James F. Ewing

In situ hybridization and immunohistochemical localization of heme oxygenase-2 mRNA and protein in normal rat brain: Differential distribution of isozyme 1 and 2.

Heme oxygenase isozymes, HO-1 (HSP32) and HO-2, stereospecifically bind and degrade the potent prooxidant, the heme molecule, and convert it to the effective antioxidant, biliverdin, and the potential cellular messenger, carbon monoxide. In the present study we have examined the pattern of expression of the two HO-2 transcripts and protein in normal rat brain by in situ hybridization and immunochemical analysis, respectively. We have found by Northern blot analysis that HO-2 isozyme is by far the most prevalent form in the brain. Analysis of HO-2 1.3- and 1.9-kb mRNAs by in situ hybridization histochemistry showed that these transcripts are abundantly expressed in many neuronal and nonneuronal cell populations in forebrain, diencephalon, cerebellum, and brain stem regions. Furthermore, the pattern of expression of HO-2 transcripts, as detected by oligonucleotide probes, is in good agreement with that of immunoreactive protein detected by immunohistochemical analysis. Impressive levels of HO-2 transcripts and immunoreactive protein were observed in Purkinje cells of cerebellum, red nucleus, superior and inferior colliculus, nucleus of the trapezoid body, cochlear neurons, and facial nucleus of brain stem. Furthermore, in certain select brain cell populations the pattern of expression of HO-1- and HO-2-immunoreactive proteins overlapped. We suggest that the high levels of heme degradation activity and the localization of HO-2 transcripts and protein in the brain may reflect the functions of this enzyme in processes such as production of cellular messenger, regulation of the activity of heme-dependent enzymes catalyzing intracellular signaling molecule synthesis, and production of antioxidants.

Glutathione depletion induces heme oxygenase-1 (HSP32) mRNA and protein in rat brain

Abstract: In mammalian systems, the heme oxygenase (HO) isozymes HO‐1 (HSP32) and HO‐2 oxidatively cleave the heme molecule to produce bile pigments and carbon monoxide. Although HO‐1 is inducible by various chemicals in systemic organs and cell culture systems, this communication reports for the first time the induction of this stress protein and its transcript by a chemical in the brain. In addition, this study demonstrates expression of HO‐1 in select populations of cells in the brain in response to GSH depletion. Specifically, treatment of adult rats with diethyl maleate (DEM; 4.7 mmol/kg) caused a pronounced decrease in brain GSH content within 1 h. GSH levels remained significantly depressed for at least 24 h postinjection. Northern blot analysis of brain poly(A)+ mRNA following DEM treatment revealed on the average a sixfold increase in the 1.8‐kb HO‐1 mRNA level compared with that of controls; concomitant with this change was a decrease in GSH levels. Total brain HO activity was not significantly altered along with the increase in HO‐1 mRNA level. The increase in transcription of HO‐1 was a direct response to GSH depletion, as judged by the observation that treatment of neonatal rats with L‐buthionine‐(S,R)‐sulfoximine (BSO) (3 mmol/kg, twice daily, for 2 days), a selective inhibitor of GSH synthesis, caused a marked depression in total brain GSH level and a concomitant increase in brain 1.8‐kb HO‐1 mRNA content. The magnitude of the increase was up to ∼ 11.5‐fold that of the control level, as evidenced by northern blot analysis. In contrast, the level of two homologous HO‐2 transcripts (1.3 and 1.9 kb) did not increase in response to either DEM or BSO treatment. Analysis of brain HO‐1‐immunoreactive protein following DEM treatment for 9 h indicated induction of HO‐1 protein in only select nonneuronal cell populations. In particular, the ependymal cells lining ventricles throughout brain, Bergmann glia of cerebellum, and leptomeninges lining brain and glia throughout brain responded to treatment by increasing the level of HO‐1‐like protein. We suggest that when GSH is depleted, an increase in HO‐1 protein content, resulting in increased capacity to form bile pigments, may be of significance to cells with compromised antioxidant capability. Bile pigments are potent antioxidants in biological systems.

Biliverdin reductase is heat resistant and coexpressed with constitutive and heat shock forms of heme oxygenase in brain

Abstract: Two heme oxygenase (HO) isozymes—HO‐1, which is a heat shock protein (HSP32), and HO‐2—catalyze the isomer‐specific production of biliverdin IXα and carbon monoxide. The latter has the potential of functioning as a neurotransmitter, whereas the reduced form of biliverdin, bilirubin, has potent antioxidant activity. Formation of bilirubin is catalyzed by biliverdin reductase (BVR). The reductase is a unique enzyme in being dual pyridine nucleotide and dual pH dependent. Here, we show that the reductase is resistant to thermal stress at both the protein and message level. We further demonstrate that the reductase is coexpressed in cells that display HO‐1 and/or HO‐2 under normal conditions, as well as in regions and cell types that have the potential to express heat shock‐inducible HO‐1 protein. Exposure of male rats to 42°C for 20 min did not decrease brain BVR activity, but caused a slight increase in NADPH‐and NADH‐dependent activities at 1 and 6 h following hyperthermia. High levels of the ∼ 1.5‐kb BVR mRNA were detected in control brain; it too displayed thermal tolerance. Similarly, the pattern of multiplicity of net charge variants of the enzyme purified from brain of heat‐shocked rats did not differ from the control pattern. Immunochemical localization of BVR protein in normal brain correlated well with the presence of HO‐1 and/or HO‐2 throughout the forebrain, diencephalon, cerebellum, and brainstem regions. There were select neuronal and nonneuronal cells in the substantia nigra and cerebellum that did express the reductase under normal conditions, wherein no HO isozymes could be detected. The same population, however, responded to heat shock by an intense increase in the level of HO‐1. We postulate that the constitutive presence of the reductase in this cell population and the overall thermal stability of the enzyme represent a safeguard mechanism in the brain for the prompt conversion of biliverdin to bilirubin under conditions when oxidation of the heme moiety of denatured hemoproteins by HO‐1 is accelerated.

Histochemical localization of heme oxygenase-2 protein and mRNA expression in rat brain.

Heme oxygenase (HO) proteins are members of the HSP30 family and consist of 2 isozymes identified to date, termed HO-1 and HO-2. Separate genes encode the isozymes and protein products which are immunochemically distinct, share less than 50% similarity at the amino acid sequence level. Each form, however, shows greater than 90% similarity among species, including human and the rat (reviewed in ref.). Furthermore, these isozymes function in a well-defined role to carry out oxidation of the heme molecule (Fe-protoporphyrin IX) in concert with NADPH-cytochrome P450 reductase. The oxidation of heme is isomer specific and results in the formation of bile pigments, carbon monoxide, and iron. The heme molecule constitutes the prosthetic moiety of hemoproteins, such as hemoglobin, myoglobin, catalase, soluble guanylate cyclase, cytochrome b5, cytochromes P450 and NO synthase. HO-1 also known as heat shock protein (HSP) 32 is encoded by a gene which is exquisitely stress-responsive and a host of stimuli that mediate oxidative stress cause induction of the protein both in vivo and in vitro. The HO-2 form shows a unique pattern of regulation from that of HO-1. HO-2 is a constitutive protein and its expression is not affected by the inducers of HO-1 tested to date; rather, the only known regulator of HO-2 yet identified is adrenal glucocorticoids. The two isozymes display vast differences in tissue distribution and under normal conditions HO-1 is present in the whole brain at the limit of immunodetection and is discreetly localized in select neuronal populations. HO-1 protein (approximately 32 kDa) and its approximately 1.8 kb transcript are increased, however, in response to stressful stimuli primarily in non-neuronal cell populations. The heme oxygenase system serves in both a catabolic and anabolic capacity in the cell. In the former capacity, it down-regulates cellular heme and hemoprotein levels. And, as such it inactivates the most effective catalyst for formation of free radicals, the heme molecule. In its anabolic role, as noted above, heme oxygenase produces bile pigments, carbon monoxide, and iron, all of which are biologically active: bile pigments function as antioxidants; the carbon monoxide generated by HO activity has been correlated with the generation of cGMP; and iron regulates expression of various genes, including that of HO-1 itself, as well as transferrin receptors, ferritin, and NO synthase. We used rabbit anti-rat HO-2 polyclonal antibody and HO-2 cDNA to localize HO-2 immunoreactive protein and the 1.3- and 1.9 kb homologous transcripts, respectively, in rodent brain as visualized by histochemical staining procedures. These protocols provide the first detailed description of methodologies successfully used to define the pattern of HO-2 expression at the transcriptional and translational levels in the adult rat brain and glucocorticoid-treated newborn rats. The procedures described herein have the virtue of being non-radioactive, as well as applicability to the systemic organs, such as the cardiovascular system and the male reproductive organs. Visualization of cellular HO-2 expression aids in assessment of potential sites of carbon monoxide, iron, and bilirubin production within the nervous system.

Specific S-nitrosothiol (thionitrite) quantification as solution nitrite after vanadium(III) reduction and ozone-chemiluminescent detection.

Increasing evidence suggests that S-nitrosothiols (thionitrites) might represent naturally occurring nitric oxide surrogates and function as intermediates in nitrogen monoxide metabolism. A facile, sensitive, and selective micromethod has been developed and validated for quantification of S-nitrosothiols as their mercury-displaceable nitrogen monoxide content. In this method, brief (5-min), room-temperature pretreatment of S-nitrosothiol with a molar excess of aqueous mercuric chloride was used to liberate into solution, quantitatively, the nitrogen monoxide moiety, which rapidly and quantitatively converted to its stable solution end-product, nitrite. Solution nitrite was reduced back to nitric oxide with vanadium(III), and the nitric oxide was detected by gas-phase chemiluminescence after reaction with ozone in a commercial nitric oxide analyzer. A linear relationship was observed between S-nitrosothiol-bound nitrogen monoxide and ozone-chemiluminescent detector response over a wide range (16.3-3500 pmol) of nitric oxide, as generated by reaction of vanadium(III) with either nitrite standard or mercury-treated S-nitrosothiol. Assay response was quantitatively identical for equivalent amounts of nitrite and S-nitrosothiol-bound nitrogen monoxide. The method displayed 96% selectivity for nitrite vs. nitrate and negligible (<2%) interference by nitrosated compounds bearing nitrogen monoxide moieties bound to either nitrogen or carbon. The lower limits of quantitative sensitivity and qualitative detection were below 50 and 20 pmol S-nitrosothiol-bound nitrogen monoxide-equivalents, respectively. The intraday and interday coefficients of variation did not exceed 8%. This technique has been applied to quantify structurally diverse natural and synthetic S-nitrosothiols with quantitative recovery from complex biological samples such as culture media and plasma at levels of nitrogen monoxide-equivalents undetectable by the popular Saville colorimetric method.

Nitric oxide and postangioplasty restenosis: pathological correlates and therapeutic potential

Balloon angioplasty revolutionized interventional cardiology as a nonsurgical procedure to clear a diseased artery of atherosclerotic blockage. Despite its procedural reliability, angioplastys long-term outcome can be compromised by restenosis, the recurrence of arterial blockage in response to balloon-induced vascular trauma. Restenosis constitutes an important unmet medical need whose pathogenesis has yet to be understood fully and remains to be solved therapeutically. The radical biomediator, nitric oxide (NO), is a natural modulator of several processes contributing to postangioplasty restenosis. An arterial NO deficiency has been implicated in the establishment and progression of restenosis. Efforts to address the restenosis problem have included trials evaluating a wide range of NO-based interventions for their potential to inhibit balloon-induced arterial occlusion. All types of NO-based interventions yet investigated benefit at least one aspect of balloon injury to a naive vessel in a laboratory animal without inducing significant side effects. The extent to which this positive, albeit largely descriptive, body of experimental data can be translated into the clinic remains to be determined. Further insight into the pathogenesis of restenosis and the molecular mechanisms by which NO regulates vascular homeostasis would help bridge this gap. At present, NO supplementation represents a unique and potentially powerful approach to help control restenosis, either alone or as a pharmaceutical adjunct to a vascular device.

Heme Oxygenase, a Likely Regulator of cGMP Production in the Brain: Induction in Vivo of HO-1 Compensates for Depression in NO Synthase Activity

To examine the role of carbon monoxide (CO) as a putative neuronal messenger and regulator of cGMP level in vivo, we exploited an animal model to increase brain capability to generate CO. The sole source of CO in mammalian systems is the alpha-meso carbon bridge of the heme molecule cleaved by heme oxygenase isozymes, HO-1 and HO-2. In adult animals, the noninducible isozyme HO-2 is the predominant form in the brain. We chose to increase, rather than inhibit, brain heme oxygenase activity because synthetic metalloporphyrins, such as Zn-protoporphyrin, which are the only known effective inhibitors of the isozymes, are also potent inhibitors of soluble guanylate cyclase, the enzyme that generates cGMP. In newborn rats both heme oxygenase isozymes were found expressed at low levels, and in the cerebellum heme oxygenase activity could be induced by treatment of 2-day-old animals with a selective depletor of glutathione, buthionine-SR-sulfoximine. The increase in activity was accompanied by marked increases in HO-1 protein and the 1.8 kb HO-1 mRNA in the cerebellum. Despite a pronounced decrease in activity of the hemoprotein nitric oxide synthase, no change in cGMP level was observed. The decrease in the synthase could not be explained by an inhibited heme biosynthesis activity. This unchanged level of cGMP suggests that NO is not the only gaseous heme ligand that can activate guanylate cyclase resulting in the generation of cGMP, but rather that CO may also function in this capacity. Increased capability of select cerebellar cell populations to generate CO, as indicated by an increase in their HO-1 protein content, points to the active role of this isozyme in maintenance of cGMP level under stress conditions, when nitric oxide production is compromised. The cell populations expressing HO-1 protein included those in pia matter and glia, such as astrocytes.

The neuroendocrine prostate: characterization and quantitation of calcitonin in the human gland.

Calcitonin was extracted from surgically-derived prostate tissue, and quantified using radioimmunoassay. Normal prostatic specimens contained 15.18 +/- 10.03 ng./gm. wet weight (mean +/- S.D., n = 20), with a range of 1.50 to 39.62 ng./gm. The result for the hyperplastic tissue samples (n = 20) averaged 0.63 +/- 0.39 ng./gm. with a range of 0.22 to 1.49 ng./gm. This difference was statistically significant (p less than .0001). Dilution profiles for the prostatic calcitonin and synthetic monomeric human calcitonin were congruent, suggesting that the two peptides are identical. A comparison of calcitonin levels and the number of immunohistochemically derived neuroendocrine cells in contiguous tissue sections showed an empiric correlation. The mean calcitonin level in normal human prostate tissue was found to exceed values previously reported for numerous other organs, with the exception of the thyroid gland, the principal source of circulating calcitonin. We propose that a subpopulation of neuroendocrine cells within the prostate gland produce calcitonin, as is the case in the gastrointestinal tract, lung, and other organs. Our findings also support the hypothesis that the calcitonin found in seminal fluid originates in the prostate. Putative roles for calcitonin in the genitourinary system are discussed.

Regulation and expression of heme oxygenase enzymes in aged-rat brain: age related depression in HO-1 and HO-2 expression and altered stress-response.

Summary.The heme oxygenase isozymes, HO-1 and HO-2, oxidatively cleave the heme molecule to produce biliverdin and the gaseous messenger, CO. The cleavage results in the release of iron, a regulator of transferrin, ferritin, and nitric oxide (NO) synthase gene expression. Biliverdin reductase (BVR) then catalyzes the reduction of biliverdin, generating the potent intracellular antioxidant, bilirubin. We report an age-related decrease in HO-1 and HO-2 expression present in select brain regions including the hippocampus and the substantia nigra, that are involved in the high order cognitive processes of learning and memory. The age-related loss of monoxide-producing potential in select regions of the brain was not specific to the HO system but was also observed in neuronal NO-generating system. Furthermore, compared to 2-month old rats, the ability of aged brain tissue to respond to hypoxic/hyperthermia was compromised at both the protein and the transcription levels as judged by attenuated induction of HO-1 immunoreactive protein and its 1.8 Kb transcript. Neotrofin™ (AIT), a cognitive-enhancing and neuroprotective drug, caused a robust increase in HO-1 immunoreactive protein in select neuronal regions and increased the expression of HO-2 transcripts. The potential interplay between regulation of HO-2 gene expression and the serum levels of the adrenal steroids is discussed. We suggest the search for therapeutic agents that reverse the decline and aberrant stress response of HO enzymes may lead to effective treatment regimens for age-associated neuronal deficits.

Corticosterone Has a Permissive Effect on Expression of Heme Oxygenase‐1 in CA1–CA3 Neurons of Hippocampus in Thermal‐Stressed Rats

Abstract: Activity of the stress protein, heme oxygenase‐1 (hsp32; HO‐1), produces carbon monoxide (CO), the potential messenger molecule for excitatory N‐methyl‐d‐aspartate receptor‐mediated events, in the hippocampus. Long‐term stress caused by elevated adrenocorticoids induces pathological changes in CA1–CA3 neurons, of the hippocampus; the adrenal hormones also exacerbate damage from stress. In rats chronically treated with corticosterone, we examined expression of HO‐1 and its response to thermal stress in the hippocampus. An unprecedented appearance of scattered immunoreactive astrocytes marked the molecular layer of the hippocampus in corticosterone‐treated rats. Steroid treatment showed no discernible effect on whole‐brain HO‐1 mRNA. When these rats were subjected to hyperthermia, neurons in the CA1–CA3 area, including pyramidal cells, exhibited intense immunoreactivity for the oxygenase and a pronounced increase (∼10‐fold) in number. HO‐1 is essentially undetectable in this area when rats are exposed to chronic corticosterone alone or thermal stress by itself, or in control rats. In contrast, similar analysis of hilar neurons showed no apparent effect on either the number or relative intensity of HO‐1‐immunostained cells after treatment. Corticosterone treatment also intensified the stress response of cerebellum, including Purkinje cells and Bergmann glia in the molecular layer. In brain, despite a pronounced reduction in NO synthase activity in corticosterone‐treated and/or heat‐stressed animals, the level of cyclic GMP was not significantly reduced. These observations are consistent with the hypothesis that responsiveness to environmental stress of CA1–CA3 neurons brought about by chronic elevation in circulating adrenocorticoids results in an increased excitatory neuronal activity and eventual hippocampal degeneration. Moreover, these findings yield further support for a role of CO in the production of cyclic GMP in the brain.