Homogeneous segmental profile of carbon monoxide-mediated pulmonary vasodilation in rats

Jay S. Naik and Benjimen R. Walker

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Carbon monoxide (CO) has been proposed to attenuate the vasoconstrictor response to local hypoxia that contributes to pulmonary hypertension. However, the segmental response to CO, as well as its mechanism of action in the pulmonary circulation, has not been fully defined. To investigate the hemodynamic response to exogenous CO, lungs from male Sprague-Dawley rats were perfused with physiological saline solution. Measurements were made of pulmonary arterial, venous, and capillary pressures. Lungs were constricted with the thromboxane mimetic U-46619. To examine the vasodilatory response to CO, 500 µl of CO-equilibrated physiological saline solution or vehicle were injected into the arterial line. Additionally, CO and vehicle responses were examined in the presence of the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 µM) or the larger conductance calcium-activated K+ (BKCa) channel blockers tetraethylammonium chloride (10 mM) and iberiotoxin (100 nM). CO administration decreased vascular resistance to a similar degree in both vascular segments. This vasodilatory response was completely abolished in lungs pretreated with ODQ. Furthermore, CO administration increased whole lung cGMP content, which was prevented by ODQ. Neither tetraethylammonium chloride nor iberiotoxin affected the CO response. We conclude that exogenous CO administration causes vasodilation in the pulmonary vasculature via a soluble guanylyl cyclase-dependent mechanism that does not likely involve activation of KCa channels.

isolated rat lungs; pulmonary hypertension; vascular resistance; heme oxygenase


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

HEME OXYGENASE (HO) exists as three isoforms: constitutive (HO-1), inducible (HO-2), and a third not yet fully characterized form (HO-3). HO-1 and HO-2 have been found in both vascular smooth muscle and endothelial cells (6, 8). HO catalyzes the conversion of heme to biliverdin, with the release of carbon monoxide (CO) as a by-product. Several investigators (7, 9, 25, 33, 51, 52) have provided evidence that CO acts as an endogenously produced vasoactive molecule analogous to nitric oxide (NO). Functional evidence for a role of endogenous CO in regulating vascular tone has come from both whole animal (24, 27, 33, 34) and isolated vessel preparations (7, 10, 23, 25, 29, 49). For example, systemic administration of zinc protoporphyrin IX, a selective HO inhibitor, increases renal vascular resistance in chronically hypoxic rats (34). Furthermore, exogenous administration of CO or the HO substrate heme-L-lysinate produced concentration-dependent increases in vessel diameter (27). Taken together, these results suggest a potential role for endogenous CO in the regulation of vascular tone.

CO-induced vasodilation has been demonstrated in a number of vascular beds, including the skeletal, hepatic, renal, and cerebral circulations (25, 27, 34, 47). Although the mechanism of CO-induced vasodilation has been ardently investigated, there is considerable variability in the proposed mechanism between the vascular beds. It has been proposed that CO produces vasodilation by both soluble guanylyl cyclase (sGC)-dependent (7-9, 23, 32, 36, 40, 41, 45, 46, 49) and -independent (11, 43, 48-50) mechanisms. Indeed, CO-induced vasodilation has been shown to occur through a cGMP-dependent mechanism in rabbit aorta (23) and in the aorta (7) and tail artery (49) of the rat. In contrast, CO has been suggested to elicit cGMP-independent vasodilation in the lamb ductus arteriosus (10) as well as in the gracilis arterioles (25) and lungs of the rat (11, 43). In addition, vascular smooth muscle large-conductance calcium-activated K+ (BKCa) channels have been shown to play a role in CO-induced vasodilation in both rat tail artery (49) and porcine pial artery (27). However, whether activation of the vascular smooth muscle BKCa channel in response to CO occurs through a direct effect on the channel itself (48, 50) or through a cGMP-dependent mechanism is unclear. To date, the one study that investigated the mechanism of CO-induced vasodilation in the intact pulmonary circulation concluded that dilation was independent of sGC activation (43). However, CO was administered via the airway, and thus the results may not be representative of what occurs in response to CO produced within the vasculature.

Although CO appears to be a pulmonary vasodilator, the specific vascular segments within the lung that are affected have not been defined. NO donors cause dilation of both arteries and veins in the pulmonary circulation (15). This is likely indicative of even segmental distribution of sGC because NO-induced dilation appears to be cGMP dependent in the pulmonary vasculature (1). Thus if CO similarly acts solely via cGMP-dependent mechanisms, then an analogous uniform profile of dilation would be expected. In contrast, evidence exists that BKCa channels may be unevenly distributed within this bed (2), which would affect the sites of dilation if these channels are an integral part of pulmonary vascular CO signaling. Thus we examined the sites of CO-induced vasodilation in the isolated, perfused rat lung and further assessed the importance of the activation of sGC and of BKCa channels in these segmental responses. We hypothesized that CO produces vasodilation in the arterial and venous segments of the pulmonary vasculature in a cGMP-dependent manner involving activation of the vascular smooth muscle cell BKCa channel.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
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REFERENCES

All protocols and surgical procedures 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 (200-250 g; Harlan Sprague Dawley) were used for all experiments.

Isolated Lung Preparation

The procedure for lung isolation has been described previously (1, 14, 21, 31). Briefly, animals were anesthetized with 25 mg of intraperitoneal pentobarbital sodium. The trachea was isolated and cannulated with a 17-gauge needle stub. A midsternal incision was made to expose the heart, and 100 U of heparin were injected directly into the right ventricle. The pulmonary artery was cannulated with a 13-gauge needle stub via an incision in the right ventricle. The left ventricle was cannulated with a 4-mm-diameter plastic tube, the preparation was quickly connected to a perfusion system containing a physiological saline solution [PSS; 290 mosmol/kgH2O; containing 126 mM NaCl, 5.4 mM KCl, 0.83 mM MgSO4, 19 mM NaHCO3, 1.8 mM CaCl2, 5.5 mM glucose, and 4% (wt/vol) albumin] and the heart and lungs were removed en bloc. For all protocols, meclofenamate (10 µg/ml) and Nomega -nitro-L-arginine (300 µM) were added to the perfusate to inhibit the actions of cyclooxygenase and NO synthase products, respectively. After removal, the heart and lungs were suspended in a humidified chamber maintained at 38°C and initially perfused at 0.8 ml/min with PSS. The perfusion rate was gradually increased to 30 ml · min-1 · kg body wt-1 and maintained at this rate throughout the experiment. The initial 20 ml of perfusate were discarded before recirculation was begun with the remaining 40 ml of solution. The lungs were ventilated at a frequency of 60 breaths/min with a gas mixture containing 21% O2-6% CO2-73% N2 for the duration of the experiment. The gas mixture was humidified before entering the trachea. Inspiratory pressure was set at 9 cmH2O, and positive end-expiratory pressure was set at 2 cmH2O. For all experiments, lungs were studied under zone 3 conditions achieved by elevating the perfusate reservoir until pulmonary venous pressure (Pv) was 3-4 mmHg. Pulmonary arterial pressure (Ppa) and Pv were measured with SpectraMed model P23XL pressure transducers and recorded on separate channels of a Gould RS3800 chart recorder. In addition, data were stored and subsequently analyzed on a microcomputer with a commercial data acquisition system (CODAS; DATAQ Instruments).

Isolated Lung Protocols

Characterization of the segmental response to exogenous CO in the pulmonary vasculature. The present protocol was designed to characterize the segmental profile of the vasodilatory response to exogenous CO in the pulmonary vasculature. Before each experiment, 10 ml of PSS stored on ice were vigorously bubbled for 15 min with 100% CO gas (Matheson Gas Products). Lungs (n = 5), prepared as described in Isolated Lung Preparations, were allowed to equilibrate for 30 min, at which time baseline capillary pressure (Ppc) was assessed by a double-occlusion procedure. The method of estimating microvascular pressure agrees with other methods (44) and has been described previously (15, 20). Briefly, the arterial inflow line and venous outflow line were simultaneously occluded. Ppa and Pv rapidly equilibrated to a new intermediate pressure representative of Ppc during the brief no-flow condition. The occlusion procedure was completed in <2 s. After baseline occlusion, the thromboxane mimetic U-46619 was added to the reservoir to elicit a pressor response of ~10 mmHg. U-46619 is known to constrict both the arterial and venous segments of the pulmonary circulation (15). Our laboratory (15) has previously shown roughly equal arterial and venous resistances in lungs treated in this fashion. Double occlusion was performed on establishment of a stable U-46619 pressor response and was followed by administration of a 500-µl bolus of CO-equilibrated PSS (4.2 ml CO/100 mol PSS) injected directly into the arterial pressure line. Double occlusion was repeated on achievement of a maximal response to CO. Occlusion was maintained until there was a plateau in both Ppa and Pv; this typically occurred in <2 s.

Effect of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one on CO-induced dilation. The following protocol was designed to examine the role of sGC in the vasodilatory response to exogenous CO. Twenty lungs were prepared as described in Isolated Lung Preparation. To examine the role of sGC in the vasodilatory response to CO, the specific sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 µM) (17) or an equal volume of vehicle (75 µl of DMSO) was added to the reservoir at the beginning of the 30-min equilibration period. Next, U-46619 was added to the reservoir to elicit a ~10 mmHg pressor response. On establishment of a stable U-46619 pressor response, 500 µl of CO-equilibrated PSS (4.2 ml CO/100 ml PSS) were administered via arterial injection. Once arterial pressure had returned to baseline, a 500-µl bolus of the PSS vehicle was administered via arterial injection. To control for a potential order effect, a second dose of CO-equilibrated PSS was given. Similar responses were observed for the two CO injections (data not shown). In separate experiments, the vasodilatory response when the beta 2-adrenergic receptor agonist isoproterenol (10 nM) was added to the reservoir was assessed in U-46619-preconstricted lungs (n = 6/group) treated with either ODQ or vehicle to demonstrate that pretreatment with ODQ did not have nonspecific effects on non-cGMP-dependent vasodilator responsiveness.

Effect of dipyridamole on CO-induced dilation. To further demonstrate the significance of sGC in CO-induced pulmonary vasodilation, the vasodilatory response to CO was assessed in lungs pretreated with the specific phosphodiesterase 5 inhibitor dipyridamole (20 µM). Lungs were isolated, perfused, and studied as described in Isolated Lung Preparation. Because phosphodiesterase inhibition augmented the duration rather than the amplitude of the CO response, the effect of dipyridamole on CO-induced vasodilation was quantified by determining the duration of the nadir of the pulmonary vascular resistance response.

Effect of CO administration on cGMP whole lung cGMP content. The experiments were designed to provide further support for the role of sGC activation in CO-induced vasodilation. Lungs were isolated, perfused, and studied as described in Isolated Lung Preparation. Briefly, on establishment of a stable U-46619-induced pressor response, either CO or its vehicle was administered via arterial injection. Additional experiments were performed on lungs that were pretreated with ODQ before administration of CO. All experiments were performed in the presence of dipyridamole (20 µM). When the CO-induced vasodilation was maximal, lungs were immediately frozen in liquid N2 and stored at -80°C. Lungs that were given vehicle or pretreated with ODQ were frozen at a time point similar to the one for lungs that dilated to CO. cGMP content was measured in whole lung homogenates via a radioimmunoassay kit (Amersham Pharmacia, Piscataway, NJ).

Effect of BKCa channel inhibition on CO-induced vasodilation. To test the involvement of BKCa channel activation in CO-induced vasodilation, the vasodilatory response to CO was assessed in lungs pretreated with either the K+ channel inhibitor tetraethylammonium chloride (TEA; 10 mM; n = 4 lungs) or the highly specific BKCa channel inhibitor iberiotoxin (100 nM; n = 2). K+ channel inhibitors were added to the reservoir at the initiation of the equilibration period. Lungs were isolated, perfused, and studied as described in Isolated Lung Preparation.

Solutions

ODQ and dipyridamole (Sigma) were dissolved in DMSO and stored at -80°C. U-46619 (Cayman Chemical) was dissolved in ethanol and stored at -80°C. TEA and iberiotoxin were dissolved in double-distilled water and stored at -80°C. Nomega -nitro-L-arginine was dissolved directly in the bovine albumin-PSS solution before perfusion of the lungs to produce a 300 µM concentration. Meclofenamate (Sigma) was dissolved in saline (4 mg/ml).

Calculations and Statistical Analyses

Because flow through the pulmonary vascular bed was kept constant throughout the experiment, any changes in Ppa were reflective of changes in pulmonary vascular resistance. For the determination of segmental responses, pulmonary arterial resistance was calculated as the difference between Ppa and Ppc divided by flow. Similarly, pulmonary venous resistance was calculated as the difference between Ppc and Pv divided by flow. For all other experiments, changes in total vascular resistance were calculated as the difference between Ppa and Pv divided by flow. The vasodilatory responses to CO were calculated as percent reversal of the resistance response to U-46619 elicited in each segment. To meet the assumption of a normal distribution necessary for parametric statistics, percent changes in vascular resistance underwent arcsine transformation before analysis. Data were analyzed with a two-way repeated-measures ANOVA, one-way ANOVA, or an unpaired 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
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Characterization of the Segmental Response to Exogenous CO in the Pulmonary Vasculature

Baseline total vascular resistances for all experimental groups ranged from 0.10 to 0.12 mmHg · ml-1 · min · kg (Table 1) and did not differ between protocols. For experiments that examined the segmental profile of CO-induced dilation, baseline arterial resistance was 0.03 ± 0.01 mmHg · ml-1 · min · kg, whereas venous resistance was 0.04 ± 0.004 mmHg · ml-1 · min · kg. Administration of U-46619 to the reservoir increased total resistance 0.32 ± 0.02 mmHg · ml-1 · min · kg above baseline, with arterial resistance elevated by 0.25 ± 0.02 mmHg · ml-1 · min · kg and venous resistance by 0.13 ± 0.007 mmHg · ml-1 · min · kg. Administration of 500 µl of CO solution directly into the arterial line resulted in a statistically significant reversal of U-46619-induced total vasoconstriction [change (Delta ) = 0.13 ± 0.01 mmHg · ml-1 · min · kg] that persisted for ~2 min, achieving a peak response in 25 ± 2.9 s. In addition, as shown in Fig. 1, total, arterial, and venous segmental resistances were uniformly reduced by CO administration.

                              
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Table 1.   Baseline and U-46619 preconstricted resistances in isolated saline-perfused lungs



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Fig. 1.   Total, arterial, and venous vasodilatory responses to CO administered intraluminally in lungs from rats (n = 5). Lungs were preconstricted with the thromboxane mimetic U-46619. Data are means ± SE. There were no differences in vasodilatory response between vascular segments.

Effect of ODQ on CO-Induced Dilation

Pretreatment with ODQ did not alter baseline vascular resistance (Table 1). The total dose of U-46619 required to elicit an ~10 mmHg pressor response was similar between lungs treated with ODQ (2.0 ± 0.14 µg) and vehicle (2.17 ± 0.31 µg). Exogenous CO administration reduced total vascular resistance from 0.43 ± 0.08 to 0.33 ± 0.05 mmHg · ml-1 · min · kg in lungs pretreated with the ODQ vehicle (DMSO). In contrast, this vasodilatory response was abolished in lungs that were pretreated with ODQ added to the reservoir (Fig. 2). Moreover, administration of 500 µl of the vehicle (PSS) for CO resulted in minimal reductions in total vascular resistance, 0.04 ± 0.009 and 0.03 ± 0.008 mmHg · ml-1 · min · kg for vehicle and ODQ conditions, respectively (Fig. 2). Furthermore, vasodilation in response to the beta 2-adrenergic receptor agonist isoproterenol was unaffected by pretreatment with ODQ (Fig. 3), suggesting that the inhibitor does not nonselectively blunt vasodilation in this preparation.


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Fig. 2.   Vasodilatory response to CO and physiological saline solution (PSS) administered intraluminally in rat lungs pretreated with either the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or with DMSO vehicle (n = 5 lungs/group). Lungs were preconstricted with the thromboxane mimetic U-46619. Data are means ± SE. *Significantly different from physiological saline solution, P <=  0.05. #Significantly different from ODQ, P <=  0.05.



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Fig. 3.   Vasodilatory response to isoproterenol in rat lungs pretreated with the soluble guanylyl cyclase inhibitor ODQ or DMSO vehicle (VEH; n = 6 lungs/group). Lungs were preconstricted with the thromboxane mimetic U-46619. Data are means ± SE. There were no differences between groups.

Effect of Dipyridamole on CO-Induced Dilation

As shown in Fig. 4, the duration of the vasodilatory response to CO was prolonged in lungs pretreated with the phosphodiesterase 5 inhibitor dipyridamole. Although phosphodiesterase inhibition did not alter the magnitude of the vasodilatory response to CO (data not shown), pretreatment with dipyridamole resulted in an approximately twofold increase in the duration of the CO response.


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Fig. 4.   Effect of phosphodiesterase 5 inhibition with 20 µM dipyridamole (n = 6 lungs/group) on the duration of the nadir of the vasodilatory response. Data are means ± SE. *Significantly different from control, P <=  0.05 (n = 5 lungs).

Effect of CO Administration on Whole Lung cGMP Content

Isolated lungs administered CO demonstrated greater cGMP content than lungs given vehicle. In addition, cGMP content in lungs pretreated with ODQ was lower than that in both CO- and vehicle-treated groups (Fig. 5).


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Fig. 5.   Effect of CO administration on cGMP content in lungs from rats. Lungs were preconstricted with the thromboxane mimetic U-46619. CO or its vehicle (n = 3 lungs/group) was administered via arterial injection. CO administration produced an increase in lung cGMP content, which was abolished in lungs pretreated with ODQ (n = 4). Data are means ± SE. *Significantly different from vehicle and CO + ODQ, P <=  0.05. #Significantly different from vehicle, P <=  0.05.

Effect of TEA and Iberiotoxin on CO-Induced Vasodilation

Figure 6 depicts the CO response in lungs pretreated with K+ channel inhibitors. The vasodilatory response to exogenous CO was unaltered in the presence of either TEA (n = 4 lungs) or iberiotoxin (n = 2 lungs). Pretreatment with these K+ channel inhibitors had no effect on baseline vascular resistance. Close injection of 500 µl of CO solution into the arterial line decreased pulmonary vascular resistance by 0.21 ± 0.02 and 0.16 ± 0.03 mmHg · ml-1 · min · kg for TEA and iberiotoxin, respectively. The decrease in vascular resistance between the two conditions was not statistically different. In addition, the CO response in the presence of K+ channel inhibitors was similar to that in parallel experiments performed without inhibitors (Figs. 1 and 6). To alleviate concerns that the concentration of iberiotoxin used (100 nM) may not have been sufficient to block BKCa channel activity, we performed additional experiments with 200 and 300 nM concentrations of iberiotoxin, neither of which was associated with altered CO-induced vasodilation (data not shown).


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Fig. 6.   Effect of K+ channel inhibition on the vasodilatory response to CO in rat lungs under control conditions (n = 5) or pretreated with either 100 nM iberiotoxin (n = 2) or 10 mM tetraethylammonium (TEA; n = 4). Lungs were preconstricted with the thromboxane mimetic U-46619. Data are means ± SE. K+ channel inhibition had no effect on the vasodilatory response to CO.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The major findings of the present study are that 1) exogenous CO produces vasodilation in the pulmonary circulation, 2) CO alters vascular resistance by acting equally on both the arterial and venous segments, 3) CO-induced vasodilation is dependent on the activation of sGC, 4) CO administration increases lung cGMP content, and 5) exogenous CO-induced vasodilation does not appear to involve activation of the BKCa channel. These findings suggest that the mechanism of CO-induced vasodilation in the pulmonary circulation depends on the activation of sGC through a signal transduction cascade that is independent of K+ channel activation.

Several lines of evidence have suggested a role for HO-1-derived CO in the altered vascular reactivity that occurs after chronic hypoxia (7, 9, 33-35, 43, 51). Indeed, chronic hypoxia has been shown to increase HO-1 mRNA, protein, and enzyme activity both in endothelial and vascular smooth muscle cells (6, 9, 32). Moreover, transcriptional activation of the HO-1 gene in response to hypoxia has been shown to be mediated by the transcription factor hypoxia-inducible factor-1 in rat aortic vascular smooth muscle cells (26). Additionally, a recent study by Christou et al. (9) provided support for CO as a protective agent in animals chronically exposed to a hypoxic environment. These investigators demonstrated that enhancement of endogenous CO production attenuated hypoxia-induced pulmonary vascular remodeling and right ventricular hypertrophy in response to 1 wk of hypoxia in Sprague-Dawley rats (9). Moreover, hypoxic exposure increases right ventricular dilation in HO-1-knockout mice compared with dilation in control animals (51). Taken together, these results suggest that the induction of HO-1 by prolonged exposure to a hypoxic environment may play a protective role in ameliorating some of the pathophysiological adaptations that occur in response to hypoxia.

In the present study, exogenous administration of CO to the pulmonary vasculature reversed the vasoconstriction induced by U-46619 in both the arterial and venous segments to a similar degree. Previous work from our laboratory (15, 37) has demonstrated that the vasodilatory response to NO donors in the pulmonary vasculature is similar to that observed in response to CO in U-46619-preconstricted lungs. For example, although NO appears to be a more potent vasodilator than CO, administration of S-nitroso-N-acetylpenicillamine (1 µM) to isolated perfused rat lungs reversed U-46619-induced constriction by ~50 and ~45% in pulmonary arterial and venous segments, respectively (38). In contrast to the present results, Villamor et al. (46) observed that the maximal vasodilatory response to CO was greater in porcine pulmonary veins than in arteries. The apparent discrepancy between these results and those of the present study could be attributed to either species differences or differences in the size of the vessels studied. Indeed, the vasoconstrictor responses to U-46619 occur preferentially in small pulmonary arteries and veins (15). Hence CO-induced vasodilation would be confined to those vessels in our experimental preparation. In contrast, Villamor et al. (46) studied larger arteries and veins. Thus differences in the observed segmental profile may be a function of the size of the vessels examined. Finally, because HO has been shown to be expressed in both pulmonary arteries and veins (30), endogenously produced CO could potentially cause dilation in both vascular segments as observed in the present study, which employed exogenous CO. Moreover, it would be anticipated that CO would act locally in the intact circulation because CO produced within the arterial segment would bind avidly to hemoglobin before transit to downstream sites.

We have demonstrated that CO produces vasodilation in both segments of the pulmonary vasculature through sGC activation. This is consistent with evidence suggesting that sGC is expressed in both arterial and venous smooth muscle cells (3, 13, 28). Indeed, CO-induced vasodilation was abolished by sGC inhibition and prolonged by inhibition of phosphodiesterase 5. Furthermore, CO increased whole lung cGMP content. Numerous studies with different vascular beds and varied experimental approaches have provided support for a role of sGC in CO-induced vasodilation. For example, HO-derived CO has been shown to increase cGMP levels in cultured rat aortic smooth muscle cells (32). In addition, treatment of isolated rat tail artery with the membrane permeable cGMP-dependent protein kinase inhibitor Rp-8-bromo-cGMPS partially inhibited CO-induced relaxation (49). Moreover, in rabbit and rat aortic rings, the vasodilatory response to exogenous CO was blocked in the presence of the sGC inhibitor ODQ (7, 23). However, Kozma et al. (25) proposed that activation of sGC is not involved in the regulation of vascular tone by CO, but these experiments were performed in the presence of ODQ, NG-nitro-L-arginine methyl ester, and the HO inhibitor chromium mesoporphyrin. Thus it is unlikely that ODQ would have an additional effect when both the production of NO and CO were inhibited.

Although sGC activation has been shown to be important in CO-induced vasodilation in a number of vascular beds (7-9, 23, 32, 45, 49), results from studies investigating the mechanism of CO-induced vasodilation in the pulmonary circulation have been inconsistent. For example, induction of HO with NiCl2 increased cGMP levels in rat lung under normoxic conditions and reduced systolic right ventricular pressure and medial wall thickness after chronic hypoxia (9). However, others (43) have shown that pretreating isolated rat lungs with methylene blue did not alter the ability of CO administered through the airway to blunt the vasoconstrictor response to hypoxia (2% O2). The cause of the disparity in these results is not apparent, especially in view of the present study of isolated perfused rat lungs. One possibility may be a result of differences in the type of perfusate (e.g., blood vs. PSS) used in these studies (39). Indeed, because hemoglobin has a high affinity for CO, the effectiveness of CO to produce vasodilation in the presence of red cells may be reduced. In addition, it is possible that this discrepancy may be due to differences in the method of CO administration. The vascular administration of CO used in the present study was chosen to more closely mimic the likely source of endogenous CO within the circulation. For example, there is evidence that CO produced within the vasculature is of endothelial origin (7). Furthermore, administration of CO directly into the vasculature would likely reduce the diffusional distance for CO to reach its target within the vascular smooth muscle cell. Results of studies that used inhaled CO to elicit pulmonary vasodilation have been inconsistent. Indeed, the studies of Cantrell and Tucker (5) and of Grover et al. (19) failed to demonstrate vasodilation to CO when administered through the airway, although Tamayo et al. (43) observed marked inhibition of hypoxic vasoconstriction with higher concentrations of CO in the inspired gas mixture. Thus the ability of CO to dilate the pulmonary vasculature may be concentration dependent. The necessity of using higher concentrations of inspired CO to elicit vasodilation may be due to the lower diffusibility of CO compared with NO (22) and the lower affinity of sGC for CO compared with that for NO (42).

The activation of vascular smooth muscle BKCa channels has been implicated in the vasodilatory response to CO (27, 49). Evidence for an involvement of K+ channel activation in CO-induced vasodilation has arisen from studies employing both isolated vessel and patch-clamp techniques. For example, CO administration produced dose-dependent increases in pial vessel diameter that were abolished in the presence of TEA, iberiotoxin, or charybdotoxin (27). Furthermore, CO has been shown to reduce the open probability of the BKCa channel in vascular smooth muscle cells (48, 50). Interestingly, these studies suggest that CO may have a direct effect on the vascular smooth muscle BKCa channel (48, 50). To date, the present study is the first to examine the effects of K+ channel inhibition on CO-induced vasodilation in the intact pulmonary vasculature. However, our data do not support an involvement of the K+ channel activation in CO-induced vasodilation in the pulmonary vasculature. Although not directly tested, it is unlikely that the failure of K+ channel inhibition to alter the vasodilatory response to CO observed in the present study is due to an incomplete block of vascular smooth muscle K+ channels. Indeed, the concentrations of K+ channel inhibitors used in this study have been shown to effectively block K+ channel activity both in vivo (12) and in vitro (4, 16, 18). Thus inconsistencies between the present study and previous investigations demonstrating a role for K+ channels in CO-induced vasodilation are most likely due to differences in mechanisms of action between vascular beds.

In summary, the current study provides evidence that CO administered directly into the pulmonary circulation of the isolated perfused rat lung causes vasodilation in both the arterial and venous segments of the vasculature through a sGC-dependent pathway that is independent of K+ channel activation. Further studies are needed to elucidate the targets downstream of cGMP production and cGMP-dependent protein kinase activation that are involved in CO-induced vasodilation.


    ACKNOWLEDGEMENTS

We thank Minerva Murphy, Anna Holmes, and Nikki Jernigan for technical assistance.


    FOOTNOTES

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).

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.

Received 12 March 2001; accepted in final form 9 August 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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