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Research Paper

Sildenafil, nifedipine and acetazolamide do not allow for blood flow through intrapulmonary arteriovenous anastomoses during exercise while breathing 100% oxygen Jonathan E. Elliott1 , Jonathan M. Friedman1 , Joel E. Futral2 , Randall D. Goodman2 and Andrew T. Lovering1 1

Experimental Physiology

2

Department of Human Physiology, University of Oregon, Eugene, OR, USA Oregon Heart & Vascular Institute, Springfield, OR, USA

New Findings r What is the central question of this study? Compared with exercise while breathing room air, blood flow through intrapulmonary arteriovenous anastomoses during exercise while breathing 100% O2 is prevented/reduced, presumably due to vasoconstriction of these vessels. We sought to investigate the effect of sildenafil, nifedipine and acetazolamide, which are known modulators of pulmonary vascular tone, on the hyperoxia-induced reduction in blood flow through intrapulmonary arteriovenous anastomoses during exercise. r What is the main finding and its importance? We show that,independently, sildenafil, nifedipine and acetazolamide do not prevent the hyperoxia-induced reduction in blood flow through intrapulmonary arteriovenous anastomoses during exercise. These data provide the first insight into the regulation of intrapulmonary arteriovenous anastomoses during exercise while breathing 100% O2 .

Blood flow through intrapulmonary arteriovenous anastomoses (IPAVAs) is known to increase in healthy humans during exercise while breathing room air, but is prevented or significantly reduced during exercise while breathing 100% O2 , potentially due to vasoconstriction of IPAVAs. Thus, pharmacological interventions that target known pathways regulating the cardiopulmonary circulation may be able to prevent the hyperoxia-induced reduction in IPAVA blood flow (Q˙ IPAVA ) during exercise. In nine healthy human subjects, we investigated the effects of sildenafil (100 mg p.o.), nifedipine (20 mg p.o.) and acetazolamide (250 mg p.o. three times a day for 3 days) on Q˙ IPAVA at rest and during cycle ergometer exercise at 50, 100, 150, 200 and 250 W, while breathing room air (normoxia) and 100% O2 (hyperoxia). Transthoracic saline contrast echocardiography and a 0–5 bubble scoring system were used to detect and assess Q˙ IPAVA qualitatively; ultrasound was used to assess the blood flow velocity oftricuspid regurgitation and the left ventricular outflow tract blood flow to calculate pulmonary artery systolic pressure (PASP) and cardiac output, respectively. Without drugs, bubble scores increased significantly to 2 at 150 W in normoxia and to 2 at 200 W in hyperoxia. Only nifedipine consistently increased cardiac output at rest and during low-intensity exercise in normoxia and hyperoxia. However, there was no detectable effect of any drug on Q˙ IPAVA ; specifically, bubble scores were the same

DOI: 10.1113/expphysiol.2014.081562

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Hyperoxic closure of intrapulmonary shunt during exercise

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during exercise in either normoxia or hyperoxia. Accordingly, the reduction in Q˙ IPAVA during exercise while breathing 100% O2 is likely not to be due to the independent pharmacological mechanisms of action associated with sildenafil, nifedipine or acetazolamide. (Received 24 June 2014; accepted after revision 19 September 2014; first published online 25 September 2014) Corresponding author A. T. Lovering: Department of Human Physiology, 1240 University of Oregon, 122c Esslinger Hall, Eugene, OR 97403-1240, USA. Email: [email protected]

Introduction Blood flow through intrapulmonary arteriovenous anastomoses (IPAVAs) is trivial or non-detectable in healthy humans at rest breathing room air (Elliott et al. 2013), yet increases during exercise and the I.V. infusion of catecholamines when breathing room air as detected by the transpulmonary passage of saline contrast (Eldridge et al. 2004; Stickland et al. 2004; Lovering et al. 2008a; Elliott et al. 2011, 2014; Bryan et al. 2012; Laurie et al. 2012). Additionally, IPAVA blood flow (Q˙ IPAVA ) is prevented or significantly reduced during exercise and the I.V. infusion of catecholamines when breathing 100% O2 (Lovering et al. 2008b; Elliott et al. 2011; Bryan et al. 2012; Laurie et al. 2012). This particular finding is controversial, in that it implies that blood flow through the pulmonary circulation can be regulated by increasing the fraction of inspired O2 (F IO2 ) and that IPAVAs appear to be regulated in an opposite manner to the conventional pulmonary circulation. Specifically, Q˙ IPAVA appears to respond to changes in the F IO2 in a similar manner to the systemic circulation, whereby blood flow is increased in response to hypoxia and reduced in response to hyperoxia. Previous work supports this supposition by demonstrating an active regulation of the pulmonary circulation as measured by an increase in perfusion heterogeneity using 15 μm microspheres, in sheep ventilated with an F IO2 = 0.4 (Melsom et al. 1999) and pigs ventilated with an F IO2 = 0.5 (Hlastala et al. 2004), both compared with room air. Additionally, the transpulmonary passage of solid microspheres has been demonstrated to decrease in a pulmonary embolization model using anaesthetized dogs ventilated with 100% O2 in comparison to room air (Niden & Aviado, 1956). There is a vital component of the fetal cardiopulmonary circulationcalled the ductus arteriosus (DA) that is regulated in a similar manner to the systemic circulation. The DA constricts and dilates in response to increases and decreases in O2 tension (P O2 ), respectively, and is therefore regulated in a similar mannerto Q˙ IPAVA . At birth, increased P O2 inhibits voltage-gated potassium channels, depolarizing DA smooth muscle cells and resulting in an increase in intracellular calcium through L-type voltage-gated calcium channels, thereby causing constriction (Tristani-Firouzi et al. 1996; Michelakis et al. 2000). Thus, L-type calcium channel blockers, such as  C 2014 The Authors. Experimental Physiology  C 2014 The Physiological Society

nifedipine, would be expected to prevent DA constriction, and they do (Michelakis et al. 2000, 2002). Conversely, nitric oxide is a potent dilator of the DA (Th´ebaud et al. 2002), so drugs such as sildenafil which augment the action of NO may allow for dilatation of the DA (Coceani et al. 1994). In utero, the tone of the DA is reflective of competing vasodilator and vasoconstrictor influences, with the primary vasodilator mechanisms being the production of endothelium-derived nitric oxide and prostaglandin E2 (Coceani & Olley, 1973; Coceani et al. 1994). If IPAVAs are remnant fetal vessels that are regulated in a similar manner to the DA, then the regulation of Q˙ IPAVA may also resemble that of the DA, although we do not suggest that these two pathways are entirely identical in either regulation or cellular composition. Specifically, the reduction in Q˙ IPAVA during exercise while breathing 100% O2 , presumably via vasoconstriction of IPAVAs, may also be regulated in part by L-type calcium channels or reductions in the bioavailability of nitric oxide. If so, pharmacological interventions known to block L-type calcium channels (nifedipine) and/or augment the action of nitric oxide (sildenafil) may reduce or prevent the potential O2 -mediated constriction of IPAVAs and thereby allow for the transpulmonary passage of saline contrast during exercise while breathing 100% O2 . Regardless of the mechanism of action, the constriction of vascular smooth muscle is dependent on an increase in intracellular calcium, and acetazolamide has also been shown to prevent intracellular calcium from increasing in pulmonary vascular smooth muscle cells by a non-L-type voltage-gated calcium channel mechanism (Shimoda et al. 2007). Thus, acetazolamide may also prevent the hyperoxia-mediated reduction in Q˙ IPAVA during exercise if a non-L-type voltage-gated calcium channel is responsible for regulating Q˙ IPAVA in hyperoxic conditions. Accordingly, the purpose of this study was to investigate the independent effects of sildenafil, nifedipine and acetazolamide on the regulation of Q˙ IPAVA during exercise while breathing 100% O2 . To this end, we studied nine healthy human subjects at rest and during submaximal exercise while breathing room air and 100% O2 , at baseline without any pharmacological intervention, with a single dose of sildenafil (100 mg P.O.), with a single dose of

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nifedipine (20 mg P.O.) and after acetazolamide (250 mg P.O., three times a day for 3 days). We hypothesized that if the hyperoxia-mediated reduction in Q˙ IPAVA was regulated primarily by L-type calcium channels then oral nifedepine would allow for Q˙ IPAVA during exercise while breathing 100% O2 . Additionally, we hypothesized that if the hyperoxia-mediated reduction in Q˙ IPAVA was regulated primarily through nitric oxide, then oral sildenafil would allow for Q˙ IPAVA during exercise while breathing 100% O2 . Lastly, we hypothesized that if the hyperoxia-mediated reduction in Q˙ IPAVA was regulated primarily by non-L-type calcium channels then oral acetazolamide would allow for Q˙ IPAVA during exercise while breathing 100% O2 . Methods The University of Oregon Office for Protection of Human Subjects approved this project, and all subjects provided verbal and written informed consent prior to participation. All studies were performed in accordance with the Declaration of Helsinki. Subjects

Nine subjects (three female) volunteered to participate in this study, all of whomwere healthy, young non-smokers without a history of cardiopulmonary or respiratory disease. According to the American Thoracic Society and European Respiratory Society standards (ATS/ERS), all subjects demonstrated normal pulmonary function, lung volumes and capacities, and diffusion capacity for carbon monoxide, evaluated as before (Duke et al. 2014). Pulmonary function was assessed using computerized spirometry (MedGraphics Ultima CardiO2 , St Paul, MN, USA) and involved forced and slow vital capacity manoeuvres (Miller et al. 2005). Lung volumes and capacities were determined using whole-body plethysmography (MedGraphics Elite Plethysmograph; Wanger et al. 2005). Lung diffusion capacity for carbon monoxide (DLCO ) was determined by the single-breath, breath-hold method (Knudson et al. 1987; Macintyre et al. 2005) using the Jones and Meade method for timing (Jones & Meade, 1961). All subjects underwent a comprehensive echocardiographic screening procedure (Philips iE33, Amsterdam, The Netherlands) performed by a registered diagnostic cardiac sonographer in both adult and paediatric echocardiography with 25 years of experience. The purpose of this screening procedure was to confirm that subjects were without cardiac abnormalities, including a patent foramen ovale. To this end, subjects were rigorously screened using transthoracic saline contrast echocardiography as described previously

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(Lovering & Goodman, 2012; Elliott et al. 2013), and none had a patent foramen ovale.

Protocol

For each visit, subjects performed two bouts of cycle ergometer exercise (Lode Excalibur, Groningen, The Netherlands), separated by a 30 min rest period, at 50, 100, 150, 200 and 250 W for 3 min each, breathing room air and 100% O2 . All subjects completed the aforementioned exercise protocol on four separate visits separated by >5 days, as follows; (i) without pharmacologicalintervention (i.e. baseline); (ii) with a single dose of 100 mg P.O. sildenafil;(iii) with a single dose of 20 mg P.O. nifedipine; and (iv) after 3 days of 250 mg P.O. acetazolamide every 8 h (i.e. three times a day). The order of pharmacological interventionsand the order of exercise bouts while breathing room air and 100% O2 were randomized. The mechanisms of action for each of these drugs have been well characterized and have previously been demonstrated to modulate pulmonary vascular tone in response to hypoxia and/or hyperoxia at the doses used in this study. An NO-mediated relaxing mechanism has been demonstrated to be present in lamb DA (Coceani et al. 1994). Sildenafil (100 mg P.O.) augments the action of nitric oxide and, by way of potentiation of the nitric oxide–cGMP pathway, causes vasodilatation. This dose has been demonstrated to prevent hypoxic pulmonary vasoconstriction (Zhao et al. 2001) and, therefore, to be protective against altitude-induced pulmonary hypertension (Ricart et al. 2005; Richalet et al. 2005). Nifedipine is a dihydropyridine that selectively blocks L-type calcium channels (Stojilkovi´c et al. 1992), which are critical to the control of vascular tone and have been demonstrated to be integral in facilitating the hyperoxia-mediated constriction of the DA (Tristani-Firouzi et al. 1996; Michelakis et al. 2000). Additionally, and also via blockade of L-type calcium channels, nifedipine has been shown to prevent hypoxic pulmonary vascoconstriction at the same dosage (20 mg P.O.) as the present study (Simonneau et al. 1981; Oelz et al. 1989; Bartsch et al. 1991). Acetazolamide is known to reduce and/or prevent increases in pulmonary artery pressure in acute hypoxia (Swenson, 2006). The prevention of hypoxic pulmonary vasoconstriction via acetazolamide has been shown not be a result of intracellular acidification or changes in membrane potential and is independent of carbonic anhydrase inhibition (Shimoda et al. 2007). Accordingly, acetazolamide prevents increases in intracellular calcium via a non-L-type calcium channel mechanism. Importantly, the dosage used in the present study (250 mg P.O. every 8 h) has also been shown  C 2014 The Authors. Experimental Physiology  C 2014 The Physiological Society

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Hyperoxic closure of intrapulmonary shunt during exercise

to prevent hypoxic pulmonary vasoconstriction in acute hypoxia (Teppema et al. 2007). Detection of blood flow through intrapulmonary arteriovenous anastomoses

Transthoracic saline contrast echocardiography was used to detect Q˙ IPAVA as before (Laurie et al. 2012; Elliott et al. 2014). In summary, agitated saline contrast was produced by combining 3 ml room air with 1 ml of normal saline and agitating for 15 sprior to injection. Each agitated saline contrast injection was visualized in the apical, four-chamber view, with subjects on the cycle ergometer in the forward leaning aerobar position, and recorded at >30 frames s−1 for 20 cardiac cycles after the appearance of saline contrast in the right ventricle. The single frame within the 20 cardiac cycle recording with the greatest density and spatial distribution of contrast was assessed qualitatively using a previously published scoring system (Lovering et al. 2008b) similar to others (Barzilai et al. 1991), as follows: 0 = no bubbles; 1 = 1–3 bubbles; 2 = 4–12 bubbles; 3 = >12 bubbles appearing as a bolus; 4 = >12 bubbles heterogeneously filling the left ventricle; and 5 = >12 bubbles homogeneously filling the left ventricle. Representative echocardiographic videos demonstrating the persistence of saline contrast in healthy humans at rest breathing room air and 100% O2 can be found online (Elliott et al. 2013). Respiratory variables

Breath-by-breath metabolic data were collected (MedGraphics, CardiO2 ) and presented as the ‘mid 5 of 7’ (i.e. the moving average of five breaths excluding the low and high). In this way, continuous measures of pulmonary O2 uptake (V˙ O2 ), CO2 output, minute ventilation (V˙ E ) and associated variables, including end-tidal P O2 and P CO2 values, were collected. During exercise while breathing 100% O2 , only ventilatory data were recorded because the metabolic rate cannot be measured, owing to a lack of N2 . Gas mixtures with known O2 and CO2 concentrations were used to calibrate the gas analyser before every exercise test. Cardiac output and pulmonary artery systolic pressure

Cardiac output was estimated using echocardiography and determined at the level of the left ventricular outflow tract (LVOT). Previously, during the screening visit, each subject’s LVOT diameter had been determined and recorded. This value was used to estimate the cross-sectional area of the outflow tract. Pulsed-wave Doppler ultrasound was used in all studies to determine the LVOT velocity time integral (LVOTVTI ) of blood flow  C 2014 The Authors. Experimental Physiology  C 2014 The Physiological Society

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through the outflow tract. Measures of the LVOTVTI- were recorded in triplicate, and the average was multiplied by the previously determined cross-sectional area of the LVOT. The product of the LVOTVTI and LVOT cross-sectional area equals the stroke volume, which, when multiplied by heart rate, provides an estimate of cardiac output. Pulmonary artery systolic pressure was determined as before (Laurie et al. 2010; Elliott et al. 2014) by measuring the peak velocity (v) of the tricuspid regurgitation jet and estimating right atrial pressure (PRA ) based on the collapsibility of the inferior vena cava, and applying these to the modified Bernoulli equation, as follows: 4v2 + PRA = PASP (Yock & Popp, 1984; Currie et al. 1985; Himelman et al. 1989; Rudski et al. 2010). Total pulmonary resistance (TPR) was calculated (Kovacs et al. 2012) as described by PASP divided by cardiac output, and has been previously published by our group (Norris et al. 2014).

Statistics

Statistical analyses were done using GraphPad Prism (version 5.0d, La Jolla, CA, USA), and all comparisons were determined a priori. Comparisons of data from rest to exercise, within an F IO2 and pharmacological intervention, were done using a one-way ANOVA with Tukey’s multiple comparison posthoc test. Comparisons of data at rest and during exercise within an F IO2 , with and without pharmacological intervention were done using Student’s paired t test with α adjusted for the number of comparisons made. The aforementioned analyses were done for non-parametric data (i.e. bubble scores) using a Kruskal–Wallis one-way ANOVA with Dunns multiple comparison posthoc test and a Wilcoxon signed rank test.

Results Subject characterization and pulmonary function

Anthropometric and pulmonary function data, lung volumes/capacities and the diffusion capacity for carbon monoxide are presented in Table 1. All subjects demonstrated normal pulmonary function, and all variables were 90% predicted. Haemodynamic and ventilatory effects of pharmacological interventions Breathing room air. The haemodynamic and ventilatory response to exercise was not different during exercise at baseline and with all pharmacological interventions, with the exceptions noted below (Table 2). With nifedipine,

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Transpulmonary passage of saline contrast

Table 1. Anthropometric and pulmonary function data

Parameter Age (years) Height (cm) Weight (kg) FVC (l) FEV1 (l) FEV1 /FVC (%) FEF25–75 SVC (l) TLC (l) DLCO (ml min−1 mmHg−1 ) DLCO /VA (ml min−1 mmHg−1 )

Absolute 24.5 162.5 72.6 5.1 4.1 81.1 3.9 5.2 6.9 39.1 5.9

± ± ± ± ± ± ± ± ± ± ±

5.5 39.6 10.1 0.1 0.6 6.6 1.0 0.7 1.1 7.2 0.9

Percentage of predicted value — — — 94.7 ± 94.1 ± 97 ± 95.6 ± 102.3 ± 105.1 ± 123.6 ± 122.5 ±

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7.0 3.9 7.9 10 9.4 6.6 21.5 23.9

Values are means ± SD. Abbreviations: DLCO , uncorrected diffusion capacity for carbon monoxide; DLCO /VA , DLCO per unit alveolar volume; FEF25–75 , forced expiratory flow from 25 to 75% of FVC; FEV1 , forced expiratory volume in 1 s; FVC, forced vital capacity; SVC, slow vital capacity; and TLC, total lung capacity determined via whole-body plethysmography.

cardiac output was significantly elevated at rest and during exercise at 50 W, concomitant with an increased heart rate at rest and throughout exercise compared with baseline. Although no difference was observed at rest with sildenafil, similar to nifedipine, heart rate was increased during exercise compared with baseline. Despite the peripheral circulatory haemodynamic changes associated with sildenafil and nifedipine, no significant ventilatory changes were observed. In contrast, no significant haemodynamic changes were observed with acetazolamide, but minute ventilation was increased at rest and during exercise. Breathing 100% O2 . The haemodynamic and ventilatory responses to exercise were not different during exercise at baseline and with all pharmacological interventions, with the exceptions noted below (Table 3). Similar to exercise while breathing room air, nifedipine resulted in an increased cardiac output at rest and during exercise at 50 and 100 W. Likewise, compared with baseline, heart rate was increased at rest and during exercise with nifedipine. This increase in cardiac output was also accompanied by an increase in PASP, yet total pulmonary resistance was not different compared with baseline. Unlike when breathing room air, with sildenafil no significant haemodynamic changes were measured. However, the augmented ventilation at rest and during exercise with acetazolamide was also present when breathing 100% O2 , and no significant haemodynamic changes were observed.

Breathing room air. During exercise at baseline and with

all pharmacological interventions, the transpulmonary passage of saline contrast progressively increased and was significantly elevated during exercise at 150, 200 and 250 W (Fig. 1A). Bubble scores during exercise with sildenafil (Fig. 1C)and acetazolamide (Fig. 1G) were similar compared with baseline. Although not statistically different from baseline, mode bubble scores were the highest during exercise with nifedipine from rest to 200 W (Fig. 1E). Furthermore, consistent with the elevated cardiac output at rest and at 50 W with nifedipine, there was a trend (P = 0.08) for bubble scores to be higher compared with baseline. Breathing 100% O2 . As expected, the transpulmonary passage of saline contrast was trivial during exercise at baseline and increased significantly from rest while breathing 100% O2 during exercise only at 200 W (Fig. 1B). During exercise with sildenafil (Fig. 1D), nifedipine (Fig. 1F) and acetazolamide (Fig. 1H), the transpulmonary passage of saline contrast was not different compared with baseline and was significantly increased only at 200 and 250 W.

Discussion The present study investigated the pharmacological influence of sildenafil, nifedipine and acetazolamideon the regulation of Q˙ IPAVA during exercise in healthy humans breathing 100% O2 . The primary finding of the present study was that the general absence of Q˙ IPAVA during exercise while breathing 100% O2 was not changed following administration of a single dose of sildenafil (100 mg P.O.), a single dose of nifedipine (20 mg P.O.), or after acetazolamide (250 mg P.O., three times a day for 3 days). We have previously shown in healthy humans hat Q˙ IPAVA is significantly reduced or absent during exercise while breathing 100% O2 (Lovering et al. 2008b; Elliott et al. 2011). The present study found similar results during exercise while breathing 100% O2 with pharmacological interventions that are known modulators of pulmonary vascular tone. All of these pharmacological interventions act through mechanisms with the potential to prevent or reduce the O2 -induced constriction of the cardiopulmonary circulation (Barnes & Liu, 1995) and the DA (Coceani et al. 1994; Michelakis et al. 2002) and could therefore potentially either constrict IPAVAs and/or redistribute blood flow such that Q˙ IPAVA is reduced. Nifedipine and acetazolamide have the potential to prevent or limit pulmonary vascular smooth muscle contraction, while sildenafil augments the potential relaxation of pulmonary vascular smooth muscle. The increase and  C 2014 The Authors. Experimental Physiology  C 2014 The Physiological Society

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Table 2. Haemodynamic and ventilatory data during exercise while breathing room air Parameter Q˙ T (l min−1 )

V˙ O2 (l min−1 )

SV (ml)

HR (beats min−1 )

S pO2 (%)

PASP (mmHg)

TPR (mmHg l−1 min−1 )

V˙ E (l min−1 )

Condition Baseline Sildenafil Nifedipine Acetazolamide Baseline Sildenafil Nifedipine Acetazolamide Baseline Sildenafil Nifedipine Acetazolamide Baseline Sildenafil Nifedipine Acetazolamide Baseline Sildenafil Nifedipine Acetazolamide Baseline Sildenafil Nifedipine Acetazolamide Baseline Sildenafil Nifedipine Acetazolamide Baseline Sildenafil Nifedipine Acetazolamide

Rest 4.3 4.5 5.6 4.6 0.4 0.4 0.5 0.4 69.0 60.0 68.0 69.0 65.0 75.0 86.0 71.0 98.4 98.5 98.7 98.7 27.9 27.4 31.5 29.5 6.7 6.5 5.7 6.9 11.4 11.9 13.1 14.7

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.9 1.2 1.3† 1.2 0.1 0.1 0.1 0.1 13.0 16.0† 20.0 18.0 18.0 15.0 18.0† 15.0 1.7 1.8 0.9 1.2 6.7 6.0 6.2 7.2 2.3 2.1 1.0 2.0 2.2 1.4 3.7 1.6†

50 W 7.3 7.7 8.2 7.9 1.3 1.2 1.2 1.2 76.0 76.0 78.0 76.0 99.0 103.0 108.0 105.0 98.1 98.1 99.0 98.8 40.0 38.4 40.2 37.8 5.7 5.3 5.0 5.0 25.7 25.3 27.5 32.7

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.5∗ 1.9 1.9† 1.5 0.2 0.1 0.1 0.1 22.0 21.0 22.0 15.0 15.0∗ 18.0∗, † 22.0∗ 13.0∗ 2.2 1.9 0.9 1.3 5.7 7.9 9.2 9.1 1.4 1.9 1.1 0.8∗ 4.4∗ 2.7∗ 3.4∗ 4.7∗, †

100 W 9.8 9.9 10.2 9.7 1.7 1.6 1.7 1.6 85.0 78.0 83.0 85.0 117.0 128.0 126.0 118.0 97.5 97.9 98.6 98.8 43.2 46.4 44.6 40.7 4.5 4.8 4.5 4.2 35.9 38.3 37.2 46.9

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.1∗ 2.6∗ 2.9∗ 1.6∗ 0.2 1.2 0.2 0.1 25.0 22.0 27.0 20.0 15.0∗ 23.0∗, † 25.0∗ 20.0∗ 2.7 2.2 0.8 1.3 8.0∗ 13.1∗ 11.7 6.6 1.0∗ 1.0 1.2 0.5∗ 5.2∗ 4.2∗ 6.4∗ 7.2∗ ,†

150 W 10.8 11.7 10.5 11.9 2.3 2.1 2.2 2.1 82.0 84.0 74.0 86.0 137.0 145.0 146.0 140.0 97.3 98.3 98.8 98.4 49.4 51.3 51.6 45.6 4.7 4.9 5.1 3.9 49.8 52.7 54.6 63.8

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.4∗ 3.5∗ 2.9∗ 2.2∗ 0.3 0.3 0.2 0.1 27.0 31.0 25.0 18.0 19.0∗ 22.0∗ ,† 23.0∗ ,† 20.0∗ 2.1 1.5 0.8 1.5 10.1∗ 8.4∗ 13.2∗ 8.8∗ 1.1 2.2 1.4 0.6∗ 5.2∗ 5.0∗ 11.7∗ 9.4∗ ,†

200 W 12.5 13.3 13.0 15.3 2.8 2.6 2.6 2.6 83.0 85.0 82.0 96.0 153.0 159.0 162.0 160.0 96.7 98.0 98.7 97.5 55.2 56.3 58.0 48.9 4.5 4.6 4.8 3.3 66.5 71.2 72.6 81.1

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.2∗ 4.2∗ 3.6∗ 2.8∗ 0.3 0.4 0.4 0.3 22.0 30.0 27.0 17.0 19.0∗ 18.0∗ ,† 18.0∗ ,† 15.0∗ 2.4 1.4 0.8 1.6 11.3∗ 8.8∗ 10.0∗ 8.8∗ 0.9∗ 1.6 1.5 1.0∗ 8.7∗ 10.5∗ 12.7∗ 11.4∗ ,†

250 W 15.0 15.1 15.1 16.5 3.4 3.1 3.2 3.1 90.0 90.0 89.0 97.0 165.0 171.0 172.0 170.0 94.5 96.1 97.0 96.3 56.6 58.4 60.3 57.1 3.9 4.2 4.2 3.5 88.2 92.1 89.8 105.2

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.4∗ 4.1∗ 4.2∗ 1.5∗ 0.2 0.5 0.4 0.1 22.0 28.0 27.0 8.0 16.0∗ 15.0∗ 18.0∗ ,† 13.0∗ 2.4 1.4 1.3 1.5 9.2∗ 8.8∗ 12.5∗ 4.4∗ 1.3∗ 1.6 1.2 0.4∗ 14.8∗ 14.2∗ 18.6∗ 17.9∗ ,†

Values are means ± SD. Abbreviations: HR, heart rate; PASP, pulmonary artery systolic pressure; Q˙ T , cardiac output; S pO2 , peripheral arterial O2 saturation; SV, stroke volume; TPR, total pulmonary resistance; V˙ E , minute ventilation; and V˙ O2 , O2 consumption. ∗ Significantly different from rest within a given pharmacological condition. P < 0.05 (one-way ANOVA with Tukey post-hoc test-description in stats section of Methods). † Significantly different from baseline without pharmacological intervention for that specified workload. P < 0.017 (Student’s paired t-test, alpha adjusted, see Methods section).

decrease in Q˙ IPAVA during exercise while breathing room air and 100% O2 may at least partly be a result of IPAVA vasodilatation and vasoconstriction, respectively. Accordingly, by preventing the potential vasoconstriction of IPAVAs or augmenting the vasodilatation of IPAVAs, these pharmacological interventions could have allowed Q˙ IPAVA to occur during exercise while breathing 100% O2 , as occurs during exercise while breathing room air. However, we found that this was not the case, and potential explanations for this are discussed below.

Sildenafil

Endothelium-derived or exogenous NO promotes smooth muscle relaxation by activation of soluble guanylate cyclase and the subsequent production of cyclic guanosine monophosphate (cGMP) and, via activation of protein kinase G, decreased intracellular calcium levels. The type 5 phosphodiesterase (PD-5) isoform, located primarily in the penis and lungs, rapidly degrades cGMP and thereby limits the NO-mediated relaxation. Sildenafil citrate inhibits PD-5, prolonging the survival of cGMP, and  C 2014 The Authors. Experimental Physiology  C 2014 The Physiological Society

thus promotes vascular relaxation by allowing the effects of NO to persist for longer, rather than increasing the bioavailability of NO. Previous work has demonstrated a correlation between increases in Q˙ IPAVA and increases in cardiac output when breathing room air (Stickland et al. 2004; Bryan et al. 2012; Laurie et al. 2012; Elliott et al. 2014) that may be due in part to the associated increase in sheer stress and increased release of NO. If so, the possibility that Q˙ IPAVA is NO mediated should be interpreted in light of recent work demonstrating that the I.V. infusion of nitroglycerin in healthy humans at rest did not increase the transpulmonary passage of saline contrast (Lozo et al. 2014). However, the I.V. infusion of nitroglycerin did not increase cardiac output, and therefore, it remains possible that IPAVAs were open but the transpulmonary passage of saline contrast did not occur due to potential factors such as mean pulmonary transit time being too long for in vivo microbubbles to survive. Furthermore, prolongation of the action of cGMP by sildenafil ingestion did not significantly change the transpulmonary passage of saline contrast during exercise while breathing room air. Nevertheless, it remains possible that during exercise

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Table 3. Haemodynamic and ventilatory data during exercise while breathing 100% O2 Parameter Q˙ T (l min−1 )

SV (ml)

HR (beats min−1 )

S pO2 (%)

PASP (mmHg)

TPR (mmHg l−1 min−1 )

V˙ E (l min−1 )

Condition Baseline Sildenafil Nifedipine Acetazolamide Baseline Sildenafil Nifedipine Acetazolamide Baseline Sildenafil Nifedipine Acetazolamide Baseline Sildenafil Nifedipine Acetazolamide Baseline Sildenafil Nifedipine Acetazolamide Baseline Sildenafil Nifedipine Acetazolamide Baseline Sildenafil Nifedipine Acetazolamide

Rest 4.9 4.4 6.3 4.4 63.0 59.0 66.0 60.0 81.0 75.0 99.0 75.0 99.3 99.4 99.3 99.3 27.6 27.6 31.0 26.2 5.8 6.6 5.1 6.2 13.4 12.8 13.5 17.2

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.7 1.3 1.4† 1.4 14.0 15.0 19.0 16.0 19.0 16.0 19.0† 17.0 0.7 0.5 0.3 0.6 4.8 7.5 7.2 4.8 1.5 1.8 1.6 1.1 2.8 2.8 2.4 3.3†

50 W 6.9 7.2 8.8 7.7 73.0 79.0 78.0 78.0 97.0 93.0 116.0 87.0 99.3 99.2 99.5 99.6 35.0 35.7 45.6 31.5 5.9 5.1 5.2 4.3 23.7 24.6 13.1 30.8

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.9 1.6 1.5† 2.0 20.0 17.0 18.0 18.0 27.0∗ 22.0∗ 25.0∗ ,† 35.0∗ 0.7 0.4 0.5 0.4 5.8 8.4 15.3† 2.6 4.0 1.2 1.9 1.0 4.4∗ 2.1∗ 3.7∗ 3.6∗

100 W 9.0 8.5 11.1 10.2 79.0 79.0 87.0 87.0 118.0 108.0 132.0 120.0 98.9 99.7 99.3 99.5 42.5 41.6 48.9 39.7 5.1 5.0 4.5 3.8 35.7 36.7 27.5 44.8

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.5∗ 2.2∗ 2.1∗ ,† 2.2∗ 31.0 19.0 24.0 23.0 16.0∗ 25.0∗ 22.0∗, † 17.0∗ 0.5 0.4 0.4 0.4 4.6∗ 10.3∗ 11.1∗ 2.2∗ 1.5 1.1 1.1 0.8 3.6∗ 3.0∗ 3.4∗ 5.1∗, †

150 W 10.9 11.7 11.7 12.4 83.0 94.0 82.0 92.0 136.0 129.0 145.0 138.0 99.2 99.5 99.5 99.5 43.2 49.4 55.1 44.5 4.0 4.5 5.1 3.7 47.8 50.3 37.2 59.2

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.2∗ 2.5∗ 2.7∗ 2.7∗ 26.0 24.0∗ 23.0 24.0∗ 21.0∗ 23.0∗ 22.0∗ ,† 19.0∗ 0.7 0.4 0.4 0.4 8.8∗ 10.4∗ 10.8∗ 8.1∗ 0.6 1.7∗ 1.9 1.0 3.7∗ 6.8∗ 6.4∗ 10.7∗ ,†

200 W 13.1 12.8 13.2 14.2 88.0 90.0 84.0 93.0 152.0 145.0 160.0 154.0 99.1 99.6 99.5 99.7 51.8 51.1 55.4 49.0 4.2 4.2 4.4 3.6 61.6 63.2 54.6 75.8

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.8∗ 3.1∗ 3.0∗ 2.7∗ 25.0 23.0∗ 23.0 21.0∗ 19.0∗ 22.0∗ 19.0∗ ,† 18.0∗ 1.1 0.4 0.5 0.5 7.7∗ 8.0∗ 7.5∗ 7.4∗ 1.2 1.3∗ 1.0 0.8 4.3∗ 8.9∗ 11.7∗ 11.9∗ ,†

250 W 13.7 15.0 15.5 17.5 84.0 96.0 93.0 106.0 166.0 156.0 168.0 166.0 99.0 99.5 99.5 99.2 55.4 58.6 61.9 56.7 4.1 4.0 4.6 3.3 79.3 81.1 72.6 91.0

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.2∗ 3.4∗ ,† 5.0∗ 2.3∗ 20.0 20.0∗ 29.0 14.0∗ 16.0∗ 18.0∗ 18.0∗ 16.0∗ 0.8 0.5 0.5 0.7 11.1∗ 6.3∗ 11.9∗ 9.0∗ 0.8 0.6∗ 1.7 0.7 11.7∗ 15.3∗ 12.7∗ 22.0∗

Values are means ± SD. See Table 2 for definitions of terms and acronyms. ∗ Significantly different from rest within a given pharmacological condition. P < 0.05 (one-way ANOVA with Tukey post-hoc test-description in stats section of Methods). † Significantly different from baseline without pharmacological intervention for that specified workload. P < 0.017 (Student’s paired t-test, alpha adjusted, see Methods section).

while breathing room air the IPAVAs are open without augmenting the vasodilatory effects of NO with sildenafil. During exercise while breathing 100% O2 , sildenafil likewise did not result in an increase in the transpulmonary passage of saline contrast. The explanation for this may be that there is an increase in the production of reactive oxygen species, known to occur during O2 breathing, which may limit the bioavailability of NO (Rubanyi & Vanhoutte, 1986). Thus, the vasodilatory effects of NO during exercise while breathing 100% O2 may be diminished, similar to hyperoxia attenuating endothelium-mediated vasodilatation in peripheral vascular beds (Yamazaki, 2007). However, these data suggest that even when augmenting the action of NO that is present, no significant increase in Q˙ IPAVA is detected.

of the DA (Tristani-Firouzi et al. 1996; Michelakis et al. 2000), and nifedipine prevents hypoxic pulmonary vascoconstriction at the same dosage as used in the present study (Simonneau et al. 1981; Oelz et al. 1989; Bartsch et al. 1991). Accordingly, considering the pervasive influence of L-type calcium channels in regulating vascular tone and their documented role in mediating both O2 -induced constriction of the DA and hypoxic pulmonary vasoconstriction, it seemed plausible that IPAVAs may be regulated in a similar manner. Nevertheless, no effect was observed during exercise while breathing 100% O2 with nifedipine, suggesting that the O2 -induced constriction of IPAVAs is not primarily mediated via L-type calcium channels. Acetazolamide

Nifedipine

Nifedipine is a dihydropyridine that blocks the activation of voltage-gated (L-type) calcium channels (Stojilkovi´c et al. 1992), which when open allow for the influx of calcium down the concentration gradient (2 mM extracellular versus 100 nM intracellular calcium). The L-type calcium channels are critical to the control of vascular tone and have been demonstrated to be integral to facilitation of the O2 -induced constriction

Acetazolamide is a carbonic anhydrase inhibitor, most commonly known for its utility in preventing acute mountain sickness (Forwand et al. 1968) owing to its respiratory-stimulating properties secondary to renal excretion of bicarbonate (i.e. metabolic acidosis) and carbonic anhydrase inhibition in central chemoreceptors (Maren, 1967; Swenson, 1998). However, a secondary property of acetazolamide is a reduction/prevention of increases in pulmonary artery pressure in acute hypoxia (Swenson, 2006). The prevention of hypoxic  C 2014 The Authors. Experimental Physiology  C 2014 The Physiological Society

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Hyperoxic closure of intrapulmonary shunt during exercise

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Figure 1. Transpulmonary passage of saline contrast (i.e. bubble score) at rest and during exercise at 50, 100, 150, 200 and 250 W while breathing room air and a fractional inspired O2 = 1.0 at baseline (A and B ), with 100 mg sildenafil P.O. (C and D ), with 20 mg nifedipine P.O. (E and F ) and after 250 mg acetazolamide P.O. three times a day (G and H) The dashed horizontal lines between bubble scores of 2 and 3 delineate bubble scores corresponding to a trivial degree of left-sided contrast (score = 0–2) and bubble scores corresponding to significant left-sided contrast (scores = 3–5). Mode bubble scores are indicated by a continuous horizontal line for each workload and for all conditions. ∗ Significantly increased compared with baseline without pharmacological intervention. Kruskal-Wallis one-way ANOVA with Dunns multiple comparison post-test. P < 0.05.  C 2014 The Authors. Experimental Physiology  C 2014 The Physiological Society

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pulmonary vasoconstriction via acetazolamide has been shown not to be a result of intracellular acidification or changes in membrane potential and is independent of carbonic anhydrase inhibition (Shimoda et al. 2007). Accordingly, acetazolamide prevents increases in intracellular calcium via a non-L-type calcium channel mechanism. Importantly, the dosage used in the present study has also been shown to prevent hypoxic pulmonary vasoconstriction in acute hypoxia (Teppema et al. 2007). Given that we found no effect with nifedipine, which works through an L-type calcium channel-dependent mechanism, we used acetazolamide for its effect on non-L-type calcium channels. Despite the efficacy of acetazolamide in preventing hypoxic pulmonary vasoconstriction, these data suggest that the O2 -induced constriction of IPAVAs is not mediated by a similar mechanism. Active regulation of IPAVAs or active redistribution of pulmonary blood flow?

Whether or not Q˙ IPAVA during exercise while breathing room air, and the lack thereof during exercise while breathing 100% O2 , represents an active vasodilatation and vasoconstriction of IPAVAs, respectively, remains unknown. Indeed, the present study in combination with that that by Lozo et al.(2014) suggest that Q˙ IPAVA may not be regulated actively, at least by these specific independent cellular pathways. Another possible explanation for these findings that does not require active regulation of IPAVAs is an active redistribution of pulmonary blood flow during exercise while breathing 100% O2 . In this theory, when breathing 100% O2 the pulmonary blood flow is actively redistributed in comparison to when breathing room air, such that blood now flows through areas of the lung without IPAVAs. Thus, Q˙ IPAVA may be a passive response to the active regulation of pulmonary blood flow. As previously mentioned, there is an increase in pulmonary perfusion heterogeneity measured using radioactive microspheres when animals are ventilated with 40–50% O2 (Melsom et al. 1999; Hlastala et al. 2004), so this theory is not without precedent. Furthermore, recent work by Bates et al.(2014), quantifying hypoxia-induced Q˙ IPAVA at rest and during exercise in humans, lends support to this theory and suggests that the resistance distal to IPAVAs may provide an important mediator of Q˙ IPAVA . However, carefully designed studies that compare Q˙ IPAVA in subjects with and without blockade of hypoxic vasoconstriction will be required to determine the effect, if any, of hypoxic vasoconstrictionon Q˙ IPAVA . Limitations

Blood flow through IPAVAs was detected using transthoracic saline contrast echocardiography, and

Exp Physiol 99.12 (2014) pp 1636–1647

previous work has discussed the associated limitations (Eldridge et al. 2004; Laurie et al. 2010; Elliott et al. 2011; Bryan et al. 2012; Norris et al. 2014). However, particularly pertinent to the present study is addressing the potential concern that Q˙ IPAVA is not detected during exercise while breathing a 100% O2 due to O2 breathing altering the partial pressure environment of in vivo microbubbles and destabilizing them. In this way, Q˙ IPAVA may be occurring during exercise while breathing 100% O2 , yet the failure to observe significant left-sided contrast reflects the failure for saline contrast to survive long enough to reach the left heart. Previous work by our group (Elliott et al. 2011) has demonstrated this theory to not be valid experimentally,and therefore, saline contrast created from room air is appropriate to usein order to detect Q˙ IPAVA in subjects breathing 100% O2 . Along these lines, it should be noted that following a peripheral injection of agitated saline contrast, right-sided contrast persists for >20 cardiac cycles, even in healthy humans at rest while breathing room air or 100% O2 (see Elliott et al. 2013 for representative videos). Accordingly, it is not possible to explain why we observe an increase in left-sided contrast during exercise (breathing room air) simply by a reduction in mean pulmonary transit time. This also does not explain why left-sided contrast is reduced or eliminated during exercise while breathing 100% O2 , which occurs within 2 min of breathing this F IO2 (Lovering et al. 2008b). Therefore, although this doesnot preclude the possibility that an excessive production of reactive oxygen species may be involved in the regulation of Q˙ IPAVA , this finding occurs before the time necessary for hyperoxia-induced CNS toxicity or other deleterious effects of breathing 100% O2 (Dean et al. 2004). Moreover, previous work from our laboratory has shown that breathing an F IO2 = 1.0 for >30 min has no effect on Q˙ IPAVA during exercise (Elliott et al. 2011). The previously published bubble scoring system (Lovering et al. 2008b; Laurie et al. 2010, 2012; Elliott et al. 2011, 2013, 2014) used in the present study is a qualitative approach intended to characterize the degree of left-sided contrast, but is not without precedent (Barzilai et al. 1991). This bubble scoring system does not represent a means to quantify Q˙ IPAVA . To validate the reproducibility of our bubble scoring system, previous work from our laboratory has demonstrated very good agreement with bubble scores assigned by one of two expert sonographers (combined >40 years experience) and a cardiologist blinded to the conditions. In one of these studies, a cardiologist reviewed 57 saline contrast recordings and reported 100% agreement on whether there was or was not left heart contrast, and 53 (93%) were assigned the same score (Elliott et al. 2013). As described in the Methods section, cardiac output was estimated using echocardiography and determined at the level of the LVOT. This method of determining cardiac  C 2014 The Authors. Experimental Physiology  C 2014 The Physiological Society

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Hyperoxic closure of intrapulmonary shunt during exercise

output is not without limitations, the majority of which depend largely on the experience of thesonographer and their ability to align the ultrasound waveform accurately during exercise. Compared with rest, making this measurement during exercise presents several additional challenges, namely the increase in heart rate and tidal volume/respiratory rate and the extra upper body movement. Although nothing can be done about the increased heart rate and ventilation, excessive upper body movement can be minimized by exercising in the forward leaning aerobar position, as previously described (Lovering & Goodman, 2012). Nevertheless, in healthy humans nearly all of these limitations will result in an underestimation of stroke volume. For example, the predicted cardiac output (4.71V˙ O2 + 5.6 = Q˙ T , determined via direct Fick, from Barker et al. 1999) using our directly measured V˙ O2 at baseline is as follows: 7.5 (rest), 11.7(50 W), 13.6(100 W), 16.4(150 W), 18.8(200 W) and 21.6 l min–1 (250 W). Indeed, when comparing our measured values of cardiac output (Table 2) with what is predicted based on the measured V˙ O2 (when breathing room air), it can be seen that our measured values are lower than what would be expected. Nonetheless, the change in cardiac output from stage to stage while breathing room air, as calculated above from the predicted values, would be 4, 2, 3, 2 and 3 l min–1 , and the changes in cardiac output that we measured were 3, 2.5, 2, 2 and 3 l min–1 , so the absolute changes are very similar. It should also be emphasized that all echocardiographic measurements in the present study were made by highly experienced sonographers (J.E.F. and R.D.G), and therefore, any error in the measurement would be expected to be consistent across all workloads.

Conclusions

As expected, at baseline without pharmacological intervention the transpulmonary passage of saline contrast occurred during exercise at 150 W while breathing room air and was prevented or reduced during exercise while breathing 100% O2 . These findings were not significantly altered following the administration of sildenafil (100 mg P.O.), nifedipine (20 mg P.O.) and acetazolamide (250 mg P.O., three times a day for 3 days). What we can learn from this study is that none of these pharmacological interventions resulted in Q˙ IPAVA during exercise while breathing 100% O2 , despite these drugs being well-known modulators of pulmonary vascular tone (Barnes & Liu, 1995). However, rather than IPAVAs being regulated in a similar manner to the DA or fetal cardiopulmonary circulation, their regulation may be similar to the systemic circulation, which is also known to dilate in response to low O2 tensions and constrict in response to high O2 tensions. Accordingly, and also like the systemic circulation, it  C 2014 The Authors. Experimental Physiology  C 2014 The Physiological Society

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remains possible that IPAVAs are not mediated by these mechanisms and/or there are redundant mechanisms facilitating O2 -induced IPAVA constriction, similar to those redundant mechanisms regulating peripheral blood flow (Casey & Joyner, 2011). Finally, it remains possible that Q˙ IPAVA occurs passively in response to the active regulation of overall pulmonary blood flow.

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Additional information Competing interests None declared. Author contributions Conception and design of the experiments: J.E.E. and A.T.L. Collection, analysis and interpretation of data: J.E.E., J.M.F., J.E.F., R.D.G. and A.T.L. Drafting the article or revising it critically for important intellectual content: J.E.E., J.M.F. and A.T.L. All authors approved the final version for publication. Funding This work was supported by the Eugene & Clarissa Evonuk Memorial Graduate Fellowship in Environmental, Cardiovascular or Stress Physiology and an American Heart Association Pre-doctoral Fellowship.

Sildenafil, nifedipine and acetazolamide do not allow for blood flow through intrapulmonary arteriovenous anastomoses during exercise while breathing 100% oxygen.

Blood flow through intrapulmonary arteriovenous anastomoses (IPAVAs) is known to increase in healthy humans during exercise while breathing room air, ...
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