Resolution of the rat brain heme oxygenase activity: Absence of a detectable amount of the inducible form (HO-1)

doi:10.1016/0003-9861(88)90503-6

Archives of Biochemistry and Biophysics

Volume 260, Issue 2, 1 February 1988, Pages 732-739

https://doi.org/10.1016/0003-9861(88)90503-6 Get rights and content

Abstract

In the present study we report on the detection of a distinct pattern of heme oxygenase isoform composition in the rat brain. In this organ only the noninducible form of heme oxygenase, HO-2, could be clearly detected. This pattern of composition distinguishes the brain from other organs tested to date, namely the liver, testis, and spleen. The rat brain microsomal fraction displayed a rather impressive rate of heme oxygenase activity. This fraction also exhibited a rate of NADPH-cytochrome P-450 reductase activity that was sufficient to fully support the oxygenase activity. The brain microsomal fraction was solubilized and subjected to ion-exchange chromatography on DEAE-Sephacel. The Chromatographie elution pattern of heme oxygenase activity was compared with those of the liver and testis. In the brain only one peak of heme oxygenase activity was detected. The peak exhibited an elution profile similar to that of HO-2 of the liver and the testis. The presence of an activity peak was not detected in the elution profile at the region where the inducible isoform of heme oxygenase, HO-1, was expected. Cross-reactivity was observed between the solubilized brain microsomal fraction and antiserum to the testis HO-2 when subjected to Ouchterlony double diffusion immunoanalysis. A reaction was not observed when antiserum to liver HO-1 was employed. The presence of HO-2 in the brain microsomal preparation was also established by Western immunoblotting analysis. A protein having a mobility that was identical to the purified testicular HO-2 (M_r 36,000) was present in the brain microsomal preparation when probed with antiserum to HO-2. However, our attempts to demonstrate the presence of HO-1 in the brain microsomal preparation by a similar technique, but using antiserum to HO-1, were not successful. It is proposed that HO-2 is responsible for the bulk, if not all, of the brain microsomal heme oxygenase activity. It is further proposed that tissue-specific regulatory mechanisms are responsible for both the refractory response of the brain heme oxygenase to known metallic inducers and the absence of a detectable amount of the HO-1 isoform.

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In the normal, unstressed rodent brain, low-level HO-1 expression is seen in scattered neurons of the cerebral cortex, hippocampal dentate gyrus, thalamus, hypothalamus, cerebellum (Purkinje cells) and brain stem (Baranano and Snyder, 2001; Bergeron et al., 1998; Matz et al., 1996; Nakaso et al., 2000; Vincent et al., 1994). HO-2 mRNA and protein, in contrast, are relatively abundant in olfactory epithelium and olfactory bulb, hippocampal pyramidal cells and dentate gyrus, and cerebellar Purkinje and granule cell layers (Dwyer et al., 1995b; Trakshel et al., 1988; Verma et al., 1993). In rat brain, there is considerable overlap between the topography of HO-2 expression and the distribution of soluble guanylate cyclase.
Under stressful conditions, cellular heme catabolism to carbon monoxide, iron and biliverdin is mediated by the 32 kDa enzyme, heme oxygenase-1 (HO-1). A wide range of pro-oxidant and inflammatory stimuli act on diverse consensus sequences within the Hmox1 promoter to rapidly induce the gene. There is ample evidence attesting to the beneficial effects of HO-1 upregulation in brain. By converting pro-oxidant heme to the antioxidants, biliverdin and bilirubin, HO-1/biliverdin reductase may help restore a more favorable tissue redox microenvironment. Contrariwise, in some cell types and under certain circumstances, heme-derived carbon monoxide and iron may amplify intracellular oxidative stress and exacerbate the disease process. This inimical side of neural HO-1 has often been ignored in biomedical literature promulgating interventions aimed at boosting central HO-1 expression for the management of diverse CNS conditions and is the focus of the current review. A comprehensive model of astroglial stress is presented wherein sustained Hmox1 induction promotes oxidative mitochondrial membrane damage, iron sequestration and mitophagy (macroautophagy). The HO-1 mediated gliopathy renders nearby neuronal constituents vulnerable to oxidative injury and recapitulates ‘core’ neuropathological features of many aging-related neurodegenerative and some neurodevelopmental brain disorders. A balanced literature should acknowledge that, in a host of chronic human CNS afflictions, the glial HO-1 response may serve as a robust transducer of noxious stimuli, an important driver of relevant neuropathology and a potentially disease-modifying therapeutic target.
Development of new HO-1 inhibitors by a thorough scaffold-hopping analysis
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HO-1 inhibition is considered a valuable anticancer approach. In fact, up-regulation of HO-1 had been repeatedly reported in many types of human malignancies, and in these clinical cases, poor outcomes are reported. To identify novel HO-1 inhibitors suitable for drug development, a scaffold-hopping strategy calculation was utilized to design novel derivatives. Different parts of the selected molecule were analyzed and the different series of novel compounds were virtually evaluated. The calculation for the linker moiety of the classical HO-1 inhibitors structure led us to compounds 5 and 6. A synthetic pathway for the two molecules was designed and the compounds were synthesized. The biological activity revealed an HO-1 inhibition of 0.9 and 54 μM for molecules 5 and 6 respectively. This study suggested that our scaffold-hopping approach was successful and these results are ongoing for further development.
Potholing of the hydrophobic heme oxygenase-1 western region for the search of potent and selective imidazole-based inhibitors
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C18H17IN2O) C, H, N. HO-1 and HO-2 were obtained, respectively, from rat spleen and brain as the microsomal fraction prepared by differential centrifugation; the dominance of HO-1 protein in the rat spleen and of HO-2 in the rat brain has been well documented [47–50]. These particular microsomal preparations were selected in order to use the most native (i.e., closest to in vivo) forms of HO-1 and HO-2.
Here we report the design, synthesis, and molecular modeling of new potent and selective imidazole-based HO-1 inhibitors in which the imidazole nucleus and the hydrophobic groups are linked by a phenylethanolic spacer. Most of the tested compounds showed a good inhibitor activity with IC₅₀ values in the low micromolar range, with two of them (1b and 1j) exhibiting also high selectivity toward HO-2. These results were obtained by the idea of potholing the entire volume of the principal hydrophobic western region with an appropriate ligand volume. Molecular modeling studies showed that these molecules bind to the HO-1 in the consolidated fashion where the imidazolyl moiety coordinates the heme iron while the aromatic groups are stabilized by hydrophobic interaction in the western region of the binding pocket. Finally, the synthesized compounds were analyzed for in silico ADME-Tox properties to establish oral drug-like behavior and showed satisfactory results.
Physiology of Cerebral Blood Vessels
2017, Brain Edema: From Molecular Mechanisms to Clinical Practice
The vasculature in the brain is an actively regulated organ which, on the one hand, is influenced by global changes of systemic parameters (systemic blood pressure, blood gases, and blood pH) and, on the other hand, is capable of locally directing the blood to the regions of demand with high spatial and temporal resolution. Together with pericytes and astrocytes, as part of the neurovascular unit, cerebral vascular endothelium forms the tight blood-brain barrier and has an important immuno-regulatory function. Within this book chapter the main principles of the vascular architecture and the cellular components at the neurovascular unit forming the blood-brain barrier will be described before summarizing the main mechanisms of global blood flow regulation to changes in systemic parameters as well as giving an overview on the local regulation according to the cellular demand during neuronal activation.
Characterization of heme oxygenase and biliverdin reductase gene expression in zebrafish (Danio rerio): Basal expression and response to pro-oxidant exposures
2016, Toxicology and Applied Pharmacology
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To this end, HMOX1 has been shown to be highly inducible by a variety of stressors in mammalian systems, and is detected at high levels in tissues involved in heme metabolism including spleen and liver (Tenhunen et al., 1969), bone marrow (Brown et al., 1988; Abraham et al., 1989) and erythroid cells (Garcia-Santos et al., 2014). The other heme degrading enzyme, HMOX2, is expressed at high levels in the testes (Trakshel et al., 1986), brain (Maines et al., 1986; Trakshel et al., 1988; Ewing and Maines, 1992), and vascular tissues (Zakhary et al., 1996). The BVR isoforms are distinct in their enzymatic actions and their evolutionary origins (Yamaguchi et al., 1993).
While heme is an important cofactor for numerous proteins, it is highly toxic in its unbound form and can perpetuate the formation of reactive oxygen species. Heme oxygenase enzymes (HMOX1 and HMOX2) degrade heme into biliverdin and carbon monoxide, with biliverdin subsequently being converted to bilirubin by biliverdin reductase (BVRa or BVRb). As a result of the teleost-specific genome duplication event, zebrafish have paralogs of hmox1 (hmox1a and hmox1b) and hmox2 (hmox2a and hmox2b). Expression of all four hmox paralogs and two bvr isoforms were measured in adult tissues (gill, brain and liver) and sexually dimorphic differences were observed, most notably in the basal expression of hmox1a, hmox2a, hmox2b and bvrb in liver samples. hmox1a, hmox2a and hmox2b were significantly induced in male liver tissues in response to 96 h cadmium exposure (20 μM). hmox2a and hmox2b were significantly induced in male brain samples, but only hmox2a was significantly reduced in male gill samples in response to the 96 h cadmium exposure. hmox paralogs displayed significantly different levels of basal expression in most adult tissues, as well as during zebrafish development (24 to 120 hpf). Furthermore, hmox1a, hmox1b and bvrb were significantly induced in zebrafish eleutheroembryos in response to multiple pro-oxidants (cadmium, hemin and tert-butylhydroquinone). Knockdown of Nrf2a, a transcriptional regulator of hmox1a, was demonstrated to inhibit the Cd-mediated induction of hmox1b and bvrb. These results demonstrate distinct mechanisms of hmox and bvr transcriptional regulation in zebrafish, providing initial evidence of the partitioning of function of the hmox paralogs.
Ischemic preconditioning protects hippocampal pyramidal neurons from transient ischemic injury via the attenuation of oxidative damage through upregulating heme oxygenase-1
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Citation Excerpt :
To reduce background staining, the membranes were incubated with 5% nonfat dry milk in TBS containing 0.1% Tween 20 for 45 min, followed by incubation with rabbit anti-HO-1 (diluted 1:1000) overnight at 4 °C and subsequently exposed to peroxidase-conjugated donkey anti-rabbit IgG and an ECL kit (Pierce Chemical). HO-1 activity in the hippocampus at 12 h, 1 day, and 5 days after ischemia–reperfusion (n=7 at each time point) was measured by bilirubin generation as described previously [28,29]. The samples were homogenized in lysis buffer (50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 250 mM sucrose, 0.4 mM PMSF, 1 g/L leupeptin, and 1 g/L aprotinin).
Ischemic preconditioning (IPC) provides neuroprotection against subsequent severe ischemic injury by activating specific mechanisms. In this study, we tested the hypothesis that IPC attenuates postischemic neuronal death via heme oxygenase-1 (HO-1). Animals used in this study were randomly assigned to 4 groups; sham-operated group, ischemia-operated group, IPC plus (+) sham-operated group and IPC+ischemia-operated group. IPC was induced by subjecting gerbils to 2 min of ischemia followed by 1 day of recovery. A significant loss of neurons was observed in pyramidal neurons of the hippocampal CA1 region (CA1) in the ischemia-operated groups at 5 days postischemia. In the IPC+ischemia-operated groups, CA1 pyramidal neurons were well protected. The level of HO-1 protein and its activity increased significantly in the CA1 of the IPC+sham-operated group, and the level and activity was maintained in all the time after ischemia–reperfusion compared with the ischemia-operated groups. HO-1 immunoreactivity was induced in the CA1 pyramidal neurons in both IPC+sham-operated- and IPC+ischemia-operated groups. We also found that levels or immunoreactivities of superoxide anion, 8-hydroxy-2′-deoxyguanosine and 4-hydroxy-2-nonenal were significantly decreased in the CA1 of both IPC+sham-operated- and IPC+ischemia-operated groups. Whereas, treatment with zinc protoporphyrin IX (a HO-1 inhibitor) into the IPC+ischemia-operated groups did not preserve the IPC-mediated increase of HO-1 and lost beneficial effects of IPC by inhibiting ischemia-induced DNA damage and lipid peroxidation. In brief, IPC protects CA1 pyramidal neurons from ischemic injury by upregulating HO-1, and we suggest that the enhancement of HO-1 expression by IPC may be a legitimate strategy for a therapeutic intervention of cerebral ischemic damage.

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Resolution of the rat brain heme oxygenase activity: Absence of a detectable amount of the inducible form (HO-1)

Abstract

Arch. Biochem. Biophys

J. Biol. Chem

J. Biol. Chem

Arch. Biochem. Biophys

Toxicol. Appl. Pharmacol

Biochem. Biophys. Res. Commun

Biochem Pharmacol

J. Biol. Chem

Arch. Biochem. Biophys

Biochem. Pharmacol

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

Biochem. Biophys. Res. Commun

J. Biol. Chem