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Am J Physiol Heart Circ Physiol 284: H838-H845, 2003. First published November 21, 2002; doi:10.1152/ajpheart.00747.2002
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Vol. 284, Issue 3, H838-H845, March 2003

Endogenous carbon monoxide is an endothelial-derived vasodilator factor in the mesenteric circulation

Jay S. Naik, Theresa L. O'Donaughy, and Benjimen R. Walker

Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Science Center, Albuquerque, New Mexico 87131-5218


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Chronic hypoxia (CH) is associated with both blunted agonist-induced and myogenic vascular reactivity, possibly due to an enhanced production of heme oxygenase (HO)-derived carbon monoxide (CO). However, the cellular location of the HO responsible for these effects has not been clearly established. Therefore, we examined the response to administration of the substrate for HO, heme-L-lysinate (HLL), in endothelium-intact and endothelium-denuded small mesenteric arteries from CH male Sprague-Dawley rats. Mesenteric arteries were isolated and mounted on glass cannulas, pressurized to 60 mmHg, and superfused with physiological saline solution. All experiments were performed in the presence of 100 µM Nomega -nitro-L-arginine. The vasodilator response to HLL or exogenous CO was examined. HLL experiments were performed in the presence and absence of the HO inhibitor zinc protoporphyrin IX (ZnPPIX). HLL administration resulted in a dose-dependent vasodilator response that was abolished in the presence of ZnPPIX or by endothelial removal. Exogenous CO produced a vasodilator response that was independent of an intact endothelium. Cellular localization of HO was verified through immunohistochemistry in sections of the gut and aorta from CH and control animals. Staining for HO-1, HO-2, and endothelial nitric oxide synthase was confined to the endothelium. Thus we conclude that CO is a product of HO located within the endothelium.

vasodilation; hypoxia; zinc protoporphrin IX; isolated vessels


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ENDOGENOUS CARBON MONOXIDE (CO) is produced by conversion of free heme to biliverdin by the enzyme heme oxygenase (HO). HO exists as three isoforms: inducible (HO-1); constitutive (HO-2); and a third, not yet fully characterized form (HO-3). Several investigators have suggested that CO acts as an endogenously produced gaseous molecule analogous to nitric oxide (NO) (25, 26, 40). Indeed, CO has been shown to have both a vasorelaxant effect (4, 6, 12, 15, 21, 22, 41) as well as an antiproliferative effect on vascular smooth muscle (VSM) cells (23). HO-1 can be induced within the vasculature by a variety of physiological stimuli, including hypoxia (14, 20), hypertension (16, 37), endotoxic shock (42), and shear stress (39). Thus CO may be an important determinant of vascular tone under certain pathophysiological settings.

Previous work from our laboratory has shown that HO-1 protein and mRNA levels are increased in both the rat kidney and aorta after chronic hypoxia (CH) (14, 33). Functional evidence of a role for endogenous CO in regulating vascular tone has come from both whole animal (15, 33, 34) and isolated vessel preparations (4, 6, 9-12, 19, 41). For example, systemic administration of zinc protoporphyrin IX (ZnPPIX) increases renal vascular resistance in CH rats but not in control rats (33). Furthermore, exogenous administration of the HO substrate heme-L-lysinate (HLL) produces concentration-dependent increases in diameter in small mesenteric arteries from CH rats (11). In addition, our laboratory has previously shown that HO-derived CO may be important in both blunted agonist-induced vasoconstriction (4, 8-11, 33) as well as blunted myogenic reactivity (10) after CH. Interestingly, both phenylephrine (PE)-induced reactivity as well as myogenic reactivity were restored upon disruption of the endothelium (4, 9, 10). Taken together, these results suggest a potential role for CO derived from an endothelial source in the regulation of vascular tone after CH.

Although there is increasing evidence that HO-derived CO plays a role in the regulation of vascular tone, the site of HO expression within the vascular wall remains controversial. Indeed, both the constitutive and inducible isoforms of HO have been demonstrated in endothelial (29, 35, 36, 42) and VSM cells (5, 32, 42). For example, HO-1 has been shown to be expressed primarily in the medial layer of the rat aorta after lipopolysaccaride treatment (42) and within the endothelium in response to S-nitroso-N-acetyl penicillamine administration (36) as well as basally in cultured aortic VSM cells (5). Moreover, HO-2 protein has been demonstrated within the endothelium of porcine pulmonary, mesenteric, basilar, and pial arteries (35, 43) as well as in cultured aortic VSM cells (5).

Therefore, the goals of the present study were to 1) demonstrate that the HLL-induced vasodilation is due to a product of the HO reaction, and 2) determine the cellular location of HO isoforms within the mesenteric circulation after CH. To this end, we hypothesized that HLL administration results in enhanced CO production through HO-1 localized within the endothelium of small mesenteric arteries from CH rats.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. All animal protocols employed in this study were reviewed and approved by the Institutional Animal Care and Use Committee of the University of New Mexico School of Medicine. Male Sprague-Dawley rats (250-300 g body wt, Harlan Industries) were used for these experiments. CH rats were exposed to hypobaric hypoxia for 48 h (barometric pressure = 380 Torr).

Isolated mesenteric resistance artery diameter measurements. Rats were anesthetized with pentobarbital sodium (50 mg ip). A midsternal incision was made to expose the heart, and 100 units of heparin were injected directly into the left ventricle. The mesenteric arcade was excised through a midline abdominal incision and placed in ice-cold physiological saline solution [PSS; containing (in mM) 129.8 NaCl, 5.4 KCl, 0.5 NaH2PO4, 0.83 MgSO4, 19 NaHCO3, 1.8 CaCl2, and 5.5 glucose] aerated with a gas mixture consisting of 21% O2-6% CO2-balance N2. The arcade was secured in a Silastic-coated petri dish containing cold, aerated PSS. After removal of veins and adipose tissue, fourth-order arteries were transferred to a beaker of cold, aerated PSS. Arteries were then cannulated and pressurized to 60 Torr as described previously (9-11). Measurements of internal diameter (ID) were made using a high-resolution videocamera (model XC-73, Sony) and a video-tracking device (Video Dimension Analyzer V 94, Living Systems). Vessels were continuously superfused (5 ml/min) with aerated PSS warmed to 37°C and allowed to equilibrate for 60 min before any experimental manipulation. Viability of the vessel was assessed by administering PE (10-4 M). After the equilibration period, sequential doses of PE were administered to the superfusion reservoir until the vessel constricted by ~30% of baseline diameter. To reduce the possibility of interactions with NO, all experiments were performed in the presence of 100 µM Nomega -nitro-L-arginine. Passive diameter was determined at the end of each experiment by superfusing the vessel in Ca2+-free PSS [containing (in mM) 129.8 NaCl, 5.4 KCl, 0.5 NaH2PO4, 0.83 MgSO4, 19 NaHCO3, 5.5 glucose, and 3 EGTA] for 30 min. For all experiments, changes in vessel diameter are expressed as the percent reversal of PE-induced vasoconstriction. Data were stored and subsequently analyzed on a microcomputer using a commercial data-acquisition system (CODAS, Dataq Instruments).

Specificity of HLL-induced vasodilation. Small mesenteric arteries (n = 5 arteries/group) were excised from CH rats, isolated, cannulated, and perfused as described above. Vessels were superfused with PSS containing 500 nM of the HO inhibitor ZnPPIX or an equal volume of drug vehicle for 1 h before experimental manipulation (n = 5 vessels/group). After the equilibration period, serial doses of HLL were administered in PE-preconstricted vessels, ranging from 10-7 to 10-5 M. Whereas ZnPPIX is a potent inhibitor of HO, at higher concentrations it may also inhibit NO synthase (NOS) and soluble guanylyl cyclase (sGC) activities (1, 13, 24). To demonstrate the lack of nonspecific effects of ZnPPIX on other heme-containing enzymes (e.g., sGC), dose-response curves to S-nitroso-N-acetyl penicillamine were performed in the presence and absence of ZnPPIX (n = 5 for the vehicle-treated group and 4 for the ZnPPIX-treated group). In addition, male Sprague-Dawley rats were treated with sodium arsenite, a known inducer of HO-1 (75 µmol/kg sc) (5), to demonstrate that the vasodilator response to HLL is due to a product of the HO reaction rather than to a factor related to hypoxia per se. Sixteen hours after sodium arsenite treatment, small mesenteric arteries (n = 5 arteries/group) were excised, isolated, and cannulated as described above. Vasodilator responses to HLL (10-7-10-5 M) were determined in the presence and absence of ZnPPIX in PE-preconstricted vessels.

Endothelial dependence of HLL-induced vasodilation. Small mesenteric arteries (n = 5 arteries/group) were excised from CH rats, isolated, cannulated, and superfused as described above. Vessels were superfused with PSS (without Nomega -nitro-L-arginine) 1 h before experimental manipulation. After the equilibration period, sequential doses of PE were administered to the superfusion reservoir until the vessel constricted by ~30% of the initial diameter. Once a stable baseline was achieved, the response to the endothelium-dependent vasodilator ACh (10-6 M) was assessed, followed by a 15-min washout period. Arteries were denuded by passing a 1-ml air bubble through the lumen as described previously (9, 10). Vascular tone was reestablished with PE (10-6 M), and the ACh response was repeated to verify endothelial removal. The vessel was then washed with PSS containing 100 µM Nomega -nitro-L-arginine for 30 min. Vascular tone was reestablished with PE, and the vasodilator response to HLL (10-6 M) was assessed. In a separate set of experiments (n = 4 vessels/group), the vasodilator response to S-nitroso-N-acetyl penicillamine (10-7.5 M) was assessed in intact and denuded vessels, demonstrating that the ability of these vessels to respond to an endothelial-independent vasodilator was unaltered by passage of an air bubble through the lumen of the vessel.

Endothelial dependence on exogenous CO-mediated vasodilation. Small mesenteric arteries (n = 5 arteries/group) were excised from control and CH rats, isolated, cannulated, and superfused as described above. Disruption of the endothelium was performed in some vessels as described above. Before each experiment, 10 ml of PSS were vigorously bubbled with 100% CO gas or 100% nitrogen gas for 10 min. Vessels were preconstricted with PE to ~30% of the baseline diameter. Once a stable level of constriction was achieved, the vessel was superfused for ~2 min with a 20-fold dilution of the saturated CO solution in PSS (210 µl CO/100 ml PSS) that contained the same concentration of PE as was used to establish vascular tone. To control for pH differences between the standard PSS and the CO solution, the response to a 20-fold diluted N2-equilibrated solution was assessed (n = 3 vessels).

Western blot analysis. Previous studies (14) have demonstrated increased HO-1 levels by Western blot in vascular tissue from CH rats compared with controls. Additional Western blots were performed to determine the level of HO-1 in the mesenteric microcirculation from control (vehicle) and sodium arsenite-treated rats (see above). Each blot consisted of the experimental group and a parallel control group. Rats were anesthetized with pentobarbital sodium (50 mg ip), and the mesenteric arcade was excised through a midline abdominal incision and placed in ice-cold dissection solution [containing 30 mM MOPS, 1.2 mM NaH2PO4, 5 mM glucose, 2 mM pyruvate, 0.02 mM EDTA (disodium salt), 145 mM NaCl, 4.7 mM KCl, 2 mM CaCl2, 1.2 mM MgSO4, and 10 g/l BSA; pH 7.4, 290-310 mosM]. After removal of veins and adipose tissue, resistance artery branches were collected and snap frozen in liquid N2. All vessels harvested from a single animal were pooled and treated as one sample. A microsomal fraction was prepared using 10 mM Tris · HCl homogenization buffer (containing 255 mM sucrose, 2 mM EDTA, 12 µM leupeptin, 1 µM pepstatin A, 0.3 µM aprotinin, and 1 mM phenylmethylsulfonyl fluoride). Samples were centrifuged at 10,000 g for 10 min at 4°C. The supernatant was collected and centrifuged at 100,000 g for 60 min at 4°C. The microsomal pellet was resuspended in homogenization buffer. Sample protein concentrations were determined by the Bradford method (Bio-Rad protein assay). Proteins (20 µg/lane) were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. The blocking solution contained 5% nonfat milk, 3% BSA, 5% goat serum, 5% rabbit serum, and 0.5% Tween 20 in Tris-buffered saline (TBS) containing 10 mM Tris · HCl and 50 mM NaCl (pH 7.5). HO-1 primary antibodies were diluted in TBS plus 5% nonfat milk and 0.05% Tween 20. The secondary antibody was diluted in TBS and 0.05% Tween 20. Blots were incubated for 1 h at room temperature with goat anti-mouse horseradish peroxidase-conjugated IgG (1:5,000 dilution, Stressgen).

Immunohistochemistry in small mesenteric arteries and aortas. Normoxic control and CH rats were anesthetized with pentobarbital sodium (50 mg ip). Heparin (500 units) was injected directly into the left ventricle. The superior mesenteric artery or ascending aorta was cannulated with a needle stub. Blood was flushed from the vasculature by perfusion of buffer (see Drugs and solutions) at a rate of 4.8 or 48 ml/min for the mesentery and aorta, respectively, followed immediately with paraformaldehyde fixative containing 10-4 M papaverine (25 or 250 ml for the mesentery and aorta, respectively). One-centimeter segments of the gut wall or the thoracic aorta were cut and placed in fixative for 4 h. Segments of tissue were cryoprotected in PBS containing sucrose (30%) overnight. The tissue was then imbedded in OCT, and 10-µm sections were cut with the use of a cryostat and placed on glass slides. To prepare sections for primary antibody, they were washed in PBS + 0.3% Triton X-100 (PBS-TX). Inhibition of endogenous peroxidases was accomplished by washing sections in a H2O2 solution (0.33%) for 30 min. Mesenteric samples were blocked with 4% horse serum [HO-1 and endothelial NOS (eNOS)] or goat serum (HO-2) in PBS-TX for 30 min. Sections were incubated in primary antibody for 2 h at room temperature [1:100 mouse monoclonal HO-1 (Stressgen OSA-111), 1:400 rabbit polyclonal HO-2 (Stressgen OSA-200)] or for 1 h with 1:2,500 mouse monoclonal eNOS antibody (Transduction Laboratories) followed by 24 h at 4°C. Sections were washed in PBS and incubated in secondary antibody for 2 h at room temperature [biotinylated horse anti-mouse IgG (1:200 HO-1 or 1:400 eNOS) or biotinylated goat anti-rabbit IgG (1:5,000 HO-2)], followed by incubation with an avidin biotinylated peroxidase complex containing 0.1% Tween 20 (ABC Elite kit, Vector Laboratories). For the aorta, sections were blocked with 15% horse serum or goat serum in PBS-TX for 30 min. Sections were incubated in primary antibody for 1 h at room temperature [1:1,000 mouse monoclonal HO-1 (Stressgen OSA-111), 1:2,000 rabbit polyclonal HO-2 (Stressgen OSA-200), or 1:1,000 mouse monoclonal eNOS (Transduction Laboratories)] followed by 24 h at 4°C. Sections were washed in PBS and incubated in secondary antibody for 2 h at room temperature (1:400 biotinylated horse anti-mouse IgG for HO-1 and eNOS; 1,500 biotinylated goat anti-rabbit IgG for HO-2). For all sections, immunoprecipitation of the antigen-peroxidase conjugate was achieved by treatment of sections with 3,3'-diaminobenzidine (DAB) tetrahydrochloride dihydrate and H2O2 (DAB kit, Vector Laboratories). To enhance contrast, NiCl2 was added to the DAB solution. Negative controls were prepared as above without the addition of primary antibody. Sections were then washed in deionized H2O, dehydrated with ethanol, and mounted.

Drugs and solutions. HLL was prepared according to the methods of Tehunen et al. (38). Briefly, 1.02 g hemin (Sigma) was dissolved in a solution containing ethanol, 1,2-propanediol, and L-lysine and stored in light-resistant glass containers at 4°C until use. ZnPPIX (Porphyrin Products) was prepared as described previously (33). ZnPPIX stock solution was aliquoted and stored at -80°C for a maximum of 1 wk. PE and ACh were dissolved in deionized H2O, aliquoted, and stored at -80°C until use. Sodium arsenite (100 µmol/ml) was dissolved in saline, filter sterilized using a 0.2-µm filter (Millipore), and stored at 4°C. S-nitroso-N-acetyl penicillamine was dissolved in ethanol on the day of the experiment and stored on ice until use. Nomega -nitro-L-arginine was dissolved in PSS on the day of the experiment. Perfusion buffer consisted of PSS containing 2,000 units heparin, 4 mg/ml BSA, and 10-4 M papaverine.

Data analysis and statistics. For all experiments, the vasodilator response to HLL, S-nitroso-N-acetyl penicillamine, or ACh was calculated as the percent reversal of PE-induced tone. To meet the assumption of a normal distribution necessary for parametric statistics, percent changes in ID underwent arcsine transformation before analysis. Data were analyzed using two-way repeated-measures ANOVA, two-way ANOVA, or an unpaired Student's t-test as appropriate. Where significant main effects occurred, statistical differences between groups were assessed with the Student-Newman-Keuls post hoc test. A probability of P <=  0.05 was accepted as statistically significant for all comparisons.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Specificity of HLL-induced vasodilation. The magnitude of constriction generated by PE administration was 41.6 ± 10.8% (2.0 ± 0.5 µM PE) and 31.0 ± 3.5% (1.1 ± 0.5 µM PE) of the initial diameter under vehicle and ZnPPIX conditions, respectively. Changes in ID in response to HLL in pressurized mesenteric resistance arteries (60 Torr) from CH rats in the presence and absence of HO inhibition are shown in Fig. 1. HLL administration produced a dose-dependent increase in ID in resistance arteries pretreated with the vehicle for ZnPPIX. In contrast, the vasodilator response to HLL was completely abolished in the presence of HO inhibition. Because ZnPPIX may have nonspecific effects on other heme-containing enzymes (e.g., sGC) (13, 24), the vasodilator response to S-nitroso-N-acetyl penicillamine was assessed in the presence and absence of HO inhibition. As illustrated in Fig. 2, S-nitroso-N-acetyl penicillamine produced a dose-dependent increase in ID that was unaltered by ZnPPIX.


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  		Fig. 1.   Effect of heme-L-lysinate (HLL; 10-7-10-5 M) on internal diameter (ID) in mesenteric arteries from chronic hypoxic (CH) rats. Experiments were performed in the presence and absence of the heme oxygenase (HO) inhibitor zinc protoporphyrin IX (ZnPPIX). Fourth-order mesenteric arteries from 48-h CH rats were pretreated with Nomega -nitro-L-arginine and either vehicle or ZnPPIX 1 h before experimentation. The change in ID is presented as the percent reversal of phenylephrine (PE)-induced constriction. Data are means ± SE (n = 5 arteries/group). #Significantly different from 10-7 M HLL (P <=  0.05); dagger  significantly different from 10-6 and 10-7 M HLL (P <=  0.05); * significantly different from vehicle (P <=  0.05).



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  		Fig. 2.   Effect of HO inhibition on the vasodilator response to the nitric oxide (NO) donor S-nitroso-N-acetyl penicillamine (SNAP). Fourth-order mesenteric arteries from 48-h CH rats were pretreated with Nomega -nitro-L-arginine and either vehicle (n = 5) or ZnPPIX (n = 4) 1 h before experimentation. The change in ID is presented as the percent reversal of PE-induced constriction. Data are means ± SE. There was no significant difference between groups.

As shown in Fig. 3, HO-1 protein was detected in small mesenteric arteries from rats treated for 16 h with sodium arsenite. However, HO-1 was not detected in arteries from control animals. HLL produced a dose-dependent increase in ID in arteries from sodium arsenite-treated animals. This response was completely abolished by HO inhibition (Fig. 4).


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  		Fig. 3.   Western blot showing HO-1 protein expression in mesenteric arteries from control and sodium arsenite-treated rats. Lane 1, HO-1 standard; lanes 2-4, normoxia; lanes 5-8, sodium arsenite treatment. Note that HO-1 was not detected in arteries from control rats.



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  		Fig. 4.   Effect of HLL (10-7-10-5 M) on ID in mesenteric arteries from sodium arsenite-treated rats. Experiments were performed in the presence and absence of the HO inhibitor ZnPPIX. Fourth-order mesenteric arteries from sodium arsenite-treated rats were pretreated with Nomega -nitro-L-arginine and either vehicle or ZnPPIX 1 h before experimentation. The change in ID is presented as the percent reversal of PE-induced constriction. Data are means ± SE (n = 5). * Significantly different from vehicle (P <=  0.05).

Endothelial dependence of HLL-induced vasodilation. The integrity of the endothelium was assessed by administration of ACh (10-6 M) before and after passage of an air bubble through the lumen of the vessel. Figure 5 shows the response to ACh in mesenteric resistance arteries constricted with PE. ACh administration completely reversed the PE-induced tone. This response was inhibited in denuded mesenteric arteries, whereas the vasodilator response to S-nitroso-N-acetyl penicillamine (3 × 10-7 M) was unaffected (Fig. 5). Figure 6 illustrates the effect of disruption of the endothelium on HLL-induced vasodilation in mesenteric arteries constricted with PE. Administration of HO substrate produced an increase in ID in arteries that were sham treated. This response was nearly eliminated in arteries in which the endothelium was disrupted.


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  		Fig. 5.   Validation of selective endothelial disruption. The vasodilator response to acetylcholine (ACh; 10-6 M) and SNAP (3 × 10-7 M) was assessed in mesenteric arteries from CH rats before and after passage of a 1-ml air bubble through the lumen of the vessel (n = 4 vessels/group). The change in ID is presented as the percent reversal of PE-induced constriction. Data are means ± SE (n = 5). * Significantly different from intact (P < 0.05).



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  		Fig. 6.   Effect of endothelial disruption on the vasodilator response to HLL (10-6 M) in mesenteric arteries from CH rats. The endothelium was disrupted by passage of a 1-ml bubble of air through the lumen of the vessel (denuded). Sham treatment consisted of perfusion of the vessel with physiological saline solution (PSS) for an equal period of time (sham). The change in ID is presented as the percent reversal of PE-induced constriction. Data are means ± SE. * Significantly different from sham (P < 0.05).

Endothelial dependence of exogenous CO-mediated vasodilation. As shown in Fig. 7, exogenous CO elicited vasodilation in small mesenteric arteries from both control and CH rats. Disruption of the endothelium did not alter this response. In contrast, superfusion with a nitrogen-equilibrated solution produced a modest transient vasoconstriction (data not shown).


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  		Fig. 7.   The effect of endothelial disruption on the vasodilator response to exogenous CO in mesenteric arteries from control and CH rats. The endothelium was disrupted by passage of a 1-ml bubble of air through the lumen of the vessel (denuded). Sham treatment consisted of perfusion of the vessel with PSS for an equal period of time (sham). Vessels were preconstricted with PE and superfused with PSS equilibrated with CO (210 µl CO/100 ml PSS). The change in ID is presented as the percent reversal of active tone. Data are means ± SE. There are no significant differences between groups.

Immunohistochemistry for eNOS, HO-1, and HO-2 in arteries from the rat small intestine. Photomicrographs of eNOS, HO-1, and HO-2 immunoreactivity in arteries lying within the muscularis mucosae of the small intestine from control, CH, and sodium arsenite-treated rats are illustrated in Fig. 8. An example of eNOS immunostaining in an artery from a control rat is shown in Fig. 8F. HO-1 staining in arteries from CH and control rats are shown in Fig. 8, A and B, respectively. Note that sections incubated with HO-1 primary antibody show a similar pattern of staining as that for eNOS, suggesting that HO-1 protein is localized to the endothelium. An example of HO-1 immunostaining in an artery from a sodium arsenite-treated animal is shown in Fig. 8E. Examples of HO-2-immunoreactive arteries from both control and CH rats are illustrated in Fig. 8, C and D, respectively. Again, staining for HO-2 was localized to the endothelium. No staining was observed in sections that were not treated with primary antibody (Fig. 8G).


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  		Fig. 8.   Immunohistochemisty for HO-1, HO-2, and endothelial NO synthase (eNOS) in mesenteric arteries from CH and control rats. A and B: examples of HO-1 staining in mesenteric arteries from CH and control rats, respectively. C and D: mesenteric arteries immunostained for HO-2 from control and CH rats, respectively. E: example of HO-1 staining in a mesenteric artery from a sodium arsenite-treated rat. F: immunostaining for eNOS in a mesenteric artery from a control rat. G: mesenteric artery from a sodium arsenite-treated rat incubated with horse anti-mouse IgG secondary antibody only. Note that HO-1 and HO-2 have a similar pattern of staining as that of eNOS. Magnification: ×40; bar = 20 µm.

Immunohistochemistry for eNOS, HO-1, and HO-2 in the aorta. Figure 9 contains photomicrographs of eNOS, HO-1, and HO-2 immunoreactivity in aortas from CH and normoxic control animals. Examples of eNOS immunostaining in aortas from both control and CH rats are shown in Fig. 9, A and B. Note that sections incubated with HO-1 or HO-2 primary antibody show a similar pattern of staining as that for eNOS, suggesting that HO protein is localized to the endothelium in the aorta (Fig. 9, C-F).


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  		Fig. 9.   Immunohistochemisty for HO-1, HO-2, and eNOS in aortas from CH and control rats. A and B: examples of HO-1 staining in aortas from CH and control rats, respectively. C and D: aortas immunostained for HO-2 from CH and control rats, respectively. E and F: immunostaining for eNOS aortas from a CH and control rats, respectively. Note that HO-1 and HO-2 have a similar pattern of staining as that of eNOS. Magnification: ×20.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The major findings of the present study are that 1) the vasodilator response to HLL administration is the result of a product of the HO reaction; 2) disruption of the endothelium nearly abolishes the vasodilator response to HLL; 3) exogenous CO elicited an endothelium-independent vasodilation; 4) sodium arsenite treatment increases HO-1 protein levels, 5) sodium arsenite treatment increase HO-1 protein expression in mesenteric arteries; 6) HLL causes vasodilation in vessels from sodium arsenite-treated rats, which is abolished by ZnPPIX; and 7) HO-1 and HO-2 protein is expressed within the endothelium of both small mesenteric arteries and aorta. These findings suggest that endogenous CO is an endothelium-derived vasodilator factor acting on vascular smooth muscle analogous to NO.

Several investigators have shown that HLL is a vasodilator in a number of vascular beds, including the mesenteric, cerebral, and skeletal muscle circulations (11, 16, 21, 22). For example, earlier experiments from our laboratory have demonstrated that HLL causes a dose-dependent vasodilator response in small mesenteric arteries isolated from rats exposed to 48 h of hypobaric hypoxia. However, in vessels from normoxic control animals, HLL had an effect only at the highest dose (11). The present study provides evidence that the vasodilator response to HLL is mediated by a product of the HO reaction. Indeed, HLL-induced vasodilation was abolished by ZnPPIX. This is in agreement with a study by Leffler et al. (21), who demonstrated in porcine pial arteries that HLL treatment caused a dose-dependent vasodilator response that was reversed to a vasoconstriction by the HO inhibitor chromium mesoporphyrin. Moreover, intraperitoneal injection of HLL produced a decrease in blood pressure in spontaneously hypertensive rats that was prevented by pretreatment with the HO inhibitor zinc deuteroporphyrin 2,4-bis glycol (16). Furthermore, hemin-induced increases in HO activity in renal microsomes was abolished by the metalloporphyrin tin mesoporphyrin (7). Taken together, the results of these studies suggest that hemin-induced vasodilation is mediated by a product of the HO reaction, presumably CO.

It is well established that catabolism of free heme by HO results in the production of equimolar quantities of CO, free iron, and biliverdin. Previous work from our laboratory has provided evidence that the vasoactive product of the HO reaction is CO. Indeed, neither free iron nor biliverdin reduced the active tension developed in PE-preconstricted aortic rings, whereas exogenous CO produced a dose-dependent reduction in tension (4). Moreover, others have shown that treatment of rat aortic VSM cells with hemin resulted in a dose-dependent increase in CO release (31). Taken together, these results suggest that the administration of HLL augments the production of CO by the enzyme HO.

The present study provides evidence that HO-1 and HO-2 are present within the endothelium of mesenteric arteries from both normoxic and hypoxic animals. Previously, HO-1 protein has been shown within the endothelial layer of small pulmonary arteries in response to hemin treatment (2) as well as in the aorta after high concentrations of S-nitroso-N-acetyl penicillamine (36). However, others have shown HO-1 within the medial layer of the aorta (42) and small pulmonary arteries (3) in response to lipopolysaccaride and hypoxia, respectively. In addition, HO-1 mRNA has been demonstrated in cultured aortic and pulmonary VSM cells in response to hypoxia (32). Furthermore, HO-1 expression has also been shown in aortic VSM cells after sodium arsenite treatment (5). These inconsistencies may be explained by differences between vascular beds, the factors used to induce HO-1, or cell culture conditions. The present study provides evidence that within the mesenteric circulation both HO-1 and HO-2 are expressed selectively within the endothelium in tissue from both control and CH animals. In contrast, others have failed to demonstrate HO-1 expression under control conditions (3, 36, 42) using immunohistochemistry. These differences as well as the inability of the present study to detect differences between control and hypoxic tissue may be due to low levels of HO-1 in these vascular beds or to a lack of adequate sensitivity of the immunohistochemistry technique.

We have previously shown in aortic rings from CH rats that the contractile response to PE after inhibition on NOS and HO was greater than in tissue treated only with Nomega -nitro-L-arginine (4). Removal of the endothelium abolished the effect of HO inhibition in this tissue, providing support for the postulate that HO-derived CO is an endothelial-derived vasoactive compound.

CO has been shown to inhibit the synthesis of endothelial-derived vasoactive compounds (18). Hence, the vasodilator response to endothelium-derived CO may be mediated through an autocrine effect on endothelial cells rather than acting directly on VSM cells. In the present study, administration of exogenous CO produced a vasodilator response in small mesenteric arteries. Removal of the endothelium did not alter this response, suggesting the CO-mediated vasodilation occurs through a direct effect on VSM cells. In addition, the vasodilator response to CO in arteries from control and CH rats was similar. Therefore, exposure to an hypoxic environment does not alter the sensitivity of these vessels to CO.

Given that ZnPPIX inhibits both HO-1 and HO-2 and that both isoforms are expressed in small mesenteric vessels, it is not possible to know with certainty which isoform is being affected in our preparation. Indeed, HO-2 has been shown to be abundant in the brain, testes, and endothelial lining of the aorta (43). However, this isoform is considered to be constitutive, although its expression can be induced by glucocorticoids (27). Furthermore, we have failed to observe any effect of hypoxic exposure on HO-2 mRNA or protein in aortic and renal tissue in earlier studies from our laboratory (34). Given that hypoxia induces HO-1 expression (3, 20, 32), and the inability of HLL to cause vasodilation in vessels from control animals except at high concentrations (11), elevated levels of this form of the enzyme are most likely responsible for the observed response to HLL in our experiments. Indeed, we have previously shown that 48 h of hypoxia induces HO-1 expression in the rat aorta (14). Moreover, in the present study, sodium arsenite treatment resulted in an increase in HO-1 protein expression within the mesenteric vasculature. Administration of HLL caused vasodilation in these vessels, but not in vessels from control animals (11), even though both HO-1 and HO-2 are expressed within the mesenteric vasculature. These results suggest that HO-1 is the isoform responsible the vasodilator response to HLL.

As stated above, we have previously shown a differential response to HLL between control and CH rats. The vasodilator response to excess substrate (i.e., HLL) implies a substrate limitation in vessels from CH rats. This may result from an increase in expression of HO-1 or an increase in enzyme activity after hypoxic exposure. Alternatively, de novo synthesis of heme within endothelial cells may be impaired in CH rats, resulting in a reduced availability of free heme.

Finally, Johnson and Johnson (17) have suggested that CO acts as a vasoconstrictor agent in endothelium-intact vessels through the inhibition of NOS, whereby the vasodilator properties of CO are unmasked when the endothelium is removed or NOS is inhibited. However, because our experiments eliminated the contribution of NO by administration of a NOS inhibitor, any potential interactions between CO and NO have been eliminated. Furthermore, we have previously shown that exogenous CO relaxed both endothelium-intact and -denuded aortic rings (4). Discrepancies between the results of the our laboratory and those of Johnson and Johnson (17) may be explained by differences in vascular beds studied or to other changes independent of HO occurring at the level of the vasculature secondary to hypoxic exposure. In addition, NOS inhibition also rules out any nonspecific actions of ZnPPIX on NOS activity in the present studies.

In summary, the current study provides evidence that HLL administered to small mesenteric arteries results in vasodilation meditated by CO produced by HO located within the endothelium. Further studies are required to better elucidate the downstream target(s) involved in CO-mediated vasodilation.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-58124, HL-63207 (to B. R. Walker), and F32-HL-08456 (to J. S. Naik) and by American Heart Association Desert/Mountain Affiliate Predoctoral Fellowship 0215248Z (to J. S. Naik).


    FOOTNOTES

Address for reprint requests and other correspondence: B. R. Walker, Dept. of Cell Biology and Physiology, Univ. of New Mexico Health Sciences Center, 915 Camino de Salud NE, Albuquerque, NM 87131-5218 (E-mail: bwalker{at}salud.unm.edu).

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.

10.1152/ajpheart.00747.2002

Received 28 August 2002; accepted in final form 12 November 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Appleton, SD, Chretien ML, McLaughlin BE, Vredrik HJ, Stevenson DK, Brien JF, Nakatsu K, Maurice DH, and Marks GS. Selective inhibition of heme oxygenase without inhibition of nitric oxide synthase or soluble guanylyl cyclase by metalloporphyrins at low concentrations. Drug Metab Dispos 27: 1214-1219, 1999[Abstract/Free Full Text].

2.   Balla, J, Nath KA, Balla G, Juckett MB, Jacob HS, and Vercellotti GM. Endothelial cell heme oxygenase and ferritin induction in rat lung by hemoglobin in vivo. Am J Physiol Lung Cell Mol Physiol 268: L231-L327, 1995.

3.   Carraway, M, Ghio A, Carter J, and Piantadosi C. Expression of heme oxygenase-1 in the lung in chronic hypoxia. Am J Physiol Lung Cell Mol Physiol 278: L806-L812, 2000[Abstract/Free Full Text].

4.   Caudill, T, Resta T, Kanagy N, and Walker BR. Role of endothelial carbon monoxide in attenuated vasoreativity following chronic hypoxia. Am J Physiol Regul Integr Comp Physiol 275: R1025-R1030, 1998[Abstract/Free Full Text].

5.   Christodoulides, N, Durante W, Kroll M, and Schafer A. Vascular smooth muscle cell heme oxygenase generate guanylyl cyclase-stimulatory carbon monoxide. Circulation 91: 2306-2309, 1995[Abstract/Free Full Text].

6.   Coceani, F, Kelsey L, and Seidlitz E. Carbon monoxide formation in the ductus arteriosus in the lamb: implications for the regulation of muscle tone. Br J Pharmacol 118: 1689-1696, 1996[ISI][Medline].

7.   Da Sliva, JL, Zand BA, Yang LM, Sabaawy HE, Lianos E, and Abrahem N. Heme oxygenase isoform-specific expression and distribution in the rat kidney. Kidney Int 59: 1448-1457, 2001[ISI][Medline].

8.   Doyle, MP, and Walker BR. Attenuation of systemic vasoreactivity in chronically hypoxic rats. Am J Physiol Regul Integr Comp Physiol 260: R1114-R1122, 1991[Abstract/Free Full Text].

9.   Earley, S, Naik JS, and Walker BR. 48-h Hypoxic exposure results in endothelium-dependent systemic vascular smooth muscle cell hyperpolarization. Am J Physiol Regul Integr Comp Physiol 283: R79-R85, 2002[Abstract/Free Full Text].

10.   Earley, S, and Walker BR. Endothelium-dependent blunting of myogenic responsiveness after chronic hypoxia. Am J Physiol Heart Circ Physiol 283: H2202-H2209, 2002[Abstract/Free Full Text].

11.   Gonzales, RJ, and Walker BR. Role of CO in attenuated vasoconstictor reactivity of mesenteric resistance arteries after chronic hypoxia. Am J Physiol Heart Circ Physiol 282: H30-H37, 2002[Abstract/Free Full Text].

12.   Hussain, A, Marks G, Brien J, and Nakatsu K. The soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) inhibits relaxation of rabbit aortic rings induced by carbon monoxide, nitric oxide, and glyceryl trinitrate. Can J Physiol Pharmacol 75: 1034-1037, 1997[ISI][Medline].

13.   Ignarro, LJ, Ballot B, and Woods KS. Regulation of soluble guanylate cyclase activity by porphrins and metalloporphyrins. J Biol Chem 259: 6201-6207, 1984[Abstract/Free Full Text].

14.   Jernigan, NL, O'Donaughy TL, and Walker BR. Correlation of HO-1 expression with onset and reversal of hypoxia-induced vasoconstrictor hyporeactivity. Am J Physiol Heart Circ Physiol 281: H298-H307, 2001[Abstract/Free Full Text].

15.   Johnson, R, Lavesa M, Askari B, Abraham N, and Nasjletti A. A heme oxygenase product, presumably carbon monoxide, mediates a vasodepressor function in rats. Hypertension 25: 166-169, 1995[Abstract/Free Full Text].

16.   Johnson, RA, Lavesa M, DeSeyn K, Scholer MJ, and Nasjletti A. Heme oxygenase substrates acutely lower blood pressure in hypertensive rats. Am J Physiol Heart Circ Physiol 271: H1132-H1138, 1996[Abstract/Free Full Text].

17.   Johnson, RA, and Johnson FK. The heme-heme oxygenase-carbon monoxide system and hypertension. In: Carbon Monoxide and Cardiovascular Functions, edited by Wang R.. Boca Raton, FL: CRC, 2002, p. 149-164.

18.   Kourembanas, S, Morita T, Liu Y, and Christou H. Mechanisms by which oxygen regulates gene expression and cell-cell interaction in the vasculature. Kidney Int 51: 438-443, 1997[ISI][Medline].

19.   Kozma, F, Johnson RA, Zhang F, Yu C, Tong X, and Nasjletti A. Contribution of endogenous carbon monoxide to regulation of diameter in resistance vessels. Am J Physiol Regul Integr Comp Physiol 276: R1087-R1094, 1999[Abstract/Free Full Text].

20.   Lee, P, Jiang B, Chin B, Iyer N, Alam J, Semenza G, and Choi A. Hypoxia-inducible factor-1 mediates transcriptional activation of the heme oxygenase-1 gene in response to hypoxia. J Biol Chem 272: 5375-5381, 1997[Abstract/Free Full Text].

21.   Leffler, C, Nasjletti A, Yu C, Johnson RA, Fedinec A, and Walker N. Carbon monoxide and cerebral microvascular tone in newborn pigs. Am J Physiol Heart Circ Physiol 276: H1641-H1646, 1999[Abstract/Free Full Text].

22.   Leffler, CW, Nasjletti A, Johnson RA, and Fedinec AL. Contributions of prostacyclin and nitric oxide to carbon monoxide-induced cerebrovascular dilation in piglets. Am J Physiol Heart Circ Physiol 280: H1490-H1495, 2001[Abstract/Free Full Text].

23.   Li Volti, G, Wang J, Tranganos F, Kappas A, and Abraham NG. Differential effect of heme oxygenase-1 in endothelial and smooth muscle cell cycle progression. Biochem Biophys Res Commun 6: 1077-1082, 2002.

24.   Luo, D, and Vincent SR. Metalloporphyrins inhibit nitric oxide dependent cGMP formation in vivo. Eur J Pharmacol 267: 263-267, 1994[ISI][Medline].

25.  Maines MD. Heme oxygenase: function, multiplicity, regulatory mechanisms, and clinical applications. FASEB J 2557-2568, 1988.

26.   Maines, MD. The heme oxygenase system: a regulator of second messenger gases. Annu Rev Pharmacol Toxicol 37: 517-554, 1997[ISI][Medline].

27.   Maines, MD, Eke BC, and Zhao X. Corticosterone promotes increased heme oxygenase-2 protein and transcript expression in the newboen rat brain. Brain Res 722: 83-92, 1996[ISI][Medline].

28.   Marks, GS, Brien JF, Nakatsu K, and McLaughlin BE. Does carbon monoxide have a physiological function? Trends Pharmacol Sci 12: 185-188, 1991[Medline].

29.   Marks, GS, McLaughlin BE, Vreman HJ, Stevenson DK, Nakatsu K, Brien JF, and Pang SC. Heme oxygenase activity and immunohistochemical localization in bovine pulmonary artery and vein. J Cardiovasc Pharmacol 30: 1-6, 1997[ISI][Medline].

30.   Morimoto, Y, Durante W, Lancaster DG, Klattenhoff J, and Tittel FK. Real-time measurement of endogenous CO production from vascular cells using an ultrasensitive laser sensor. Am J Physiol Heart Circ Physiol 280: H483-H488, 2001[Abstract/Free Full Text].

31.   Morita, T, Perrella MA, Lee M, and Kourembanas S. Smooth muscle cell-derived carbon monoxide is a regulator of vascular cGMP. Proc Natl Acad Sci USA 92: 1475-1479, 1995[Abstract/Free Full Text].

32.   Motterlini, R, Gonzales A, Foresti R, Clark JE, Green CJ, and Winslow RM. Heme oxygenase-1-derived carbon monoxide contributes to the suppression of acute hypertension in vivo. Circ Res 83: 568-577, 1998[Abstract/Free Full Text].

33.   O'Donaughy, TL. Role of Heme Oxygenase in the Regulation of Vascular Tone During Chronic Hypoxia (PhD dissertation). Albuquerque, NM: Univ. of New Mexico, 2001.

34.   O'Donaughy, TL, and Walker BR. Renal vasodilatory influence of endogenous carbon monoxide in chronically hypoxic rats. Am J Physiol Heart Circ Physiol 279: H2908-H2915, 2000[Abstract/Free Full Text].

35.   Parfenova, H, Neff RA, Alonso JS, Shlopov BV, Jamal CN, Sarksova SA, and Leffler CW. Cerebral vascular endothelial heme oxygenase: expression, localization, and activation by glutamate. Am J Physiol Cell Physiol 281: C1954-C1963, 2001[Abstract/Free Full Text].

36.   Sammut, IA, Foresti R, Clark JE, Exon DJ, Vesely MJ, Sarathchandra P, Green CJ, and Motterlini R. Carbon monoxide is a major contributor to the regulation of vascular tone in aortas expressing high levels of haeme oxygenase-1. Br J Pharmacol 125: 1437-1444, 1998[ISI][Medline].

37.   Seki, T, Naruse M, Naruse K, Toshimoto T, Tanabe A, Tsuchiya K, Hirose S, Imake H, Nihei H, and Demura H. Roles of heme oxygenase/carbon monoxide system in genetically hypertensive rats. Biochem Biophys Res Commun 241: 574-578, 1997[ISI][Medline].

38.   Tehunen, R, Tokala O, and Linden B. Haem arginate: a new stable haem compound. J Pharm Pharmacol 39: 780-786, 1987[ISI][Medline].

39.   Wagner, CT, Durante W, Christodoulides N, Hellums JD, and Schafer AI. Hemodynamic forces induce the expression of heme oxygenase in cultured vascular smooth muscle cells. J Clin Invest 100: 589-596, 1997[ISI][Medline].

40.   Wang, R. Resurgence of carbon monoxide: an endogenous gaseous vasorelaxing factor. Can J Physiol Pharmacol 76: 1-15, 1998[ISI][Medline].

41.   Wang, R, Wang Z, and Wu L. Carbon monoxide-induced vasorelaxation and the underlying mechanisms. Br J Pharmacol 121: 927-934, 1997[ISI][Medline].

42.   Yet, SF, Pellacani A, Patterson C, Tan L, Folta SC, Foster L, Lee WS, Hsieh CM, and Perella MA. Induction of heme oxygenase-1 expression in vascular smooth muscle cells. A link to endotoxic shock. J Biol Chem 272: 4295-4301, 1997[Abstract/Free Full Text].

43.   Zakhary, R, Gaine S, Dinerman J, Ruat M, Flavahan N, and Snyder S. Heme oxygense-2: endothelial and neuronal localization and role in endothelium-dependent relaxation. Proc Natl Acad Sci USA 93: 795-798, 1996[Abstract/Free Full Text].

Am J Physiol Heart Circ Physiol 284(3):H838-H845
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