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American Journal of Physiology - Heart and Circulatory Physiology logoLink to American Journal of Physiology - Heart and Circulatory Physiology
. 2014 Mar 21;306(11):H1495–H1506. doi: 10.1152/ajpheart.00978.2013

Vasodilator responses to acetylcholine are not mediated by the activation of soluble guanylate cyclase or TRPV4 channels in the rat

Edward A Pankey 1, Modar Kassan 2, Soo-Kyoung Choi 3, Khalid Matrougui 4, Bobby D Nossaman 5, Albert L Hyman 1, Philip J Kadowitz 1,✉
PMCID: PMC4042198  PMID: 24658016

Abstract

The effects of 1H-[1,2,4]-oxadizaolo[4,3-]quinoxaline-1-one (ODQ), an inhibitor of the activation of soluble guanylate cyclase (sGC) on responses to NO donors acetylcholine (ACh) and bradykinin (BK) were investigated in the pulmonary and systemic vascular beds of the rat. In these studies the administration of ODQ in a dose of 5 mg/kg iv attenuated vasodilator responses to five different NO donors without inhibiting responses to ACh and BK in the systemic and pulmonary vascular beds of the rat. Vasodilator responses to ACh were not inhibited by l-NAME or the transient receptor vanilloid type 4 (TRPV4) antagonist GSK-2193874, which attenuated vasodilator responses to the TRPV4 agonist GSK-1016790A. ODQ did not inhibit vasodilator responses to agents reported to act in an NO-independent manner or to vasoconstrictor agents, and ODQ did not increase blood methemoglobin levels, suggesting that off target effects were minimal. These results show that ODQ in a dose that inhibited NO donor-mediated responses did not alter vasodilator responses to ACh in the pulmonary and systemic vascular beds and did not alter systemic vasodilator responses to BK. The present results indicate that decreases in pulmonary and systemic arterial pressures in response to ACh are not mediated by the activation of sGC or TRPV4 channels and that ODQ can be used to study the role of the activation of sGC in mediating vasodilator responses in the rat.

Keywords: pulmonary vascular bed, transient receptor vanilloid type, vasodilation

cyclic guanosine monophosphate (cGMP) has an important role in the regulation of vascular smooth muscle tone and is synthesized from guanosine triphosphate (GTP) by soluble guanylate cyclase (sGC), the receptor for nitric oxide (NO) (2, 29, 32, 41). sGC is a heterodimeric enzyme, and binding of NO to the heme-containing subunit activates sGC (2). In contrast, oxidation of the heme-containing subunit reduces the sensitivity of the enzyme to NO (7, 25, 45, 48, 49). Pharmacological agents and genetic alterations that selectively modulate sGC activity are useful in the study of the physiological function of the enzyme in regulating vascular tone and mediating vasodilator responses (19, 35, 43).

Although the role of NO in the regulation of baseline tone is well established, the role of NO in mediating vasodilator responses to acetylcholine (ACh) and bradykinin (BK) is controversial (4, 12, 34). A large number of studies show that vasorelaxant responses to ACh are mediated by the NO-cGMP pathway; however, other studies indicate that NO is not involved (13, 18, 20). It has been hypothesized that vessel size, vascular segment studied, method used, and species may account for differences in the role of NO in mediating responses to ACh and BK reported in the literature (17, 34). Studies in isolated first-order gracilis muscle arterioles from endothelial NOS (eNOS) knockout mice show that responses to ACh were only slightly attenuated, and studies in eNOS and cGMP-dependent protein kinase type-1 (cGKI) knockout mice show that vasodepressor responses to ACh are preserved, suggesting that NO and G kinase do not play a major role in mediating these responses (18, 28). Inasmuch as eNOS- and cGKI-deficient rats have not yet been developed, current studies are limited to the use of pharmacological inhibitors in this species.

1H-[1,2,4]-oxadizaolo[4,3-]quinoxaline-1-one (ODQ) is an agent that has been shown to inhibit NO activation of sGC by oxidizing the heme moiety on the β-subunit of the enzyme (9, 11, 39, 52). ODQ has been used to study activation of sGC by NO in biochemical and isolated vessel experiments and has been used in a small number of in vivo cardiovascular studies (9, 11, 39, 52). Studies with ODQ in the rat show that vasodilator responses to the NO donor sodium nitroprusside (SNP) are inhibited, whereas vasodilator responses to chemical activators of sGC are enhanced (35, 43). The present study was undertaken to investigate the effects of ODQ on cardiovascular responses in the pulmonary and systemic vascular beds of the intact rat. In these experiments the effect of ODQ on responses on NO-dependent and NO-independent vasodilator agents and responses to vasoconstrictor agents were investigated. These results show that ODQ did not attenuate decreases in pulmonary and systemic arterial pressures in response to ACh in a dose that inhibits vasodilator responses to a variety of NO donors without inhibiting responses to NO-independent vasodilator agents or responses to vasoconstrictor agents. In these studies, ODQ did not alter blood methemoglobin levels.

It has been reported that vasodilator responses to ACh are mediated by hyperpolarization, and it has been hypothesized that transient receptor vanilloid type 4 (TRPV4) channels are involved in mediating vasodilator responses to ACh (10, 40, 51). In the present study the effect of TRPV4 blockade on vasodilator responses to ACh was investigated. The present results suggest that ODQ can be used to study the role of NO and sGC in mediating vasodilator responses to NO donors and that vasodilator responses to ACh are not mediated by NO and the activation of sGC or the activation of TRPV4 receptors in the rat.

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 100 mg/kg ip Inactin (Sigma-Aldrich) and 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 radio-opaque marker was passed from the right jugular vein into the main pulmonary artery (PA) under fluoroscopic guidance (Picker-Surveyor Fluoroscope) as previously described (22, 36). 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 from the left carotid artery. The indicator dilution curve data were stored on the PC.

Each experimental series was carried out in a separate group of rats, and, in the first set of experiments, the effect of ODQ in a dose of 5 mg/kg iv on responses to the NO donors SNP, sodium nitrite (NaNO2), glyceryl trinitrate (GTN), diethylamine NONOate (DEA/NO), and S-nitrosoglutathione (SNGT) was investigated.

In the second set of experiments, the effect of ODQ on responses to vasodilator agents that act by NO-independent mechanisms was investigated. In the third set of experiments, the effect of ODQ on responses to vasoconstrictor agents was evaluated. In the fourth set of experiments, the effect of ODQ on responses to ACh and BK was investigated, and, in the fifth set of experiments, the effects of l-NAME, and a combination of l-NAME and ODQ on responses to ACh were investigated. The effect of ODQ on the vasodilator response to a subthreshold dose of the sGC activator BAY 60-2770 was also investigated.

In another group of experiments, the effect of the TRPV4 antagonist GSK-2193874 on responses to ACh was investigated.

In ex vivo experiments, PA were isolated and dissected from the lung and cut into rings (∼2 mm in length) from large, medium, and small pulmonary arterial branches (∼300, 200, and 100 μm in diameter) and bathed in PSS solution (containing in mM): 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 KH2PO4, 1.2 MgSO4·7H2O, 25 NaHCO3, and 11 glucose at pH = 7.4. The PA rings were mounted in a small-vessel dual-chamber myograph, bathed in PSS solution at 37°C, and oxygenated with a gas mixture of 95% O2 and 5% CO2. Isometric tension was measured as previously described (18, 27, 33, 44). The rings were initially stretched with a resting tension equivalent to a luminal pressure of 30 mmHg (3.9 kPa). The normalization was performed by distending the vessel and measuring micrometer and force readings. The wall tension is the force divided by the wall length, and the LaPlace relationship can be used to determine the effective pressure, which was set to 30 mmHg or 3.9 kPa. After precontraction to ∼80% of maximum tension induced by the thromboxane A2 mimic U-46619 (10−6 M) and a steady contraction was achieved, cumulative dose-response curves were then obtained for ACh (10−8 to 10−5 M) and SNP (10−8 to 10−5 M) in the presence or absence of ODQ (10−5 M).

For determination of methemoglobin levels, a 200-μl arterial blood sample was withdrawn from a femoral artery and analyzed with a Radiometer NPT7 (Copenhagen, Denmark) blood gas analyzer.

For determination of cGMP levels, PA segments from the rats were dissected and cut into rings and incubated with ODQ (10−5 M) or SNP (10−4 M) or the combination of ODQ and SNP. After 1 h incubation, the PA rings were frozen in liquid nitrogen. The cGMP measurements were performed using an enzyme-linked immunosorbent assay kit from Cayman Chemical (Ann Arbor, MI) according to the manufacturer's protocol and as previously described (1, 6).

Drugs.

ODQ (Cayman Chemical), ACh, BK, fasudil, PGE1, angiotensin II, phenylephrine, histamine, albuterol, sodium meclofenamate, SNP, NaNO2, GTN, DEA/NO, SNGT, l-NAME, Nω-nitro-l-arginine methyl ester hydrochloride, and HOE-140 (Sigma-Aldrich) were dissolved in 0.9% NaCl, and apocynin was dissolved in dimethyl sulfoxide (DMSO). BAY 60-2770 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) (42). U-46619 (Cayman Chemical) was dissolved in 95% ethyl alcohol and diluted in 0.9% NaCl solution. The TRPV4 agonist GSK-1016790A (Smith Klein Glaxo) and the TRPV4 antagonist GSK-2193874 (Smith Klein Glaxo) were prepared in 1% DMSO/20% Captisol (sulfobutyl ether-β-cyclodextrin; CyDex, Lenexa, KS) and saline. The doses of GSK-1016790A and GSK-2193874 were determined from previous studies and pilot data (37, 47).

The hemodynamic data are expressed as means ± SE and were analyzed using paired and group t-tests and ANOVA for repeated measures. The criteria used for statistical significance was P < 0.05.

RESULTS

Effect of ODQ on NO donor responses.

The effect of ODQ on responses to SNP, NaNO2, GTN, DEA/NO, and SNGT was investigated in the intact chest rat, and the data are summarized in Fig. 1. The intravenous injections of the five NO donors produced dose-related decreases in pulmonary and systemic arterial pressure and no change or small increases in cardiac output (Fig. 1). After administration of ODQ in a dose of 5 mg/kg iv, the decreases in pulmonary and systemic arterial pressures in response to the NO donors were significantly attenuated, whereas changes in cardiac output were not altered (Fig. 1).

Fig. 1.

Fig. 1.

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Bar graphs showing the effects of iv injection of sodium nitroprusside (SNP), diethylamine NONOate (DEA/NO), S-nitrosoglutathione, sodium nitrite, and glyceryl trinitrate on pulmonary and systemic arterial pressure and cardiac output before and after treatment with 1H-[1,2,4]-oxadizaolo[4,3-]quinoxaline-1-one (ODQ, 5 mg/kg iv). n, No. of experiments. *P < 0.05 compared with control, paired analysis.

Effect of ODQ on responses to NO-independent vasodilator agents.

The effects of ODQ on responses to vasodilator agents that act by NO-independent mechanisms were investigated, and the intravenous injections of albuterol, isoproterenol, histamine, fasudil, and PGE1 produced decreases in systemic and pulmonary arterial pressures with small effects on cardiac output, and responses to these agents were not altered after administration of 5 mg/kg iv ODQ (Fig. 2A).

Fig. 2.

Fig. 2.

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A: bar graphs showing responses to the iv injections of the vasodilators albuterol, isoproterenol, fasudil, histamine, and PGE1. B: iv injections of the vasoconstrictors U-46619, phenylephrine, and angiotensin II and acute ventilatory hypoxia (10% O2-90% N2 gas) on pulmonary and systemic arterial pressure and cardiac output before and after treatment with ODQ (5 mg/kg iv). n, No. of experiments. *P < 0.05 compared with control, paired analysis.

Effect of ODQ on responses to vasoconstrictor agents.

The intravenous injections of angiotensin II and phenylephrine produced dose-related increases in pulmonary and systemic arterial pressures, and exposure to acute ventilatory hypoxia (10% O2) produced an increase in pulmonary arterial pressure and a decrease in systemic arterial pressure. The hemodynamic responses to phenylephrine and ventilatory hypoxia were not altered after administration of 5 mg/kg iv ODQ, whereas increases in pulmonary arterial pressure in response to angiotensin II were significantly increased (Fig. 2B). The enhanced response to angiotensin II in the pulmonary vascular bed after ODQ treatment was not reduced by treatment with the NADPH oxidase inhibitor apocyanin (300 μmol/kg iv) (Fig. 3). The intravenous injections of U-46619 increased pulmonary arterial pressure in a dose-related manner, had no consistent effect on systemic arterial pressure, and decreased cardiac output (Fig. 2B). The increases in pulmonary arterial pressure in response to intravenous injections of U-46619 were not changed after administration of ODQ (Fig. 2B).

Fig. 3.

Fig. 3.

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Bar graphs showing the effects of iv injection of angiotensin II (0.3 μg/kg) on pulmonary and systemic arterial pressure and cardiac output in control settings, after treatment with apocynin (300 μmol/kg), after ODQ (5 mg/kg) treatment, and after combined treatment with ODQ (5 mg/kg) and apocynin. n, No. of experiments. *P < 0.05.

Effect of ODQ on responses to BAY 60-2770.

The effect of ODQ on the response to a subthreshold or threshold dose of the sGC activator BAY 60-2770 was investigated, and the intravenous injection of BAY 60-2770 in a dose of 1 μg/kg had little or no effect on pulmonary or systemic arterial pressure (0 ± 0 and −4 ± 2 mmHg, respectively) in the control period but produced significant decreases in these pressures after treatment with 5 mg/kg iv ODQ (Fig. 4).

Fig. 4.

Fig. 4.

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Bar graphs showing the effects of iv injection of a subthreshold dose (1 mg/kg) of the soluble guanylate cyclase (sGC) activator BAY 60-2770 on pulmonary and systemic arterial pressure and cardiac output before and after treatment with ODQ (5 mg/kg iv). *P < 0.05 compared with control.

Effect of ODQ on responses to ACh and BK.

The effect of ODQ on responses to ACh and BK was investigated in the rat, and these data are summarized in Figs. 5 and 6. The intravenous injections of ACh in doses of 0.3–3 μg/kg produced dose-related decreases in systemic arterial pressure and at higher doses studied decreases in pulmonary arterial pressure with no change or small increases in cardiac output that were probably baroreceptor reflex mediated (Fig. 5). The increase in cardiac output is most likely caused by the decrease in afterload and a reflex increase in heart rate. The decreases in pulmonary and systemic arterial pressures in response to intravenous injections of ACh were not altered after administration of 5 mg/kg iv ODQ (Fig. 5).

Fig. 5.

Fig. 5.

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Bar graphs showing the effects of iv injections of ACh (0.3 to 3 μg/kg) on pulmonary and systemic arterial pressure and cardiac output before and after treatment with ODQ (5 mg/kg iv). n, No. of experiments.

Fig. 6.

Fig. 6.

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Bar graphs showing the effects of iv injections of bradykinin on pulmonary and systemic arterial pressure and cardiac output before and after treatment with ODQ (5 mg/kg iv) (A), HOE-140 (0.1 mg/kg iv) (B), sodium meclofenomate (5 mg/kg iv) (C), l-NAME (50 mg/kg iv) (D), and phentolamine (0.5 mg/kg iv) (E). n, No. of experiments. *P < 0.05.

The intravenous injections of BK produced dose-related decreases in systemic arterial pressure and dose-related increases in pulmonary arterial pressure with small changes in cardiac output (Fig. 6A). The decreases in systemic arterial pressure and increases in pulmonary arterial pressure in response to intravenous injections of BK were not altered after administration of 5 mg/kg iv ODQ (Fig. 6A). The decreases in systemic arterial pressure and increases in pulmonary arterial pressure in response to BK were significantly reduced after administration of the BK B2-receptor antagonist HOE-140 in a dose of 0.1 mg/kg iv (Fig. 6B) but were not altered after administration of the cyclooxygenase inhibitor sodium meclofenamate in a dose of 5 mg/kg iv or l-NAME in a dose of 50 mg/kg iv (Fig. 6, C and D). The increases in pulmonary arterial pressure in response to BK were significantly reduced after administration of the α-receptor antagonist phentolamine in a dose of 0.5 mg/kg iv that significantly reduced the pressor response intravenous injection of the α1-receptor agonist phenylephrine (Fig. 6E).

Effect of ODQ on SNP and ACh responses in isolated PA.

The effects of ODQ on vasorelaxant responses to ACh and SNP were investigated in large (∼300-μm)-, medium (∼200-μm)-, and small (∼100-μm)-diameter PA rings precontracted with U-46619. The endothelium-dependent relaxation response to ACh was greater in large arteries (87%) compared with medium (51%) and small (27%) arteries. The incubation with ODQ (10−5 M) had no significant effect on endothelium-dependent relaxation responses to ACh in large, medium, and small PA segments (Fig. 7, A, B, and C). Interestingly, endothelium-independent relaxation responses to SNP were independent of vessel size and were attenuated by ODQ. The incubation with 10−5 M ODQ shifted the dose-response curve for SNP to the right in large artery rings, indicating a reduction in sensitivity to the NO donor. In medium- and small-sized arteries, ODQ also reduced the maximal response to SNP (Fig. 7, A, B, and C).

Fig. 7.

Fig. 7.

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Graphs showing SNP- and ACh-induced vessel relaxation after precontraction with U-46619 on large (∼300 μm diameter) (A), medium (∼200 μm diameter) (B), and small (∼100 μm diameter) (C) pulmonary artery rings before and after treatment with 10 μM ODQ. n, No. of experiments.

The incubation with ODQ had no significant effect on baseline tension in the isolated PA ring segments.

Effect of l-NAME and ODQ on responses to ACh in the rat.

The effects of l-NAME alone and in combination with ODQ on responses to ACh in the rat were investigated, and these data are summarized in Fig. 7. The administration of l-NAME in a dose of 50 mg/kg iv significantly increased pulmonary and systemic arterial pressures and decreased cardiac output (Table 1). However, l-NAME treatment did not attenuate decreases in pulmonary and systemic arterial pressures in response to intravenous injections of ACh, and deceases in pulmonary and systemic arterial pressures were enhanced, an effect that was probably related to the increase in tone (Fig. 8A). Previous studies have shown that decreases in pulmonary arterial pressures in response to vasodilator agents are dependent on the level of baseline vasoconstrictor tone (23, 24). The effect of l-NAME in combination with ODQ on the response to ACh was investigated, and, after intravenous administration of 50 mg/kg l-NAME and 5 mg/kg iv ODQ, decreases in pulmonary and systemic arterial pressures in response to intravenous injections of ACh were not reduced (Fig. 8B).

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 105 ± 4 19 ± 1 113 ± 7
l-NAME (50 mg/kg iv) (n = 10) 146 ± 4* 31 ± 2* 73 ± 7*
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Values are means ± SE; n, no. of eperiments. l-NAME, NG-nitro-l-arginine methyl ester.

*

P < 0.05 compared with corresponding control.

Fig. 8.

Fig. 8.

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Bar graphs showing the effect of ACh (0.3–3 μg/kg iv) on pulmonary and systemic arterial pressure before and after treatment with l-NAME (50 mg/kg iv) (A) and after treatment with l-NAME (50 mg/kg iv) and a combination of l-NAME (50 mg/kg iv) and ODQ (5 mg/kg iv) (B). n, No. of experiments. *P < 0.05.

Effect of ODQ on baseline hemodynamic values and methemoglobin levels.

The intravenous injection of ODQ in a dose of 5 mg/kg iv had no significant effect on baseline systemic and pulmonary arterial pressures or cardiac output and on blood methemoglobin levels (Table 2).

Table 2.

Effect of ODQ on hemodynamic parameters and methemeoglobin formation

Systemic Arterial Pressure, mmHg Pulmonary Arterial Pressure, mmHg Cardiac Output, ml/min %MetHb, g/dl
Control 102 ± 4 19 ± 1 115 ± 3 0.94 ± 0.3
ODQ (5 mg/kg iv) (n = 10) 112 ± 5 19 ± 1 108 ± 4 2.18 ± 0.5
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Values are means ± SE; n, no. of eperiments.

MetHb, methemoglobin.

Effect of ODQ and SNP on cGMP levels.

Colorimetric determination of cGMP content was performed in the lysate from PA. The cGMP level in arteries incubated with SNP (10−4 M) was significantly higher compared with values in control arteries or arteries incubated with ODQ (10−5 M) or arteries incubated with ODQ and SNP (Fig. 9).

Fig. 9.

Fig. 9.

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Bar graph showing cGMP levels in pulmonary arterial segments under control conditions, following treatment with 10 μM ODQ, 100 μM SNP, and combined treatment with 100 μM SNP and 10 μM ODQ. n, No. of experiments. *P < 0.05 compared with control.

Role of TRPV4 channels.

It has been suggested that vasodilator responses to ACh are mediated in part by hyperpolarization, and it has been hypothesized that TRPV4 channels are involved in mediating the response to ACh in resistance arteries from the mouse mesentery (40, 46, 51). To investigate the role of TRPV4 channels in mediating decreases in pulmonary and systemic arterial pressures in response to ACh in the rat, the effect of the TRPV4 antagonist GSK-2193874 was evaluated. The intravenous injection of the TRPV4 agonist GSK-1016790A (6 μg/kg), SNP (1 μg/kg), and ACh (1 μg/kg) produced decreases in pulmonary and systemic arterial pressures (Fig. 10). The decreases in pulmonary and systemic arterial pressures in response to intravenous injections of the TRPV4 agonist GSK-1016790A were significantly attenuated by the intravenous administration of the TRPV4 antagonist GSK-2193874 (300 μg/kg), whereas decreases in pulmonary and systemic arterial pressures in response to intravenous injections of ACh were not changed (Fig. 10).

Fig. 10.

Fig. 10.

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Bar graphs showing the effects of the transient receptor vanilloid type 4 (TRPV4) antagonist GSK-2192874 (300 μg/kg iv) on changes in pulmonary and systemic arterial pressure and cardiac output in response to iv injections of SNP (1 μg/kg), the TRPV4 agonist GSK-1016790A (6 μg/kg), and ACh (1 μg/kg). n, No. of experiments. *P < 0.05 compared with control.

DISCUSSION

ODQ is an agent that inhibits the activation of sGC by NO and provides a useful probe for studying the role of sGC activation in mediating vasorelaxant responses to NO (9, 11, 39, 52). ODQ has been used in many biochemical and isolated blood vessel studies but has only been used in few cardiovascular studies in the intact animal (9, 11, 14, 35, 39, 52). ODQ has recently been shown to inhibit vasodilator responses to the NO donor SNP in the rat (35). In the present study, ODQ was used to investigate responses to five different NO donors, endothelium-dependent and -independent vasodilator agents, and vasoconstrictor agents in the intact chest rat. The results of these studies show that decreases in pulmonary and systemic arterial pressures in response to intravenous injections of the NO donors DEA/NO, NaNO2, GTN, SNGT, and SNP are attenuated by ODQ in a dose that had no significant effect on responses to vasodilator agents that act by NO-independent mechanisms. These data provide support for the concept that decreases in pulmonary and systemic pressures in response to the NO donors are mediated by the activation of sGC in resistance vessel elements in the pulmonary and systemic vascular beds in the intact chest rat and that ODQ is useful for investigating the role of sGC activation in mediating NO-dependent responses in vivo in the rat. In addition to not altering responses to vasodilator agents that act by NO-independent mechanisms, ODQ did not inhibit responses to vasoconstrictor agents, providing support for the hypothesis that the inhibitory effect of ODQ is selective for NO-sGC-cGMP pathway-mediated responses. Although ODQ attenuated responses to the NO donors in the rat in a selective manner, decreases in pulmonary and systemic arterial pressures in response to intravenous injections of ACh were not decreased by ODQ. These data are consistent with results of studies with the NOS inhibitor l-NAME, indicating that decreases in systemic arterial pressure and regional vascular resistance in response to ACh in the rat are not attenuated, suggesting that responses are not mediated by NO (8, 12–14, 17). The results with ODQ in the pulmonary vascular bed of the intact rat are consistent with results in isolated pulmonary vessels where vasorelaxant responses to the NO donor SNP were attenuated, whereas vasorelaxant responses to ACh were not altered by ODQ in various-sized arterial rings precontracted with U-46619. These results are in agreement with studies in cGKI and eNOS knockout mice in which vasodepressor responses to SNP were attenuated in cGKI knockout mice, whereas vasodepressor responses to ACh are not altered in cGKI or eNOS knockout mice (19, 28). The results of pharmacological studies in the rat and studies in knockout mice provide support for the hypothesis that the sGC-cGMP-cGK pathway has an important role in mediating the vasodilator response to the NO donors but is not involved in mediating decreases in pulmonary and systemic arterial pressure in response to ACh in the intact mouse or rat (8, 12–14, 17, 28).

The present results are not in agreement with a number of studies in the literature, including previous studies from our own laboratory in several laboratory species (3, 8, 31). The reason for the different results with ACh is uncertain and is complicated by the large increases in baseline pulmonary and systemic arterial pressures induced by the NOS inhibitor l-NAME and by species (21, 23, 24). In this regard, when systemic arterial pressure was restored to baseline values after l-NAME treatment with an SNP infusion, the decreases in systemic arterial pressure in response to ACh were not altered by l-NAME in the rat (8). In previous studies in the rat hindlimb vascular bed, vasodilator responses to ACh were attenuated by apamin and charybdotoxin, providing support for the hypothesis that they were mediated by the opening calcium-activated K+ channels (8).

The intravenous injection of BK produced an increase in pulmonary arterial pressure and a decrease in systemic arterial pressure, and these responses were not altered by ODQ. The increase in pulmonary arterial pressure and decrease in systemic arterial pressure were attenuated by HOE-140, indicating that these opposite responses were mediated by BK B2 receptors (26). Responses to BK were not altered by sodium meclofenamate or l-NAME, and the increase in pulmonary arterial pressure was attenuated by phentolamine, suggesting a role for α-adrenergic receptors in mediating the pulmonary pressor response. The observation that responses to BK were not inhibited by sodium meclofenamate, l-NAME, or ODQ suggests that the release of cyclooxygenase products or the activation of sGC by NO was not involved in mediating or modulating responses to the peptide.

Treatment with ODQ reduced responses to NO donors without altering vasodilator responses to NO-independent agents. The inhibition of sGC by ODQ is induced by oxidation of the heme iron on the enzyme reducing the sensitivity to NO and increasing responses to sGC activator agents (35, 43, 52). The present data are consistent with previous results and show that responses to a low or subthreshold dose of the sGC activator BAY 60-2770 are markedly enhanced by ODQ, suggesting that the inactivated or oxidized enzyme is stimulated by a subthreshold dose sGC activator (30, 35). The enhanced pressor response to angiotensin II in the pulmonary vascular bed observed after treatment with ODQ was not antagonized by apocyanin, suggesting that acute responses were not dependent on NADPH oxidase activation and may involve an effect of the peptide on NO formation, which is counterregulatory and can be observed when NO-sGC-cGMP signaling is inhibited. These data suggest that the acute pressor response to angiotensin II is not modulated by the activation of NADPH oxidase (15). Although it has been reported that ODQ can oxidize other heme-containing enzymes, the observation that methemoglobin levels were not significantly increased suggests that off-target effects were minimal in the present experiments.

Treatment with ODQ had no significant effect on baseline pulmonary and systemic arterial pressures; are consistent with previous studies in the fetal lamb, ovine fetus, and rat knockout models; and are consistent with the normal systemic arterial pressure in cGKI knockout mice (5, 35, 42). A transient elevation in pulmonary and systemic arterial pressure occurred during the infusion; however, these effects were short-lived and were not present after 10–15 min. In contrast, the intravenous administration of 50 mg/kg iv l-NAME produced elevations of systemic and pulmonary arterial pressure and decreases in cardiac output that persist for several hours.

The observation that systemic arterial pressure was not different in wild-type and cGKI knockout mice is supported by the present data with ODQ in the rat (28). The lack of effect of ODQ on baseline pressure may be explained in part by the observation that ODQ had no effect on basal sGC activity but inhibited the activation of the enzyme by NO (52).

It has been reported that ACh-induced vasodilation is mediated by hyperpolarization, and it has been suggested that TRPV4 channels are involved in mediating the endothelium-dependent vasodilator response to the muscarinic agonist (10, 40, 51).

Recent studies support the hypothesis that TRPV4 activation plays an important role in mediating agonist-induced vasodilation (50). Studies in TRPV4−/− mice show that cholinergic agonist-induced vasodilator responses in isolated resistance arteries and decreases in systemic arterial pressure in response to intravenous injections of ACh were attenuated (10, 51). In contrast, it has been reported that vasodilation in response to ACh in isolated carotid arteries from TRPV4−/− mice was not attenuated compared with responses in wild-type mice (16). In addition vasorelaxant responses to ACh were not attenuated by TRPV4 antagonists in isolated PA from the rat (46). In the present study, the hypothesis that vasodilator responses to ACh are mediated by activation of TRPV4 channels was investigated in experiments with the TRPV4 antagonist GSK-2193874. The TRPV4 antagonist reduced vasodilator response to the TRPV4 agonist GSK-1016790A but had no significant effect on vasodilator responses to ACh. These data indicate that decreases in pulmonary and systemic arterial pressures in response to intravenous ACh are not mediated by the activation of TRPV4 channels in the rat and are consistent with results in isolated vessel studies (16, 46). The reason for the difference in results in regard to the role of TRPV4 in mediating cholinergic vasodilator responses is uncertain.

Recent studies have shown a cGMP-independent mechanism for activating PKG1α that involves enzyme dimerization (38). It is possible that cGMP-independent activation of PKG may be involved in mediating decreases in pulmonary and systemic arterial pressure in response to ACh in the present study in the rat.

In summary, results of the present study show that ODQ inhibits the vasodilator response to NO donors without altering responses to vasodilator agents that act by NO-independent mechanisms in the rat. ODQ did not inhibit vasoconstrictor responses or increase methemoglobin blood levels, suggesting that off target effects were minimal. ODQ did not inhibit responses to ACh and BK, suggesting the vasodilator responses are not mediated by the activation of sGC and are similar to results with ACh in cGKi knockout mice (28). Although it has been hypothesized that TRPV4 channels mediate vasodilator responses to ACh, responses to the muscarinic agonist in isolated resistance vessel were not altered by the TRPV4 antagonist (40, 46).

The present results are consistent with the hypothesis that vasodilator responses to ACh are mediated by membrane hyperpolarization. However, the activation of sGC or TRPV4 channels is not involved in mediating vasodilator responses to ACh in the pulmonary and systemic vascular beds in the rat.

Limitations

In regard to limitations of the present in vivo study in the rat, the observation that decreases in pulmonary and systemic arterial pressures in response to intravenous injection of ACh are not inhibited by ODQ or the TRPV4 antagonist GSK-2192874 do not exclude a role for sGC activation or TRPV4 channels in mediating vasodilator responses to ACh in some vascular segments or beds in the rat or in vessel segments isolated from large and small arteries. The present results do indicate, however, that the overall physiological response to ACh, which is a decrease in pulmonary and systemic arterial pressure in the rat in vivo in response to systemic (iv) injection of ACh, is not mediated in large part by activation of sGC or TRPV4 channels. It is quite possible that vasodilator responses in some vascular segments or domains in the intact rat are inhibited. However, this contribution is difficult to evaluate when overall decreases in systemic or pulmonary arterial pressures are measured.

GRANTS

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

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Author contributions: E.A.P. and P.J.K. conception and design of research; E.A.P., M.K., and S.-K.C. performed experiments; E.A.P., M.K., S.-K.C., K.M., and P.J.K. analyzed data; E.A.P., S.-K.C., K.M., B.D.N., and P.J.K. interpreted results of experiments; E.A.P., M.K., and B.D.N. prepared figures; E.A.P., B.D.N., and P.J.K. drafted manuscript; E.A.P., K.M., B.D.N., A.L.H., and P.J.K. edited and revised manuscript; E.A.P., M.K., S.-K.C., B.D.N., A.L.H., and P.J.K. approved final version of manuscript.

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