Heme Oxygenase-2 Immunoreactivity Research Articles

Heme oxygenase is necessary for the excitatory response of cultured neonatal rat rostral ventrolateral medulla neurons to hypoxia by D'Agostino D, Mazza E, and Neubauer JA

the effect of hypoxia to stimulate breathing and the cardiovascular system has been well known for more than a century (for a review, see Ref. [11][1]). In 1938, Heymans was awarded the Nobel Prize for Physiology or Medicine for demonstrating the importance of carotid body chemoreceptors. However,

Immunocytochemical evidence for nitric oxide‐ and carbon monoxide‐producing neurons in the stomatogastric nervous system of the crayfish Cherax quadricarinatus

Nitric oxide (NO) and carbon monoxide (CO) have been shown to serve neuromodulatory roles in both vertebrates and invertebrates. Here, we use antibodies to their respective biosynthetic enzymes, nitric oxide synthase (NOS) and heme oxygenase 2 (HO-2), to map the distribution of putative gas-producing neurons in the stomatogastric nervous system (STNS) of the crayfish Cherax quadricarinatus. In this species, NOS immunolabeling is found in the neuropil of the stomatogastric ganglion (STG). This staining originates from two immunopositive axons that project to the STG through the superior oesophageal and stomatogastric nerves, presumably from cell bodies located in the commissural ganglia (CoGs). HO-2 immunoreactivity is present in small diameter fibers and varicosities in the periphery of nerves located in the anterior portion of the STNS. This labeling originates from approximately 12 somata in each CoG. Transmission electron microscopy done on the nerves of the anterior STNS shows they contain a neuroendocrine plexus. Collectively, our results indicate that NO- and CO-producing neurons are likely to exist in the crayfish STNS. Moreover, these gases appear to be produced by distinct subsets of the neurons present there. The localization of NO to the STG neuropil suggests that it serves as a locally released modulator or is involved in the local release of other substances within this ganglion. The presence of CO in the neurohemal plexus of the anterior STNS suggests that it serves as a circulating hormone or is involved in the control of neuroendocrine release from this plexus.

Distribution of heme oxygenase-2 in nerves and interstitial cells of Cajal in the normal pylorus and in infantile hypertrophic pyloric stenosis.

Abstract Context.—Interstitial cells of Cajal (ICCs) are pacemaker cells, which are of fundamental importance in regulating gastrointestinal motility. Recent evidence suggests that carbon monoxide is a neurotransmitter involved in neurotransmission between ICC and smooth muscle cells. Heme oxygenase-2 (HO-2) is the major physiological mechanism for the generation of carbon monoxide in the enteric nervous system. Objective.—To investigate the immunocolocalization of HO-2 and ICCs in the normal pylorus and in infantile hypertrophic pyloric stenosis (IHPS). Design.—Specimens from 18 infants with IHPS and 8 control specimens were examined using double-immunostaining with c-Kit and HO-2 antibodies. The immunolocalization was detected with the help of confocal laser scanning microscopy. Results.—Abundant HO-2 immunoreactivity was found in ICCs in the smooth muscle layer of normal pylorus. There was a decrease in the number of ICCs and lack of HO-2 immunoreactivity in ICCs in IHPS. Conclusions.—The results of th...

Immunocolocalization of the heme oxygenase-2 and interstitial cells of Cajal in normal and aganglionic colon

Purpose: Interstitial cells of Cajal (ICCs) are pacemaker cells that play an important role in the control of gut motility. Carbon monoxide (CO) has been proposed as an endogenous messenger molecule between ICC and smooth muscle cells in the gastrointestinal tract (GIT). Heme oxygenase-2 (HO-2) is the main physiologic mechanism for generating CO in human cells. The aim of this study was to investigate the immunocolocalization of the HO-2 and ICCs in normal and aganglionic bowel of Hirschsprung's disease (HD). Methods: Full-thickness specimens were obtained from aganglionic colon during pull-through operation from 10 patients diagnosed as having HD. Normal control large bowel specimens were collected from 4 patients during bladder augmentation procedures. Double immunostaining was carried out using c-kit and HO-2 antibodies. Immunolocalization was detected by means of confocal laser scanning microscopy. Results: HO-2 immunoreactivity (IR) was found in many ICCs present around the myenteric plexus, within the longitudinal and circular muscle layers and at the innermost part of the circular muscle layer in normal colon. In the aganglionic colon there was absence of HO-2 IR in the sparsely found ICCs. In the transitional zone of HD bowel the colocalization of HO-2 IR and ICCs was much reduced compared with controls. Conclusions: The results of this study provide the first evidence for the presence of HO-2 immunoreactivity in the ICCs in normal human colon and absence of HO-2 immunoreactivity in sparsely appearing ICCs in the bowel of HD patients. The lack of HO-2 in the ICCs in the bowel of HD patients may result in impaired intracellular communication between ICCs and SMCs causing motility dysfunction. J Pediatr Surg 38:73-77. Copyright 2003, Elsevier Science (USA). All rights reserved.

Heme oxygenase-2 immunoreactivity in developing and mature bovine olfactory epithelium

In the present study the localization of heme oxygenase-2 (HO-2) in developing and mature olfactory epithelium of the bovine is investigated using immunohistochemistry and post embedding immunogold labelling. HO-2 immunoreactivity is first seen in epithelial cells localized along the luminal surface of the olfactory pit. Up to midgestation the number of HO-2 immunoreactive cells increases throughout all layers of the developing olfactory epithelium. From midgestation through adulthood immunostaining is restricted to perinuclear cytoplasm and axons of mature olfactory receptor neurons localized in intermediate epithelial regions. The temporal and spatial expression patterns of HO-2 immunohistochemistry support the notion that CO plays a role in neuronal differentiation while its presence in mature neurons might be functionally related to olfactory transduction.

A new antioxidant compound H-290/51 attenuates upregulation of constitutive isoform of heme oxygenase (HO-2) following trauma to the rat spinal cord.

Influence of a new antioxidant compound H-290/51 on carbon monoxide (CO) production following spinal cord injury was examined using immunohistochemistry of the constitutive isoform of heme oxygenase-2 (HO-2) in a rat model. Subjection of rats to 5 h spinal cord injury by making an incision into the right dorsal horn of the T10-11 segments resulted in upregulation of HO-2 in the injured and adjacent T9 and T12 segments. At this time, disruption of the blood-spinal cord barrier (BSCB) permeability, edema formation and cell injury were more pronounced. Pretreatment with H-290/51 (50 mg/kg, p.o., 30 min before trauma) significantly attenuated the HO-2 immunoreactivity along with breakdown of the BSCB permeability, edema and cell injury. These results for the first time demonstrate that the antioxidant compound H-290/51 is capable of attenuating HO-2 expression and thereby influencing CO production. Furthermore, our observations indicate that oxidative stress is involved in CO production, as reflected by HO-2 expression, which is injurious to the cord and H-290/51 exerts powerful neuroprotective effects in spinal cord injury.

Topical application of brain derived neurotrophic factor influences upregulation of constitutive isoform of heme oxygenase in the spinal cord following trauma an experimental study using immunohistochemistry in the rat.

Influence of brain derived neurotrophic factor (BDNF) on carbon monoxide (CO) production following spinal cord injury was examined using expression of the constitutive isoform of the enzyme hemeoxygenase-2 (HO-2) in a rat model. A longitudinal incision of the right dorsal horn on the T10-11 segment markedly increased the HO-2 immunostaining in the cord at 5 h. At this time period, breakdown of the blood-spinal cord barrier (BSCB) and edema formation were quite prominent. Repeated topical application of BDNF (20 microliters of a 1 microgram/ml solution) over the exposed surface of the cord significantly attenuated the edematous expansion of the cord and the disturbances in the BSCB permeability. In BDNF-treated rats, expression of HO-2 immunoreactivity was considerably reduced. These results strongly suggest that BDNF is neuroprotective in spinal trauma and this growth factor has the capacity to attenuate CO production by downregulating HO-2 expression.

CO-LOCALIZATION OF CARBON MONOXIDE AND NITRIC OXIDE SYNTHESIZING ENZYMES IN THE HUMAN URETHRAL SPHINCTER

Purpose Ineffective relaxation of the urethral sphincter during micturition can result in obstructive voiding symptoms. Several studies suggest carbon monoxide (CO) acts alongside nitric oxide (NO) as an inhibitory neurotransmitter, and may regulate NO production. We have investigated the distribution of the constitutive CO producing enzyme, heme oxygenase-2 (HO-2) and the NO producing enzyme, neuronal nitric oxide synthase (nNOS), in the human urethra. Materials and Methods Transverse cryostat sections (12 micro m.) were cut from three male membranous and six female urethrae. Double immunofluorescence was carried out for colocalization of HO-2 with nNOS using standard methodology. Results Nerve trunks showing both HO-2 and nNOS immunoreactivities were identified in the urethrae in both sexes. In the female urethrae, of 152 ganglionic cell bodies expressing HO-2 or nNOS immunoreactivity, 74.3% exhibited both HO-2 and nNOS immunoreactivities, 25% exhibited only HO-2 immunoreactivity and 0.7% exhibited only nNOS immunoreactivity. Conclusions Immunoreactivity for HO-2 has been demonstrated in neuronal structures innervating the male and female urethral sphincters. The dual expression of HO-2 with nNOS immunoreactivity in cell bodies and nerves suggests that there is an interaction between the CO and NO generating systems. Abnormality in these systems may play a role in urethral dysfunction.

Effect of vasospasm on heme oxygenases in a rat model of subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH)-induced heme oxygenase-1 (HO-1) in glia throughout the rat brain without affecting heme oxygenase-2 (HO-2). However, the relationship between cerebral vasospasm and the expression of heme oxygenases after SAH is thus far unknown. The purpose of the present study was to clarify the effect of vasospasm on the expression of heme oxygenases in a rat model of SAH. Endothelin, hemolysate, hemolysate saturated with carbon monoxide (CO-hemolysate), and saline were injected into the cisterna magna of adult rats. Angiography was repeated before each injection and 15 and 60 minutes and 24 hours after each injection. Immunocytochemistry for HO-1, HO-2, and glial fibrillary acidic protein (GFAP) was performed 24 hours after the injection. A significant vasospasm occurred in the basilar artery after the injection of endothelin, hemolysate, and CO-hemolysate. The degree of vasospasm was most prominent 15 minutes after each injection. There was no significant difference in the degree of vasospasm among injections. The HO-1 was induced exclusively in the glial cells throughout the brain after injection of hemolysate and CO-hemolysate; however, it was not induced by endothelin and saline. In the dentate gyrus of the hippocampus and the molecular layer of the cerebellum, the HO-1-positive cells were also stained for GFAP, suggesting astrocytic glial cells. On the other hand, HO-2 immunoreactivity was abundant in neurons and was not affected by endothelin, hemolysate, CO-hemolysate, or saline. It is suggested that heme per se, rather than ischemia induced by vasospasm, plays a pivotal role in the expression of HO-1 in this rat model.

Corticosterone promotes increased heme oxygenase-2 protein and transcript expression in the newborn rat brain

Heme oxygenase-2 (HO-2) is the predominant heme oxygenase isozyme in neurons in the brain, the enzyme cleaves the heme molecule at the a-meso carbon bridge to form CO, Fe and biliverdin. Recently, in the promotor region of the HO-2 gene a consensus sequence of the glucocorticoid response element (GRE) has been identified. Presently, we have investigated the potential relevance of the GRE to the expression of the isozyme, at the transcript and protein levels, in the 14 day old rat brain, by examining the effect of postparturition corticosterone treatment (4 days, starting 24–36 h after birth) on the developmental pattern of HO-2 expression. Northern blot analysis showed that HO-2 transcripts (∼ 1.3 and ∼ 1.9 kb) in brain increase with age. In many brain nuclei, HO-2 protein, as visualized by immunohistochemistry, was detected at low levels in neurons in the 14 day old rat brain. Postparturition exposure to corticosterone resulted in a marked enhancement of HO-2 immunoreactivity in several neuronal populations, including, among others, the cerebellum, the hippocampal formation, and the oculomotor and red nuclei. The response to elevated levels of corticosterone was particularly striking in the Purkinje neurons of the cerebellum and the CA3 region of the hippocampus. This was linked to an increase in gene transcription, as indicated by in situ hybridization analysis, which revealed an increase in the signal for HO-2 transcripts in these regions. Elevated levels of heme oxygenase activity and HO-2 protein were consistent with an increase in catalytically active protein expression. These data point to the intimate involvement of the adrenal steroids in developmentally-linked HO-2 expression in the neurons involved in motor function and cognition, and hence, identify a potentially important aspect of the adrenal steroids' effect on brain growth and differentiation.

Direct demonstration of a physiological role for carbon monoxide in olfactory receptor neurons

Recent evidence suggests that, like nitric oxide (NO), carbon monoxide (CO), another activator of soluble guanylyl cyclase, may serve as an intercellular messenger in the brain. Heme oxygenase, which converts heme to biliverdin and CO, is abundantly expressed in the brain and is localized to discrete neuronal populations. However, evidence for the actual generation of CO by neurons is lacking. Heme oxygenase-2 immunoreactivity is abundantly present in olfactory receptor neurons where it essentially colocalizes with immunoreactivity to soluble guanylyl cyclase, the target of CO action. To examine the generation of CO by neurons, we measured CO production directly and determined its relationship to cyclic GMP levels in cultured rat olfactory receptor neurons. This system has the advantage of not having measurable NO production, which could confound results since NO is a more potent activator of guanylyl cyclase than CO. Metabolic labeling experiments permitted the direct measurement of 14CO production by neurons in vitro. CO release parallels endogenous cyclic GMP concentrations with its peak at the immature stage of neuronal differentiation in culture. Cyclic GMP production is inhibited by zinc protoporphyrin-9 and zinc deuteroporphyrin IX 2,4-bis glycol, inhibitors of heme oxygenase, indicating that CO is an endogenous regulator of soluble guanylyl cyclase activities in these cells. Transforming growth factor-beta 2, an olfactory neurogenic factor, specifically shows a negative effect on CO release in olfactory receptor neurons. These results indicate that CO may serve as a gaseous neuronal messenger linked to cyclic GMP production and suggests its involvement in developmental processes of the olfactory receptor neuron.