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Am J Physiol Heart Circ Physiol 288: H1139-H1146, 2005. First published October 28, 2004; doi:10.1152/ajpheart.01139.2003
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Nitric oxide contributes to right coronary vasodilation during systemic hypoxia

Rodolfo R. Martinez, Srinath Setty, Pu Zong, Johnathan D. Tune, and H. Fred Downey

Department of Integrative Physiology, University of North Texas Health Science Center, Fort Worth, Texas

Submitted 1 December 2003 ; accepted in final form 20 October 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
As arterial partial pressure of O2 (PaO2) is reduced during systemic hypoxia, right ventricular (RV) work and myocardial O2 consumption (MO2) increase. Mechanisms responsible for maintaining RV O2 demand/supply balance during hypoxia have not been delineated. To address this problem, right coronary (RC) blood flow and RV O2 extraction were measured in nine conscious, instrumented dogs exposed to normobaric hypoxia. Catheters were implanted in the right ventricle for measuring pressure, in the ascending aorta for measuring arterial pressure and for sampling arterial blood, and in an RC vein. A flow transducer was placed around the RC artery. After recovery from surgery, dogs were exposed to hypoxia in a chamber ventilated with N2, and blood samples and hemodynamic data were collected as chamber O2 was reduced progressively to ~8%. After control measurements were made, the chamber was opened and the dog was allowed to recover. N{omega}-nitro-L-arginine (L-NNA) was then administered (35 mg/kg, via RV catheter) to inhibit nitric oxide (NO) production, and the hypoxia protocol was repeated. RC blood flow increased during hypoxia due to coronary vasodilation, because RC conductance increased from 0.65 ± 0.05 to 1.32 ± 0.12 ml·min–1·100 g–1. L-NNA blunted the hypoxia-induced increase in RC conductance. RV O2 extraction remained constant at 64 ± 4% as PaO2 was decreased, but after L-NNA, extraction increased to 70 ± 3% during normoxia and then to 78 ± 3% during hypoxia. RV MO2 increased during hypoxia, but after L-NNA, MO2 was lower at any respective PaO2. The relationship between heart rate times RV systolic pressure (rate-pressure product) and RV MO2 was not altered by L-NNA. To account for L-NNA-mediated decreases in RV MO2, O2 demand/supply variables were plotted as functions of MO2. Slope of the conductance-MO2 relationship was depressed by L-NNA (P = 0.03), whereas the slope of the extraction-MO2 relationship increased (P = 0.003). In summary, increases in RV MO2 during hypoxia are met normally by increasing RC blood flow. When NO synthesis is blocked, the large RV O2 extraction reserve is mobilized to maintain RV O2 demand/supply balance. We conclude that NO contributes to RC vasodilation during systemic hypoxia.

right ventricular function; right ventricular oxygen balance; myocardial oxygen consumption

REDUCING THE FRACTION OF O2 in inspired air lowers the O2 content of arterial blood and also activates reflexes that increase myocardial O2 consumption (MO2). Vasodilation of the coronary arteries in response to hypoxia is an important physiological mechanism that helps to match increases in coronary blood flow and delivery of O2 to increases in myocardial O2 demand. Hypoxia has been reported to cause left coronary vasodilation by enhancing the opening of ATP sensitive-K+ (KATP) channels (11, 29, 41) as well as by stimulating the production of adenosine (3, 29, 41), prostacyclin (30), and nitric oxide (NO) (8, 31). Although numerous investigations have focused on delineating the mechanisms responsible for hypoxic left coronary vasodilation, little attention has been directed toward defining the function of these mechanisms in the right ventricle.

NO has been demonstrated to exert effects on right ventricular (RV) O2 supply and demand under various experimental conditions. Several studies have shown that NO has a tonic vasodilatory influence on resting RC blood flow (13, 34, 37, 43); however, the effects of NO on resting canine left coronary blood flow at rest are modest (40). In anesthetized dogs, Setty et al. (34) demonstrated that NO increases RC blood flow while reducing RV MO2 in response to intracoronary norepinephrine-infusion and during RC hypoperfusion (35). In conscious dogs, Zong et al. (43) found that NO regulates RC blood flow during pulmonary hypertension. It is, therefore, conceivable that NO contributes to hypoxia-induced vasodilation in the RC circulation and thus may be an important factor in maintaining RV myocardial O2 demand/supply balance during acute hypoxia.

With regard to hypoxia, an earlier study by Audibert et al. (2) found that inhibition of NO synthesis decreased flow to the right ventricle in conscious dogs at 2- and 4-h exposure to hypoxia. Systemic inhibition of NO synthesis elevates arterial pressure and reflexly decreases heart rate (2, 43), an important determinant of RV myocardial O2 demand; however, Audibert et al. (2) did not measure RV MO2, so they could not determine to what extent the observed decreases in RC blood flow were the result of reduced RV O2 demand. Clearly, it is important to normalize RC flow and conductance data for changes in RV MO2. Thus experiments were designed so that RV MO2 as well as variables that determine RV O2 supply, i.e., RC blood flow and RV O2 extraction could be measured at rest and during acute, graded hypoxia. In this manner, the relative contributions of increases in RC flow and RV O2 extraction in meeting elevated RV O2 requirements could be defined. This investigation then tested the hypothesis that NO contributes to hypoxia-induced RC vasodilation. We also investigated whether or not RV O2 demand/supply balance could be sustained if NO synthesis was inhibited.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Surgical preparation. This investigation was approved by the Institutional Animal Care and Use Committee and was conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication No. 85–23, Revised 1996). Nine adult mongrel dogs of either sex, free of clinically evident disease, were used to examine the role of NO in the RC vasodilatory responses to graded hypoxia. Four additional dogs were used to examine the repeatability of responses to systemic hypoxia in absence of treatment to block NO synthesis.

Dogs were preanesthetized with acepromazine (0.03 mg/kg sc). Thirty minutes after administration of this preanesthetic, anesthesia was induced by thiopental sodium (5 mg/kg iv). After endotracheal intubation, a surgical plane of anesthesia was maintained by mechanical ventilation with 1–3% isoflurane gas and supplemental O2. With the use of sterile technique, a right lateral thoracotomy was performed in the fourth right intercostal space. A 17-gauge pressure-monitoring catheter was placed in the ascending aorta to measure aortic blood pressure and to obtain systemic arterial blood samples. A 1- to 2-cm section of the RC artery was dissected free for implanting a 2-mm diameter, perivascular flow transducer (Transonic Systems). A coronary venous catheter prepared from Micro-Renathane tubing was inserted into a superficial vein draining the RV myocardium, as described previously (5). For measuring pressure, a coextruded polyurethane catheter was inserted into the right ventricle through a stab wound.

At the conclusion of instrumentation, the catheters and the flow transducer cable were tunneled subcutaneously and exteriorized between the scapulae. The chest was closed, and the pneumothorax was evacuated through a chest tube. The incision was infiltrated with 2.5% bupivacaine, and buprenorphine (Buprenex; 0.3 mg im) was administered to minimize postoperative pain. Clavamox (6.25 mg/lb.) and aspirin (81 mg) were administered by mouth twice a day for 10 days after surgery. A nylon jacket (Alice King, Chatham Medical Arts, Hawthorne, CA) was placed on the animals to protect the catheters and the flow transducer cable. An elastomeric balloon pump (Access Technologies) was connected to the RC venous catheter, so that heparinized saline (10 U/ml) could be continuously infused at 5 ml/h.

Pressure and flow measurements. An external pressure transducer (model 1290C; Hewlett-Packard) was positioned at midheart level and connected to the aortic catheter to measure systemic arterial pressure. A high-fidelity 3-Fr micro-tip catheter pressure transducer (Millar Instruments) was inserted through the RV catheter at the time of the experiment to measure RV pressure. This pressure transducer was introduced into the polyurethane RV catheter through a hemostatic control valve (Tuohy-Borst; Mallinckrodt Medical), which allowed infusion of N{omega}-nitro-L-arginine (L-NNA) while maintaining a fluid-tight seal around the catheter. Mean aortic pressure, RV pressure, heart rate, as well as, phasic and mean RC blood flows were continuously measured throughout the experimental protocol. RC conductance was calculated from RC blood flow per mean aortic pressure.

Blood sampling. Arterial and coronary venous blood samples were collected simultaneously in heparinized syringes that were immediately sealed and placed on ice. These samples were analyzed for pH, PCO2, PO2, hematocrit, and O2 content with an Instrumentation Laboratories automatic blood gas analyzer (Synthesis 30) and CO-oximeter (model 682). RC MO2 (ml O2·min–1·100 g–1) was calculated by multiplying RC coronary blood flow per gram of perfused tissue by the RC coronary arteriovenous difference in O2 content. RV O2 extraction (%) was calculated by dividing the RC coronary arteriovenous difference in O2 content by the arterial O2 content and multiplying by 100.

Experimental protocols. Hemodynamic variables were measured while the dogs stood quietly in a sling suspended within a Plexiglas chamber. These variables were recorded with a Hewlett-Packard 7758A recorder and analyzed with an EMKA Technologies IOX 1.7.0 data acquisition system. Basal normoxic arterial and RC venous blood samples were obtained. The chamber was then closed, and N2 was introduced to progressively reduce the O2 within the chamber. The percent O2 within the chamber was monitored by using an Alpha Omega Instruments O2 analyzer (Series 2000). Arterial and RC venous blood samples were collected when hemodynamic variables were stable at each of four degrees of hypoxia, with FIO2 = ~15%, ~12% ~10%, and ~8% . This protocol required ~20 min. After blood samples and data were collected at the most severe of hypoxia (FIO2 = ~8%), the chamber was opened and the dogs were allowed to breath room air.

L-NNA (35 mg/kg) was then administered over 10 min via the RV catheter to block NO synthesis. This dose of L-NNA was previously found to reduce vasodilation to acetylcholine by >60% in conscious dogs (36). The experimental protocol described above was repeated.

To examine the repeatability of responses to graded systemic hypoxia, four dogs were subjected to a second exposure to systemic hypoxia without L-NNA treatment. In these experiments, RC venous blood was not collected, because these animals were either not instrumented with a RC venous catheter or the catheter was not functional.

After all experiments were completed, the animals were euthanized with an overdose of pentobarbital sodium, and the RC artery perfusion territory was dyed with Evans blue. The dyed tissue was weighed, so RC blood flow could be expressed per 100 grams of perfused myocardium.

Statistical analyses. All values are presented as means ± SE. RV hemodynamic and arterial blood gas variables before and after L-NNA treatment were evaluated by a two-way ANOVA (Factor A = L-NNA treatment; Factor B = degree of hypoxia). When significance (P < 0.05) was found with ANOVA, a Student-Newman-Keuls multiple comparison test was performed. Analysis of covariance (ANCOVA) was used to compute and compare the slopes of response variables (RC blood flow, coronary conductance, and RV O2 delivery) plotted versus MO2. If the slopes of the regression lines were not significantly different, ANCOVA was used to test for differences in elevation. Statistical computations were performed by using SigmaStat 2000 and GBSTAT version 9.0 software.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Repeatability of responses to graded hypoxia. Figure 1 illustrates RC blood flow measured during the second graded hypoxia plotted against flow measured during the first graded hypoxia. The relationship was highly linear (P < 0.001), with the slope = 1.05 ± 0.08 and r2 = 0.91. These data demonstrate that the RC flow response to the second exposure to hypoxia was not altered by prior exposure. With regard to RV mechanical function, heart rate, RV systolic pressure, and RV dP/dtmax also responded similarly to repeated hypoxia.



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  		Fig. 1. Right coronary (RC) blood flow measured during repeated, graded hypoxia. Flow during the second exposure to hypoxia is plotted as a function of flow during first exposure. Flow response to the second exposure to hypoxia was not affected by the prior exposure.

 
Effects of graded hypoxia before blocking NO synthesis. RV function, hemodynamic, blood gas, and metabolic data collected during normoxia and during graded hypoxia are presented in Table 1. Each hypoxia data point was assigned to one of three arbitrarily chosen hypoxia ranges (69–50, 49–30, or 29–10 mmHg) according to its corresponding arterial PO2 value.


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  		Table 1. Blood gas, hemodynamic, and metabolic variables during normoxia and graded hypoxia before and during NOS inhibition

 
As the chamber atmosphere became progressively more hypoxic, arterial PO2 and O2 content fell to 25 ± 1 mmHg and 8.9 ml O2/100 ml blood, respectively. Hyperventilation caused arterial PCO2 to fall and arterial pH to rise. Arterial lactate was unchanged during all degrees of hypoxia, except at the most severe condition when a significant increased was observed.

Hypoxia caused significant increases in aortic pressure (24%), RV systolic pressure (26%), heart rate (24%), and RV dP/dtmax (60%). RC blood flow increased 135%, and RC conductance increased 103%.

RV MO2 increased 35% to 9.2 ± 1.2 ml O2·min–1·100 g–1 at the most severe hypoxia, whereas RC venous PO2 fell to 15 ± 1 mmHg. RV O2 extraction was 64 ± 4% during normoxia and did not change during hypoxia. RC venous lactate increased only at the most severe hypoxia.

Effects of graded hypoxia after blocking NO synthesis. In the normoxic condition, NO synthesis inhibition had no significant effects on arterial PO2, PCO2, pH, or hematocrit, but arterial O2 content was slightly lower (Table 1). The dogs were exposed to a similar hypoxic stress after L-NNA, because the fall in arterial PO2 and O2 content were similar to that observed in the control state. During hypoxia, arterial PCO2 declined and pH increased. Arterial lactate was significantly elevated at the most severe hypoxia.

L-NNA produced peripheral vasoconstriction, elevated mean aortic pressure, and caused a reflex decrease in heart rate compared with the untreated control condition (Table 1). Hypoxia after L-NNA caused significant increases in aortic pressure (19%), RV systolic pressure (26%), heart rate (44%), and RV dP/dtmax (46%). RC blood flow increased 188%, and RC conductance increased 123%.

RV MO2 increased 82% to 8.2 ± 1.4 ml O2·min–1·100 g–1 at the most severe hypoxia, whereas RC venous PO2 fell to 11 ± 0 mmHg. RV O2 extraction increased significantly, from 70 ± 3% during normoxia to 78 ± 3% during hypoxia. RC venous lactate concentration increased only at the most severe hypoxia.

Effects of blocking NO synthesis on RV O2 demand/supply balance during hypoxia. Figure 2, A and B, demonstrates that after NO synthesis inhibition, RC blood flow and conductance were reduced at any degree of hypoxia compared with the untreated group. After L-NNA, RC conductance increased less steeply as arterial PO2 fell during hypoxia (interaction = 0.02; Table 1), as illustrated in Fig. 2B.



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  		Fig. 2. Average results of coronary blood flow (A), conductance (B), myocardial O2 consumption (MO2) (C), and RC venous PO2 (D) at rest and during graded hypoxia (as indexed by decreasing arterial PO2) before and after treatment with N{omega}-nitro-L-arginine (L-NNA; L-NNA). With reductions in arterial PO2 there was an increase in RC blood flow, conductance, and right ventricular (RV) MO2. RC venous PO2 decreased with reductions in arterial PO2. Inhibition of nitric oxide (NO) synthesis of RC blood flow, conductance, MO2, and RC venous PO2 were lower than respective control values. *P < 0.05 vs. normoxia, same treatment; {dagger}P < 0.05 vs. control, same degree of hypoxia; {ddagger}P < 0.05 vs. previous degree of hypoxia, same treatment.

 
Decreases in heart rate and RV contractility after L-NNA were associated with a decrease in RV MO2 during normoxia and at any given degree of hypoxia compared with the untreated condition, as shown in Fig. 2C. However, the difference between treated and untreated RV MO2 was less during severe hypoxia than during normoxia.

After L-NNA, normoxic RC venous PO2 decreased by 11% from the untreated normoxia condition and was significantly decreased at any given degree of hypoxia, compared with corresponding untreated values (Fig. 2D). Correspondingly, RV O2 extraction was greater during normoxia and during hypoxia compared with the untreated condition, although RV MO2 was lower. Arterial lactate concentration increased during severe hypoxia after NO synthesis inhibition, and although RC venous lactate increased, the arterial-venous lactate difference remained positive at 0.31 mmol/l.

To normalize for changes in RV metabolism due to both graded hypoxia and to NO synthesis blockade, RC blood flow and conductance were plotted as functions of RV MO2 (Fig. 3). Figure 3A shows that after NO synthesis inhibition, there was a tendency for RC blood flow to be less responsive to increases in RV MO2 compared with the untreated control condition (ANCOVA, P = 0.09 slope). RC conductance is plotted as a function of RV MO2 in Fig. 3B to normalize for L-NNA-induced increases in arterial pressure. Figure 3B shows that the slope of the RC conductance-MO2 relationship was significantly depressed after L-NNA treatment (ANCOVA, P = 0.03 slope). These conductance data demonstrate that NO contributes to the RC vasodilatory response induced by hypoxia.



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  		Fig. 3. RC blood flow (A) and RC conductance (B) as functions of RV MO2 during hypoxia before and after treatment with L-NNA. After L-NNA, the RC blood flow response tended to be less sensitive to increases in RV MO2 [analysis of covariance (ANCOVA), P = 0.09 slope]. There was a significant decrease in the slope of the RC conductance vs. MO2 relationship (ANCOVA, P = 0.03 slope), indicating that NO contributes to RC vasodilation during hypoxia.

 
The relationship between RV myocardial O2 delivery (RC blood flow times arterial O2 content) and MO2 is plotted in Fig. 4. For both the untreated and L-NNA conditions, RV O2 delivery increased during hypoxia as MO2 increased. NO synthesis inhibition significantly decreased the slope of this relationship (ANCOVA, P = 0.003 slope), and at any comparable MO2, RV O2 delivery was reduced. Increased RV O2 extraction after L-NNA caused a downward shift in the relationship between O2 delivery and MO2 toward the line reflecting 100% O2 extraction, i.e., O2 consumed equal to O2 delivered.



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  		Fig. 4. Relationship between RV O2 delivery and MO2 before and after treatment with L-NNA. L-NNA significantly decreased the slope of this relationship, indicating that NO contributes to hypoxia-induced coronary vasodilation in the RV. The point at which the O2 delivery equals the O2 consumed (i.e., 100% extraction) is represented by the dash-dot-dash line.

 
In animals instrumented as described in Surgical procedures, additional experiments were conducted in which glibenclamide to block KATP channels (1 mg/kg iv) and 8-phenyltheophylline to block adenosine receptors (3 mg/kg iv) were administered along with L-NNA. Data were collected after L-NNA alone in two of these experiments and after triple blockade in three experiments. Figure 5 illustrates the relationship between RC blood flow and arterial PO2 for these triple-block experiments, as well as RC blood flow data from the nine single-block experiments. In the triple-block experiments, L-NNA alone reduced RC flow to values similar to those observed in the single-block experiments. Additional blockade of KATP channels and adenosine receptors caused no further reduction in RC blood flow at rest or during graded hypoxia. Thus data from these additional experiments indicate that neither adenosine nor KATP channels contribute to normoxic RC vascular tone or to hypoxia-induced RC vasodilation after inhibition of NO synthesis.



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  		Fig. 5. Average results for RC coronary blood flow at rest and during graded hypoxia (as indexed by decreasing arterial PO2) under control conditions, after treatment with L-NNA (single blockade) and after combined treatment with L-NNA + 8-phenyltheophylline (8-PT) + glibenclamide (triple blockade). Triple blockade was administered to 3 dogs, whose control data are illustrated. RC flow was measured in 2 of these dogs after L-NNA, and those data are illustrated. For comparison, data from the 9 dogs treated only with L-NNA are also illustrated. The RC blood flow response to hypoxia after triple blockade closely matched the response observed after L-NNA alone.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study is the first to examine mechanisms of RV O2 demand/supply balance during systemic hypoxia. In this condition, RV O2 consumption increases due to elevated heart rate and RV systolic pressure and dP/dtmax, resulting from increased sympathetic activation (14, 16, 18). Hypoxia-induced pulmonary vasoconstriction also contributes to the rise in RV systolic pressure (6, 43). In the face of this increased RV O2 consumption, arterial O2 content falls during hypoxia. To meet increased RV O2 requirements during systemic hypoxia, RC blood flow must increase. In the present study, this was accomplished by increased RC conductance and elevated arterial blood pressure. NO contributed significantly to the hypoxia-induced RC vasodilation. However, when NO synthesis was inhibited, RV O2 demand during basal conditions and during hypoxia was adequately met by increased O2 extraction in the presence of elevated arterial pressure.

Repeatability of responses to graded hypoxia. In this investigation dogs were exposed sequentially to graded hypoxia, first with no treatment, and then after L-NNA. Due to the prolonged action of L-NNA, it was not possible to reverse this order. Thus it was important to ascertain that differing responses after L-NNA were, in fact, due to drug treatment, and not due to the prior exposure to hypoxia. To address this concern, four dogs were subjected to repeated hypoxia with no drug treatment. Figure 1 illustrates that repeated, graded hypoxia similarly affected RC blood flow. RV mechanical function was also found to respond in a similar manner to repeated hypoxia. RC venous blood was not collected in these experiments, so comparative data on repeatability of RV oxygen extraction and MO2 are not available. However, we did find a high correlation between heart rate times RV systolic pressure (rate-pressure product) and RV MO2 in experiments in which RC venous blood was collected (Fig. 6) independent of treatment. Taken together, these data indicate that differences observed after L-NNA were due to inhibition of NO synthesis and not due to prior exposure to hypoxia.



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  		Fig. 6. RV rate-pressure product is plotted as a function of RV MO2 for the untreated control condition ({bullet}) and after NO synthesis blockade with L-NNA ({square}). L-NNA had no significant effect on the relationship between RV rate-pressure product and RV MO2.

 
A limitation of these repeatability experiments must be acknowledged. Because RV MO2 was not determined in these experiments, it was not possible to compare the repeatability of the mechanical variables as functions of MO2. If hypoxia had caused an upregulation of NO synthase activity, this relationship might have been altered (21, 33).

Mechanisms of RV O2 demand/supply balance during hypoxia. Myocardial O2 supply is a function of coronary blood flow and O2 extraction. Under normal resting conditions, the right ventricle extracts ~45 to 55% of the O2 delivered to it (19, 24, 28, 43). Therefore, the right ventricle has a large extraction reserve as well as a significant flow reserve (24, 28); both are available to increase the supply of O2 when RV O2 demand increases. Hart et al. (19) found that the RV extraction reserve is the primary mechanism for increasing O2 supply when RV O2 demand increases during light-to-moderate exercise. They reported that the RV flow reserve was not mobilized until RC venous PO2 fell to ~20 mmHg, an apparent threshold for RC vasodilation. In contrast, Zong et al. (43) found that during pulmonary hypertension, increases in RV O2 demand were met primarily by RC vasodilation at RC venous PO2 values well above 20 mmHg. To date, the relative contributions of the RC flow and RV O2 extraction reserves during hypoxia have not been defined.

It should be noted that basal RC flow and RV MO2 values reported here are higher than the resting data reported by Hart et al. (19) and the basal data reported by Zong et al. (43). This likely reflected excitement associated with the dog being placed in the chamber. In fact, baseline normoxic values presented here are similar to those observed by Hart et al. (19) during mild exercise. In the present investigation, increases in RV demand during graded hypoxia were met by increases in RC blood flow such that O2 extraction did not increase during hypoxia. This degree of O2 extraction was not maximal, because after blockade of NO synthesis, extraction increased significantly. Although O2 extraction expressed as a percentage of arterial O2 content remained constant in the untreated condition, RC venous O2 content and PO2 did fall with hypoxia. Thus directly or indirectly, a corresponding decline in RV tissue PO2 was likely responsible for the observed RC vasodilation.

Effect of NO on RV O2 demand/supply balance during hypoxia. NO has little or no influence on left coronary blood flow at rest or during exercise (1, 4, 9, 15, 36, 40). In contrast to findings in the left ventricle, results of this investigation, in agreement with previous reports (13, 37), demonstrated that NO is an important determinant of resting RC blood flow. The contribution of NO to the control of normal resting RC blood flow was evident in this investigation not only from reduced RC blood flow, conductance, and RC venous PO2 (Fig. 2 and Table 1), but also when RC conductance and RV O2 extraction were plotted as functions of RV MO2 (Figs. 3 and 4).

Earlier studies from this laboratory demonstrated that NO modulates RC blood flow in response to intracoronary norepinephrine infusion (34) and RC hypoperfusion (35) and during acute pulmonary hypertension (43). In the present investigation, hypoxia-induced RC hyperemia was blunted when NO synthesis was inhibited. This observation agrees with findings of an earlier study by Audibert et al. (2), who measured RC blood flow after more prolonged hypoxia (at 2 and 4 h). A complication of systemic inhibition of NO synthesis as done in the present study and by Audibert et al. (2) is systemic hypertension and reflex bradycardia (Table 1; Refs. 1, 4, 15, 40, 43). Because afterload of the right ventricle is increased moderately at only the most severe hypoxia and heart rate falls markedly, RV O2 demand decreases after inhibition of NO synthesis. Thus it is important to normalize RC flow and conductance data for changes in RV MO2. Because Audibert et al. (2) did not measure RV MO2, they could not determine to what extent the observed decreases in RC flow were the result of decreased RV O2 demand. By utilizing a technique developed in this laboratory to collect RC venous blood samples from the conscious dog (5), we were able to address this issue in the current investigation.

As noted by others (1, 4, 15, 40, 43), L-NNA produced peripheral vasoconstriction, elevated mean aortic pressure, and caused a reflex decrease in heart rate compared with the untreated control condition. These changes would have impacted RV myocardial oxygen demand, so it was important to compare key variables at corresponding RV MO2. The RC blood flow response to hypoxia tended to be reduced as a function of RV MO2 after inhibition of NO synthesis (Fig. 3A, ANCOVA, P = 0.09 slope). However, when the systemic hypertension that accompanied inhibition of NO synthesis with L-NNA was taken into account by examining RC conductance, a significant blunting of the response was evident (Fig. 3B). Furthermore, L-NNA significantly decreased the slope of the relationship between RV O2 delivery (which takes into account both arterial O2 content and RC flow) and MO2 (Fig. 4), indicating that O2 delivery to the right ventricle was impaired in the absence of NO as RV MO2 was increased during hypoxia. Taken together, these data demonstrate that NO exerts a vasodilatory influence on the RC circulation at rest and during systemic hypoxia. This vasodilation appears to be a key factor in adjusting RC flow, so that a large RV O2 extraction reserve is maintained at rest and during systemic hypoxia.

We acknowledge that in plots of RC flow and conductance (Fig. 3) and RV O2 delivery (Fig. 4) as functions of RV MO2, the x- and y-axis variables are not completely independent. For example, flow, the dependent variable of Fig. 3A, is one of the determinants of MO2, the x-axis variable. However, it is well accepted that coronary flow and O2 delivery are functions of myocardial metabolic rate. We assessed RV myocardial metabolic rate directly by measuring RV MO2, and having done so, we have used this variable for the x-axis in Figs. 3 and 4. As long as metabolic rate is the true independent variable, we suggest that absence of complete independence between the x- and y-axis variables does not obscure the intended and important comparisons of effects of NO synthesis blockade presented in Figs. 3 and 4. An alternative approach would have been to use a less closely related index of metabolic rate, the RV rate-pressure product, which, however, for these experiments correlated closely with RV MO2 (Fig. 6).

Although a role for NO-mediated RC vasodilation during hypoxia was identified, NO was not essential for RV O2 demand/supply balance under the conditions of these experiments. The marked increase in RV O2 extraction that occurred after blockade of NO synthesis sustained RV O2 supply, and there was no evidence of RV ischemia. RV function was well maintained, as was the positive RC arteriovenous difference in lactate. However, it must be recognized that continued myocardial uptake of lactate evaluated by differences in arterial and coronary venous lactate does not exclude regional release of lactate and the possibility of regional ischemia (17). For example, release of lactate by more severely hypoxic subendocardium could have been obscured by lactate uptake by other regions.

Effect of NO on the relationship between RV mechanical function and RV MO2 during hypoxia. NO has been shown to depress left and RV MO2 (1, 4, 33–36), although this topic is controversial (10). In the right ventricle, moderate RC hypoperfusion, a condition which causes NO release (21), reduces RV MO2 with no effect on RV mechanical function (35). Blockade of NO synthesis increased RV MO2, but did not significantly affect RV mechanical function. In contrast, Heusch et al. (21) reported that mechanical function of hypoperfused left ventricle was further depressed after blockade of NO synthesis, whereas MO2 was unchanged. To date no studies have examined the relationship between ventricular mechanical function and MO2 during systemic hypoxia.

Figure 6 shows the RV rate-pressure product plotted as a function of RV MO2 for the untreated condition and after L-NNA. A high linear correlation between the rate-pressure product and MO2 is clearly evident in Fig. 6. L-NNA had no significant effect on this relationship, i.e., both RV MO2 and RV mechanical function were reduced after NO synthesis inhibition, according to the relationship observed between these variables in the untreated, control state. Thus the effects of NO on the relationship between mechanical function and MO2 during hypoxia differ from the effects found in either RV or left ventricle during hypoperfusion. Maintenance of normal or elevated RC perfusion pressure values during systemic hypoxia may account for this difference, although this interesting topic merits further investigation.

Compensatory mechanisms after blocking NO synthesis. Recent studies (22, 26, 27, 38) suggest that multiple pathways are involved in regulation of coronary blood flow and that when one mechanism is inhibited, another may compensate. To address this possibility we conducted three additional experiments in which adenosine receptors and KATP channels were also blocked to determine whether these vasodilator mechanisms are involved in control of RC blood flow when NO synthesis is inhibited. Increase in RC blood flow during hypoxia after this triple blockade closely matched the increase in flow observed after L-NNA alone (Fig. 5). These data indicate that neither adenosine nor KATP channels contribute to basal RC tone or to hypoxic-induced RC vasodilation when NO synthesis is inhibited.

In summary, this report presents the first data describing the contribution of NO to O2 demand/supply balance in the right ventricle during graded systemic hypoxia. Additionally, new data are provided on the relative contributions of increases in RC flow and RV O2 extraction required to meet increased RV O2 requirements during hypoxia. In the untreated state, RV O2 requirements during hypoxia were met by increased RC blood flow with no increase in O2 extraction. NO is an important factor in this hypoxia-induced RC vasodilation. However, when NO is blocked, increased O2 extraction compensated for reduced vasodilation and RV ischemia was avoided. In addition, combined blockade of other metabolic vasodilators (adenosine, KATP channels) failed to further suppress the RC hyperemic response to hypoxia. NO blockade had no effect on the relationship between RV rate-pressure product and RV MO2 during hypoxia.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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REFERENCES
 
This study was completed by Rodolfo Martinez in partial fulfillment of the requirements for the Master of Science degree at the University of North Texas Health Science Center.

This work was supported by National Heart, Lung, and Blood Institute Grant HL-64785.


   ACKNOWLEDGMENTS
 
We are grateful to Arthur G. Williams and Drs. Wei Sun and Jian Bi for expert technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. Fred Downey, Dept. of Integrative Physiology, Univ. of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107-2699 (E-mail: fdowney{at}hsc.unt.ed)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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