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. 2010 Jul 16;299(4):H1153–H1159. doi: 10.1152/ajpheart.01101.2009

Pulmonary and systemic vasodilator responses to the soluble guanylyl cyclase stimulator, BAY 41-8543, are modulated by nitric oxide

Adeleke M Badejo Jr 1, Vaughn E Nossaman 1, Edward A Pankey 1, Manish Bhartiya 1, Chandrika B Kannadka 1, Subramanyam N Murthy 1, Bobby D Nossaman 1,2, Philip J Kadowitz 1,✉
PMCID: PMC2957355  PMID: 20639220

Abstract

BAY 41-8543 is a nitric oxide (NO)-independent stimulator of soluble guanylyl cyclase (sGC). Responses to intravenous injections of BAY 41-8543 were investigated under baseline and elevated tone conditions and when NO synthase (NOS) was inhibited with Nω-nitro-l-arginine methyl ester (l-NAME). Under baseline conditions, intravenous injections of BAY 41-8543 caused small decreases in pulmonary arterial pressure, larger decreases in systemic arterial pressure, and increases in cardiac output. When pulmonary arterial pressure was increased to ∼30 mmHg with an intravenous infusion of U-46619, intravenous injections of BAY 41-8543 produced larger dose-dependent decreases in pulmonary arterial pressure, and the relative decreases in pulmonary and systemic arterial pressure in response to the sGC stimulator were similar. Treatment with l-NAME markedly decreased responses to BAY 41-8543 when pulmonary arterial pressure was increased to similar values (∼30 mmHg) in U-46619-infused and in U-46619-infused plus l-NAME-treated animals. The intravenous injection of a small dose of sodium nitroprusside (SNP) when combined with BAY 41-8543 enhanced pulmonary and systemic vasodilator responses to the sGC stimulator in l-NAME-treated animals. The present results indicate that BAY 41-8543 has similar vasodilator activity in the systemic and pulmonary vascular beds when pulmonary vasoconstrictor tone is increased with U-46619. These results demonstrate that pulmonary and systemic vasodilator responses to BAY 41-8543 are significantly attenuated when NOS is inhibited by l-NAME and show that vasodilator responses to BAY 41-8543 are enhanced when combined with a small dose of SNP in l-NAME-treated animals. The present results are consistent with the concept that pulmonary and systemic vasodilator responses to the sGC stimulator are NO-independent; however, the vasodilator activity of the compound is greatly diminished when endogenous NO production is inhibited with l-NAME. These data show that BAY 41-8543 has similar vasodilator activity in the pulmonary and systemic vascular beds in the rat.

Keywords: soluble guanylyl cyclase stimulator, pulmonary and systemic vascular beds, U-46619, Nω-nitro-l-arginine methyl ester, sodium nitroprusside

the heme-protein soluble guanylyl cyclase (sGC) is the intracellular receptor for nitric oxide (NO) (16–20, 23, 25, 26). The sGC is a heterodimetric enzyme with α- and β-subunits (3, 6, 22, 26, 30). The enzyme contains a heme moiety that is essential for the binding of NO and activation of the enzyme (2, 7, 19, 31). The activation of sGC enhances the conversion of GTP to cGMP that mediates physiological responses, including smooth muscle relaxation and inhibition of platelet aggregation (1, 4, 16, 21, 27, 28). Compounds have been developed that can directly stimulate sGC and increase cGMP formation in pathophysiological conditions when NO formation and bioavailability are impaired or when NO tolerance has developed (14, 35, 38). The pyrazolopyridine compound BAY 41-8543 is a NO-independent stimulator of sGC that has been shown to reduce systemic and pulmonary arterial pressure and relax isolated vessels from a variety of organ systems (33, 37, 38). BAY 41–2272 is closely related to BAY 41-8543, and this sGC stimulator has been shown to have significant pulmonary vasodilator activity in a variety of species (9, 10, 13, 15, 34, 37). BAY 41–2272 has been shown to reduce right ventricular hypertrophy and pulmonary vascular remodeling in a chronic hypoxia-induced model of pulmonary hypertension (8). It has been reported that, when either BAY 41–2272 or BAY 41-8543 was administered by inhalation to awake lambs, the pyrazolopyridine compounds had selective pulmonary vasodilator activity, and BAY 41-8543 could enhance the magnitude and prolong the duration of the vasodilator response to inhaled NO (9, 13). The present study was undertaken to investigate responses to BAY 41-8543 in the pulmonary and systemic vascular beds of the rat and to determine the role of endogenous and exogenously administered NO in mediating vasodilator responses to the sGC stimulator. The results of these studies indicate that BAY 41-8543 has similar vasodilator activity in the pulmonary and systemic vascular beds of the intact-chest rat; that vasodilator responses are markedly attenuated when NO synthase (NOS) is inhibited by Nω-nitro-l-arginine methyl ester (l-NAME); and that vasodilator responses can be enhanced by coadministration of a NO donor.

METHODS

The Institutional Animal Care and Use Committee of the Tulane University School of Medicine approved the experimental protocol employed in these studies, and all procedures were conducted in accordance with institutional guidelines. In these experiments, adult male Sprague-Dawley rats (Charles Rivers) weighing 325–450 g were anesthetized with Inactin (100 mg/kg ip) (Sigma-Aldrich) and were placed in the supine position on an operating table. Supplemental doses of Inactin were administered intraperitoneally to maintain a uniform level of anesthesia. Body temperature was maintained with a heating lamp. The trachea was cannulated with a short segment of PE-240 tubing to maintain a patent airway. The animals spontaneously breathed room air. A femoral artery was catheterized with PE-50 tubing for measurement of systemic arterial pressure. The left jugular and femoral veins were catheterized with PE-50 tubing for intravenous injections and infusions of agents. For pulmonary arterial pressure measurement, a specially designed 3-Fr single-lumen catheter with a curved tip and with radio-opaque marker was passed from the right jugular vein into the main pulmonary artery under fluoroscopic guidance (Picker-Surveyor Fluoroscope) as previously described (5). Pulmonary and systemic arterial pressures were measured with Namic Perceptor DT transducers (Boston Scientific), digitized by a Biopac MP100 data acquisition system (Biopac Systems), and stored on a Dell personal computer (PC). Cardiac output was measured by the thermodilution technique with a Cardiomax II computer (Columbus Instruments). A known volume (0.2 ml) of room temperature 0.9% NaCl solution was injected in the jugular vein catheter with the tip near the right atrium, and changes in blood temperature were detected by a 1.5-Fr thermistor microprobe catheter (Columbus Instruments) positioned in the aortic arch advanced from the left carotid artery. The indicator dilution curve data were stored on the PC.

In the first set of experiments, the effects of intravenous injections on the sGC stimulator BAY 41-8543 on pulmonary and systemic arterial pressures and on cardiac output were investigated in the anesthetized intact-chest rat under baseline conditions.

In the second set of experiments, responses to intravenous injections of BAY 41-8543 were investigated when pulmonary arterial pressure was increased to ∼30 mmHg by continuous intravenous infusion of the thromboxane receptor agonist U-46619. After an initial high priming rate, the U-46619 infusion was adjusted (200–400 ng/min) to maintain pulmonary arterial pressure at ∼30 mmHg.

In the third set of experiments, the effects of the NOS inhibitor l-NAME in a dose of 25 mg/kg iv on responses to BAY 41-8543 in the pulmonary and systemic vascular beds were investigated.

In the fourth set of experiments, responses to BAY 41-8543 were compared in the pulmonary vascular bed when baseline pulmonary arterial pressure was increased to a similar value (∼30 mmHg) in U-46619-infused animals and in l-NAME-treated rats in which an infusion of U-46619 was used to increase pulmonary arterial pressure from a value of 27 to 30 mmHg (Table 1) so that responses were compared at the same level of tone.

Table 1.

Effect of l-NAME on systemic and pulmonary arterial pressure and on cardiac output

Systemic Arterial Pressure, mmHg Pulmonary Arterial Pressure, mmHg Cardiac Output, ml/min
Control 117 ± 8 21 ± 1 129 ± 8
l-NAME (25 mg/kg) 151 ± 3* 27 ± 2* 69 ± 8*
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Values are means ± SE; n = 6–10 experiments. l-NAME, Nω-nitro-l-arginine methyl ester.

*

P < 0.05 compared with control.

In the last set of experiments, the interaction between BAY 41-8543 and the NO donor sodium nitroprusside (SNP) was investigated in the pulmonary and systemic vascular beds. In these experiments, responses to intravenous injections of BAY 41-8543 (100 μg/kg) and SNP (0.3 μg/kg) where compared when injected separately and together in l-NAME-treated rats.

BAY 41-8543 {2-[1-(2-fluorophenylmethyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5(4-morpholinyl)-4,6-pyrimidinediamine} was obtained from Dr. Johannes-Peter Stasch of the Institute of Cardiovascular Research, Pharma Research Centre, Bayer, Wuppertal, Germany, and was dissolved in Transcutol-Cremophor EL-0.9% NaCl solution (10:10:80) (36). U-46619 (Cayman Chemical) was dissolved in 95% ethyl alcohol and diluted in 0.9% NaCl solution. SNP and l-NAME (Sigma-Aldrich) were dissolved in 0.9% NaCl.

The hemodynamic data are expressed as means ± SE and were analyzed using paired and group t-tests and with an ANOVA with Dunnett's post hoc test. The criteria used for statistical significance was P < 0.05.

RESULTS

Responses to BAY 41-8543 under baseline conditions.

Responses to intravenous injections of BAY 41-8543 in the systemic and pulmonary vascular beds were investigated under baseline conditions in the anesthetized rat, and these data are summarized in Fig. 1. The intravenous injections of the sGC stimulator in doses of 10, 30, and 100 μg/kg produced small decreases in pulmonary arterial pressure, larger dose-dependent decreases in systemic arterial pressure, and increases in cardiac output (Fig. 1).

Fig. 1.

Fig. 1.

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Bar graphs showing changes in pulmonary and systemic arterial pressures and cardiac output in responses to iv injections of BAY 41-8543 under baseline conditions. n, No. of experiments. *P < 0.05.

Responses to BAY 41-8543 under elevated tone conditions.

Responses to BAY 41-8543 were investigated under elevated pulmonary vascular tone conditions, and these data are summarized in Fig. 2. When pulmonary arterial pressure was increased to ∼30 mmHg with an intravenous infusion of the thromboxane receptor agonist U-46619, the intravenous injections of BAY 41-8543 in doses of 10, 30, and 100 μg/kg produced dose-related decreases in pulmonary and systemic arterial pressures and increases in cardiac output (Fig. 2). The percent decreases in systemic arterial pressure in response to BAY 41-8543 were similar in U-46619-infused animals compared with decreases in control animals, whereas the percent decreases in pulmonary arterial pressure were significantly greater when pulmonary arterial pressure was increased to a high steady level with U-46619 (Fig. 3).

Fig. 2.

Fig. 2.

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Bar graphs showing changes in pulmonary and systemic arterial pressures and cardiac output in response to BAY 41-8543 in experiments in which pulmonary arterial pressure was increased to a high steady level (∼30 mmHg) with U-46619. n, No. of experiments. *P < 0.05.

Fig. 3.

Fig. 3.

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Dose-response curves for BAY 41-8543 comparing %decreases in pulmonary and systemic arterial pressure in baseline (Control) and U-46619-infused animals. n, No. of experiments. *P < 0.05.

Effect of l-NAME on responses to BAY 41-8543.

The administration of l-NAME in a dose of 25 mg/kg iv produced a significant increase in pulmonary and systemic arterial pressures and a significant decrease in cardiac output (Table 1). The intravenous administration of BAY 41-8543, in doses of 10, 30, and 100 μg/kg, produced dose-related decreases in pulmonary and systemic arterial pressures and increases in cardiac output in l-NAME-treated animals (Fig. 4). The decreases in pulmonary and systemic arterial pressures in response to BAY 41-8543 were smaller in l-NAME-treated animals compared with responses in U-46619-infused rats (Figs. 2 and 4). Because pulmonary arterial pressure was lower in l-NAME-treated animals (27 ± 2 mmHg; Table 1) than in U-46619-infused rats, another series of experiments was carried out in l-NAME-treated rats that were infused with U-46619 in concentrations sufficient to increase pulmonary arterial pressure to ∼30 mmHg. The comparison of decreases in pulmonary and systemic arterial pressures in response to intravenous injections of BAY 41-8543 in these two groups of rats is shown in Fig. 5. The decreases in pulmonary and systemic arterial pressures in the U-46619 plus l-NAME treatment group in response to BAY 41-8543 were significantly smaller than decreases observed in U-46619-infused animals when pulmonary arterial pressure was increased to ∼30 mmHg in both groups of rats (Fig. 5). The increases in cardiac output in response to BAY 41-8543 were similar in both groups of rats (Fig. 5).

Fig. 4.

Fig. 4.

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Bar graphs showing changes in pulmonary and systemic arterial pressures in response to BAY 41-8543 in animals treated with Nω-nitro-l-arginine methyl ester (l-NAME, 25 mg/kg iv). n, No. of experiments.

Fig. 5.

Fig. 5.

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Bar graphs comparing changes in pulmonary and systemic arterial pressures and cardiac output in response to BAY 41-8543 in experiments in which pulmonary arterial pressure is increased with U-46619 (filled bars) and U-46619 and l-NAME (25 mg/kg iv) (open bars) to the same level (∼30 mmHg). n, No. of experiments. *P < 0.05.

Interactions of BAY 41-8543 and SNP.

The interaction between BAY 41-8543 and the NO donor SNP was investigated in l-NAME-treated rats. In these experiments, responses to intravenous injections of BAY 41-8543 (100 μg/kg) and SNP (0.3 μg/kg) were compared when injected separately and when injected together in l-NAME-treated rats. The separate intravenous injections of BAY 41-8543 (100 μg/kg) and SNP (0.3 μg/kg iv) produced significant decreases in pulmonary and systemic arterial pressures (Fig. 6). The combined intravenous injection of BAY 41-8543 (100 μg/kg) plus SNP (0.3 μg/kg) produced significantly greater decreases in pulmonary and systemic arterial pressure than when the separate responses to BAY 41-8543 and SNP were added together (Fig. 6).

Fig. 6.

Fig. 6.

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Bar graphs showing changes in pulmonary and systemic arterial pressures in response to iv injections of BAY 41-8543 (100 μg/kg) and sodium nitroprusside (SNP, 0.3 μg/kg) when injected separately and when injected together. The decreases in pressure in response to the combined injection of BAY 41-8543 and SNP are significantly greater than responses to either agent alone. n, No. of experiments. *P < 0.05.

DISCUSSION

The heterochimeric heme-protein sGC is the intracellular receptor for NO (16–20, 23, 25, 26). NO-independent stimulators for sGC have been developed for use in pathophysiological conditions when NO formation and bioavailability are impaired or when NO tolerance has developed (14, 35, 38). BAY 41-8543 is a NO-independent stimulator of sGC that has been shown to have vasodilator activity in a number of vascular beds and to have selective pulmonary vasodilator activity in the awake lamb when inhaled in a microparticle formulation (13, 37). The results of the present study show that intravenous injections of BAY 41-8543 under control conditions produced small decreases in pulmonary arterial pressure, larger dose-dependent decreases in systemic arterial pressure, and increases in cardiac output. The intravenous injections of BAY 41-8543 produced larger dose-dependent decreases in pulmonary arterial pressure when tone in the pulmonary vascular bed was increased with the thromboxane receptor agonist U-46619. The percent decreases in pulmonary and systemic arterial pressures in response to intravenous injections of the BAY 41-8543 in U-46619-infused animals were not different, suggesting that the sGC stimulator had similar vasodilator activity in the pulmonary and systemic vascular beds in the rat.

The effect of NOS inhibition with l-NAME on vasodilator responses to BAY 41-8543 was investigated, and, following administration of the NOS inhibitor, decreases in pulmonary and systemic arterial pressures in response to intravenous injections of BAY 41-8543 were reduced compared with responses in U-46619-infused animals as shown in Figs. 4 and 5. However, because pulmonary arterial pressure was higher in U-46619-infused animals (∼30 mmHg) compared with l-NAME-treated animals (∼27 mmHg; Table 1), another set of experiments was performed in l-NAME-treated rats in which pulmonary arterial pressure was additionally increased to ∼30 mmHg by infusion of U-46619. The comparison of responses to BAY 41-8543 at the same level of pulmonary arterial pressure indicates that decreases in pulmonary arterial pressure in response to the sGC stimulator are reduced by >50% in l-NAME-treated animals. These results are consistent with the concept that responses to the sGC stimulator are NO-independent; however, in the absence of endogenous NO formation, vasodilator responses to the sGC stimulator were markedly attenuated. It has been reported that stimulators of sGC have a dual role of action in that they directly stimulate the native form of the enzyme and render it more sensitive to endogenously produced NO or augment the action of exogenously administered NO (9, 12). Our results are consistent with these findings, since the results of the present study show that vasodilator responses to BAY 41-8543 are attenuated when endogenous NO formation is inhibited.

The role of BAY 41-8543 synergy with exogenous NO was examined in experiments with the NO donor SNP. The separate administration of BAY 41-8543 and SNP produced significant decreases in pulmonary and systemic arterial pressures. The coadministration of a small dose of the NO donor along with BAY 41-8543 produced decreases in pulmonary and systemic arterial pressures that were significantly greater than the sum of responses to either agent when administered alone. These results suggest that BAY 41-8543 synergizes with NO in mediating vasodilator responses to the sGC stimulator in the pulmonary and systemic vascular beds in the intact rat.

BAY 41-8543 and BAY 41–2272 were synthesized based upon analysis of the structure of YC-1 (11, 14, 24). These pyrazolopyridine stimulators activate sGC in a manner independent of NO (14, 34, 38). These compounds activate purified sGC and strongly synergize with NO, reflecting stabilization of the nitrosyl-heme complex of the enzyme (32). Both BAY 41-8543 and BAY 41–2272 relax vascular smooth muscle and have vasodilator activity in the pulmonary and systemic vascular beds (12, 13, 34–37). The increase in cardiac output in response to intravenous injections of BAY 41-8543 may be because of the decrease in afterload.

Although BAY 41-8543 had beneficial effects in experimental models of pulmonary hypertension, this agent does not have favorable pharmacokinetic properties and cannot be used in clinical trials (29). In contrast, BAY 63–2521 (Riociguat), a heme-dependent sGC stimulator closely related to BAY 41-8543, has a better pharmacokinetic profile and has been used in clinical studies (29). In respect to interesting similarities and differences between the actions of BAY 41-8543 and other NO-independent stimulators of sGC, it has been reported that BAY 41–2272, which is chemically similar to BAY 41-8543, produced greater decreases in pulmonary than systemic arterial pressure and that pulmonary vasodilator responses were not attenuated by l-NAME (9). In the present study, BAY 41-8543 produced similar decreases in pulmonary and systemic arterial pressures in U-46619-infused animals, and decreases in both pulmonary and systemic arterial pressures were attenuated by l-NAME treatment. In an ovine fetal model of pulmonary hypertension, chronic infusion of BAY 41–2272 produced potent sustained decreases in pulmonary arterial pressure that were not attenuated by a NOS inhibitor (Nω-nitro-l-arginine), and, when infused at higher rates, systemic arterial pressure was decreased (10). The main differences in response to stimulators of sGC in the awake sheep, the ovine fetal circulation, and intact-chest rat are the relative differences in vasodilator activity in the pulmonary and systemic vascular beds and the role of endogenously produced NO in mediating these responses (9, 10). The reason for the differences in results in the different experimental models may involve differences in species, experimental design and preparation, the BAY compound studied, or, more importantly, the mechanisms involved in the stimulation of sGC. In the present study, BAY 41-8543 had similar vasodilator activity in the preconstricted pulmonary vascular bed and the systemic vascular bed, and vasodilator responses in both beds were attenuated when NOS was inhibited with l-NAME. These data suggest that the role of endogenous NO in the activation of sGC is similar in both circulations in the intact-chest rat model and may differ from sGC activation mechanisms in the pulmonary and systemic vascular beds in the awake sheep and ovine fetal preparation (9, 10).

Although pulmonary vasodilator response to the sGC stimulator BAY 41–2272 was not dependent on the formation of endogenous NO in the awake sheep model, the response strongly synergized with inhaled NO (9). Therefore, the ability of the sGC stimulator to synergize with exogenous NO was in some respects similar in the awake sheep and intact rat models, although the synergism was much greater in the awake sheep when NO was administered by inhalation (9). The present results are consistent with the concept that sGC stimulators can be given along with inhaled NO to produce maximum pulmonary vasodilation (9, 12).

In summary, the results of the present study show that the sGC stimulator BAY 41-8543 has vasodilator activity in the pulmonary and systemic vascular beds in the rat and that vasodilator responses are similar in both vascular beds when tone is increased with U-46619. Vasodilator responses to BAY 41-8543 were significantly attenuated by treatment with the NOS inhibitor l-NAME. In l-NAME-treated animals, vasodilator responses to BAY 41-8543 were enhanced by simultaneous injection of a small dose of SNP. These results show that vasodilator responses to BAY 41-8543 are NO-independent; however, in the absence of endogenous NO formation, vasodilator responses are markedly attenuated. The present data also demonstrate that the sGC stimulator BAY 41-8543 synergizes with exogenous NO in the pulmonary and systemic vascular beds of the rat.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-62000 and HL-77421.

DISCLOSURES

No conflicts of interest are declared by the authors.

ACKNOWLEDGMENTS

We thank Dr. Johannes-Peter Stasch and Bayer for providing the BAY 41-8543 compound.

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