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NMR Laboratory for Physiological Chemistry, Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Endogenous
nitric oxide (eNO) modulates tissue respiration. To test whether eNO
modulates myocardial O2 consumption
(M
O2), ATP synthesis, and metabolic
efficiency, we used isolated isovolumic guinea pig hearts perfused at a
constant flow. N
-nitro-L-arginine
(L-NNA; 5 × 10
5 mol/l) was used to
inhibit eNO production. MO2 was measured at different levels of cardiac work, estimated as the rate-pressure product (RPP). ATP content and synthesis rate were determined using
31P NMR and magnetization transfer during high cardiac
work. L-NNA increased coronary vascular resistance (19 ± 3%, P < 0.05) and MO2 (12 ± 3%, P < 0.05) without an increase in the RPP. In contrast, vehicle infusion
resulted in insignificant changes in coronary vascular resistance
(3 ± 2%, P > 0.05) and
MO2 (
2 ± 1%, P > 0.05). Compared with vehicle, L-NNA caused a higher
MO2 both during KCl arrest
(L-NNA 5.6 ± 0.5 vs. vehicle 3.0 ± 0.4 µmol · min1 · mg dry wt1,
P < 0.05) and during increased cardiac work elicited
by elevating perfusate Ca2+, indicating an upward shift in
the relationship between contractile performance (measured as RPP) and
MO2. However, neither ATP contents nor
ATP synthesis rates were different in the two groups during high
cardiac work. Thus, because inhibition of eNO production by
L-NNA increased MO2 without
a change in the ATP synthesis rate, these data suggest that eNO
increases myocardial metabolic efficiency by reducing
MO2 in the heart.
nuclear magnetic resonance; N-nitro-L-arginine
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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ENDOGENOUS NITRIC
OXIDE (eNO) derived from vascular endothelial cells plays a
crucial role in the control of blood vessel tone and blood flow
(9, 14, 21). Recently, it has been reported that eNO also
functions to regulate tissue O2 consumption (27). In skeletal muscle, both at rest (17,
26) and at various levels of work produced by exercise
(28) in the conscious dog, inhibiting eNO synthesis
increased tissue O2 consumption, thereby reducing the
reserve for tissue O2 utilization. Whether eNO is also
involved in the regulation of myocardial O2 consumption
(MO2), however, remains controversial.
Evidence supporting a role for eNO in the regulation of
MO2 was obtained from a study
(35) using noncontracting myocardial strips. However,
inconsistent observations have been reported from in vivo studies. In
one study using conscious dogs, MO2
increased after inhibition of eNO production (3). In
contrast, in other studies using hearts in open-chest dog preparations
as well as in instrumented conscious dogs,
MO2 decreased after inhibition of eNO
synthesis (25, 29).
The first goal of the present study was to determine whether eNO
modulates MO2. Studies (2,
27) in the in vivo setting have shown that the systemic
inhibition of eNO synthesis results in peripheral vasoconstriction and
hypertension, which triggers a series of cardiovascular reflexes
leading to changes in cardiac preload, afterload, and heart rate. In
addition, it has been reported that inhibition of eNO synthesis causes
a significant change in myocardial substrate selection and utilization
(3, 24). It is important to recognize that
MO2 is directly related to cardiac performance and metabolic substrate utilization (4, 30,
36). To avoid these confounding factors, we used an isolated
isovolumic guinea pig heart preparation with constant flow perfusion,
in which exogenous substrate supply was limited to glucose only. N-nitro-L-arginine
(L-NNA), a specific potent NO synthesis inhibitor (10, 22), was used to inhibit eNO production in these
guinea pig hearts.
If MO2 is modulated by eNO, then the
next relevant question is whether the ATP synthesis rate is also
affected by eNO, which has not been addressed experimentally. Because
previous studies (4, 15, 16, 18, 19) have demonstrated
that changes in the rate of ATP synthesis estimated from O2
consumption and 31P NMR magnetization transfer are in good
agreement, our goal was to use 31P NMR techniques to
directly assess the ATP synthesis rate in the intact heart with and
without inhibition of eNO synthesis. Finally, to understand the role of
eNO in the regulation of metabolic efficiency in the heart, we also
evaluated myocardial metabolic efficiency, determined from the coupling
between MO2 and cardiac performance and
the coupling between MO2 and ATP synthesis.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Heart Preparation and Constant Flow Perfusion
Experiments were performed on isolated isovolumic buffer-perfused hearts. Guinea pigs weighing 350-500 g were anesthetized by intraperitoneal injection of 65 mg/kg pentobarbital sodium. The heart was rapidly excised, arrested in ice-cold Krebs-Henseleit buffer, and cannulated by the aorta to a constant-flow heart perfusion apparatus. Retrograde perfusion began through the aorta at 37°C. The flow was adjusted from 18-22 ml/min to maintain coronary perfusion pressure (CPP) at above 60 mmHg. The perfusion catheter was connected to a strain-gauge transducer (Statham P23 ID; Newark, NJ) to record CPP. This was proportional to changes in coronary vascular resistance because the flow was held constant during the entire experimental protocol. Hearts were perfused with phosphate-free Krebs-Henseleit buffer containing (in mmol/l) 118 NaCl, 4.7 KCl, 1.75 CaCl2, 25 NaHCO3, 1.2 MgSO4, 0.5 EDTA, and 11 glucose. The perfusate was equilibrated with 95% O2-5% CO2 to maintain a pH of 7.4.Measurement of Isovolumic Contractile Performance
The heart was surrounded by its own perfusate in a water-jacketed reservoir, and the perfusate level was kept above the pulmonary outflow tract by continuous suction. After a small part of the left atrium was removed, a polyethylene (PE)-90 drain was placed through the apex of the left ventricule (LV) for drainage of the Thebesian veins. A water-filled latex balloon attached to a Statham strain-gauge pressure transducer (P23 XL, Spectra-Med) was inserted through the mitral valve into the LV for measurement of LV pressure (LVP). The balloon volume was adjusted to set the LV end-diastolic pressure to 8-10 mmHg; balloon volume was not changed during the experiment. To define isovolumic contractile performance, we used the LV developed pressure (the difference between systolic and diastolic pressures), rate-pressure product (RPP; product of LV developed pressure and heart rate), and the first derivative of the pressure development (dP/dtmax).Measurement of MO2
31P NMR Spectroscopy
ATP content in the whole heart was measured using 31P NMR spectroscopy. Guinea pig hearts were placed in a 20-mm NMR sample tube and inserted into an Oxford 9.4-T superconducting magnet connected to a GE-400 Omega spectrometer (Freemont, CA). 31P NMR spectra were collected with the resonance frequency of 161.94 MHz on the GE-400. Spectra were obtained over a 4-min period by signal averaging 104 scans with a pulse width of 27 µs, pulse angle of 60°, recycle time of 2.14 s, and sweep width of 6,000 Hz. Individual free induction decays were zero filled and weighed with a 20-Hz line broadening decaying exponential before Fourier transformation.In aerobic tissue, ATP synthesis occurs predominantly in the
mitochondria through oxidative phosphorylation. In the intact tissue,
-P resonances of mitochondrial and cytosolic ATP are expected to
have approximately the same chemical shift. Consequently, irradiating
the ATP- resonance position during 31P NMR magnetization
transfer measurement will saturate both the mitochondrial and the
cytosolic ATP- spins. In this case, the rate determined by NMR is
primarily the rate of incorporation of cytosolic Pi into
mitochondrial ATP. In previous studies (4, 15, 16, 18,
19), it has been demonstrated that changes in the rate of ATP
synthesis estimated from 31P NMR magnetization transfer
were in good agreement with changes in the rates calculated from
O2 consumption.
In the current study, ATP synthesis rates were measured using the
two-site saturation transfer technique, saturating the [-P]ATP resonance and observing changes in the Pi resonance area
(4, 18). Magnetization transfer was performed by applying
a low-power narrow-band radiofrequency pulse (B1
field of 30-35 Hz) at the [-P]ATP resonance for either
0.0 s (M0) or 4.8 s
(M
). For each experiment, the low-power pulse
was calibrated by irradiating the [-P]ATP peak to ensure complete
saturation, followed by irradiation upfield at a frequency equidistant
from the Pi resonance to ensure that there was negligible
off-resonance saturation of the Pi resonance (data not
shown). Magnetization transfer spectra were obtained by signal
averaging 192 scans of high-power broad-band 90° read pulses (41 µs) after the saturation pulse, each separated by a constant delay of
7 s, including the saturation pulse time.
M0 and M spectra were
collected in an interleaved fashion: 24 free induction decays of each
type were collected, and the final spectra were obtained by signal
averaging eight of these cycles. A complete saturation transfer
measurement was acquired in ~50 min.
Experimental Protocols
Protocol 1: the effect of eNO on
MO2.
CPP, isovolumic contractile performance, and coronary perfusate
effluent O2 tension were monitored simultaneously.
L-NNA dissolved in H2O was introduced (at a
rate of 1% coronary flow) via a fine cannula into the aortic perfusion
catheter; the final concentration of L-NNA supplied to the
heart was 5 × 105 mol/l. One-half of the guinea pig
hearts were randomly selected as control; for these hearts,
H2O only was supplied via the cannula. Baseline data were
collected before and after treatment with either L-NNA or
H2O. One of the following experiments was then performed: 1) Cardiac performance was changed by varying the perfusate
[Ca2+]. Initially, the perfusate [Ca2+] was
switched from a baseline value of 1.75 to 1.25 mmol/l to decrease
cardiac performance; the perfusate [Ca2+] was then
increased in steps of 0.5 or 1.0 mmol/l from 1.25 to 5.75 mmol/l to
increase cardiac performance. Hearts were perfused at each
[Ca2+] for 5 min, during which time they achieved steady
state in terms of LV performance. 2) The heart was arrested
by increasing the [KCl]. The [KCl] in the perfusate was increased
to 15 mmol/l to arrest the heart, and the high KCl perfusate perfusion
was maintained for 10 min to reach a steady state in terms of
MO2 and perfusion pressure. The
perfusate was then switched back to the regular perfusate for another
15 min for assessment of recovery.
Protocol 2: the effect of eNO on the ATP synthesis rate.
Guinea pig hearts were randomly divided into L-NNA- and
vehicle-treated groups. CPP, isovolumic contractile performance, and 31P NMR spectroscopy were measured simultaneously. After
stabilization, baseline one-pulse spectra were acquired before and 15 min after perfusion with L-NNA (5 × 105
mol/l) for the L-NNA-treated group or H2O for
the vehicle-treated group. Isovolumic contractile performance was then
increased by increasing the perfusate [Ca2+] to 4 mmol/l,
and the magnetization transfer experiment was done. Additional
one-pulse spectra were acquired immediately before and after the
magnetization transfer experiment.
Data Analysis
MO2 (in
µmol · min1 · g dry wt1)
was calculated according to the following formula: (perfusate
PO2 difference across the heart) × (solubility of O2/mmHg) × (coronary flow)/ (dry
heart wt). The ratio of MO2 to
RPP was calculated to represent mechanical efficiency.
The first step in determining the cytosolic concentrations of
Pi, phosphocreatine (PCr), and ATP was to normalized the
absolute resonance area corresponding to [
-P] ATP in the
31P NMR spectra by heart weight. We made the assumption
that the fractional volume of intracellular H2O in the
myocytes of hearts of both groups are similar and equal to values
typical of the well-perfused rodent heart (0.48 µl/mg wet wt). In
this case, area units per milligrams of wet weight are directly
proportional to the absolute intracellular concentrations. The value of
10 mM for [ATP] was used to calibrate the [-P]ATP peak areas of the 31P NMR initial baseline spectrum (2).
[Pi] and [PCr] were calculated by multiplying the ratio
of their resonance peak areas to the [-P]ATP peak area of the
initial baseline spectrum by 10 mmol/l. Intracellular pH was determined
by comparing the chemical shift of Pi and PCr to values
from a standard curve.
Magnetization transfer measurement of the unidirectional velocity of
the reaction Pi
ATP was analyzed according to the
two-site chemical exchange model of Forsen and Hoffman
(8), providing estimates of the pseudo first-order rate
constant (Kfor) for the synthesis of ATP and the
intrinsic longitudinal relaxation time for Pi
(T1). Because values for
T1 were indistinguishable between L-NNA- and vehicle-treated hearts, magnetization transfer
experiments with saturation times of 0 s
(M0) and 4.8 s
(M) were performed using the measured
T1 value (2 s) for Pi to calculate
Kfor
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(1) |
ATP was
calculated by multiplying the rate constant Kfor
by the substrate [Pi].
Statistical Analyses
Data are expressed as mean ± SE. A paired Student's t-test was used to compare differences in vascular resistance and O2 consumption before and after L-NNA or H2O treatments at baseline. An unpaired Student's t-test was used to compare differences in L-NNA- and vehicle-treated groups. Measurements made sequentially (e.g., during high cardiac working performance) were compared by ANOVA for repeated measures, and Tukey post hoc test for multiple comparisons was used. The relationship between MO2 and RPP was fitted with an
exponential curve. All statistical analyses were performed with the use
of StatView (Brainpower), and changes were considered significant at
the P < 0.05 level.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Increase in MO2 and Reduction of
Cardiac Mechanical Efficiency After L-NNA
O2, L-NNA was supplied to
inhibit the production of eNO in isolated hearts, and
MO2 was then measured as a function of
contractile performance.
L-NNA increased MO2 at
baseline.
The responses of coronary vessels, contractile performance, and
MO2 of hearts supplied with either
L-NNA or vehicle at baseline are summarized in Table
1. Compared with vehicle, after 15 min of
treatment, there was an increase in coronary vascular resistance (by
21 ± 3%, P < 0.05) in the
L-NNA-treated group, showing that supplying
L-NNA inhibited eNO synthesis in the guinea pig heart. There was an increase in MO2 by 12 ± 2.6% (P < 0.05) in the L-NNA-treated
group but no change in the vehicle group. Isovolumic contractile
performance, assessed as RPP, slightly decreased with time and was not
different between the groups. Mechanical efficiency, represented by the
MO2-to-RPP ratio, increased by 23 ± 2% (P < 0.05) in the L-NNA-treated
group, but, again, there was no change in the vehicle-treated group.
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L-NNA increased MO2 in
KCl-arrested hearts.
The coronary vascular response and MO2
measured in the KCl-arrested heart are shown in Fig. 1.
Increasing the [KCl] in the perfusate from 4.7 to 15 mmol/l led to a
cessation in cardiac mechanical work. The coronary vascular resistance
increased by 41 ± 5% in the KCl-arrested heart supplied with
L-NNA, and this increase in coronary vascular resistance
was reversible on withdrawal of KCl. In contrast, there was
no significant change in the coronary vascular resistance in the
vehicle group (Fig. 1A).
MO2 was higher (by 87%) in the
L-NNA-treated group (5.6 ± 0.5 µmol · min1 · g dry wt1,
P < 0.05; Fig. 1B) than in the
vehicle-treated group (3.0 ± 0.4 µmol · min1 · g dry wt1).
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L-NNA increased MO2
during lower and higher levels of cardiac performance.
Values for coronary vascular resistance and
MO2 as a function of contractile
performance altered by changing the perfusate [Ca2+] in
both L-NNA- and vehicle-treated groups are shown in Fig. 2. Coronary vascular resistance in the
L-NNA-treated group was significantly higher than that in
controls supplied with vehicle; this increase persisted during the
entire study.
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O2 and the ratio of
MO2 to RPP were higher in the
L-NNA-treated hearts than for the vehicle-treated hearts
(MO2: 20.7 ± 1.0 vs. 15.5 ± 1.0 µmol · min1 · g dry
wt1 and MO2-to-RPP ratio:
1.74 ± 0.12 vs. 1.41 ± 0.04, respectively, both
P < 0.05).
Increasing the perfusate [Ca2+] from 1.25 to 5.75 mmol/l
resulted in gradually increasing contractile performance and,
concomitantly, increasing MO2 in both
groups. When the perfusate [Ca2+] reached 5.75 mmol/l, LV
developed pressure increased to 104 ± 4 mmHg (by 72 ± 15%)
and to 104 ± 3 mmHg (by 76 ± 12%) in the L-NNA- and vehicle-treated groups, respectively, with only
small increases in heart rate. dP/dtmax
increased to 3,222 ± 167 mmHg/s (by 93 ± 7%) and to
3,093 ± 182 mmHg/s (by 98 ± 14%), and RPP increased to
25.5 ± 0.9 × 103 mmHg/min (by 116 ± 17%)
and to 23.1 ± 1.1 × 103 mmHg/min (by 114 ± 25%), respectively, in the L-NNA- and vehicle-treated groups. Thus contractile performance, estimated from LV developed pressure, RPP, and dP/dtmax, was not different
between groups. In contrast, MO2 and the
ratio of MO2 to RPP were higher in the
L-NNA-treated hearts than the vehicle-treated hearts
(MO2: 38.6 ± 2.6 vs. 29.1 ± 0.8 µmol · min1 · g dry
wt1 and MO2-to-RPP ratio:
1.51 ± 0.81 vs. 1.27 ± 0.06, respectively, both
P < 0.05).
The MO2 at each level of cardiac
contractile performance was plotted against RPP. Figure
3 shows the
MO2-RPP relationships fitted to
exponential equations. The results show that the relationship for the
L-NNA-treated group was shifted upward.
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O2 at all levels of cardiac contractile
performance, including cardiac arrest, was significantly increased
after inhibition of eNO synthesis with L-NNA.
No Change in Myocardial ATP Content and Synthesis Rate After L-NNA
To determine whether the increase in MO2 caused by inhibiting eNO synthesis
increased the myocardial ATP synthesis rate, we used 31P
NMR spectroscopy to measure the myocardial ATP content and ATP synthesis rate during high levels of contractile performance in guinea
pig hearts after L-NNA and vehicle treatment.
L-NNA has no effect on myocardial ATP content.
At baseline, the ATP resonance areas for hearts in the two groups were
indistinguishable (L-NNA 37.6 ± 1.6 vs. vehicle
36.0 ± 2.1 area units/mg wet wt). The mean values for metabolite
concentrations, including [ATP], [PCr], and [Pi] and
intracellular pH measured or calculated from 31P NMR
spectroscopy, as well as indexes of cardiac performance at baseline and
during high work states in L-NNA- and vehicle-treated groups, are summarized in Table 2.
Compared with vehicle, L-NNA treatment did not cause
significant changes in [ATP], [PCr], [Pi], intracellular pH, and cardiac performance except for an increase in
coronary vascular resistance.
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L-NNA did not change myocardial ATP synthesis rate.
Figure 4 shows representative
31P magnetization transfer spectra obtained during high
work states induced by increasing the perfusate [Ca2+].
The change in the resonance areas of Pi is proportional to the rate constant for the reaction Pi ATP. Note that, in
Fig. 4, bottom (showing a magnification of the
Pi region in the spectrum including phosphomonoesters,
Pi, and phosphodiesters resonances), only the
Pi resonance area changed when the -P of ATP was
selectively saturated.
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1, P > 0.05) and fluxes
(L-NNA 3.8 ± 0.7 vs. vehicle 3.5 ± 0.4 mM/s,
P > 0.05) for the reaction Pi ATP were
indistinguishable between the L-NNA- and vehicle-treated
groups (Fig. 5).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The important findings of the current study are as follows:
1) MO2 was significantly
elevated after inhibition of eNO synthesis by L-NNA in the
isolated guinea pig heart at all levels of contractile performance
ranging from arrest to high levels of isovolumic contractile performance; and 2) despite the increase in
MO2, neither the [ATP] nor the ATP
synthesis rate measured using 31P magnetization transfer
differed. Thus these results provide the first direct evidence
suggesting that eNO plays an important role modulating myocardial
metabolic efficiency by enhancing both the coupling between
MO2 and cardiac performance and the
coupling between MO2 and ATP synthesis.
Choice of Experimental Model
The question of whether eNO is involved in the regulation of O2 consumption in the heart has not yet been resolved. With the use of an in vivo heart preparation, some investigators have concluded that MO2 increases
after inhibition of eNO synthesis (3), whereas others have
concluded the opposite (25, 29). With the inhibition of
eNO synthesis, peripheral vasoconstriction and hypertention cause
activation of many cardiovascular reflexes, which in turn leads to
changes in cardiac preload, afterload, heart rate, and contractility
(3, 26-28). These cardiovascular responses have a
direct impact on MO2. Moreover, the
inhibition of eNO synthesis resulted in a significant change in
myocardial substrates selection and utilization, i.e., increases
glucose uptake and decrease in fatty acid uptake (3, 24),
which also affect MO2. Thus these
conflicting reports from in vivo studies are likely due to the
difficulties of controlling the complex experimental condition
associated with the cardiovascular responses and myocardial selective
substrate utilization after inhibition of eNO production. We used the
isolated isovolumic heart preparation in the current study. Using the
isolated heart allowed us to maintain a constant flow, cardiac preload,
and afterload throughout the experiment, thereby limiting their
influence on MO2 both before and after
inhibition of eNO. In addition, it has recently been reported that
inhibition of eNO resulted in a reduction of utilization of fatty acid
with a concomitant increase in utilization of glucose and lactate in
myocardial tissue (3, 24). This change in myocardial
substrate selection could lead to underestimate of the effect of NO on
MO2. To limit any potential impact on
MO2 due to a change in myocardial
substrate selection, we chose glucose as the sole exogenous substrate
for the heart in the current study.
Inhibition of eNO Synthesis by L-NNA
L-NNA, a L-arginine analog, is a potent and effective inhibitor of NO synthase (10, 22). It has been shown that L-NNA exerts an inhibitory effect on coronary endothelium-dependent vascular relaxation in response to acetylcholine and bradykinin in guinea pig hearts (6, 13). In the current study, the perfusion pressure and coronary vascular resistance were monitored and used as a bioassay to test whether eNO was inhibited by L-NNA in our experimental preparation. Consistent with literature reports, L-NNA resulted in a significant increase in coronary vascular resistance in the isolated guinea pig hearts studied here, showing that supplying L-NNA inhibited eNO synthesis. We also observed a reversible coronary vasoconstriction in the L-NNA-treated KCl-arrested hearts. The vasoconstriction induced by a high [K+] is most likely due to the depolarization of the vascular smooth muscle membrane resulting from a high [K+], leading to increase intracellular Ca2+. The high [K+]-related vasoconstriction was completely compensated for by NO in the vehicle-treated group but not in the L-NNA-treated group. This provides further evidence that eNO synthesis was significantly inhibited by L-NNA.NO and MO2
O2 at all levels of
work studied, ranging from KCl arrest to high work states. Furthermore,
the MO2-cardiac performance curve was
shifted upward without a change in slope. Because the increase in
MO2 was similar in arrested and beating hearts, ~2-2.6 µmol · min1 · g
dry wt1, these results indicate that the increased
MO2 after inhibition of eNO synthesis is
not related to contractile performance. This observation is important
because it suggests that the mechanism by which NO changes
MO2 in the intact heart is a
direct biochemical effect on the tissue. Our results also suggests that
there is sufficient eNO produced in the heart to have a measurable
effect on MO2. If so, then NO is likely
to have an important regulatory effect on
MO2 in vivo.
The work of others has identified the likely molecular targets of NO. In 1982, Granger and Lehninger (12) showed that a substance produced from activated macrophages had an inhibitory effect on cellular O2 consumption. Subsequently, Hibbs et al. (7, 11) confirmed that NO was the agent responsible. The mechanism for reduced O2 consumption is inhibition of the activity of a number of mitochondrial enzymes, including aconitase and complex I and complex II in the electron transport chain (7, 11, 12). Recently, it has been reported that a low (nanomolar) concentration of NO specifically and reversibly inhibits cytochrome oxidase in competition with O2, indicating that the NO inhibition of cytochrome oxidase may be involved in the physiological and/or pathological regulation of tissue respiration rate and its affinity for O2 (5, 31).
Peroxinitrate, formed from the interaction of NO with superoxide, has also been shown to promote suppression of respiration (23, 34). However, the production of peroxinitrate is associated with a high cellular concentration of NO and superoxide, which contribute to inhibiting respiration in pathological conditions related to inflammatory processes or to hypoxia/reoxygenation and ischemia-reperfusion (33, 34). Unlike eNO, the inhibition of cellular respiration by peroxinitrate is not reversible. Thus it is unlikely to act as a potent physiological regulator for the tissue O2 consumption and mitochondrial function.
NO and the ATP Synthesis Rate
In our study, the inhibition of eNO synthesis resulted in a higher MO2 for the heart to accomplish the same
level of contractile performance as the control heart with normal eNO
synthesis. This observation raises two possibilities regarding ATP
synthesis and utilization: 1) the increase in
MO2 could reflect an increased ATP
synthesis rate in response to an increased ATP utilization, or
2) if the myocardium requires the same amount of ATP to
accomplish same amount of work, then the increase in
MO2 after inhibition of eNO indicates a
partial uncoupling of mitochondrial oxidative phosphorylation. Here, we
tested the first possibility. In the current study, ATP content was
determined by standard one-pulse 31P NMR techniques, and
the ATP synthesis rate was estimated from the unidirectional flux for
the reaction Pi ATP using magnetization transfer
techniques. It should be pointed out that a reliable measurement of ATP
synthesis by 31P magnetization transfer can be made only
for hearts at moderate to high levels of cardiac performance. This is
because the [Pi] in hearts at low levels of performance
is too small to be accurately measured (4). Additional
aspects of the experimental design used here provide confidence that we
have not underestimated or missed an increase in
MO2. These include the use of glucose as
the sole exogenous substrate to further maximize the Pi
area and the observation that the areas of the two resonances on either side of the Pi peak changes by <10% during the
magnetization transfer experiment.
We found that infusion of L-NNA resulted in a significant
elevation of MO2 without changes in
either the ATP content or the ATP synthesis rate. Thus the myocardial
P:O ratio decreased after inhibition of eNO synthesis. This indicates
an uncoupling of oxidative phosphorylation or a change of the
carbon-based substrate utilized. It is well known that switching
metabolic substrate selection from glucose to fatty acid could result
in a decrease in the myocardial P:O ratio (24, 30, 36).
However, in our model, this seems highly unlikely because 1)
glucose was the sole exogenous carbon substrate used, 2) the
endogenous fatty acids were consumed relatively rapidly (in 30-60
min) in isolated perfused (glucose only) rodent hearts (Odiet JA,
unpublished data), and 3) the contribution of the glycolysis
to the Pi to ATP unidirctional velocity is likely to be
small (15). Thus these results provide the first
evidence suggesting that eNO affects the coupling of MO2 and the ATP synthesis rate. We can
only speculate as to the fate of the increased
MO2. On the basis of previous
observations that body temperature was increased after inhibition of
eNO in conscious dogs (27), it is possible that the
increased O2 consumption is associated with tissue heat
production. The underlying mechanisms are not known.
NO and Metabolic Efficiency
Compared with hearts with normal NO production, for the hearts treated with L-NNA, the relationship between MO2 and RPP shifted upward, without a
change in the relationship between the rate of ATP synthesis
(Pi ATP) and RPP. This indicates that the elevation of
MO2 for a given RPP could be attributed
entirely to the effect of eNO (direct or indirect) on
mitochondria. Thus inhibition of eNO resulted in decreased myocardial
metabolic efficiency, including both coupling between
MO2 and cardiac performance (MO2-to-RPP ratio) and coupling
of MO2 and the ATP synthesis rate.
Endothelial NO synthase is present in coronary arterial, venous, and capillary endothelial cells in the normal heart (1, 32). There is a tonic production of NO from vascular endothelial cells in response to stimulation from shear stress when circulating blood flows across the surface of vascular endothelial cells in vivo (14). When this occurs in capillaries, the parenchymal cells are so close to capillaries that NO is likely to have actions on nonvascular cells in the microcirculation. It has been shown that, in canine skeletal muscle and myocardium, a NO-dependent inhibition of respiration is elicited by activation of muscarinic cholinergic and bradykinin B2 receptors, which are thought to be primarily localized on this cell type (26, 35). Thus NO acts as a paracrine signal released from the vascular endothelium to regulate adjacent cellular respiration and enhance myocardial metabolic efficiency.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Ralph A. Kelly for stimulating discussion and to Dr. Jeffrey Odiet for sharing unpublished results.
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FOOTNOTES |
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This study was supported by National Heart, Lung, and Blood Institute Grants HL-09669 (to W. Shen), HL-09259 (to K. W. Saupe), and HL-52350 (to J. S. Ingwall) and by a Deutsche Forschungsgemeinschaft Research Fellowship (to M. Spindler).
Present address of K. W. Saupe: Cardiac Muscle Research laboratory, Boston Univ. School of Medicine, 650 Albany St., Boston, MA 02118.
Present address of M. Spindler: Medizinische Universitaetsklinik, Josef-Schneider-Strasse 2, 97080 Wuerzburg, Germany.
Present address and address for reprint requests: W. Shen, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285.
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.
Received 17 April 2000; accepted in final form 18 April 2001.
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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, and ATP-