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Am J Physiol Heart Circ Physiol 288: H962-H970, 2005. First published October 14, 2004; doi:10.1152/ajpheart.01218.2003
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Interaction between endothelial heme oxygenase-2 and endothelin-1 in altered aortic reactivity after hypoxia in rats

Vasanthi Govindaraju,1 Hwee Teoh,2 Qutayba Hamid,1 Peter Cernacek,3 and Michael E. Ward2

2Division of Respirology and Department of Critical Care, St Michael's Hospital, University of Toronto, Toronto, Ontario; and 1Meakins Christie Laboratories and 3Royal Victoria Hospital, McGill University, Montreal, Quebec, Canada

Submitted 22 December 2003 ; accepted in final form 5 October 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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 µM) 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.

vascular reactivity; oxygen delivery; blood flow regulation

THE ENDOTHELINS ARE POTENT vasoconstrictor peptides released by endothelial cells in response to a variety of stimuli. Hypoxia is a particularly potent activator of endothelin-1 (ET-1) production (19, 47), and recent studies indicate that ET-1 plays a central role in adaptation to hypoxia, regulating peripheral chemoreceptor sensitivity (36) and potentiating the vascular responses that enhance pulmonary gas exchange and tissue oxygen extraction (5, 34, 48, 52). Given the need to balance these important functions with the potential for exacerbation of ischemic injury through excessive vasoconstriction (6), it is not surprising that mechanisms have evolved to modulate the local bioavailability and potency of ET-1 to preserve the relationship between regional oxygen delivery and metabolic demand (20, 46).

Heme oxygenase (HO), the rate-limiting enzyme in the heme catabolic pathway that yields bilirubin as the final product, catalyzes the formation of biliverdin through the elimination of the {alpha}-methene carbon bridge of the porphyrin ring as CO (24). Both biliverdin and bilirubin possess antioxidant properties and, therefore, the capacity to suppress intracellular concentrations of the reactive oxygen species that regulate ET-1 precursor mRNA expression (18, 37) and that serve as second messengers in ET-1 signaling (22). CO mimics many NO functions including cGMP-dependent and -independent inhibition of agonist-induced vascular smooth muscle contraction (29, 44, 45). The products of the reaction catalyzed by HO, therefore, have the capacity to attenuate ET-1-mediated effects at multiple levels. HO expression is hypoxia inducible in vascular cells (30) and modulates arterial reactivity in some vascular beds. Accordingly, the current study was carried out to determine whether HO contributes to the change in aortic contractile responses after exposure to hypoxia (2) and the extent to which this is due to modulation of the effect of locally produced ET-1.


   MATERIALS AND METHODS
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RESULTS
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Studies were carried out in male Sprague-Dawley rats (200–250 g). All protocols were in accordance with standards set by the Canadian Council on Animal Care and were approved by the institutional animal care committee. As described previously (2), rats were placed in a Plexiglas chamber (30 cm x 18 cm x 15 cm) into which the flows of air and nitrogen were controlled independently. Oxygen concentration in the chamber was monitored continuously with an oximeter (Oxan 1501, Engineered Systems Design, Wilmington, DE), and air and nitrogen flows were adjusted such that animals exposed to hypoxia breathed a mixture containing 10% O2 throughout the hypoxia exposure period, whereas control animals breathed air only under otherwise identical conditions. Thoracic aortas were excised immediately after decapitation and mounted in organ bath myographs, frozen in liquid nitrogen for later protein extraction, or fixed for immunohistochemical studies.

Tin protoporphyrin IX (SnPP IX) and copper protoporphyrin IX (CuPP IX) were purchased from Porphyrin Products (Logan, UT). BQ-123 was from American Peptide (Sunnyvale, CA). All other chemicals were purchased from Sigma (St. Louis, MO). Monoclonal HO-1 and polyclonal HO-2 antibodies were obtained from StressGen (Victoria, BC, Canada). Rabbit anti-ET-1 antiserum was from Peninsula Laboratories (Belmont, CA). Immunohistochemical reagents were bought from DAKO (Carpinteria, CA). Electrophoresis reagents were from Bio-Rad (Mississauga, ON, Canada). Donkey anti-rabbit-horseradish peroxidase (HRP) secondary antibody and enhanced chemiluminescence reagents and film were from Amersham (Oakville, ON, Canada).

Functional studies. Thoracic aortas were cleaned of connective tissue and cut into 4-mm segments. The endothelium was removed from some segments by gentle abrasion of the luminal surface. Segments were suspended in organ bath myographs containing Krebs-Henseleit solution (in mM: 120 NaCl, 25 NaHCO3, 11.1 glucose, 4.76 KCl, 1.18 MgSO4, 1.18 KH2PO4, 2.5 CaCl2) aerated with 95% O2-5% CO2 at 37°C (2). Baseline tension was adjusted to 2 g, the optimum tension for maximal responses under our experimental conditions (2), and equilibrated for 1 h. Failure of acetylcholine (1 µM) to elicit relaxation after contraction with phenylephrine (1 µM) was taken as evidence of functional endothelial ablation (2, 50).

Cumulative concentration-response relationships for phenylephrine (10–9–10–5 M) were generated in endothelium-intact and -denuded aortic rings from normoxic rats and from rats exposed to hypoxia for 16 and 48 h after 30-min incubation with, and in the continuous presence of, 0.1% DMSO (vehicle) or the HO inhibitor SnPP IX (10 µM). Phenylephrine concentration-response relationships were also generated in the presence of the inactive protoporphyrin cogener CuPP IX (30 µM) in endothelium-intact and -denuded rings from rats exposed to hypoxia for 16 h, the time point at which the SnPP IX effect was found to be maximal, to test for nonspecific effects.

To establish the relative importance of HO and NO pathways in inhibition of aortic reactivity after hypoxia and to ensure that the effects of SnPP IX were not due to nonspecific inhibition of NO synthesis (1), concentration-response relationships for phenylephrine were generated in the presence of vehicle (DMSO), the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 10 µM), SnPP IX (10 µM), or both L-NAME and SnPP IX in endothelium-intact aortic rings from rats exposed to hypoxia for 16 h, the time point at which maximum HO-2 expression was noted (see below),

To evaluate the role of effects on endothelin A receptor (ETAR) activation and/or signaling in the enhancement of aortic contraction by HO inhibition observed in hypoxia-exposed rats, concentration-response relationships for phenylephrine were generated in a separate group of endothelium-intact aortic rings from rats exposed to normoxia or hypoxia for 16 and 48 h in the presence of vehicle (DMSO), the ETAR-specific antagonist BQ-123 (1 µM), SnPP IX (10 µM), or both BQ-123 and SnPP IX. As reported in a previous study (52), this concentration of BQ-123 completely inhibited the response to ET-1 in rat aorta, with no effect on phenylephrine-induced contraction in endothelium-intact or -denuded aortic rings from normoxic rats or in endothelium-denuded aortic rings from rats exposed to hypoxia.

To confirm that the failure to detect an effect of SnPP IX on phenylephrine-induced contraction in endothelium-denuded rings was due to the dependence of this effect on the presence of endothelial HO rather than to removal of the source of endogenous ET-1 production, concentration-response relationships for ET-1 (10–11-10–7 M) were generated in endothelium-intact and -denuded aortic rings from rats exposed to normoxia and hypoxia for 16 h in the presence of DMSO, SnPP IX (10 µM), and CuPP IX (30 µM). The 16-h time point was chosen for these studies because this is the duration of hypoxic exposure at which HO protein levels reached their maximum (see below) and the effect of SnPP IX on phenylephrine responses in endothelium-intact aortic rings was most apparent.

ET-1 radioimmunoassay. Rat aortic ET-1 content was determined as previously described (38). Briefly, ET-1 was extracted from the supernatant of rat aortic homogenates with Sep-Pak C18 cartridges. The dried extracts and standards were reconstituted in the radioimmunoassay buffer (in mM: 19 NaH2PO4, 81 Na2HPO4, and 50 NaCl, with 0.1% BSA, 0.01% NaN3, and 0.1% Triton X-100, pH 7.4) and incubated with rabbit anti-ET-1 serum for 24 h at 4°C followed by a 24-h incubation with 4,000 counts/min of 125I-labeled ET-1. Samples were then incubated at room temperature with 1:50 normal rabbit serum and 1:25 goat anti-rabbit IgG before precipitation with 6.25% polyethylene glycol. After centrifugation, samples were aspirated and examined for gamma radioactivity emitted by the pellets.

Western blots. Rat aortas were bisected, and one-half was denuded of endothelium by gentle abrasion before being frozen in liquid nitrogen. Aortic proteins were extracted, resolved on 10–20% tricine gels, and electrotransferred onto nitrocellulose overnight at room temperature. Membranes were blocked for 2 h in 7% skimmed milk containing 1% fetal bovine serum and 0.1% Tween-Tris-buffered saline before being probed with a specific monoclonal HO-1 antibody (1:1,500) or polyclonal HO-2 antibody (1:1,000). In all cases, protein concentration was determined by the Bradford assay and appropriate volumes of extraction buffer to produce constant protein loading in each lane were mixed with SDS loading buffer. Control and hypoxic samples were always paired on each gel to control for interexperimental variation. Protein loading and transfer efficiency were verified after transfer by full-lane densitometry of the Ponceau red-stained membranes. Immunoblots were probed with HRP-donkey anti-rabbit IgG (Amersham) and visualized by enhanced chemiluminescence. Band intensity was quantified by densitometry.

Immunohistochemistry. Localization of HO protein expression within the aortic wall was carried out by immunohistochemistry in aortas from normoxic rats and from rats exposed to hypoxia for 16 h, the time at which Western blot analysis indicated that both HO-1 and HO-2 isoforms are maximally upregulated. For HO-1 analysis, 7-µm transverse sections from liquid nitrogen-frozen rat aortas were fixed in acetone-methanol (60:40). Deparaffinated and rehydrated sections (7 µm) from rat aortas that had been fixed in 4% paraformaldehyde and soaked in 30% sucrose were used for the corresponding HO-2 studies. Tissue peroxidase activity was quenched by incubating all of the sections with 3% H2O2 for 30 min before immersion in 0.1% Triton X-100 for 30 min and then in a universal blocking solution. Sections were immunolabeled with primary antibodies (HO-1 1:100; HO-2 1:500) overnight at 4°C, probed with biotinylated secondary antibodies, incubated with a HRP-streptavidin complex and placed in 3,3'-diaminobenzidine substrate solution before counterstaining with Mayer's hematoxylin and mounting.

Data analysis. On completion of the contractility experiments, rings were dried overnight at 50°C and weighed to express tension as gram per milligram of dry weight. Results obtained in aortic segments studied under a given condition from a given animal were averaged, and the averaged value was used as a single data point in the statistical analysis. Paired means were compared by two-tailed Student's t-test. Differences among multiple means were evaluated by ANOVA corrected for multiple measures where appropriate, and, when overall differences were detected, individual means were compared post hoc with the Student-Newman-Keuls procedure. Results are presented as means ± SE for n samples, with P < 0.05 representing statistical significance.


   RESULTS
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Functional studies. The effects of SnPP IX on the concentration-response relationship for phenylephrine in endothelium-intact and -denuded aortic rings from normoxic rats and rats exposed to hypoxia are illustrated in Fig. 1. The maximum tensions generated and the concentrations of phenylephrine that elicited a 50% maximal response (EC50) are presented in Table 1. SnPP IX did not alter phenylephrine-evoked responses in rings from normoxic rats or in rings from hypoxia-exposed rats that had been denuded of endothelium. In contrast, endothelium-intact rings from rats exposed to hypoxia for 16 and 48 h produced greater tension and demonstrated greater sensitivity (decreased EC50) to phenylephrine in the presence of SnPP IX than in the presence of vehicle only (Table 1). CuPP IX, the inactive cogener, had no effect on the response to phenylephrine (data not shown).



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  		Fig. 1. Phenylephrine concentration-response curves in endothelium-intact and -denuded aortic rings from normoxic rats (A and B, respectively) and rats exposed to hypoxia for 16 h (C and D, respectively) and 48 h (E and F, respectively) in the presence of tin protoporphyrin IX (SnPP IX, 10 µM) or vehicle alone (0.1% DMSO); n = 7/group. *P < 0.05 for difference from corresponding value in DMSO-treated rings.

 

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  		Table 1. Maximum tensions and EC50 values for phenylephrine responses in rings treated with SnPP IX or vehicle (DMSO) alone

 
The effects of treatment with SnPP IX, L-NAME, and both SnPP IX and L-NAME on phenylephrine-induced contraction in endothelium-intact aortic rings from rats exposed to hypoxia for 16 h are illustrated in Fig. 2. The maximum tensions generated during phenylephrine-induced contraction in the presence of SnPP IX (1.426 ± 0.055 g/mg dry wt) and L-NAME (1.491 ± 0.031 g/mg dry wt) did not differ. The maximum tension generated in rings treated with both SnPP IX and L-NAME (1.93 ± 0.045 g/mg dry wt) was greater than that recorded in the presence of either agent alone, and the increase in tension in rings treated with both agents over that recorded in rings exposed to vehicle alone (1.036 ± 0.071 g/mg dry wt) did not differ from the sum of the corresponding increases resulting from treatment with the individual antagonists.



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  		Fig. 2. Phenylephrine concentration-response curves of endothelium-intact and -denuded aortic rings from rats exposed to hypoxia for 16 h in the presence of SnPP IX, NG-nitro-L-arginine methyl ester (L-NAME), both L-NAME and SnPP IX, and vehicle (DMSO, 0.1%) alone; n = 6/group. *P < 0.05 for difference from corresponding value in DMSO-treated rings or for differences between rings treated with either L-NAME or SnPP IX and those treated with both L-NAME and SnPP IX.

 
Concentration-response relationships for phenylephrine obtained in endothelium-intact aortic rings from rats exposed to hypoxia for 16 and 48 h in the presence of vehicle (DMSO) alone, SnPP IX, BQ-123 (1 µM), and both SnPP IX and BQ-123 are presented in Fig. 3. In endothelium-intact rings from these groups the phenylephrine concentration-response relationship recorded in the presence of both SnPP IX and BQ-123 did not differ from that obtained in the presence of BQ-123 alone. As expected, given that neither SnPP IX nor BQ-123 had any effect on phenylephrine-induced contraction in aortic rings from normoxic animals, the combination of these two antagonists also did not alter the phenylephrine response in rings from normoxic rats (data not shown).



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  		Fig. 3. Phenylephrine concentration-response relationships in endothelium-intact aortic rings from rats exposed to hypoxia for 16 and 48 h in the presence of SnPP IX, BQ-123, both SnPP IX and BQ-123, and vehicle (DMSO, 0.1%) alone; n = 7/group. *P < 0.05 for difference from corresponding value in DMSO-treated rings.

 
The effect of SnPP IX (10 µM) on ET-1-induced contraction is illustrated in Fig. 4. In endothelium-intact but not -denuded aortic rings from rats exposed to hypoxia for 16 h, SnPP IX enhanced the contractile response. The inactive cogener, CuPP IX (30 µM), had no effect. SnPP IX had no effect on ET-1-induced contraction in either endothelium-intact or -denuded aortic rings from normoxic animals (data not shown).



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  		Fig. 4. Concentration-response relationships for endothelin-1 (ET-1) in endothelium-intact and -denuded aortic rings from rats exposed to hypoxia for 16 h in the presence of SnPP IX (10 µM), copper protoporphyrin IX (CuPP IX, 30 µM), or vehicle (0.1% DMSO); n = 7/group. *P < 0.05 for difference from rings treated with DMSO alone.

 
Aortic ET-1 levels. The results of the ET-1 radioimmunoas say in endothelium-intact and -denuded aortic segments from rats exposed to normoxia and to hypoxia for 16 and 48 h are presented in Fig. 5. Hypoxia increased ET-1 levels in intact rat aortic segments (P < 0.01, 16- and 48-h hypoxic vs. normoxic control group). In endothelium-denuded aortic segments ET-1 levels did not differ between hypoxia-exposed and normoxic animals, localizing the increase in ET-1 production to the endothelium.



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  		Fig. 5. ET-1 levels in endothelium-intact and -denuded thoracic aortas from rats exposed to normoxia and to hypoxia for 16 and 48 h; n = 10/group; *P < 0.05 compared with corresponding value in the normoxic control group.

 
Western blots. Levels of both HO isoforms were increased in aortic homogenates after 16 h of hypoxia and had returned to near-control levels after 48 h of hypoxic exposure (Fig. 6). Removal of the endothelium had no effect on the increase in HO-1 protein level recorded after 16 h of hypoxia, indicating that the majority of this increase occurred within the aortic media. In contrast, no increase in aortic HO-2 protein was detected in endothelium-denuded segments from hypoxia-exposed compared with normoxic rats, localizing the increase in this isoform to the endothelial cell layer.



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  		Fig. 6. Top: heme oxygenase-1 (HO-1) protein levels in endothelium-intact (EC intact) and -denuded (EC removed) thoracic aortas from rats exposed to normoxia or to hypoxia for 16 and 48 h; n = 4/group. Bottom: heme oxygenase-2 (HO-2) protein levels in endothelium-intact and -denuded thoracic aortas from rats exposed to normoxia and to hypoxia for 16 and 48 h; n = 5/group. *P < 0.05 compared with corresponding value in the normoxic control group.

 
Immunohistochemistry. Immunohistochemical staining with HO-1- and HO-2-specific antisera failed to detect either isoform in aortic sections from normoxic animals. In aortas from rats exposed to hypoxia, HO-1 immunoreactivity was observed in both endothelium and smooth muscle. HO-2 was detected only in the endothelium in aortas from hypoxia-exposed animals (Fig. 7).



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  		Fig. 7. A: HO-1 immunoreactivity (brown 3,3'-diaminobenzidine staining) is apparent in the aortic smooth muscle (and endothelium in some sections) in rats exposed to hypoxia for 16 h. B: HO-2 staining is seen in the endothelium but not in the smooth muscle of aortic sections from rats exposed to hypoxia for 16 h.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We found that 1) HO inhibition with SnPP IX increases the contractile responses to phenylephrine and ET-1 in endothelium-intact but not -denuded aortic rings from hypoxia-exposed rats; 2) SnPP IX enhancement of phenylephrine contraction in aortic rings from rats exposed to hypoxia is completely inhibited by the ETAR-specific antagonist BQ-123; 3) in endothelium-intact aortic rings from rats exposed to hypoxia for 16 h, the increase in tension after treatment with SnPP IX is comparable in magnitude and additive to the effect of NOS inhibition with L-NAME; 4) in contrast to aortic ET-1 levels, which increase with the duration of hypoxic exposure, HO-1 and HO-2 protein levels are increased after 16 h of hypoxia and return to near-control levels after 48 h; and 5) HO-1 is increased in the smooth muscle of aortas from rats exposed to hypoxia, whereas increases in ET-1 and HO-2 immunoreactivity are localized to the endothelium.

The contributions of smooth muscle- and endothelium-dependent mechanisms to the alteration in systemic vasoreactivity after hypoxia have varied among vessels from different anatomic sites, in different species, and with different durations of the hypoxic epoch. In rat aorta (2, 51) and diaphragmatic arterioles (42) after 16–48 h of hypoxia and in rat iliac artery (3, 4) and ovine uterine artery (16) after 4–6 wk of hypoxia, the primary locus of contractile impairment is the vascular smooth muscle. In keeping with our current findings, a potentially compensatory alteration in the function of the endothelium, in that it becomes an agency of vasoconstriction as opposed to its normal role as a source of vasorelaxing factors, was also noted in these preparations (2–4, 42, 52). In rat mesenteric artery strips, in contrast, 48 h of hypoxia results in endothelium-dependent hyperpolarization of the smooth muscle, which contributes to impaired pressure and phenylephrine-induced contraction (11).

A large body of evidence now supports a role for HO-derived CO in the alterations in pulmonary (8, 32, 43, 50) and systemic (7) vascular reactivity that develop during hypoxia. HO-1 and -2 have been detected in both systemic vascular endothelial and smooth muscle cells (9, 29, 30, 31, 53). However, because HO inhibition had no effect on contractile responses in endothelium-denuded aortic segments in the current study (Fig. 1, D and F; Table 1), our results suggest that physiologically relevant CO production after exposure to hypoxia is primarily localized to the endothelium. Consistent with this conclusion, prior studies demonstrated that the effect of HO inhibition on contraction and membrane depolarization in mesenteric arteries and on contraction in aortic segments from chronically hypoxic rats is eliminated by endothelial ablation (7, 11, 31). Because vascular cell expression of HO-1 has been shown to be hypoxia inducible (29, 30), whereas that of HO-2 has not, these previous findings were attributed, by inference, to upregulation of HO-1 (7). In the present study, however, we found that the increase in HO-2, rather than HO-1, immunoreactivity was eliminated by endothelial ablation and concluded that it is the HO-2 isoform that primarily accounts for the increase in sensitivity of contractile responses to HO inhibition seen in endothelium-intact aortic segments after hypoxia.

The biochemical mechanisms that mediate arterial smooth muscle dysfunction after hypoxia are not completely understood; however, studies in rat aorta indicate that enhanced targeting of type 1 phosphatase activity to the contractile myofilaments with attendant dephosphorylation of the 20-kDa myosin light chain (40) and increased expression of the inhibitory thin-filament proteins caldesmon and calponin (51) may contribute. Because hypoxic incubation activates HO-1 gene transcription and enhances cGMP production in vascular smooth muscle cells (29), and because HO-1 protein levels correlate with reduced vascular reactivity during hypoxia in vivo (17), a role for this isoform in the loss of smooth muscle reactivity after hypoxia has also been proposed. Our finding that SnPP IX has no effect on contractile responses in endothelium-denuded aortic rings from hypoxia-exposed animals (Fig. 1, D and F), despite robust induction of aortic smooth muscle HO-1 protein expression, was therefore unexpected. Although the concentration of SnPP IX used in the current study is effective in inhibiting HO activity in vascular smooth muscle cells (29), we cannot rule out the possibility that a functional effect might have been seen at higher concentrations. The usefulness of such experiments, however, is limited because the specificity of metalloprotoporphyrin inhibitors is lost at higher levels (1). Moreover, the lack of an acute effect of its inhibition on agonist-induced contraction does not exclude a vasoregulatory role for this isoform. One known function of HO is the degradation of heme proteins, some of which, including members of the cytochrome P-450 family of enzymes and NOS, catalyze the basal production of potent vasoregulatory molecules. The role of HO-1 in regulating vascular tone through effects on the enzymatic capacity of these pathways therefore requires evaluation.

HO-1 expression is primarily regulated at the transcriptional level (29), and its 5' genomic region is enriched in known hypoxia regulatory elements (15, 21, 49). In contrast, analysis of the region 5' to the rat HO-2 open reading frame reveals a glucocorticoid regulatory element but no sites corresponding to the transcription factors known to participate in hypoxic or other cellular stress responses (27, 35). Not surprisingly, therefore, hypoxic incubation has not resulted in upregulation of HO-2 transcription or mRNA levels in any of the cell culture systems in which it has been evaluated (29, 47). Despite the lack of evidence for transcriptional regulation, HO-2 protein expression is not entirely constitutive. Development stage-specific changes in HO-2 protein levels have been noted (12, 39), and cigarette smoke increases the number of lung cells expressing HO-2 protein (23). In the brain and testis, alternate HO-2 transcripts have been identified that differ in their 3'-and 5'-untranslated regions (25, 26, 39), and the dissociation between regional HO-2 protein and mRNA expression in testicular germ cells of differing stages suggests that HO-2 mRNA is sequestered from translation in these cells (12). Thus mRNA sequestration, alternate promoter usage, or posttranscriptional modification of mRNA structure may provide the key to understanding changes in HO-2 protein expression observed in the current study and in other instances in which a basis for transcriptional regulation is not apparent.

In endothelium-intact aortic rings from rats exposed to hypoxia for 16 h, we found that SnPP IX enhanced contraction to approximately the same extent as L-NAME and that these effects are additive. Classically, this would be taken to indicate that NOS- and HO-mediated inhibition of contraction are of approximately equal importance and independent of each other. CO is significantly less potent than NO as an activator of soluble guanylyl cyclase (sGC) (13), however, and may function as a partial sGC agonist (52). Therefore, the effect of HO inhibition may be enhanced under conditions of NO deficiency, and an interaction between these two pathways will be masked by this method of evaluation. These results do confirm that the SnPP IX effect is not attributable to NOS inhibition.

Hypoxia induces ET-1 precursor transcription in endothelial cells through the formation of a hypoxia-responsive complex that involves binding of adjacent hypoxia-inducible factor-1{alpha}, activator protein-1, GATA-2, and NF-1 sites and that is further modulated by interaction with the activator protein p300/CBP (47). This mechanism is inhibited, however, by CO and NO (20, 28), so our current finding that aortic ET-1 protein increases progressively concomitant with an increase in endothelial HO levels is somewhat surprising. Rat aortic endothelial expression of NOS is decreased during hypoxia (41), however, and because CO is less diffusible than NO (13), the increase in CO production may be insufficient to compensate for the loss of NO-mediated suppression. Increased levels of endothelin-converting enzyme-1 have also been reported in aortas from rats exposed acutely to hypoxia (10); therefore, the increase in aortic ET-1 protein noted in the present study may be the result of enhanced posttranslational processing and insensitive to regulation at the transcriptional level.

In endothelium-intact aortic rings from hypoxia-exposed rats, we found that BQ-123 abrogated the effect of HO inhibition on phenylephrine-induced contraction. The products of the HO-catalyzed reaction, therefore, exert their effect through interference with ETAR-mediated events rather than through impairment of the function and/or signaling of the {alpha}1-adrenoceptor itself. Known mechanisms by which CO can influence vascular contraction include stimulation of cGMP formation and Ca2+-activated K+ channel-mediated hyperpolarization of the smooth muscle cell membrane (44, 45). Neither of these is agonist specific, however, and if they were the predominant active mechanisms, impairment of phenylephrine contraction would be independent of effects on other receptor types. Our findings therefore suggest an additional mechanism of action that involves either impairment of the specific pathways by which activation of the ETA receptor potentiates the response to adrenoceptor stimulation (48) or alteration of the affinity of its interaction with ET-1. Further studies to delineate the exact nature of this interaction are now required.

In summary, we present evidence that endothelial modulation of rat aortic contraction to phenylephrine is mediated by HO-2 and that this is due to inhibition of ET-1 potentiation of adrenoceptor-induced contraction. In addition to suggesting the existence of an unrecognized mechanism by which HO may inhibit systemic vascular reactivity, this may have pathophysiological significance in cardiopulmonary diseases associated with hypoxemia, in which upregulation of endothelial HO activity may serve to counteract excessive ET-1-mediated vasoconstriction in vascular beds vulnerable to hypoxic injury.


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This study was funded by a grant from the Canadian Institutes of Health Research. H. Teoh holds a fellowship from the Heart and Stroke Foundation of Canada/Richard Lewar Centre of Excellence.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. E. Ward, Rm. 4-015, Bond Wing, St Michael's Hospital, 30 Bond St., Toronto, Ontario, Canada M5B 1W8 (E-mail: wardm{at}smh.toronto.on.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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