elevated levels of endogenous CO contribute to NO dysfunction in salt-induced hypertensive DS rats. Male DS rats were placed on high-salt (8% NaCl, HS) or low-salt (0.3% NaCl, LS) diets for 4 weeks. With respect to the LS group, the HS group's blood pressure and carboxyhemoglobin levels were elevated, and abdominal aortas showed 6-fold higher HO-1 protein levels. Experiments used isolated pressurized first-order gracilis muscle arterioles superfused with oxygenated modified Krebs buffer. An inhibitor of NO synthase, Nomega-nitro-L-arginine methyl ester (L-NAME), caused concentration-dependent vasoconstriction in both groups, with attenuated responses in HS arterioles. HS arterioles also showed attenuated vasodilatory responses to an endothelium-dependent vasodilator, acetylcholine. Acute pretreatment with an inhibitor of HO, chromium mesoporphyrin, enhanced vascular responses to L-NAME and acetylcholine in both groups but abolished the differences between HS and LS arterioles. These data show that HO-1 protein levels and CO production are increased in HS rats. Arteriolar responses to L-NAME and acetylcholine are impaired in HS rats compared with LS animals, and this difference can be abolished by an inhibitor of endogenous CO production. These results suggest that elevated levels of endogenous CO contribute to arteriolar NO dysfunction in DS rats with salt-induced hypertension.

Vascular effects of a heme oxygenase inhibitor are enhanced in the absence of nitric oxide

Background Vascular endothelium and smooth muscle express heme oxygenase (HO) that metabolizes heme to biliverdin, iron and carbon monoxide (CO). Carbon monoxide promotes endothelium-independent vasodilation, but also inhibits nitric oxide formation. This study examines the hypothesis that an inhibitor of HO promotes endothelium-independent vasoconstriction, which is attenuated in the presence of unabated nitric oxide formation. Methods In vivo studies were conducted in anesthetized male Sprague-Dawley (SD) rats instrumented with flow probes and arterial catheters. In vitro experiments were performed on pressurized first-order gracilis muscle arterioles isolated from male SD rats superfused with oxygenated modified Krebs buffer. Results Vascular smooth muscle and endothelium showed positive HO-1 and HO-2 immunostaining. In anesthetized rats the HO inhibitor chromium mesoporphyrin (CrMP; 45 μmol/kg intraperitoneally) had minimal effect on hindlimb resistance. However, in animals pretreated with N ω-nitro- l-arginine methyl ester (L-NAME; 300 mg/kg intraperitoneally), CrMP substantially increased hindlimb resistance. In contrast, in rats infused with phenylephrine to increase blood pressure and vascular tone, CrMP had no effect on hindlimb resistance. In isolated arterioles denuded of endothelium, CrMP (15 μmol/L) caused a powerful vasoconstriction, which was abolished in the presence of a functional endothelium. In arterioles with intact endothelium pretreated with L-NAME (1 mmol/L), or with L-NAME and sodium nitroprusside (10 to 30 nmol/L), CrMP promoted a similarly powerful vasoconstriction as in vessels denuded of endothelium. Conclusions These results suggest that smooth muscle-derived CO may contribute to endothelium-independent regulation of vascular tone by providing a vasodilatory influence. Furthermore, the dilatory effects of endogenous CO are offset by a unique interaction between the CO and nitric oxide systems.

Effects of chronic administration of a heme oxygenase substrate or inhibitor on progression of the estrous cycle, pregnancy and lactation of Sprague-Dawley rats

The purpose of this study was to investigate whether a heme oxygenase substrate (hemin) or an inhibitor (chromium mesoporphyrin IX; CrMP) had any effect on the normal course of the estrous cycle, pregnancy, parturition or lactation in rats. The hypothesis was that these agents, acting on HO to increase or decrease endogenous production of carbon monoxide (CO) respectively, would disrupt these reproductive processes. The results showed that hemin administered s.c. at 30 μmoles/kg for 10 or 11 days, did not markedly influence the estrous cycle; whereas CrMP blocked the estrous cycle in a dose dependant fashion. At 2 and 4 μmoles/kg for 11 days CrMP significantly reduced the occurrence of estrus phase of the estrous cycle and the effect continued after the treatments were discontinued, while a dose of 1 μmole/kg produced no significant effects. CrMP, administered at 4 μmoles/kg during days 5-14 of pregnancy, led to massive fetal resorption with no live births from 14 successfully mated rats. Administration of hemin at 30 μmoles/kg for 10 days during lactation did not have any effect on milk production, whereas administration of CrMP at 4 μmoles/kg significantly decreased lactational performance which was attributed to milk production and not to suckling intensity of the pups. From these observations we conclude that heme oxygenase, and presumably endogenous CO, play positive roles in female reproductive processes and lactation in the rat.

Spinal cord heme oxygenase participates in glutamate-induced pain-related behaviors

Heme oxygenase catalyzes the formation of CO, Fe 2+ and biliverdin from the substrate heme. In these studies, we attempted to define the roles heme oxygenase play in pain-related behaviors induced by intrathecal injection of the spinal neurotransmitter glutamate. The intrathecal injection of glutamate or the more selective agonists N-methyl- d-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) in C57Bl/6 mice lead to caudally directed pain behaviors which were sensitive to the heme oxygenase inhibitors tin protoporphyrin (Sn-protoporphyrin) and chromium mesoporphyrin (Cr-mesoporphyrin). Intrathecal injections of glutamate in heme oxygenase type 2 (HO-2) null-mutant animals resulted in reduced pain-related behaviors when compared with wild type animals. Glutamate, NMDA and AMPA stimulated cGMP accumulation in mouse spinal cord slices, which was blocked by heme oxygenase inhibitors. Glutamate did not stimulate cGMP production in HO-2 null-mutant animals. Our data are consistent with the hypothesis that pain-related behaviors induced by spinal glutamate rely on the activation of HO-2 and subsequent production of cGMP.

Role of heme oxygenases in sepsis-induced diaphragmatic contractile dysfunction and oxidative stress.

Heme oxygenases (HOs), essential enzymes for heme metabolism, play an important role in the defense against oxidative stress. In this study, we evaluated the expression and functional significance of HO-1 and HO-2 in the ventilatory muscles of normal rats and rats injected with bacterial lipopolysaccharide (LPS). Both HO-1 and HO-2 proteins were detected inside ventilatory and limb muscle fibers of normal rats. Diaphragmatic HO-1 and HO-2 expressions rose significantly within 1 and 12 h of LPS injection, respectively. Inhibition of the activity of inducible nitric oxide synthase (iNOS) in rats and absence of this isoform in iNOS(-/-) mice did alter sepsis-induced regulation of muscle HOs. Systemic inhibition of HO activity with chromium mesoporphyrin IX enhanced muscle protein oxidation and hydroxynonenal formation in both normal and septic rats. Moreover, in vitro diaphragmatic force generation declined substantially in response to HO inhibition both in normal and septic rats. We conclude that both HO-1 and HO-2 proteins play an important role in the regulation of muscle contractility and in the defense against sepsis-induced oxidative stress.

Selective regulation of blood pressure by heme oxygenase-1 in hypertension.

Heme oxygenase (HO) and carbon monoxide (CO) participate in the homeostatic control of cardiovascular functions, including the regulation of blood pressure (BP). Upregulation of the HO/CO system has been shown to lower BP in young (8 weeks) but not in adult (20 weeks) spontaneously hypertensive rats (SHR). The underlying mechanism for this selective effect, however, has been unknown and was investigated in the present study. The administration of hemin resulted in a marked decrease in BP (from 148.6+/-3.2 to 125.8+/-2.6 mm Hg, P<0.01) in young but not in prehypertensive (4 weeks) or adult SHR or Wistar-Kyoto rats at all ages. The inhibition of HO with chromium mesoporphyrin abrogated the BP-lowering effect of hemin. Significantly lower expression levels of HO-1 and soluble gyanylyl cyclase (sGC) as well as reduced cGMP content were detected in 8-week SHR but not in adult SHR or Wistar-Kyoto rats of all ages. These deficiencies were all corrected by hemin treatment. The expression of HO-2 protein was not different among all animal groups tested and not affected by hemin treatment. Desensitization of the sGC/cGMP pathway in adult SHR was demonstrated by the reduced vasorelaxant potency of the sGC activator 3-(5' -hydroxymethyl-2-'furyl)-1-benzylindazole. Thus, in young and prehypertensive SHR, a defective HO/CO-sGC/cGMP system might constitute a pathogenic mechanism for the development of hypertension. The HO/CO-sGC/cGMP system appears normal in adult SHR, but desensitization of the downstream targets of the system to sGC/cGMP may endow SHR at this stage a persistent hypertension status.

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.

Inhibition of haem oxygenase activity increases leukocyte accumulation in the liver following limb ischaemia-reperfusion in mice.

The role of haem oxygenase (HO) in the hepatic accumulation of leukocytes in mice during the initiation of remote organ injury following normotensive limb ischaemia-reperfusion (I-R) was investigated. Remote organ injury was initiated by 1 h bilateral hindlimb ischaemia followed by either 1 or 1.5 h reperfusion (I-R) in male C57BL/6 mice. Mice were randomly assigned to either sham (no I-R, n = 4), I-R (n = 4 for both time points), I-R plus chromium mesoporphyrin (CrMP, n = 4) to inhibit HO or I-R plus haemin (n = 4) to increase HO. Leukocyte accumulation and leukocyte-endothelial interaction were directly measured using fluorescence intravital microscopy. Leukocytes were labelled via an injection of rhodamine 6G. In sinusoids the total number and the number of stationary leukocytes were assessed. In postsinusoidal venules the number of adherent and rolling leukocytes and the velocities of both red blood cells and leukocytes were measured. The total number of leukocytes increased in sinusoids of I-R mice reaching a plateau within 1 h compared with sham animals, while the number of stationary leukocytes progressively increased over the entire study period. Stationary leukocytes in sinusoids increased after 1 and 1.5 h of I-R following CrMP, while they were significantly reduced following haemin treatment compared to animals treated with I-R only. In postsinusoidal venules a progressive increase in adherent leukocytes also occurred. As observed in sinusoids, CrMP significantly increased, while haemin significantly reduced leukocyte adhesion. The number of rolling leukocytes increased after CrMP in both I-R groups (1 and 1.5 h). The velocities of rolling leukocytes declined following 1.5 h of I-R compared with sham. Haemin treatment of 1.5 h I-R animals restored the velocities back to sham levels. The calculated wall shear rates in postsinusoidal venules were significantly lower in all I-R groups in comparison to sham animals. Combination of 1.5 h I-R with CrMP resulted in the lowest shear rates of all I-R groups. The number of stationary leukocytes within sinusoids and adherent leukocytes in postsinusoidal venules were correlated to the corresponding alanine aminotransferase (ALT) levels. In conclusion, endogenous HO reduces leukocyte-endothelial interactions within the liver. Thus, endogenous HO activity provides an important mechanism controlling the hepatic inflammatory response during the initiation of remote organ injury following normotensive limb ischaemia-reperfusion.