Heme oxygenase-1 induction may explain the antioxidant profile of pentaerythrityl trinitrate

The organic nitrate pentaerythrityl tetranitrate (PETN) is known to exert long-term antioxidant and antiatherogenic effects by as yet unidentified mechanisms. In cultured endothelial cells derived from human umbilical vein, the active PETN metabolite PETriN (0.01-1 mM) increased heme oxygenase (HO)-1 mRNA and protein levels in a concentration-dependent fashion. HO-1 induction was accompanied by a marked increase in catalytic activity of the enzyme as reflected by enhanced formation of carbon monoxide and bilirubin. Pretreatment with PETriN or bilirubin at low micromolar concentrations protected endothelial cells from hydrogen peroxide-mediated toxicity. HO-1 induction and endothelial protection by PETriN were not mimicked by isosorbide dinitrate, another long-acting nitrate. The present study demonstrates that PETriN stimulates mRNA and protein expression as well as enzymatic activity of the antioxidant defense protein HO-1 in endothelial cells. Increased HO-1 expression and ensuing formation of cytoprotective bilirubin may contribute to and explain the specific antioxidant and antiatherogenic actions of PETN.     

Heme oxygenase-1 induction may explain the antioxidant profile of aspirin

Aspirin is known to exert antioxidant effects by as yet unidentified mechanisms. In cultured endothelial cells derived from human umbilical vein, aspirin (30-300 microM) increased heme oxygenase-1 (HO-1) protein levels in a concentration-dependent fashion up to fivefold over basal levels. HO-1 induction was accompanied by a marked increase in catalytic activity of the enzyme as reflected by enhanced formation of both carbon monoxide and bilirubin. Pretreatment with aspirin or bilirubin at low micromolar concentrations protected endothelial cells from hydrogen peroxide-mediated toxicity. HO-1 induction and endothelial protection by aspirin were not mimicked by indomethacin, another inhibitor of cyclooxygenase. The nitric oxide (NO) synthase blocker L-NAME prevented aspirin-dependent HO-1 induction. These findings demonstrate that aspirin targets HO-1, presumably via NO-dependent pathways. Induction of HO-1 expression and activity may be a novel mechanism by which aspirin prevents cellular injury under inflammatory conditions and in cardiovascular disease.     

Natriuretic peptides and nitric oxide stimulate cGMP synthesis in different cellular compartments

Cyclic nucleotide-gated (CNG) channels are a family of ion channels activated by the binding of cyclic nucleotides. Endogenous channels have been used to measure cyclic nucleotide signals in photoreceptor outer segments and olfactory cilia for decades. Here we have investigated the subcellular localization of cGMP signals by monitoring CNG channel activity in response to agonists that activate either particulate or soluble guanylyl cyclase. CNG channels were heterologously expressed in either human embryonic kidney (HEK)-293 cells that stably overexpress a particulate guanylyl cyclase (HEK-NPRA cells), or cultured vascular smooth muscle cells (VSMCs). Atrial natriuretic peptide (ANP) was used to activate the particulate guanylyl cyclase and the nitric oxide donor S-nitroso-n-acetylpenicillamine (SNAP) was used to activate the soluble guanylyl cyclase. CNG channel activity was monitored by measuring Ca2+ or Mn2+ influx through the channels using the fluorescent dye, fura-2. We found that in HEK-NPRA cells, ANP-induced increases in cGMP levels activated CNG channels in a dose-dependent manner (0.05-10 nM), whereas SNAP (0.01-100 microM) induced increases in cGMP levels triggered little or no activation of CNG channels (P < 0.01). After pretreatment with 100 microM 3-isobutyl-1-methylxanthine (IBMX), a nonspecific phosphodiesterase inhibitor, ANP-induced Mn2+ influx through CNG channels was significantly enhanced, while SNAP-induced Mn2+ influx remained small. In contrast, we found that in the presence of IBMX, both 1 nM ANP and 100 microM SNAP triggered similar increases in total cGMP levels. We next sought to determine if cGMP signals are compartmentalized in VSMCs, which endogenously express particulate and soluble guanylyl cyclase. We found that 10 nM ANP induced activation of CNG channels more readily than 100 muM SNAP; whereas 100 microM SNAP triggered higher levels of total cellular cGMP accumulation. These results suggest that cGMP signals are spatially segregated within cells, and that the functional compartmentalization of cGMP signals may underlie the unique actions of ANP and nitric oxide.     

Specific binding activities and cyclic GMP responses by atrial natriuretic polypeptide in kidney epithelial cell line (LLC-PK1)

Receptor binding activities and cyclic GMP responses by alpha-human atrial natriuretic polypeptide (alpha-hANP) and its fragments were studied in a kidney epithelial cell line (LLC-PK1). Binding of 125I-alpha-hANP to the cells at 0 degrees C was saturable, time-dependent and reversible, indicating the presence of a single class of binding sites. alpha-hANP (7-23)NH2 fragment inhibited most effectively the specific binding of 125I-alpha-hANP to the LLC-PK1 cells, followed by alpha-hANP (17-28) and alpha-hANP (8-22), while alpha-hANP (1-6) and alpha-hANP (24-28) did not. alpha-hANP stimulated the formation of cyclic GMP in the LLC-PK1 cells dose-dependently. Although no fragments of alpha-hANP used were effective for cyclic GMP formation in the LLC-PK1 cells, alpha-hANP (7-23) NH2 antagonized the action of alpha-hANP on cyclic GMP formation. These data suggest that the LLC-PK1 cells retain specific receptors for atrial natriuretic polypeptide (ANP) and respond to ANP by stimulating cyclic GMP formation, and therefore this cell line may be useful for studying the mechanism of action for ANP in renal tubular cells.     

11,12-epoxyeicosatrienoic acid stimulates heme-oxygenase-1 in endothelial cells

As epoxyeicosatrienoic acids (EETs), particularly 11,12-EET, and the heme oxygenase/carbon monoxide (HO/CO) system share overlapping biological activities, we examined a possible link between 11,12-EET and HO activity in endothelial cells. Confocal microscopy analysis of immunostaining of HO-1 and HO-2 in cultured endothelial cells treated with 11,12-EET (1 microM) showed an increase in florescence of HO-1 protein in the various cellular compartments, but not of HO-2. Incubation of endothelial cells with 11,12-EET (1 microM) for 24 h increased the level of HO-1 protein by about three-fold. Similarly, incubation of endothelial cells with 8,9-EET and sodium nitroprussiate, a known inducer of HO-1, increased HO-1 protein without any effect on HO-2. Upregulation of HO-1 by 11,12-EET, as well as 8,9-EET, was associated with an increase in HO activity, which was inhibited by stannous mesoporphirin (10 microM). Incubation of rat aortas with 11,12-EET (1 microM for 60 min) increased HO activity. These findings identify a novel effect of EETs on endothelial HO-1 and indicate that the signaling pathway of EETs in endothelial cells is possibly via an increase in HO-1 expression and activity.     

Role of human heme oxygenase-1 in attenuating TNF-alpha-mediated inflammation injury in endothelial cells

Heme oxygenase (HO) is the rate-limiting enzyme in the formation of bilirubin, an antioxidant, and carbon monoxide (CO), a cell cycle modulator and a vasodilator. Cyclooxygenase (COX) is a hemeprotein that catalyzes the conversion of arachidonic acid (AA) to various prostanoids, which play an important role in the regulation of vascular endothelial function in normal and disease states. The influence of suppression or overexpression of HO isoforms on COX expression and synthesis of prostanoids is of considerable physiological importance. Consequently, the goal of the present study was to determine whether the heme-HO system regulates COX enzyme expression and activity in vascular endothelial cells in the absence and presence of TNF-alpha (100 ng/ml). Endothelial cells stably transfected with the retrovirus containing the human HO-1 gene exhibited a several-fold increase in HO-1 protein levels, which was accompanied by an increase in HO activity and a marked decrease in PGE(2) and 6-keto PGF(1alpha) levels. We also assessed the effect of retrovirus-mediated HO-1 gene transfer in the sense and antisense orientation on HO-1 expression and cell cycle progression in human endothelial cells. The levels of CO and HO activity were increased in cells transduced with the HO-1 sense and were greatly suppressed in cells transduced with HO-1 antisense as compared to control sham-transduced cells (P < 0.05). The percentage of the G(1)-phase in cells transduced with HO-1 significantly increased (41.4% +/- 9.1) compared with control endothelial cells (34.8% +/- 4.9). We measured COX activity by determining the levels of PGI(2) and PGE(2). The levels of PGI(2) decreased in cells transduced with HO-1 sense and increased in cells transduced with HO-1 in antisense orientation. The expression of p27 was also studied and showed a marked decrease in cells transduced with HO-1 sense and a marked increase in the HO-1 antisense transduced cells. Cell cycle analysis of endothelial cell DNA distributions indicated that the TNF-alpha-induced decrease in the proportion of G(1)-phase cells and increase in apoptotic cells in control cultures could be abrogated by transfection with HO-1 in the sense orientation. Tin mesoporphyrin (SnMP) reversed the protective effect of HO-1. These results demonstrate that overexpressing HO-1 mitigated the TNF-alpha-mediated changes in cell cycle progression and apoptosis, perhaps by a decrease in the levels of COX activity.     

Concurrent expression of heme oxygenase-1 and p53 in human retinal pigment epithelial cell line

Heme oxygenase-1 (HO-1) is a stress-responsive protein that is known to regulate cellular functions such as cell proliferation, inflammation, and apoptosis. Here, we investigated the effects of HO activity on the expression of p53 in the human retinal pigment epithelium (RPE) cell line ARPE-19. Cobalt protoporphyrin (CoPP) induced the expression of both HO-1 and p53 without significant toxicity to the cells. In addition, the blockage of HO activity with the iron chelator DFO or with HO-1 siRNA inhibited the CoPP-induced expression of p53. Similarly, zinc protoporphyrin (ZnPP), an inhibitor of HO, suppressed p53 expression in ARPE-19 cells, although ZnPP increased the level of HO-1 protein while inhibiting HO activity. Also, CoPP-induced p53 expression was not affected by the formation of reactive oxygen species (ROS). Based on these results, we conclude that HO activity is involved in the regulation of p53 expression in a ROS-independent mechanism, and also suggest that the expression of p53 in ARPE-19 cells is associated with heme metabolites such as biliverdin/bilirubin, carbon monoxide, and iron produced by the activity of HO.     

Heme oxygenase-1 and heme oxygenase-2 have distinct roles in the proliferation and survival of olfactory receptor neurons mediated by cGMP and bilirubin,respectively

Heme oxygenase (HO) is implicated in protection against oxidative stress, proliferation and apoptosis in many cell types, including neurons. We utilized olfactory receptor neurons (ORNs) as a model to define the roles of HO-1 and HO-2 in neuronal development and survival, and to determine the mediators of these effects. The olfactory system is a useful model as ORNs display neurogenesis post-natally and do not contain nitric oxide synthase (NOS) activity, which could confound results. HO isoforms were expressed in ORNs during embryogenesis and post-natally. Mice null for either HO-1 or HO-2 displayed decreased proliferation of neuronal precursors. However, apoptosis was increased only in HO-2 null mice. Cyclic GMP immunostaining was reduced in ORNs in both genotypes, providing direct evidence that HO mediates cGMP production in vivo. Bilirubin immunostaining was reduced only in HO-2 null mice. These roles for HO-1 and HO-2 were confirmed using detergent ablation of the epithelium to observe increased neurogenesis of ORNs after target disruption in HO null mice. Primary cultures of ORNs revealed that proliferative and survival effects of HO were mediated through cGMP and bilirubin, respectively. These results support a role for HO, the CO-cGMP signaling system and bilirubin in neurodevelopment and in response to injury.     

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 microM AA or 100 microM 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.     

Effect of glucose and oxygen deprivation on heme oxygenase expression in human chorionic villi explants and immortalized trophoblast cells

Although hypoxia induces heme oxygenase (HO)-1 mRNA and protein expression in many cell types, recent studies in our laboratory using human placental tissue have shown that a preexposure to hypoxia does not affect subsequent HO enzymatic activity for optimized assay conditions (20% O2; 0.5 mM NADPH; 25 microM methemalbumin) or HO-1 protein content. One of the consequences of impaired blood flow is glucose deprivation, which has been shown to be an inducer of HO-1 expression in HepG2 hepatoma cells. The objective of the present study was to test the effects of a 24-h preexposure to glucose-deprived medium, in 0.5 or 20% O2, on HO protein content and enzymatic activity in isolated chorionic villi and immortalized HTR-8/SVneo first-trimester trophoblast cells. HO protein content was determined by Western blot analysis, and microsomal HO enzymatic activity was measured by assessment of the rate of CO formation. HO enzymatic activity was increased (P < 0.05) in both placental models after 24-h preexposure to glucose-deficient medium in 0.5 or 20% O2. Preexposure (24 h) in a combination of low O2 and low glucose concentrations decreased the protein content of the HO-1 isoform by 59.6% (P < 0.05), whereas preexposure (24 h) to low glucose concentration alone increased HO-2 content by 28.2% in chorionic villi explants (P < 0.05). In this preparation, HO enzymatic activity correlated with HO-2 protein content (r = 0.825). However, there was no correlation between HO-2 protein content and HO enzymatic activity in HTR-8/SVneo trophoblast cells preexposed to 0.5% O2 and low glucose concentration for 24 h. These findings indicate that the regulation of HO expression in the human placenta is a complex process that depends, at least in part, on local glucose and oxygen concentrations.     

Endothelial protection by pentaerithrityl trinitrate: bilirubin and carbon monoxide as possible mediators

Pentaerithrityl tetranitrate (PETN) is a long-acting donor of nitric oxide (NO) and has recently been characterized as an antianginal agent that, in contrast with other nitric acid esters, does not induce oxidative stress and is therefore free of tolerance. Moreover, animal experiments have revealed that PETN actively reduces oxygen radical formation in vivoand specifically prevents atherogenesis and endothelial dysfunction. Because heme oxygenase-1 (HO-1) has been described as an antiatherogenic and cytoprotective gene in the endothelium, our aim was to investigate the effect of the active PETN metabolite pentaerithrityl trinitrate (PETriN) on HO-1 expression and catalytic activity in endothelial cells. Endothelial cells derived from human umbilical vein were incubated with PETriN (0.01-1 mM) for 8 hr. PETriN increased HO-1 mRNA and protein levels in a concentration-dependent fashion up to 3-fold over basal levels. Elevation of HO-1 protein was accompanied by a marked increase in catalytic activity of the enzyme as reflected by enhanced formation of both carbon monoxide and the endogenous antioxidant, bilirubin. Pretreatment of endothelial cells with PETriN or bilirubin at low micromolar concentrations protected endothelial cells from hydrogen peroxide-mediated toxicity. HO-1 induction and endothelial protection by PETriN were not mimicked by isosorbide dinitrate, another long-acting nitrate. The present study demonstrates that the active PETN metabolite, PETriN, stimulates mRNA and protein expression as well as enzymatic activity of the antioxidant defense protein, HO-1, in endothelial cells. Increased HO-1 expression and ensuing formation of bilirubin and carbon monoxide may contribute to and explain the specific antioxidant and antiatherogenic actions of PETN.     

trans-Arachidonic acids induce a heme oxygenase-dependent vasorelaxation of cerebral microvasculature

Nitrative stress is an important regulator of vascular tone. We have recently described that trans-arachidonic acids (TAA) are major products of NO(2)(.)-mediated isomerization of arachidonic acid in cell membranes and that nitrative stress increases TAA levels leading to neural microvascular degeneration. In the present study, we explored whether TAA exert acute effects on neuromicrovascular tone and investigated potential mechanisms thereof. TAA induced an endothelium-dependent vasorelaxation of rat brain pial microvasculature. This vasorelaxation was independent of nitric oxide, prostanoids, lipoxygenase products, and CYP(450) metabolite trans-hydroxyeicosatetraenoic acids. However, inhibition of heme oxygenase (using zinc protoporphyrin IX) and of dependent soluble guanylate cyclase (sGC; using ODQ) significantly diminished (by approximately 70%) the TAA-induced vasorelaxation. Consistent with these findings, TAA stimulated heme oxygenase (HO)-2-dependent bilirubin (using siRNA HO-2) and cGMP formation, and the HO product carbon monoxide (using CO-releasing CORM-2) reproduced the sGC-dependent cGMP formation and vasorelaxation. Further exploration revealed that TAA-induced vasorelaxation and bilirubin formation (HO activation) were nearly abrogated by large-conductance calcium-dependent potassium channels (BK(Ca)) (using TEA and iberiotoxin). Opening of BK(Ca) with the selective activator NS1619 induced a concentration-dependent vasorelaxation, which was inhibited by HO and sGC inhibitors. Coimmunoprecipitation suggested a molecular complex interaction between BK(Ca) and HO-2 (but not HO-1). Collectively, these findings identify new properties of TAA, specifically cerebral vasorelaxation through interactive activation of BK(Ca) with HO-2 and, in turn, sGC. Our findings provide new insights into the characterization of nitrative stress-derived TAA products, by showing they can act as acute mediators of nitrative stress on neurovascular tone.     

Atrial natriuretic peptide inhibits the phosphoinositide hydrolysis in murine Leydig tumor cells

The ability of ANP to inhibit the hydrolysis of phosphoinositides was examined in [³H] myoinositol-labeled intact murine Leydig tumor (MA-10) cells. Arginine vasopressin (AVP) stimulated the formation of inositol monophosphate (IP₁), inositol bisphosphate (IP₂), and inositol trisphosphate (IP₃) both in a time- and dose- dependent manner in MA-10 cells. ANP inhibited the AVP-induced formation of IP₁, IP₂, and IP₃ in these cells. The inhibitory effect of ANP on the AVP-stimulated formation of IP₁, IP₂, and IP₃ accounted for 30%, 38% and 42%, respectively, which was observed at the varying concentrations of AVP. ANP caused a dose-dependent attenuation in AVP-stimulated production of IP₁, IP₂ and IP₃ with maximum inhibition at 100 nM concentration of ANP. The production of inositol phosphates was inhibited in the presence of 8- bromo cGMP in a dose-dependent manner, whereas dibutyryl-cAMP had no effect on the generation of these metabolites. The LY 83583, an inhibitor of guanylyl cyclase and cGMP production, abolished the inhibitory effect of ANP on the AVP-stimulated production of inositol phosphates. Furthermore, 10 M LY 83583 also inhibited the ANP-stimulated guanylyl cyclase activity and the intracellular accumulation of cGMP by more than 65–70%. The inhibition of eGMP-dependent protein kinase by H-8, significantly restored the levels of AVP-stimulated inositol phosphates in the presence of either ANP or exogenous 8-bromo cGMP. The results of this study suggest that ANP exerts an inhibitory effect on the production of inositol phosphates in murine Leydig tumor (MA-10) cells by mechanisms involving cGMP and cGMP-dependent protein kinase.Established Investigator of the American Heart Association     

Tin-protoporphyrin potentiates arsenite-induced DNA strand breaks, chromatid breaks and kinetochore-negative micronuclei in human fibroblasts

Numerous reports have shown that oxidative stress is involved in arsenite-induced genetic damage. Arsenite is also a potent inducer of heme oxygenase (HO)-1. To understand whether HO-1 could function as a cellular antioxidant and protect cells from arsenite injury, the effects of tin-protoporphyrin (SnPP), a competitive inhibitor of HO-1, on arsenite-induced genetic damage were examined in human skin fibroblasts (HFW). In the present study, we found that SnPP at 100 microM significantly potentiated arsenite-induced cytotoxicity, DNA strand breaks (assayed by alkaline single cell gel electrophoresis(SCGE)), and chromatid breaks. Although arsenite alone mainly induced kinetochore-plus micronuclei (K(+)-MN), SnPP only synergistically enhanced kinetochore-negative micronuclei (K(-)-MN). The increase in K(-)-MN by SnPP cotreatment was consistent with the increase in DNA strand breaks and chromatid breaks caused by SnPP. However, at higher arsenite doses, K(+)-MN was significantly reduced by SnPP. Pretreatment of HFW cells with hemin, an inducer of HO-1, significantly attenuated the cytotoxicity of arsenite. Therefore, the present results suggest that HO-1 induction by arsenite plays certain roles in protecting cells from arsenite-induced injury.     

Effect of heme oxygenase-1 on the vulnerability of astrocytes and neurons to hemoglobin

The heme oxygenase (HO) enzymes catalyze the rate-limiting step of heme breakdown. Prior studies have demonstrated that the vulnerability of neurons and astrocytes to hemoglobin is modified in cells lacking HO-2, the constitutive isoform. The present study assessed the effect of the inducible isoform, HO-1. Wild-type astrocytes treated for 3-5 days with 3-30 microM hemoglobin sustained no loss of viability, as quantified by LDH and MTT assays. The same treatment resulted in death of 25-50% of HO-1 knockout astrocytes, and a 4-fold increase in protein oxidation. Cell injury was attenuated by transfer of the HO-1 gene, but not by bilirubin, the antioxidant heme breakdown product. Conversely, neuronal protein oxidation and cell death after hemoglobin exposure were similar in wild-type and HO-1 knockout cultures. These results suggest that HO-1 induction protects astrocytes from the oxidative toxicity of Hb, but has no effect on neuronal injury.     

Effects of natriuretic peptides (ANP, BNP, CNP) on catecholamine synthesis and TH mRNA levels in PC12 cells

Atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) are present in adrenal chromaffin cells, and are co-secreted with catecholamines suggesting that these natriuretic peptides (NPs) may modulate functions of chromaffin cells in an autocrine and/or paracrine manner. Therefore, we investigated the effects of NPs on tyrosine hydroxylase (TH: a rate-limiting enzyme in biosynthesis of catecholamine) mRNA in rat pheochromocytoma PC12 cells. It was also determined whether the cyclic GMP/cGMP-dependent protein kinase (cGMP/PKG) pathway was involved in theses effects. Finally, we examined the effects of NPs on intracellular catecholamine content to confirm increase of catecholamine synthesis following TH mRNA induction. NPs (0.1 microM) induced significant increases of the TH mRNA (ANP= BNP> CNP). Also, the effects of NPs on TH mRNA were mimicked by 8-bromo cyclic GMP (1mM), and were blocked by KT5823 (1 microM) (inhibitor PKG) or LY83583 (1 microM) (guanylate cyclase inhibitor). Moreover, NPs were shown to induce significant increases of intracellular catecholamine contents (ANP= BNP> CNP). These findings suggest that NPs induced increases of TH mRNA through cGMP/PKG dependent mechanisms, which, in turn, resulted in stimulation of catecholamine synthesis in PC12 cells.