Heme oxygenase is necessary for the excitatory response of cultured neonatal rat rostral ventrolateral medulla neurons to hypoxia

Heme oxygenase has been linked to the oxygen-sensing function of the carotid body, pulmonary vasculature, cerebral vasculature, and airway smooth muscle. We have shown previously that the cardiorespiratory regions of the rostral ventrolateral medulla are excited by local hypoxia and that heme oxygenase-2 (HO-2) is expressed in the hypoxia-chemosensitive regions of the rostral ventrolateral medulla (RVLM), the respiratory pre-B?tzinger complex, and C1 sympathoexcitatory region. To determine whether heme oxygenase is necessary for the hypoxic-excitation of dissociated RVLM neurons (P1) cultured on confluent medullary astrocytes (P5), we examined their electrophysiological responses to hypoxia (NaCN and low Po(2)) using the whole-cell perforated patch clamp technique before and after blocking heme oxygenase with tin protoporphyrin-IX (SnPP-IX). Following the electrophysiological recording, immunocytochemistry was performed on the recorded neuron to correlate the electrophysiological response to hypoxia with the expression of HO-2. We found that the responses to NaCN and hypoxia were similar. RVLM neurons responded to NaCN and low Po(2) with either depolarization or hyperpolarization and SnPP-IX blocked the depolarization response of hypoxia-excited neurons to both NaCN and low Po(2) but had no effect on the hyperpolarization response of hypoxia-depressed neurons. Consistent with this observation, HO-2 expression was present only in the hypoxia-excited neurons. We conclude that RVLM neurons are excited by hypoxia via a heme oxygenase-dependent mechanism.     

Effects of hypoxia on heme oxygenase expression in human chorionic villi explants and immortalized trophoblast cells

Although hypoxia induces heme oxygenase (HO)-1 protein and mRNA expression in many cell types, hypoxia has also been shown to decrease HO-1 mRNA and protein expression. We tested the hypothesis that 24-h preexposure to hypoxia in human placental preparations suppresses HO protein expression and enzymatic function. Immortalized HTR-8/SVneo first-trimester trophoblast cells and explants of normal human chorionic villi (CV) from term placentas were cultured for 24 h in 1%, 5%, or 20% O(2). HO protein levels were determined by Western blot analysis, and microsomal HO activity was measured. HO-2 protein content was decreased by 17% and 5% in human trophoblast cells after 24-h exposure to 1% and 5% O(2), respectively, versus 20% O(2). In contrast, HO-2 protein content in CV explants was unaffected by changes in oxygenation. HO-1 protein content, which was barely detectable in both biological systems, was not affected by changes in oxygenation. Similarly, HO enzymatic activity was unchanged in both preparations after 24-h exposure to 1%, 5%, or 20% O(2). The above data do not support the hypothesis that hypoxia in the human placenta suppresses both HO protein content and HO protein function. The present observations reinforce the necessity to determine both HO protein expression and function.     

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.     

Interaction between endothelial heme oxygenase-2 and endothelin-1 in altered aortic reactivity after hypoxia in rats

The aim of this study was to determine whether increased expression of heme oxygenase (HO) contributes to impairment of aortic contractile responses after hypoxia through effects on reactivity to endothelin-1 (ET-1). Thoracic aortas from normoxic rats and rats exposed to hypoxia (10% O2) for 16 or 48 h were mounted in organ bath myographs for contractile studies, fixed in paraformaldehyde, or frozen in liquid nitrogen for protein extraction. In rings from normoxic rats, the HO inhibitor tin protoporphyrin IX (SnPP IX, 10 microM) did not alter the response to phenylephrine or ET-1. In rings from rats exposed to 16-h hypoxia, maximum tension generated in response to these agonists was higher in endothelium-intact but not -denuded rings in the presence of SnPP IX. In rings from rats exposed to 48-h hypoxia SnPP IX increased contraction in endothelium-intact but not -denuded rings. In endothelium-intact aortic rings from rats exposed to 16-h hypoxia incubated with endothelin A receptor-specific antagonist BQ-123 (10(-7) M), SnPP IX did not alter phenylephrine-induced contraction. Aortic ET-1 protein levels, measured by radioimmunoassay, were increased in rats exposed to hypoxia for 16 and 48 h. Western blotting showed that HO-1 and HO-2 protein were increased after 16 h of hypoxia and returned to near-control levels after 48 h. Increase in HO-1 protein was detected in endothelium-intact and -denuded rings. Removal of endothelium abolished the increase in HO-2 immunoreactivity. Immunohistochemistry localized expression of HO-1 protein to vascular smooth muscle, whereas HO-2 was only detected in endothelium. HO-2 is expressed by aortic endothelial cells early during hypoxic exposure and impairs ET-1-mediated potentiation of contraction to alpha-adrenoceptor stimulation.     

Opposite effects of prostaglandin-J2 on VEGF in normoxia and hypoxia: role of HIF-1

The vascular endothelial growth factor (VEGF) is produced in response to hypoxia or inflammatory cytokines. In normoxia VEGF synthesis is upregulated by 15-deoxy-Delta(12,14)-prostaglandin-J(2) (15d-PGJ(2)) via induction of heme oxygenase-1 (HO-1). Here we compared the influence of 15d-PGJ(2) on VEGF expression in human microvascular endothelial cells in normoxia (approximately 20% O(2)) and hypoxia ( approximately 2% O(2)). Regardless of the oxygen concentration, 15d-PGJ(2) inhibited activity of hypoxia inducible factor-1 (HIF-1), the major hypoxic regulator of VEGF. However, in normoxic conditions 15d-PGJ(2) (1-10microM) activated the VEGF promoter and increased synthesis of the VEGF protein. Concomitantly, it strongly induced expression of HO-1. In contrast, in hypoxia, 15d-PGJ(2) decreased VEGF promoter activity and reduced VEGF release by 50%. Inhibition of HO-1 activity additionally attenuated VEGF synthesis in hypoxia. We conclude that induction of HO-1 by 15d-PGJ(2) results in augmentation of VEGF synthesis in normoxia. In hypoxia, however, the stimulatory effect of HO-1 is outweighed by 15d-PGJ(2)-mediated inhibition of the HIF-1 pathway.     

Exposure to hypoxia primes the respiratory and metabolic responses of the epaulette shark to progressive hypoxia

The majority of vertebrates are not tolerant to hypoxia but epaulette sharks (Hemiscyllium ocellatum) living on shallow reef platforms appear to tolerate hypoxic periods during tidal fluctuations. The effects of progressive hypoxia on the metabolic and ventilatory responses of these elasmobranchs were examined in a closed respirometer. In order to determine whether repeated exposure to hypoxia primes these sharks to alter their metabolism, one group of sharks was exposed to repeated sub-lethal hypoxia, at 5% of air saturation, prior to respirometry. In response to falling oxygen concentration [O(2)], the epaulette shark increased its ventilatory rate and maintained its O(2) consumption rate (VO(2)) down to 2.2 mg O(2) l(-1) at 25 degrees C. This is the lowest critical [O(2)] ([O(2)](crit)) ever measured for any elasmobranch. After reaching the [O(2)](crit), the shark remained in the respirometer for a further 4-5 h of progressive hypoxia. Only after the [O(2)] fell to 1.0 mg l(-1) was there a decrease in the ventilatory rate followed by a rise in blood lactate levels, indicating that the epaulette shark responds to severe hypoxia by entering a phase of metabolic and ventilatory depression. Interestingly, hypoxia tolerance was dynamic because hypoxic pre-conditioning lowered the VO(2) of the epaulette shark by 29%, which resulted in a significantly reduced [O(2)](crit) (1.7 mg O(2) l(-1)), revealing that hypoxic pre-conditioning elicits an enhanced physiological response to hypoxia.     

Heme oxygenase activity in placenta: direct dependence on oxygen availability

Carbon monoxide (CO), which is formed endogenously from heme catalyzed by heme oxygenase (HO), is proposed to play a role in vascular control. The mRNA and protein expression of the inducible isoform of HO (HO-1) increases in response to hypoxia, and it has been assumed that HO activity also increases. This assumption requires evaluation because the catalytic activity of HO requires three molecules of O(2) for each molecule of CO formed from heme, and HO activity may be limited by O(2) availability. To test the hypothesis that low physiological O(2) concentrations limit HO activity, heme-derived CO formation by microsomal fractions of homogenates of chorionic villi of human placentas was determined after exposure to 0, 1, 5, or 21% O(2). Results revealed that HO activity was directly dependent on O(2) concentration. Thus, although hypoxia may increase HO protein and mRNA expression, there is a progressive decrease in HO activity with decreasing O(2) concentration and the dependence of HO activity on O(2) concentration is similar in chorionic villi from noninfarcted areas of preeclamptic and normotensive placenta.     

Hypoxemia and blunted hypoxic ventilatory responses in mice lacking heme oxygenase-2

Heme oxygenase (HO) catalyzes physiological heme degradation and consists of two structurally related isozymes, HO-1 and HO-2. Here we show that HO-2-deficient (HO-2(-/-)) mice exhibit hypoxemia and hypertrophy of the pulmonary venous myocardium associated with increased expression of HO-1. The hypertrophied venous myocardium may reflect adaptation to persistent hypoxemia. HO-2(-/-) mice also show attenuated ventilatory responses to hypoxia (10% O2) with normal responses to hypercapnia (10% CO2), suggesting the impaired oxygen sensing. Importantly, HO-2(-/-) mice exhibit normal breathing patterns with normal arterial CO2 tension and retain the intact alveolar architecture, thereby excluding hypoventilation and shunting as causes of hypoxemia. Instead, ventilation-perfusion mismatch is a likely cause of hypoxemia, which may be due to partial impairment of the lung chemoreception probably at pulmonary artery smooth muscle cells. We therefore propose that HO-2 is involved in oxygen sensing and responsible for the ventilation-perfusion matching that optimizes oxygenation of pulmonary blood.     

Heme oxygenase activity in fetal and adult sheep is not altered by acclimatization to high altitude hypoxia

Hypoxic stress has been reported to induce the expression of stress proteins such as heme oxygenase (HO), which catalyze the breakdown of heme to generate biliverdin, ferrous iron, and carbon monoxide. These degradation products play a role in the regulation of a variety of processes such as vascular tone, inflammation, and central nervous system function. In mammals, there are 2 catalytically functional HO isozymes, HO-1 (inducible) and HO-2 (constitutive). HO-1 expression is regulated by an array of nonphysiological and physiological stimuli including acute hypoxemia. As relatively little is known of the HO response to prolonged hypoxia in whole animals other than small laboratory rodents, the aim of this work was to examine the effect of long-term hypoxia on total HO activity in fetal and adult ovine tissue. Sheep were maintained at high altitude (3820 m), after which the following tissues were harvested from near-term fetal and non-pregnant ewes for in vitro measurement of HO activity: left ventricle, renal papilla, lung apex, pulmonary artery, carotid artery, mesenteric artery, placental cotyledon, spleen, and brain frontal cortex. There were no significant differences between HO activities in tissues from hypoxic fetal and adult sheep compared with their normoxic controls. Fetal heart HO activities were higher than those of adult tissue (p < 0.05), whereas adult spleen HO activity was significantly higher than that of fetal tissue (p < 0.05). In conclusion, these data indicate that long-term exposure to high altitude hypoxia does not have a persistent effect on HO activity in ovine tissues. Also, except for the spleen where there is a high expression of HO-1 under normal conditions, tissue HO activity is correlated with the expression of HO-2, the constitutive isozyme.     

Hypoxia activates nucleus tractus solitarii neurons projecting to the paraventricular nucleus of the hypothalamus

Peripheral chemoreceptor afferent information is sent to the nucleus tractus solitarii (nTS), integrated, and relayed to other brain regions to alter cardiorespiratory function. The nTS projects to the hypothalamic paraventricular nucleus (PVN), but activation and phenotype of these projections during chemoreflex stimulation is unknown. We hypothesized that activation of PVN-projecting nTS neurons occurs primarily at high intensities of hypoxia. We assessed ventilation and cardiovascular parameters in response to increasing severities of hypoxia. Retrograde tracers were used to label nTS PVN-projecting neurons and, in some rats, rostral ventrolateral medulla (RVLM)-projecting neurons. Immunohistochemistry was performed to identify nTS cells that were activated (Fos-immunoreactive, Fos-IR), catecholaminergic, and GABAergic following hypoxia. Conscious rats underwent 3 h normoxia (n = 4, 21% O(2)) or acute hypoxia (12, 10, or 8% O(2); n = 5 each). Hypoxia increased ventilation and the number of Fos-IR nTS cells (21%, 13 ± 2; 12%, 58 ± 4; 10%, 166 ± 22; 8%, 186 ± 6). Fos expression after 10% O(2) was similar whether arterial pressure was allowed to decrease (-13 ± 1 mmHg) or was held constant. The percentage of PVN-projecting cells activated was intensity dependent, but contrary to our hypothesis, PVN-projecting nTS cells exhibiting Fos-IR were found at all hypoxic intensities. Notably, at all intensities of hypoxia, ～75% of the activated PVN-projecting nTS neurons were catecholaminergic. Compared with RVLM-projecting cells, a greater percentage of PVN-projecting nTS cells was activated by 10% O(2). Data suggest that increasing hypoxic intensity activates nTS PVN-projecting cells, especially catecholaminergic, PVN-projecting neurons. The nTS to PVN catecholaminergic pathway may be critical even at lower levels of chemoreflex activation and more important to cardiorespiratory responses than previously considered.     

Expression of heme oxygenase-1 in the lung in chronic hypoxia

Heme oxygenase (HO)-1 is an oxygen-dependent enzyme that may regulate vascular tone and cell proliferation through the production of carbon monoxide (CO). We tested the hypothesis that HO-1 is upregulated in the lung in chronic hypoxia by exposing male Sprague-Dawley rats to 17,000 feet (395 Torr) for 0, 1, 3, 7, 14, or 21 days. After exposure, blood gases, carboxyhemoglobin (COHb) levels, and hematocrit were measured, and the lungs were either inflation fixed for immunohistochemistry or frozen for later measurement of HO enzyme activity, Western blot for HO-1 protein, and RT-PCR for HO-1 mRNA. The heart was excised and weighed, and the right-to-left heart weight ratio was determined. During hypoxia, the hematocrit increased progressively, reaching significantly higher values than the control value after 3 days. COHb levels increased above the control value after 1 day of hypoxia and increased progressively between 14 and 21 days, whereas arterial PO(2) and arterial PCO(2) did not vary significantly. HO-1 protein determined by Western blot increased for the first 7 days and declined thereafter; however, enzyme activity was elevated only after 1 day. Changes in HO-1 during hypoxia were localized by immunohistochemistry to inflammatory cells (early) and newly muscularized arterioles (later). Lung HO-1 mRNA normalized to glyceraldehyde-3-phosphate dehydrogenase was increased after 1 and 21 days. The data indicate that lung HO-1 protein and activity are upregulated only during early chronic hypoxia, whereas persistent COHb elevations indicate high endogenous CO production rates at nonpulmonary sites. If CO has antiproliferative properties, the lack of HO enzyme activity in the lung may be permissive for pulmonary vascular proliferation in hypoxia.     

Regional hypoxic pulmonary vasoconstriction in prone pigs.

Hypoxic pulmonary vasoconstriction (HPV) is known to affect regional pulmonary blood flow distribution. It is unknown whether lungs with well-matched ventilation (V)/perfusion (Q) have regional differences in the HPV response. Five prone pigs were anesthetized and mechanically ventilated (positive end-expiratory pressure = 2 cmH2O). Two hypoxic preconditions [inspired oxygen fraction (FI(O2)) = 0.13] were completed to stabilize the animal's hypoxic response. Regional pulmonary blood Q and V distribution was determined at various FI(O2) (0.21, 0.15, 0.13, 0.11, 0.09) using the fluorescent microsphere technique. Q and V in the lungs were quantified within 2-cm3 lung pieces. Pieces were grouped, or clustered, based on the changes in blood flow when subjected to increasing hypoxia. Unique patterns of Q response to hypoxia were seen within and across animals. The three main patterns (clusters) showed little initial difference in V/Q matching at room air where the mean V/Q range was 0.92-1.06. The clusters were spatially located in cranial, central, and caudal portions of the lung. With decreasing FI(O2), blood flow shifted from the cranial to caudal regions. We determined that pulmonary blood flow changes, caused by HPV, produced distinct response patterns that were seen in similar regions across our prone porcine model.     

Repression of heme oxygenase-1 by hypoxia in vascular endothelial cells

Heme oxygenase 1 (HO-1), a rate-limiting enzyme in heme catabolism, has been reported to be induced by hypoxia. Unexpectedly, here we show that expression of HO-1 mRNA is repressed by hypoxia in primary cultures of human umbilical vein endothelial cells (HUVECs), but is increased by cobalt chloride (CoCl(2)) that is known to mimic hypoxia. Under the culture conditions used, the DNA-binding and transactivation activities of hypoxia-inducible factor 1 were increased in HUVECs by hypoxia or CoCl(2). Therefore, hypoxia and cobalt showed opposing effects on HO-1 mRNA expression, despite activation of hypoxia-inducible factor 1. The half-life of HO-1 mRNA was not changed by hypoxia, but was significantly prolonged by CoCl(2). Hypoxia also represses HO-1 mRNA expression in human coronary artery endothelial cells and astrocytes. The repression of HO-1 expression may represent the adaptation to hypoxia in certain cell types.     

Intermittent hypoxia increases ventilation and Sa(O2) during hypoxic exercise and hypoxic chemosensitivity.

The purpose of this study was 1) to test the hypothesis that ventilation and arterial oxygen saturation (Sa(O2)) during acute hypoxia may increase during intermittent hypoxia and remain elevated for a week without hypoxic exposure and 2) to clarify whether the changes in ventilation and Sa(O2) during hypoxic exercise are correlated with the change in hypoxic chemosensitivity. Six subjects were exposed to a simulated altitude of 4,500 m altitude for 7 days (1 h/day). Oxygen uptake (VO2), expired minute ventilation (VE), and Sa(O2) were measured during maximal and submaximal exercise at 432 Torr before (Pre), after intermittent hypoxia (Post), and again after a week at sea level (De). Hypoxic ventilatory response (HVR) was also determined. At both Post and De, significant increases from Pre were found in HVR at rest and in ventilatory equivalent for O2 (VE/VO2) and Sa(O2) during submaximal exercise. There were significant correlations among the changes in HVR at rest and in VE/VO2 and Sa(O2) during hypoxic exercise during intermittent hypoxia. We conclude that 1 wk of daily exposure to 1 h of hypoxia significantly improved oxygenation in exercise during subsequent acute hypoxic exposures up to 1 wk after the conditioning, presumably caused by the enhanced hypoxic ventilatory chemosensitivity.     

Role of heme oxygenase-1 in hypoxia-reoxygenation: requirement of substrate heme to promote cardioprotection

Heme oxygenase-1 (HO-1) catalyzes the enzymatic degradation of heme to carbon monoxide, bilirubin, and iron. All three products possess biological functions; bilirubin, in particular, is a potent free radical scavenger of which its antioxidant property is enhanced at low oxygen tension. Here, we investigated the effect of severe hypoxia and reoxygenation on HO-1 expression in cardiomyocytes and determined whether HO-1 and its product, bilirubin, have a protective role against reoxygenation damage. Hypoxia caused a time-dependent increase in both HO-1 expression and heme oxygenase activity, which gradually declined during reoxygenation. Reoxygenation of hypoxic cardiomyocytes produced marked injury; however, incubation with hemin or bilirubin during hypoxia considerably reduced the damage at reoxygenation. The protective effect of hemin is attributable to increased availability of substrate for heme oxygenase activity, because hypoxic cardiomyocytes generated very little bilirubin when incubated with medium alone but produced substantial bile pigment in the presence of hemin. Interestingly, incubation with hemin also maintained high heme oxygenase activity levels during the reoxygenation period. Reactive oxygen species generation was enhanced after hypoxia, and hemin and bilirubin were capable once again to attenuate this effect. These results indicate that the HO-1-bilirubin pathway can effectively defend hypoxic cardiomyocytes against reoxygenation injury and highlight the issue of heme availability in the cytoprotective action afforded by HO-1.     

Carbon monoxide as an endogenous vascular modulator

Carbon monoxide (CO) is produced by heme oxygenase (HO)-catalyzed heme degradation to CO, iron, and biliverdin. HO has two active isoforms, HO-1 (inducible) and HO-2 (constitutive). HO-2, but not HO-1, is highly expressed in endothelial and smooth muscle cells and in adjacent astrocytes in the brain. HO-1 is expressed basally only in the spleen and liver but can be induced to a varying extent in most tissues. Elevating heme, protein phosphorylation, Ca(2+) influx, and Ca(2+)/calmodulin-dependent processes increase HO-2 activity. CO dilates cerebral arterioles and may constrict or dilate skeletal muscle and renal arterioles. Selected vasodilatory stimuli, including seizures, glutamatergic stimulation, hypoxia, hypotension, and ADP, increase CO, and the inhibition of HO attenuates the dilation to these stimuli. Astrocytic HO-2-derived CO causes glutamatergic dilation of pial arterioles. CO dilates by activating smooth muscle cell large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels. CO binds to BK(Ca) channel-bound heme, leading to an increase in Ca(2+) sparks-to-BK(Ca) channel coupling. Also, CO may bind directly to the BK(Ca) channel at several locations. Endothelial nitric oxide and prostacyclin interact with HO/CO in circulatory regulation. In cerebral arterioles in vivo, in contrast to dilation to acute CO, a prolonged exposure of cerebral arterioles to elevated CO produces progressive constriction by inhibiting nitric oxide synthase. The HO/CO system is highly protective to the vasculature. CO suppresses apoptosis and inhibits components of endogenous oxidant-generating pathways. Bilirubin is a potent reactive oxygen species scavenger. Still many questions remain about the physiology and biochemistry of HO/CO in the circulatory system and about the function and dysfunction of this gaseous mediator system.