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Am J Physiol Heart Circ Physiol 280: H2350-H2356, 2001;
0363-6135/01 $5.00
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Vol. 280, Issue 5, H2350-H2356, May 2001

Nitric oxide reduces energy supply by direct action on the respiratory chain in isolated cardiomyocytes

Thomas Stumpe, Ulrich K. M. Decking, and Jürgen Schrader

Department of Physiology, Heinrich-Heine University, D-40225 Düsseldorf, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

To investigate the effect of nitric oxide (NO) on cardiac energy metabolism, isolated cardiomyocytes of Wistar rats were incubated in an Oxystat system at a constant ambient PO2 (25 mmHg) and oxygen consumption (VO2); free intracellular Ca2+ (fura 2), free cytosolic adenosine [S-adenosylhomocysteine (SAH) method], and mitochondrial NADH (autofluorescence) were measured after application of the NO donor morpholinosydnonimine (SIN-1). In Na+-free medium (contracting cardiomyocytes), VO2 increased from 7.9 ± 1.2 to 26.4 ± 3.1 nmol · min-1 · mg protein-1. SIN-1 (100 µmol/l) decreased VO2 in contracting (-21 ± 3%) and in quiescent cells (-24 ± 7%) by the same extent. Inhibition of VO2 was dose dependent (EC50: 10-7 mol/l). S-nitroso-N-acetyl-penicillamine, another NO donor, also inhibited VO2, whereas SIN-1C (100 µmol/l), the degradation product of SIN-1, displayed no inhibitory effect. Intracellular Ca2+ remained unchanged, and inhibition of protein kinases G, A, or C did not antagonize the effect of NO. Mitochondrial NADH increased with NO, indicating a reduced flux through the respiratory chain. In quiescent but not in contracting cardiomyocytes, NO significantly increased adenosine, indicating a reduced energy status. These data suggest the following. 1) NO decreases cardiac respiration, most likely via direct inhibition of the respiratory chain. 2) Whereas in quiescent cardiomyocytes the inhibition of aerobic ATP formation by NO causes reduction in energy status, contracting cells are able to compensate for the NO-induced inhibition of oxidative phosphorylation, maintaining energy status constant.

oxygen consumption; NADH; adenosine; Oxystat system; calcium


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

NITRIC OXIDE (NO), in addition to its vasodilatory action, is known to modulate myocardial contractile function. Early in vivo studies (26, 41) revealed a positive inotropic action of organic nitrates on the ventricular myocardium. The interpretation of in vivo studies is difficult, because vasodilatation alters cardiac loading conditions and peripheral and vascular tone and therefore indirectly influences cardiac performance. However, when the regional effect of organic nitrates and spontaneous NO-generating drugs were tested, a small flow-independent positive inotropic effect was demonstrated in the in vivo dog heart (31).

Negative inotropic effects of NO have been reported for cardiomyocytes (2, 4, 11) and isolated perfused hearts (15, 23) and under in vivo conditions after stimulation with isoproterenol (17). An increase in cGMP was proposed as a possible mechanism (28). This is supported by the finding that bromo-cGMP, a cell-permeable cGMP analog, induces a negative inotropic effect (4, 17, 29) and that NO is known to activate soluble guanylate cyclase (1). Furthermore, studies performed in cardiomyocytes after endotoxin shock (2, 10, 22) or with electrical stimulation (19) found an increase in cGMP. Recently, a biphasic inotropic effect of cGMP was reported: In the lower concentration range, cGMP augments cardiac contractile force, whereas at higher concentrations, the negative inotropic effect prevails (25, 28).

However, a cGMP-independent effect of NO on contractility was also shown by several investigators, e.g., in the perfused heart (21), papillary muscle preparation (46), or isolated cardiomyocytes (34). In these studies, a direct inhibitory effect of NO on cardiac energetics was proposed. In line with this interpretation, studies in isolated mitochondria have shown that NO exerts a direct inhibition of the respiratory chain (6), most likely by inhibition of cytochrome oxidase (14, 37). This inhibition is competitive to the binding of oxygen (5), with a very low inhibitory constant of 27 nmol/l (24). Inhibition of the respiratory chain most likely can explain the cytotoxic effect of NO when released by activated macrophages (9, 18). Additionally, peroxynitrite, the product of the two radical species superoxide (O<UP><SUB>2</SUB><SUP>−</SUP></UP>·) and nitric oxide (·NO), can irreversibly inhibit complex I of the respiratory chain (7, 32). It is conceivable that inhibition of the respiratory chain by NO may be also involved in the negative inotropic effect observed in the in vivo heart (39). In line with this concept, endothelial NO synthase was found to be present in mitochondria (3, 45). Furthermore, evidence for control of oxygen consumption (VO2) by NO in the in vivo heart has recently been shown in chronically instrumented conscious dogs (39, 40, 44).

In this study, the use of an Oxystat system allowed the exact control of ambient PO2 in parallel with the measurement of VO2 and the energy status of contracting cardiomyocytes while NO was administered by the spontaneous NO donors morpholinosydnonimine (SIN-1) or S-nitroso-N-acetyl-penicillamine (SNAP). PO2-related effects on contractility or VO2 can be excluded in this system. Furthermore, the parallel determination of VO2, NADH, and energy status allowed us to identify whether a decrease in VO2 was secondary to a fall in contractile activity (constant energy status and NADH) or directly due to inhibition of the respiratory chain (possible decreased energy status and increased NADH).


    MATERIAL AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

Incubation of Cardiomyocytes in the Oxystat System

The cardiomyocytes of Wistar rats were prepared as described previously (42). After the preparation, the cell pellet was resuspended in a Tris buffer [containing (in mmol/l) 137 NaCl , 5.4 KCl , 1.0 NaH2PO4, 0.8 Mg2SO4, 2.0 CaCl2, 5.5 glucose, and 5.0 Tris; pH 7.4] or in a Na+-free medium, forcing the cells to periodically contract due to intracellular calcium elevation (42). In this setting, the organic cation choline served as a counter ion within a Tris-buffered Krebs-Henseleit buffer [containing (in mmol/l) 139 choline chloride, 4.3 KHCO3, 1.0 KH2PO4, 1.6 MgCl2, 5.0 Tris, 11 glucose, and 2.0 CaCl2]. Ca2+ elevation was comparable to the increase of mean intracellular Ca2+ when cardiomyocytes were electrically stimulated with a frequency of 9 Hz.

The isolated cardiomyocytes were incubated in the Tris buffer or in the Na+-free medium at 37°C in a continuously stirred chamber, the so-called Oxystat system (Hugo Sachs Electronic; March-Hugstetten, Germany) (42). In this feedback control system, a PO2 electrode (Eschweiler; Kiel, Germany) serves as the sensor. The PO2 is transmitted to a control unit, which reads the sensor signal and compares it to a preselected PO2 value (Impulsomat 614, Metrohm; Herisau, Switzerland). If the actual PO2 is below the preselected value, the control unit activates a motor-driven burette (Dosimat 665, Metrohm), which pumps oxygen-rich medium (equilibrated with air) into the chamber. When chamber PO2 again reaches the preselected value, infusion is stopped. Thereby, ambient chamber PO2 is maintained in a steady state close to the preselected PO2.

Experimental Protocol

Four different protocols were performed 1) to measure VO2 and the energy status of contracting and quiescent cardiomyocytes after addition of NO, 2) to determine the effect of inhibition of protein kinases on VO2 during NO application, and 3) to investigate the free intracellular calcium concentration (free [Ca2+]i) or 4) the mitochondrial NADH when NO was added. To measure VO2 and energy status, isolated cardiomyocytes were incubated in the Oxystat system at constant ambient PO2 of 25 mmHg. Quiescent cardiomyocytes were incubated in a Tris-buffered Krebs-Henseleit solution (see Incubation of Cardiomyocytes in the Oxystat System), and, in parallel experiments, contraction was induced by the Na+-free buffer. A bolus of the NO-generating substance SIN-1 or SNAP, respectively, was added after 6 min of control equilibrium. VO2 was determined throughout the experiments. Samples of cell suspension were withdrawn from the Oxystat via a sideport for the measurement of S-adenosylhomocysteine (SAH) and cellular protein content at the end of control and SIN-1 incubation. In parallel experiments, inhibitors of protein kinase G (PKG; inhibitor KT5823, 2 µmol/l), protein kinase A (PKA; inhibitor H-89, 1 µmol/l), and protein kinase C (PKC; inhibitor calmoduline, 0.2 µmol/l) were given after 6 min of incubation with SIN-1. For a description of the experimental protocols of the measurements of free [Ca2+]i and mitochondrial NADH, please refer to Analytical Procedures.

Analytical Procedures

Oxygen consumption. Continuous recordings of the chamber PO2 and the volume of the medium supplied to maintain the PO2 were used for the calculation of the VO2 of the isolated cardiomyocytes according to the following equation
<A><AC>V</AC><AC>˙</AC></A><SC>o</SC><SUB><IT>2</IT></SUB><IT>=</IT>[(V<IT>×</IT>S<SUB>i</SUB>)<IT>−</IT>(V<IT>×</IT>S<SUB>e</SUB>)]<IT>/</IT>mg protein (1)
where V is the volume added or released and Si and Se are the oxygen saturations of the infused and released medium, respectively, corrected for the actual cellular protein (42).

Fluorometric measurement of free intracellular Ca2+. Freshly isolated cardiomyocytes were incubated with 5 µmol/l fura 2-acetoxymethyl ester for 30 min in the dark at room temperature and gassed with oxygen. After fura 2 was loaded, cells were briefly washed by centrifugation (30 s, 10 g). The fluorescence of fura 2 was measured (Perkin-Elmer LS 5B) in a stirred 3-ml cuvette at an excitation wavelength switched between 340 and 380 nm and an emission wavelength of 509 nm. Data were collected every second for a 20-min interval and stored in a personal computer with the use of the program Fura2 (Perkin-Elmer). Fura 2 fluorescence of contracting cardiomyocytes stimulated with Na+-free medium were collected for 400 s. Subsequently, SIN-1 was added, with end concentrations of 1, 10, and 500 µmol/l, respectively, and data collection was continued. The free [Ca2+]i was calculated with the use of the following equation (16)
Free [Ca<SUP>2+</SUP>]<SUB>i</SUB><IT>=</IT>(F<IT>−</IT>R<SUB>min</SUB>)<IT>/</IT>(R<SUB>max</SUB><IT>−</IT>F)<IT>×</IT>sfb<IT>×K</IT><SUB>d</SUB> (2)

where F is the ratio of the fluorescence at an excitation wavelength of 340/380 nm, Rmax is the fluorescence ratio in the presence of 0.5% Triton X-100 and 2 mmol/l Ca2+, and Rmin is the respective value with 50 mmol/l EGTA. Kd (135 nmol/l) is the dissociation constant of Ca2+ bound to fura, and sfb is the ratio of fluorescence values at 380 nm with and without EGTA.

Free cytosolic adenosine. We determined the free cytosolic adenosine with the SAH method described by Deussen et al. (8). Cardiomyocytes were incubated in the Oxystat system with 0.4 mmol/l homocysteine thiolactone. Under this condition, the equilibrium of the SAH hydrolase is shifted towards synthesis, and the rate of SAH accumulation is proportional to the free cytosolic concentration of adenosine as follows
adenosine<IT>+</IT>homocysteine <LIM><OP><ARROW>↔</ARROW></OP><UL>SAH hydrolase</UL></LIM> SAH (3)
The formed SAH is not further metabolized and cannot pass the sarcolemmal membrane, so that SAH accumulates intracellularly. Samples (0.8 ml) were withdrawn from the Oxystat after equilibration and after the bolus injection of SIN-1 (see above).

To analyze the cellular content of SAH, the samples were immediately layered on 1 ml of ice-cold 1-bromododecane and centrifuged into 100 µl of 2 mol/l HClO4. With the use of this procedure, it was possible to separate cells from supernatant in <45 s (42). For the intracellular measurements, the perchloric acid extract was neutralized with 1 mol/l K3PO4, centrifuged, and used for HPLC analysis.

Protein content in the pellet was measured according to Lowry, and the concentration of SAH was analyzed using a HPLC method according to Stumpe and Schrader (42). In brief, samples were injected on a reverse-phase C18 column (Bondapak, 10 µm, Waters), which was equilibrated with an ammonium acetate buffer (26 mmol/l; pH 5.0) containing 5% (vol/vol) methanol. Nucleosides were eluted using a concave methanol (70%, vol/vol) gradient, and peaks were monitored using an ultraviolet detector at 254 nm.

Mitochondrial NADH. Autofluorescence at an excitation and emission wavelength of 360 and 460 nm, respectively, is known to be predominantly caused by mitochondrial NADH (38). In the Oxystat system, autofluorescence was measured using a light guide system directly connected to the Oxystat chamber. A fluorimeter (Perkin-Ellmer, LS 50B) provided a constant excitation of the cells at 360 nm. Emission was measured through a different fiber system at wavelengths varied between 390 and 500 nm. The scans were registered by a personal computer using the software of the manufacturer.

To improve sensitivity, autofluorescence was measured in a separate experimental series at a constant emission wavelength of 480 nm using a modular fluorescence detection system by Oriel (Stratford, CT). This setup omits the sensitivity decrease of the diffraction grating as it is used in a multiwavelength detection system. For this setup, a light guide with a superior ultraviolet transmittance, optimized for both wavelengths in this setup, was used to illuminate the cardiomyocytes directly in the Oxystat chamber. The light source was a 100-W Hg arc lamp (using the specific irradiance peak at 365 nm of this lamp type) with a condenser system to focus the light in the Oxystat chamber. Another light guide was used to register the fluorescence with an end-on photomultiplier tube. An interference filter of 360 nm defined the wavelength of the excitation light, and a filter of 480 nm defined the excitation wavelength (both filters had bandwidths of 10 nm). Data of the photomultiplier were registered by a personal computer (program DTVEE). Because the emission scans demonstrated a clear NADH signal at an emission wavelength of 480 nm, the latter setup could be used and achieved a more than 10-fold higher sensitivity. After 6 min of control equilibrium, SIN-1 (100 µmol/l) was added. After another 6 min of incubation, carbonylcyanid-3-chlorophenylhydrazon (CCCP; 10 µmol/l), a potent decoupler of the respiratory chain, was added. Decoupling of the respiratory chain caused a dramatic increase in the VO2 of the cardiomyocytes, and the fluorescence signal was decreased by more than 95%, indicating that the total NAD in the cells is oxidized. The remaining fluorescence signal is therefore caused by fluorescence of the medium or other cellular substances. Autofluorescence data were corrected using this value. Subsequently, 1 mmol/l sodium azide was added to totally block oxidative phosphorylation. Fluorescence signal was increased within 2 min to a maximum value, indicating that produced NADH was not further oxidized in the respiratory chain. The corrected autofluorescence data (decoupler) were shown as a percentage of the maximum fluorescence (inhibition of the respiratory chain).

Data Analysis

Data are expressed as means ± SD. To compare groups of experimental data, Student's paired t-tests were used to compare control and experimental intervention. P values of <0.05, <0.01, and <0.001 were considered to be significantly different.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

Isolated rat cardiomyocytes were incubated in the Oxystat system, which allowed the simultaneously measurement of VO2 and energy status at a constant ambient PO2 of 25 mmHg. NO was added using the spontaneously NO-generating substances SIN-1 or SNAP, respectively.

The VO2 of quiescent cardiomyocytes was determined to be 7.9 ± 1.2 nmol · min-1 · mg protein-1 (n = 6). Stimulation of cardiomyocytes using Na+-free medium increased the VO2 threefold to 26.4 ± 3.1 nmol · min-1 · mg protein-1 (n = 10). As shown in Fig. 1, SIN-1 at a final concentration of 100 µmol/l significantly decreased VO2 to 6.2 ± 0.9 and 20.1 ± 2.6 nmol · min-1 · mg protein-1 in quiescent and contracting cardiomyocytes, respectively (n = 6 and 10 separate myocyte preparations, P < 0.001). This corresponds to an inhibition of 21 ± 8% in the case of quiescent cardiomyocytes and 24 ± 7% in contracting cells. The reduction of VO2 by 100 µmol/l SIN-1 was reversible when the cells were reperfused for at least 45 min. The dose-response curve of the inhibitory action of SIN-1 showed that maximal inhibitory effect was obtained at a concentration of ~10 µmol/l. The calculated half-maximal reduction in VO2 by SIN-1 was at ~100 nmol/l (Fig. 2).


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  		Fig. 1.   Oxygen consumption (VO2) of quiescent (left) and contracting cardiomyocytes (right) after addition of morpholinosydnonimine (SIN-1; 100 µmol/l). The VO2 of contracting cardiomyocytes was increased threefold (different scale of the axis). Open bars, without SIN-1; hatched bars, with SIN-1. Results are expressed as means ± SD; n, number of preparations. ***P < 0.001.



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  		Fig. 2.   Dose-response curve of the inhibitory effect of SIN-1 on the VO2 of contracting cardiomyocytes. The half-maximal effect of SIN-1 at a concentration of ~100 nmol/l is shown. Results are expressed as means ± SD; numbers in parentheses are numbers of preparations.

To test whether the observed reduction of VO2 is due to NO generated by SIN-1 or by unspecific effects of the agent itself, contracting cardiomyocytes were incubated with SIN-1C, the breakdown product of SIN-1. As shown in Table 1, SIN-1 at 100 µmol/l reduced VO2 by 24 ± 7% (n = 12), whereas SIN-1C at the same concentration did not significantly alter VO2. However, SIN-1C at 500 µmol/l decreased VO2 by 18 ± 3% (P < 0.01, n = 3). Similarly, SNAP, another NO donor, decreased VO2 in a dose-dependent manner. Inhibition at 50 µmol/l SNAP was 24 ± 11% (P < 0.01, n = 5). A comparable effect was obtained when 10 µmol/l bromo-cGMP, a cell-permeable cGMP derivative, was added to the contracting cardiomyocytes; VO2 was reduced by 29 ± 7% (P < 0.001, n = 5).

                              
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  		Table 1.   VO2 of contracting cardiomyocytes as influenced by SIN-1, SIN-1C (SIN-1 after release of NO), SNAP (a different NO donor), and bromo-cGMP

To test whether cGMP or cAMP and Ca2+-related protein phosphorylation might have caused the inhibitory effect of NO on respiration, PKG, PKA, and PKC were blocked by specific inhibitors. Neither inhibition of PKG using KT5823 (2 µmol/l) nor PKA using H-89 (1 µmol/l) nor PKC using calmodulin (0.2 µmol/l) antagonized the inhibitory action of NO on contracting cardiomyocytes (Fig. 3).


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  		Fig. 3.   VO2 of contracting cardiomyocytes as influenced by SIN-1 (100 µmol/l; hatched bar) and additional inhibition of protein kinase G (PKG; inhibitor KT5823, 2 µmol/l), protein kinase A (PKA; inhibitor H-89, 1 µmol/l), and protein kinase C (PKC; inhibitor calmodulin, 0.2 µmol/l). Results are expressed as means ± SD; the numbers in parentheses are numbers of experiments. Open bar, without SIN-1.

Free [Ca2+]i in quiescent cardiomyocytes was found to be 128 ± 37 nmol/l (n = 4). As expected, the mean free [Ca2+]i significantly increased to 260 ± 42 nmol/l (n = 4, P < 0.001) when cells were stimulated with Na+-free medium. SIN-1, however, did not change intracellular Ca2+ of contracting cardiomyocytes at a concentration of 10 or 500 µmol/l, as depicted in Fig. 4.


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  		Fig. 4.   Free intracellular Ca2+ concentration of contracting cardiomyocytes after addition of SIN 1. A: original time trace of a representative experiment. The addition of SIN-1 and end concentrations of the nitric oxide donor are indicated in the graph. B: means ± SD of 4 experiments. Open bar, without SIN-1; hatched bar, with SIN-1.

To test whether NO directly inhibit respiratory chain, the substrate of the respiratory chain, NADH, was measured using a fluorescence method. Figure 5A illustrates the effect of SIN-1 on the emission spectrum of isolated cardiomyocytes in the Oxystat at an excitation wavelength of 360 nm. Autofluorescence decreased by a factor of 10 after stimulation in Na+-free medium. SIN-1 (100 µmol/l) did not change the autofluorescence of stimulated cardiomyocytes; however, it caused a pronounced increase in NADH in quiescent cardiomyocytes. To improve the sensitivity of the fluorescence measurements, the autofluorescence of cardiomyocytes was monitored at a constant emission wavelength. Autofluorescence of quiescent cardiomyocytes was 33.5 ± 8.2% of maximal fluorescence after blocking the respiratory chain. SIN-1 (100 µmol/l) caused an increase in autofluorescence of quiescent cardiomyocytes to 73.3 ± 14.8% of maximal fluorescence (n = 4, P < 0.01), indicating a significant increase in mitochondrial NADH (Fig. 5B).


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  		Fig. 5.   Autofluorescence of cardiomyocytes as an index for mitochondrial NADH after addition of SIN-1. A: emission wavelength spectrum of quiescent (top) and contracting cardiomyocytes (bottom) at an excitation wavelength of 360 nm. +SIN-1 and -SIN-1, traces with and without SIN-1 (100 µmol/l), respectively. B: mean autofluorescence (excitation wavelength 360 nm, emission wavelength 480 nm) of quiescent cardiomyocytes after addition of SIN-1 (100 µmol/l; hatched bar). Autofluorescence is depicted as a percentage of maximal fluorescence (inhibition of the respiratory chain with 1 mmol/l sodium azide) after correction with basal fluorescence and after decoupling with carbonylcyanid-3-chlorophenylhydrazon (10 µmol/l). Open bar, without SIN-1.

In a separate experimental series, changes in the energy status of isolated rat cardiomyocytes were measured using the SAH method (8) to detect changes in intracellular free adenosine. The SAH accumulation rate in quiescent cardiomyocytes was 1.42 ± 0.05 pmol · min-1 · mg protein-1 (n = 4), and stimulation of cardiomyocytes with Na+-free medium did not significantly alter the level of this nucleoside, as indicated by an unchanged rate of SAH accumulation (1.01 ± 0.61 pmol · min-1 · mg protein-1, n = 4). As shown in Fig. 6, SIN-1 (100 µmol/l) did not alter the SAH accumulation in contracting cardiomyocytes (1.39 ± 0.38 pmol · min-1 · mg protein-1, n = 4). However, in quiescent cells, SIN-1 substantially increased the SAH accumulation to 3.23 ± 1.42 pmol · min-1 · mg protein-1 (P < 0.05, n = 4), indicating a decrease in energy status.


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  		Fig. 6.   Effect of SIN-1 (100 µmol/l) on the S-adenosylhomocysteine (SAH) accumulation rate of quiescent (left) and contracting cardiomyocytes (right). Results are expressed as means ± SD; n, number of preparations. ns, Not significant. *P < 0.05. Open bars, without SIN-1; hatchbars, with SIN-1.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

The major conclusion of this study is that NO reduced cardiac VO2 by a direct inhibitory effect on the respiratory chain. This is supported by the following lines of evidence.

First, in both contracting and quiescent cardiomyocytes, NO reduced VO2 by the same extent (Fig. 1). In principal, a reduction in VO2 could be due to a decrease in contractile activity induced by NO-mediated formation of cGMP (28). Such a negative inotropic effect of bromo-cGMP was demonstrated in several studies (17, 29, 30). In the present study, bromo-cGMP at a concentration of 10 µmol/l also decreased the VO2 of contracting cardiomyocytes by 29 ± 7% (n = 5), similar to SIN-1 (Table 1). However, quiescent cardiomyocytes, by definition, do not contract, and in this situation bromo-cGMP (in contrast to SIN-1) did not change VO2 (101%, n = 2). These findings suggest that elevation of cGMP by NO may not be a necessary prerequisite for the inhibitory action of NO on cellular respiration.

Second, NO induced a major increase in autofluorescence in quiescent cardiomyocytes (Fig. 5). This autofluorescence signal is mainly caused by mitochondrial NADH (13, 38). Inhibition of the respiratory chain by severe hypoxia (ambient PO2 of ~0.1 mmHg) caused as comparable an increase in autofluorescence as 100 µmol/l SIN 1 did, strongly indicating that NO directly blocks the respiratory chain.

Third, the NO-induced reduction in VO2 was unchanged when PKG, PKA, or PKC were inhibited (Fig. 3). This makes it rather unlikely that inhibition of contractile force by NO is mediated by phosphorylation of contractile proteins (33).

Finally, NO did not alter free [Ca2+]i in the present study at a concentration that inhibited VO2 by 50% of its maximal effect or at a supramaximal concentration (Fig. 4). This finding does not support the proposal that NO directly or via cGMP may alter phospholamban and Ca2+ transporter activity or Ca2+ channels, all known to modulate cytosolic Ca2+ concentration (22, 27).

SIN-1 is well known to exert unspecific side effects, which are caused by the formation of the breakdown product SIN-1C (35, 36). In our experiments, SIN-1C at 100 µmol/l did not significantly change VO2, whereas SIN-1 inhibited respiration by 24% (Table 1). At higher concentrations, however, SIN-1C also decreased VO2. The specificity of the SIN-1 effect in the chosen concentration is further supported by experiments with SNAP, which inhibited the VO2 of stimulated cardiomyocytes similarly to SIN-1.

The concentration of bioactive NO generated by SIN-1 or SNAP is difficult to assess. It is well known that NO is avidly inactivated by reaction with buffer oxygen or scavenged in the cytosol of the cardiomyocytes by myoglobin, other proteins, or membrane lipids (12, 20). Furthermore, the NO donors used are capable of penetrating through cell membranes and therefore may contribute to an unknown extent to the intracellular formation of NO. Therefore, the high concentrations of the NO donors were not equivalent to a similar high NO concentration.

To investigate the energy status of NO-treated cardiomyocytes, free cytosolic adenosine was measured using the SAH method. Because the free concentration of adenosine in the well-oxygenated heart is very low, changes in SAH serve as a very sensitive index of cardiac energy status (40). We found SAH accumulation to be unchanged when quiescent cardiomyocytes were stimulated; similar to previous results, the enhanced ATP demand of contraction did not change energy status when oxygenation was adequate (40). In the present study, NO reduced VO2 by ~25%. Although this effect appears to be small, the following quantitative considerations demonstrate that the influence on cardiac energy metabolism is much more pronounced. Oxidative phosphorylation, calculated on the basis of the measured VO2, was 30.6 µmol ATP · min-1 · gww-1, taking into account a P:O ratio of 3 (no mitochondrial uncoupling). At a normal ATP concentration of 4 µmol/gww (42), a discrepancy between ATP formation and consumption in the order of 7 µmol · min-1 · gww-1, which is 25% reduction, would cause a total depletion of ATP within only 30 s. This should be associated with a massive increase in the formation of free cytosolic adenosine. In line with this interpretation, in quiescent cardiomyocytes, free cytosolic adenosine clearly increased in the presence of NO-induced inhibition of oxidative phosphorylation (Fig. 6). However, there was no change in free cytosolic adenosine by SIN-1 in contracting cardiomyocytes (Fig. 6). This suggests that the ATP consumption of contracting cardiomyocytes was actively downregulated to match its decreased formation and to maintain a constant energy status. Such a downregulation of ATP consumption in cardiac muscle cells most likely results from a decrease of contractile activity (43). It appears possible that the negative inotropic effect of NO, which has been shown previously (4), is not solely due to an increased cGMP level but also due to an adaptation of the cardiomyocytes to an NO-induced inhibited oxidative phosphorylation (11, 15).

In summary, this study demonstrates that NO can effectively inhibit cellular respiration and that, in this experimental system, factors such as Ca2+- or cGMP-induced phosphorylation are unlikely to mediate the NO-mediated effects. It therefore appears that the primary action of NO is on respiration. The decrease in ATP synthesis by NO observed by others and also in the present study is likely to be compensated for by a decrease in ATP consumption so that energy status remains unchanged.


    ACKNOWLEDGEMENTS

This study was supported by the Deutsche Forschungsgemeinschaft (Schr 154/6-2).


    FOOTNOTES

Address for reprint requests and other correspondence: T. Stumpe, Dept. of Physiology, Heinrich-Heine Univ. of Düsseldorf, Universitätsstrasse 1, D-40225 Düsseldorf, Germany (E-mail: stumpe{at}uni-duesseldorf.de).

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 6 June 2000; accepted in final form 8 January 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 280(5):H2350-H2356
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