Function and Production of Nitric Oxide in the Coronary Circulation of the Conscious Dog During Exercise
This study determined the changes in NO production from the coronary circulation of the conscious dog during exercise. The role of endogenous NO as it relates to coronary flow, myocardial work, and metabolism was also studied. Mongrel dogs were chronically instrumented for measurements of coronary blood flow (CBF), ventricular and aortic pressures, and ventricular diameter, with catheters in the aorta and coronary sinus. Acute exercise (5 minutes at 3.6, 5.9, and 9.1 mph) was performed, and hemodynamic measurements and blood samples were taken at each exercise level. Nitro-l-arginine (NLA, 35 mg/kg IV) was given to block NO synthesis, and the exercise was repeated. Blood samples were analyzed for oxygen, plasma nitrate/nitrite (an index of NO), lactate, glucose, and free fatty acid (FFA) levels. Acute exercise caused significant elevations in NO production by the coronary circulation (46±23, 129±44, and 63±32 nmol/min at each speed respectively, P<.05). After NLA, there was no measurable NO production at rest or during exercise. Blockade of NO synthesis resulted in elevations in myocardial oxygen consumption and reductions in myocardial FFA consumption for comparable levels of CBF and cardiac work. The metabolic changes after NLA occurred in the absence of alterations in myocardial lactate or glucose consumptions. NO production by the coronary circulation is increased with exercise and blocked by NLA. The absence of NO in the coronary circulation during exercise does not affect levels of CBF, because it shifts the relationship between cardiac work and myocardial oxygen consumption, suggesting that endogenous NO modulates myocardial metabolism.
- nitric oxide
- endothelium-derived relaxing factor
- coronary resistance
- myocardial metabolism
- oxygen consumption
Nitric oxide is an important mediator of vascular tone in the peripheral circulation and functions to maintain the vasculature in a dilated state.1 Studies in perfused hearts and anesthetized animals revealed an elevation of vascular resistance in response to blockade of NO synthesis.2 3 4 Studies from our laboratory5 6 have demonstrated that systemic blockade of NO synthesis has no effect on the changes in CBF in response to acute exercise in the conscious dog. This is consistent with studies by Duncker and Bache,7 who showed that intracoronary blockade of NO synthesis does not alter the changes in CBF in response to acute exercise. Additional studies by Altman et al8 have shown that intracoronary blockade of NO synthesis actually increases CBF during acute exercise in the conscious dog. In fact, in all these studies, the fall in coronary vascular resistance was greater during exercise after blockade of NO synthesis.5 6 7 8 Studies in humans demonstrated an increase in coronary resistance with blockade of NO synthesis and a reduction in the vasodilation produced by cardiac pacing.9 These studies seem to contradict the common perception that exercise-induced vasodilation may be partially due to shear stress–induced release of NO triggered by increases in flow.10 11 Whereas these studies in conscious animals used an established inhibitor of NO synthesis12 and tested this blockade with acetylcholine, a known agonist for NO release13 whose vasodilation is only incompletely blocked under the best circumstances, there has been no direct measure of NO production, the magnitude of NO production, or the precise degree of NO synthesis inhibition in the coronary circulation of the conscious animal.
The direct measurement of NO production from an in vivo coronary vascular bed has remained difficult. Kelm and Schrader14 used the oxidation of oxyhemoglobin to methemoglobin to detect production of NO from the coronary circulation of isolated guinea pig hearts. Work by Pohl and Busse15 on isolated rabbit hearts and further work by Kelm and Schrader16 in the isolated guinea pig heart have used elevated cGMP levels in platelets to demonstrate a basal production of NO by the coronary circulation. This basal production increased with exogenous stimulation from either acetylcholine or bradykinin. However, because of the limitations of these preparations, it is difficult to show alterations in NO production in response to endogenous physiological stimuli.
For a number of years, we have been interested in the role of NO in the regulation of coronary vascular tone during exercise. Zeballos et al17 demonstrated not only the ability to measure plasma NOX as an index of NO production but also a basal level of NO production across the intact coronary circulation of the conscious resting dog. The levels of NO metabolites were significantly higher in the CS blood than in the aorta. When this difference was multiplied by mean CBF, a significant basal production of NO was evident. Since it is well established that changes in shear stress or CBF are potent endogenous stimuli for NO production,10 11 we hypothesized that exercise-induced increases in CBF should produce measurable increases in NO production by the coronary circulation. Therefore, the goals of the present study were to determine whether exercise increased NO production from the coronary circulation of the conscious dog, the magnitude of NO production, and the relationship between cardiac work, metabolism, and NO production.
Materials and Methods
Adult male mongrel dogs weighing 20 to 30 kg were used in these studies. The dogs (n=17) were weighed, sedated with acepromazine (0.3 mg/kg IM, Ayerst), and anesthetized with sodium pentobarbital (30 mg/kg IV or to effect). The dogs were ventilated with room air via a positive-pressure respirator (Harvard Apparatus) with 5 cm H2O end-expiratory pressure. A thoracotomy in the left fifth intercostal space was performed under sterile conditions. Separate catheters (Tygon, Cardiovascular Instruments) were placed in the descending thoracic aorta and the left atrial appendage. A third catheter was placed in the CS, with the tip leading away from the right atrium. During the surgery, a blood sample was taken from the CS catheter and immediately measured on a pH/blood gas analyzer (Corning model 170). If the Po2 was >25 mm Hg, the catheter was repositioned and tied into place. A solid-state pressure gauge (model p6.5, Konigsberg Instrument) was placed in the LV via a stab wound in the apex of the LV. A Doppler flow transducer (Parks Electronics) was placed around the left circumflex coronary artery. In five dogs, a pair of 3-MHz piezoelectric crystals was placed on opposing endocardial surfaces at the base of the LV. The wires and catheters were run subcutaneously to the intrascapular region. The chest was closed in layers, and the pneumothorax was reduced.
The dogs were allowed to fully recover. Antibiotics were given postoperatively. Heart rate, body temperature, and weight were monitored daily. Approximately 2 weeks after surgery, the animals were trained to run at increasing speeds on a treadmill (Safe-T-Mill, Good Horsekeeping) and to lie quietly on a laboratory table. The protocols were approved by the Institutional Animal Care and Use Committee of New York Medical College and conform to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (Department of Health and Human Services Publication No. [NIH] 80-23, revised, Office of Science and Health Reports, Bethesda, Md).
On the day of the experiment, the dog was weighed, brought to the laboratory, and placed on the table. The previously implanted aortic catheter was attached to a strain-gauge transducer (Statham P23ID) for the measurement of aortic pressure. LVP was measured using the solid-state pressure gauge. The first derivative of LVP, LV dP/dt, was obtained using an operational amplifier (National Semiconductor LM 324), and maximum dP/dt was recorded for statistical purposes. Triangular wave signals with known slopes were substituted for the pressure signals to calibrate the differentiators directly. CBF was measured with a pulsed Doppler flowmeter (Triton Instruments). LV diameter was measured using the implanted piezoelectric crystals and a Transit Time ultrasonic dimension gauge. The instrument generates a voltage linearly proportional to the transit time of ultrasound traveling between the previously implanted transducers (1.55×103 m/s), thus giving a continuous measurement of LV internal diameter. We have used this method previously.18 All data were recorded on a 14-channel tape recorder (Bell and Howell 3700B) and played back on an eight-channel direct-writing oscillograph (Gould RS 3800). Heart rate was measured using a cardiotachometer (model 9857B, Beckman Instruments) from the LVP pulse interval. Mean values for coronary flow and aortic pressure were derived using 2-Hz resistance capacitance filters. LDCR was calculated as the late-diastolic aortic pressure divided by late-diastolic CBF. Any electronic drift was corrected by frequent calibrations during the experiment.
The dogs were trained to run at speeds of 3.6, 5.9, and 9.1 mph on the treadmill for a period of at least 5 minutes each. The training regimen was 5 to 10 minutes at each speed, twice a day, lasting <5 days. This regimen was chosen to reduce the likelihood of exercise-induced changes in coronary vascular regulation. The control data point was taken with the dog standing on the treadmill. The speed of the treadmill was then gradually increased to 3.6, 5.9, and 9.1 mph and held constant at each of those speeds for ≈5 minutes. Hemodynamic measurements were continuous throughout each experiment. Data for analysis were obtained at periods just before blood sampling, which occurred when steady state exercise levels were obtained, ≈3 minutes after the onset of exercise.
Coronary and Cardiac Dynamics
To determine the role of NO in the coronary circulation during exercise, NLA (35 mg/kg IV, Aldrich Chemical Co) was given. This dose has previously been shown to significantly block NO-mediated responses in the coronary circulation.6 19 Twenty to 30 minutes after administration of NLA, the exercise protocol was repeated.
Cardiac Oxygen Consumption
Blood samples were taken ≈3 minutes after the onset of each treadmill speed or when the hemodynamics stabilized, whichever came later. All blood samples were collected into syringes (Becton Dickinson & Co), treated with either heparin or EDTA, immediately capped, and stored on ice until measured. The measurements for blood gases were performed using a blood gas analyzer, and the hemoglobin analysis was performed (CO-Oximeter, Instruments Laboratory). Hematocrits were obtained from the aortic and CS samples by centrifugation. Special care was taken to withdraw blood slowly from the CS catheter to decrease the potential for contamination of the sample with right atrial blood. Total oxygen content of the blood was calculated as the addition of the oxygen content measured by the hemoglobin analyzer and 0.003 multiplied by the Po2. Oxygen uptake was calculated as the arterial-CS oxygen difference. Oxygen extraction was calculated as the oxygen uptake across the heart divided by the arterial oxygen content. Oxygen consumption was calculated as oxygen uptake multiplied by mean CBF.
Lactate was measured from whole blood taken from the aortic and CS catheters using a YSI 1500 sport lactate analyzer (Yellow Springs Instrument Co). FFA and glucose analysis was performed using blood samples collected into separate EDTA-treated syringes and stored on ice until the completion of the experiment. Upon completion of the experiment, blood samples for FFA and glucose were spun at 1000g for 15 minutes at 0°C. FFA analysis was performed on the plasma using a colorimetric assay (NEFA C kit from WAKO Pure Chemical Industries, Ltd) and a spectrophotometer (Kontron Instruments). Plasma glucose was determined using a Beckman Glucose 2 analyzer (Beckman Instruments).
In all dogs, cardiac function was estimated using the triple product. The triple product was calculated as the product of the LV systolic pressure, LV dP/dt, and heart rate. In five of the animals, cardiac minute work was measured as the integral of the pressure-diameter loops multiplied by heart rate. We have used this method previously.18
Changes in NO Levels
Plasma NOX was determined using a method previously described.17 Briefly, the blood that was collected into the heparinized syringes was centrifuged at 1000g for 15 minutes to remove formed elements, and the plasma was frozen until the day of analysis. Through incubation with Aspergillus nitrate reductase and acidification, the NOX in the plasma sample was converted to NO. The gaseous NO was injected into a NO chemiluminescence analyzer (Sievers, Inc). In the analyzer, NO combines with ozone to produce photons, the number of which are directly proportional to the amount of NO injected, and thus the NOX from the plasma sample.
Calculation of Vessel NO Production In Vitro and In Vivo
Using previously published data from our laboratory,18 calculations involving capillary surface area, oxygen consumption, and blood flow in the canine heart were performed to determine if NO production is constant in the blood vessels or if it varies with changes in flow. If the maximum flow in the heart is 5 mL/min per gram and we assume a 71.5-g LV free wall, a maximum coronary flow can be obtained through the following equation:Using an 80% extraction and an arterial oxygen content of 20 mL O2/100 mL blood, the maximum oxygen uptake by the heart is ≈16 mL O2/100 mL blood. Thus, the maximum oxygen consumption by the myocardium is as follows:Our highest level of oxygen consumption was 25 mL/min, or ≈50% of V̇o2max. Using capillary density and capillary surface area of the dog myocardium,18 the volume and surface area of the capillaries in the LV free wall (71.5 g tissue) can be obtained. To obtain the volume of the myocardium of the LV free wall, divide the mass of the tissue by the density (1.06 g/cm3) of the tissue:Capillary surface area equals 57 mm2/mm3 myocardium:
A study by Bassingthwaighte et al20 calculated an average capillary surface area for the dog LV myocardium of 504 cm2/g myocardium. This produces a capillary surface area of 36 036 cm2 for a 71.5-g LV free wall. Both numbers will be used in our calculations.
Because the capillary CSA accounts for 90% of the total blood vessel volume, we use this as an estimate of total coronary blood vessel CSA and its weight. Equation 4 gives us the maximum CSA at maximum blood flow. If we assume that the maximum blood flow and the maximum oxygen consumption are concurrent and that there is a linear relationship between capillary perfusion fraction and myocardial oxygen consumption, the CSA at a given level of oxygen consumption can be calculated according to the following equation:orThe measured myocardial oxygen consumption will be used in conjunction with Equation 5 to determine the perfused CSA at each level of exercise. NOX production will be divided by calculated CSA at each exercise level to determine if NO production per unit CSA changes with exercise. The calculated CSA will also be used to determine the weight of microvessels producing NO and will be compared with our previously published in vitro data21 using agonists to stimulate NO production.
All data are expressed as mean±SEM. The responses are those obtained after ≈3 minutes at a particular speed. All graphs were generated using a commercially available graphing program (SlideWrite 2.0 for Windows, Advanced Graphics Software) on a 486×33 personal computer (Everex). The linear fits represent the best fit using the least squares regression analysis. Comparisons between groups of samples were performed by paired Student's t test or repeated measures ANOVA, as appropriate. A value of P<.05 was considered statistically significant. Release or uptake of the various compounds measured in this study (oxygen, FFA, lactate, glucose, and NOX) was calculated as the difference between the arterial and CS levels. Production or consumption of the various substrates was calculated as the arterial-CS difference multiplied by mean CBF.
Coronary and Cardiac Dynamics
A representative tracing of one of the dogs standing at rest and at each of the three treadmill speeds is shown in Fig 1⇓. With each increase in treadmill speed, there were increases in all of the measured hemodynamic parameters. Table 1⇓ summarizes the hemodynamic results for all dogs during exercise before and after NLA. There were significant increases in LV systolic pressure, LV dP/dt, mean arterial pressure, mean CBF, and heart rate associated with exercise. There was also a significant fall in LDCR with exercise. These hemodynamic changes occurred both before and after blockade of NO synthesis with NLA.
Blockade of NO synthesis with NLA caused significant increases in LV systolic pressure and mean arterial pressure at rest, with a significant reduction in heart rate. There was no significant change in CBF but a marked increase in LDCR after blockade of NO synthesis.
During each level of exercise, after NLA administration, there was a significant reduction in the LV dP/dt compared with control and a significant elevation in mean arterial pressure. There was also an elevation of LDCR after blockade of NO synthesis, which was present at the lowest exercise level, even though the change from the resting state was significantly larger (P<.05). It is important to note that in the absence or presence of NLA, there was no difference between the levels of CBF during exercise.
Cardiac Oxygen Consumption
The metabolic effects of NLA on exercise-induced changes in myocardial oxygen handling are shown in Table 2⇓. Neither exercise nor NLA produced a change in the arterial oxygen levels. Myocardial oxygen uptake, extraction, and consumption all increased significantly with exercise and were not altered with NLA. When the CS Po2 was plotted versus myocardial oxygen consumption (Fig 2⇓), there was a significant reduction (n=8, P<.05) in the CS Po2 with increasing myocardial oxygen consumption before and after the blockade of NO synthesis with NLA. After NLA, however, there was a significant (P<.05) reduction from control in the CS Po2 at rest and during the lowest exercise speed.
Lactate consumption increased significantly (n=8, P<.05) during exercise and was not affected by treatment with NLA (Table 3⇓). Using a two-way repeated measures ANOVA, both the effects of speed and NLA on plasma FFA consumption were significantly different (n=7, P<.05). Taken as a group, FFA consumption increased with speed and was lower after NLA. Using paired comparisons, the basal level of FFA consumption was significantly (P<.05) lower after blockade of NO synthesis. There was no demonstrable level of glucose uptake (no value different from zero) by the myocardium. The plasma glucose in the aortic samples tended to increase during exercise (from 90±4 mg/dL at rest to 105±8 mg/dL at the highest speed, n=6, P=NS) and was not affected by NLA (from 104±10 mg/dL at rest to 119±16 mg/dL at the highest speed, P=NS). The plasma glucose levels of the CS were not different from the aortic levels, resulting in no measurable glucose production by the myocardium (Table 3⇓).
The triple product and minute work (loop area multiplied by heart rate) are compared with the measured values for myocardial oxygen consumption (Fig 3A and 3B⇓⇓); a significant effect of NO inhibition is evident. At rest, statistically, there was no difference between either the level of cardiac work or oxygen consumption after NLA. However, after blockade of NO synthesis with NLA, at any given triple product or minute work, there was a higher oxygen consumption. This caused a significant (P<.05) elevation in the slope of the work–oxygen consumption relationship after blockade of NO synthesis. Thus, the myocardium was using more oxygen for a given level of work after blockade of NO synthesis. Additionally, there was a linear relationship between the triple product and cardiac minute work (Fig 3C⇓). This relationship was not affected by blockade of NO synthesis.
Changes in NO Levels
The levels of CS NOX are slightly, but not significantly, lower after NLA (Fig 4A⇓). However, when the differences between the CS and aortic plasma NOX levels are compared (Fig 4B⇓), NOX release by the coronary circulation increases significantly with acute exercise (n=12, ANOVA, P<.05). After blockade of NO synthesis with NLA, there was no change from the resting state of NOX release from the coronary circulation.
When CBF is taken into account (Fig 4C⇑), myocardial NOX production increases significantly with exercise (n=12, ANOVA, P<.05). Administration of NLA caused a blockade of NO production, as there was no change from the resting state in NOX production with exercise in the presence of NLA.
Calculation of Vessel NO In Vitro and In Vivo
When the values for myocardial oxygen consumption from Table 2⇑ are combined with Equation 5 using data generated from our laboratory (CSA1) or from the study by Bassingthwaighte et al20 (CSA2), the perfused CSA at each level of exercise could be estimated (Table 4⇓). Since there is only a 6% difference between the numbers calculated from our laboratory and those from the study by Bassingthwaighte et al, the data from our laboratory were used to calculate vessel NO production per unit area. In order to determine if the increases in NO production were simply due to an increase in the number of perfused blood vessels releasing NO or an actual increase in NO production by the individual blood vessels, the NO production at each speed was divided by the CSA at each speed. This produced increasing levels of NO production per CSA for the two lower speeds. Thus, a major portion of the increased NO production seen was due to an increased production of NO independent of changes in calculated CSA.
The major finding of the present study is that NO production across the coronary circulation of the dog heart increases during exercise. As previously reported by Zeballos et al,17 there is a basal level of NO production by the coronary circulation of the dog heart. This production is increased with exercise, perhaps the most potent physiological stimulus for CBF. NO production has been demonstrated indirectly in a number of isolated whole-heart14 15 16 preparations using the oxidation of oxyhemoglobin to methemoglobin or elevation of cGMP in platelets. Those studies demonstrated a basal level of production that was increased with various agonists, such as acetylcholine or bradykinin. Ours is the first study to demonstrate that exercise can increase NO production from an in vivo coronary vascular bed.
During the present study, the plasma NOX from aortic and CS blood was measured with the dog standing at rest on the treadmill and at three successively increasing exercise speeds. Although not statistically different, the aortic plasma NOX levels tended to decline during exercise, whereas the CS plasma NOX levels tended to increase during exercise. This produced a widening of the difference in plasma NOX across the heart. It is important to note that plasma NOX is measured as a concentration (1 μmol/L). If the molar production was to remain constant in the presence of increasing blood volume (flow), the concentration would actually drop. Because both the concentration of plasma NOX and the level of CBF increase with each level of exercise, clearly there is an increase in the molar production by the coronary circulation.
Although there are numerous studies showing that plasma NOX is the primary breakdown product of NO,22 23 24 to validate that the increased plasma NOX seen in these studies was indeed due to NO production, NLA was used. Administration of NLA reduced the basal level of NO production by the coronary circulation to a level that was statistically not different from zero. Additionally, NLA abolished any change in the arterial-CS plasma NOX difference or production from baseline seen with exercise. Therefore, the elevated levels of plasma NOX production seen during exercise are due to an increase in NO synthesis.
The calculations of blood vessel surface area and NO production reveal that NO is not released at a constant rate from the individual blood vessels but can be released at various rates, depending on the intensity of exercise. Previous work from our laboratory has demonstrated a similar level of NO production from isolated coronary microvessels.21 Seyedi et al21 found that a basal NO production (27±2 pmol/mg) of microvessel increased to 145±17 pmol/mg with a maximum stimulation from angiotensin II during a 20-minute incubation. Using the technique of Gerritsen and Printz25 to isolate coronary microvessels from the LV free wall, ≈3.3 g of microvessels was obtained from a 40-g LV free wall (only midmyocardium). Using these numbers, a basal NO production by the microcirculation of the LV free wall would be 9 nmol/min, and the maximum stimulated level would be 49 nmol/min during agonist-induced stimulation. Although these numbers are slightly smaller than those obtained in the present study (range, 13.3 to 82 nmol/min), the NO production measured in the present study is from the entire coronary circulation of an intact in vivo dog heart during exercise-induced increases in coronary flow and shear stress. Recent reports have demonstrated constitutive NO synthase in isolated cardiac myocytes.26 This source of NO may contribute to the slightly higher levels of NO production seen in that study compared with our isolated microvessels. However, because of the conflicting influences from the autonomic nervous system, the magnitude of NO from this source is unclear. That NO production from the whole heart and per unit blood vessel surface area falls at the highest exercise rate is puzzling. This may be due to a reduction in shear stress at the higher flow rate, which would decrease the stimulus for NO production. At higher flows, coronary dilation could increase to a level where shear stress is reduced. This could account for the reduction in NO production seen at the highest exercise level.
The second major finding of the present study was the demonstration of an alteration in the myocardial handling of oxygen. After blockade of NO synthesis at rest and during the lowest level of exercise, there were significant reductions in CS Po2 for identical levels of myocardial oxygen consumption. Miyashiro and Feigl27 demonstrated alterations in CS Po2 at identical levels of myocardial oxygen consumption in anesthetized open-chest dogs in the presence of α- and β-adrenergic blockade. The underlying mechanism was proposed to be feedback and feedforward control of the coronary circulation, matching CBF to myocardial metabolism. The present study demonstrates reductions in CS Po2 at low levels of myocardial oxygen consumption in the absence of NO. A replotting of CS Po2 and myocardial oxygen consumption data from the paper by Altman et al8 demonstrates a similar effect of inhibition of NO synthesis at rest and during moderate exercise. This effect is consistent with the near maximal oxygen extraction at rest and theoretical maximum oxygen extraction at moderate to high cardiac metabolic rates. Thus, in the intact coronary circulation of the awake exercising dog, NO appears to play a role in matching CBF to myocardial metabolism. Since CBF is not different before or after blockade of NO synthesis, it would appear that NO may function more to modulate metabolism than flow in the coronary circulation.
Blockade of NO synthesis with NLA also caused a significant alteration in the relationship between myocardial work and oxygen consumption. Myocardial work was calculated directly from the pressure-volume loop area, and function was estimated using the triple product. Exercise produced increases in both myocardial work/triple product and oxygen consumption. Exercise after blockade of NO synthesis also produced increases in myocardial work and oxygen consumption; however, for any level of cardiac work, there was an increased myocardial oxygen consumption after blockade of NO synthesis. This produced a statistically significant elevation in the slope of the relationship between myocardial work and oxygen consumption. These results are consistent for both our direct measure of cardiac work and the triple product. The triple product is linearly proportional to cardiac work in conscious dogs. This has been previously shown in anesthetized dogs.28
After blockade of NO synthesis, the myocardium consumed significantly less FFA without changes in either glucose or lactate consumption. That the changes in oxygen consumption and FFA consumption seen with NLA move in opposite directions seems to be counterintuitive: the heart is consuming less fuel while burning more oxygen. Since the heart is performing comparable levels of work before and after NLA, the absence of a change in lactate or glucose consumptions implies that there is not a shift toward anaerobic metabolism. Therefore, it would appear that the heart, in the absence of NO, is still primarily using FFA in metabolism for energy. We have previously demonstrated29 in measurements of total body oxygen consumption that the elevation in oxygen consumption seen with blockade of NO synthesis is not entirely for the production of ATP. When NO synthesis was blocked systemically in the resting dog, there was an elevation in total body oxygen consumption, which was accompanied by an elevation in body temperature. This elevation in temperature indicates that at least part of the extra oxygen consumed was being used to produce heat. Although more detailed studies need to be performed in vitro, the alterations in oxygen and FFA consumption would seem to suggest that cardiac efficiency has been altered in the absence of NO.
There is an increasing body of evidence from our laboratory29 30 31 and others32 33 34 demonstrating that the basal release of NO has an inhibitory effect on tissue oxygen consumption. This has been demonstrated in vitro in both cardiac and skeletal muscle,30 31 32 33 in vivo in skeletal muscle,30 32 and from whole-body oxygen consumption.29 Previous work by Altman et al8 demonstrated alterations in oxygen handling with blockade of NO synthesis. However, the fundamental difference between that study and the present study is the intracoronary versus intravenous injection of NLA. A major drawback of the intracoronary injection of NLA is that NO synthesis is only blocked in the area perfused by that specific coronary vascular bed, at most only about half of the LV free wall. Through systemic blockade of NO synthesis with an intravenous injection of NLA, NO production from the entire heart is blocked. Even with that caveat, Altman et al demonstrated elevated levels of myocardial oxygen consumption for essentially identical levels of myocardial work (using the triple product). This is consistent with our hypothesis that endogenous NO inhibits cellular respiration.
A consistent but troubling finding in most in vivo studies was that inhibition of NO synthase did not markedly alter coronary vascular dynamics, ie, CBF regulation. Since systemic administration of NLA or spillover of NLA to the systemic circulation after intracoronary administration results in bradycardia, hypertension, and perhaps alterations in preload, it is not surprising that there were no pronounced changes in CBF regulation. Obviously, the workload of the heart was different after administration of NLA. The present study clearly shows that both the relationships between CS Po2 and myocardial oxygen consumption and between loop area times heart rate and oxygen consumption were altered after NLA. This might be partially reflected in the fall in LDCR during exercise, which is even greater during exercise after NLA. One would predict a greater production of a metabolic vasodilator during exercise after NLA because of the increased myocardial oxygen consumption for given levels of cardiac work. This reasoning underscores the secondary role of NO in the control of blood flow, particularly in the heart, since blood flow has to be altered before shear can stimulate NO production.
In summary, acute exercise caused increases in both the release and production of NO from the coronary circulation of the exercising dog. This elevation in NO release and production was blocked by NLA, a potent inhibitor of NO biosynthesis. The magnitude of NO produced is consistent with in vitro studies of isolated canine coronary microvessels and appears to be due to both increases in coronary vessel CSA and an increase in the release of NO per unit CSA. This endogenous release of NO alters myocardial metabolism, probably through a partial inhibition of mitochondrial electron transport as we have previously proposed.29 30 31
Selected Abbreviations and Acronyms
|CBF||=||coronary blood flow|
|FFA||=||free fatty acid|
|LDCR||=||late-diastolic coronary resistance|
|LV||=||left ventricle (ventricular)|
This study was supported by grants PO1-HL-43023, RO1-HL-50142, and RO1-HL-53053 from the National Heart, Lung, and Blood Institute.
This manuscript was sent to Bradford C. Berk, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
- Received February 7, 1996.
- Accepted June 14, 1996.
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