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INVITED REVIEW: POINT-COUNTERPOINT
1Department of Physiology and 2Department of Comparative Medicine, New York Medical College, Valhalla, New York 10595
ABSTRACT
We investigated the role of nitric oxide (NO) in the control of myocardial O2 consumption in the hearts of female Xenopus frogs, which lack a coronary vascular endothelium and in which the endocardial endothelium is the only source of NO to regulate cardiac myocyte function. Hence, frogs are an ideal model in which to explore the role of diffusion of NO from the endocardial endothelium (EE) without vascular endothelial or cardiac cell NO production. In Xenopus hearts we examined the regulation of cardiac O2 consumption in vitro at 25°C and 37°C. The NO-mediated control of O2 consumption by bradykinin or carbachol was significantly (P < 0.05) lower at 25°C (79 ± 13 or 73 ± 11 nmol/min) than at 37°C (159 ± 26 or 201 ± 13 nmol/min). The response to the NO donor S-nitroso-N-acetyl penicillamine was also markedly lower at 25°C (90 ± 8 nmol/min) compared with 37°C (218 ± 15 nmol/min). When Triton X-100 was perfused into hearts, the inhibition of myocardial O2 consumption by bradykinin (18 ± 2 nmol/min) or carbachol (29 ± 4 nmol/min) was abolished. Hematoxylin and eosin slides of Triton X-100-perfused heart tissue confirmed the absence of the EE. Although endothelial NO synthase protein levels were decreased to a variable degree in the Triton X-100-perfused heart, NO2 production (indicating eNOS activity) decreased by >80%. It appears that the EE of the frog heart is the sole source of NO to regulate myocyte O2 consumption. When these cells are removed, the ability of NO to regulate O2 consumption is severely limited. Thus our results suggest that the EE produces enough NO, which diffuses from the EE to cardiac myocytes, to regulate myocardial O2 consumption. Because of the close proximity of the EE to underlying myocytes, NO can diffuse over a distance and act as a messenger between the EE and the rest of the heart to control mitochondrial function and O2 consumption.
nitric oxide; Western blot analysis; Triton X-100; mitochondria; Griess reaction
As a model to determine the significance of the diffusion of NO in the control of cardiac cell O2 consumption, the frog heart is of much interest because it has no coronary circulation and can therefore be used to study the interaction between the EE and the myocardium without interference from the vascular endothelium (13, 24). This striking absence of the coronary vasculature is characteristic of all amphibians (13) and may be a consequence of a change in respiratory patterns from water environment to air (8, 19). The frog ventricle is highly trabeculated and has a much higher endothelial surface-to-myocardial volume ratio than higher vertebrates (24). The EE of the avascular frog heart is unique because it is the only endothelial barrier between blood and the myocardial environment and is the only source of NO. Thus the EE mediates the inotropic effect of luminal cholinergic stimuli via the NO-cGMP pathway (11).
In 1997, Sys et al. (24) suggested that the EE is the sole NO source in the heart of the frog and that NO produced by the EE may act as a short-distance, bidirectional messenger between EE cells and myocytes (11). Brutsaert (3, 4) has also suggested that the endothelial cell NOS-NO-cGMP signaling pathway could be involved in the control of trans-EE-permeability. Thus it appears that NO must diffuse from the EE of the frog heart to affect myocardial performance. In our study, we removed the EE in hearts from Xenopus frogs using a detergent to elucidate the role of the EE as an NO source. It has also been shown previously that eNOS is located only in the EE of the frog heart (24). Given the close proximity of the EE and underlying myocytes, eNOS activity in the EE directly affects myocardial contractility (3, 24). Our studies examined the role of eNOS in hearts both with and without an intact EE. Activity of eNOS in hearts without an EE was, however, greatly decreased. Thus it seems that an intact EE is necessary for the proper function of eNOS. The ability of NO to diffuse across the heart has been highly debated. Although some have suggested that NO can diffuse (11, 21), others have disagreed (25) and claimed that local production/concentrations of NO are important. The aim of our work was to examine the possibility that NO can diffuse from one cell type to another to regulate myocardial O2 consumption in the absence of the confounding effects of multiple sources of NO, i.e., myocyte NOS or a vascular eNOS.
MATERIALS
Bradykinin (BK), carbachol, S-nitroso-N-acetyl penicillamine (SNAP), N
-nitro-L-arginine methyl ester (L-NAME), and Triton X-100 were purchased from Sigma (St. Louis, MO).
Animal tissue. Female Xenopus frogs were obtained from NASCO (Ft. Atkinson, WI). All experimental protocols were approved by the Institutional Animal Care and Use Committee and conform to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and American Physiological Society "Guidelines for the Use and Care of Laboratory Animals." Before death for in vitro studies, the frogs were anesthetized with dissolved 3-aminobenzoic acid ethyl ester (1.7 g/l), and the heart was removed and cut into 8 or 12 pieces.
Preparation of myocardial tissue and measurement of O2 consumption. The frog hearts were separated, freed of connective tissue and fat, and cut into pieces weighing 20–40 mg. Tissue was incubated in Krebs bicarbonate solution containing (in mmol/l) 118 NaCl, 4.7 KCl, 1.5 CaCl2, 25 NaHCO3, 1.2 KH2PO4, 1.1 MgSO4, and 5.6 glucose at 37°C and bubbled with 21% O2-5% CO2-74% N2 (pH 7.4) to equilibrate for at least 1.5 h. At the end of the incubation period, two pieces of tissue were placed in a continuously stirred chamber with 3.0 ml of air-saturated Krebs bicarbonate solution containing 10 mmol/l HEPES (pH 7.4). The chambers were sealed using Clark-type platinum O2 electrodes (Yellow Springs Instruments) that were connected to O2 monitors (model YSI 5331) to continuously measure the O2 concentration in the buffer. The drop of O2 levels in the buffer is caused by and is directly related to uptake of O2 by the tissue. This methodology has been used by us (1) and by others (16, 17) in previous studies.
Effect of BK and carbachol on myocardial O2 consumption at 37°C and 25°C. The effect of cumulative doses of the B2 kinin receptor agonist BK (10–8–10–4; Sigma) and the muscarinic receptor agonist carbachol (10–8–10–4; Sigma) on myocardial O2 consumption was examined in tissue from frog hearts (n = 9 and 6, respectively). O2 consumption studies were performed at two temperatures, 37°C and 25°C, to determine the effect of temperature on the ability of NO to compete with O2 at its cytochrome c oxidase binding site and thus confirm its effect on mitochondrial respiration. These responses were also examined in the presence of the NOS inhibitor L-NAME (10–4 mol/l; Sigma) to verify the role of NO production by NOS in the regulation of myocardial O2 consumption.
Effect of SNAP on myocardial O2 consumption at 37°C and 25°C. Cumulative doses of the NO donor SNAP (10–7–10–4 mol/l) on myocardial O2 consumption were examined in frog heart tissue (n = 7) to test the sensitivity of the frog heart to NO. These responses were also examined in the presence of L-NAME and at both temperatures.
Effect of Triton X-100 on myocardial O2 consumption. To remove the source of the NO production in the Xenopus hearts, Triton X-100 (0.5% in saline), a detergent, was used. Triton X-100 dissolves lipids and therefore the endocardial endothelium of the heart, which has been shown to be the sole source of myocardial NO (24). The heart was exposed and Triton X-100 was introduced through the apex of the heart using a 23-gauge needle. The heart was perfused for 2 min and then removed and used for O2 consumption studies as described above. The effect of Triton X-100 on myocardial O2 consumption was examined in frog heart tissue (n = 4 each) in the presence of cumulative doses of BK, carbachol, and SNAP.
Hematoxylin and eosin slides. Hearts were removed from Xenopus frogs (n = 10) or perfused with Triton X-100 (n = 4) and preserved in a 10% formalin solution for 48 h to visualize the disruption of the EE by Triton. Hematoxylin and eosin slides were then prepared using standard methods from the Manual of Histologic and Special Staining Technics as well as an H2500 microwave processor (Energy Beam Sciences; Agawam, MA).
Immunoblotting. Myocardial tissue (both with and without Triton X-100, n = 5 each) was snap frozen in liquid nitrogen and stored at –80°C. For preparation of extracts, tissue was pulverized in liquid nitrogen, followed by homogenization in 5 vol of lysis buffer (0.05 M Tris·HCl, pH 7.2, 1 mM EDTA, 10 mM dithiothreitol, 1 mg/ml PMSF, 100 µg/ml leupeptin, 100 µg/ml soybean trypsin inhibitor, and 20 µg/ml aprotinin) for 15 s x 3 at 4°C and sonication for 1 min. The resulting lysate was centrifuged at 10,000 g for 10 min at 4°C and stored at –80°C before use. Protein concentrations of supernatants were measured using a standard protein assay (Bio-Rad Laboratories).
Samples of tissue lysate (75 µg of protein) were loaded onto individual lanes of a 8% polyacrylamide gel containing the detergent SDS prepared using standard techniques. Proteins were separated according to their molecular size. Proteins in the gel were transferred from the gel to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech) using a semidry transfer cell (Bio-Rad). Membranes were blocked for 1 h with 5% milk-PBS to block all other free protein-binding sites on the membrane. Purified polyclonal antibody to eNOS (1:1,000 dilution, Affinity Bioreagents) and inducible NOS (1:1,000 dilution, Affinity Bioreagents) in 1% milk-PBS was added, and incubation continued at 4°C overnight to allow the antibody to bind to its respective proteins. After incubation with a secondary anti-rabbit antibody (1:5,000 dilution, Chemicon) conjugated to horseradish peroxidase, sites of antibody-antigen reaction were visualized using Super Signal West Pico Chemiluminescent Substrate (Pierce; Rockford, IL), followed by exposure to X-ray film (Kodak; Rochester, NY).
Quantification of proteins. The relative intensities of the bands in the exposed film were determined by scanning on a documentation and analysis system (Alphaimager 2000, Imgen Technologies; Alexandria, VA), followed by analysis with Image software. Band intensity correlates directly with amounts of the original protein present in the gel.
Effect of BK and Triton X-100 on nitrite production in heart tissue.
Frog heart tissue sections, 
30–60 mg/section (wet wt), perfused with Triton X-100 (n = 5) or control (n = 5), were incubated and bubbled with 95% O2-5% CO2 in Krebs for 35 min at 37°C in 5-ml plastic tubes that contained BK (10–4 M) or L-NAME plus BK (Griess reaction). At the end of the incubation time, the tubes were removed from the tissue bath, and sulfanilamide (450 µl of a 1% solution) and N-(1-naphthyl)ethylenediamine (50 µl of a 0.2% solution) were added to each tube for diazotization of sulfanilic acid by NO. After 5–10 min of incubation at room temperature for full color (pink) development, the supernatant was removed from each tube. Formation of NO was measured as nitrite. Nitrite release was measured with a spectrophotometer (Uvikon 930 spectrophotometer, Kontron Instruments) as the increase in absorbance at 540 nm and compared with known concentrations of nitrite. Absorbance was measured and converted to a straight line with the use of linear regression analysis (y = ax + b, R > 0.99). We have described these methods recently (26, 27).
Statistical analysis. The data are shown as means ± SE. Comparisons between groups were made using one-way ANOVA, followed by correction for multiple comparisons with Bonferroni's t-test using statistical software. Statistical significance was accepted at P < 0.05.
RESULTS
Baseline O2 consumption in myocardial tissues from Xenopus hearts at 25°C and 37°C. Baseline myocardial O2 consumption was half the rate at 25°C [263 ± 22.8 nmol O2·min–1·g–1 (n = 10)] compared with 37°C [459 ± 45.9 nmol O2·min–1·g–1 (n = 7); P > 0.05]. These baselines were not affected by L-NAME [426 ± 50.1, 303 ± 39.8 nmol O2·min–1·g–1 (n = 8 each) at 25°C and 37°C, respectively, P > 0.05].
Effect of BK on myocardial O2 consumption at 37°C and 25°C. BK (10–8–10–4 mol/l) caused concentration-dependent decreases in myocardial O2 consumption (Fig. 1A) at 37°C (n = 9) (from 53.8 ± 3.6 to 159 ± 25.7 nmol/min) and at 25°C (n = 6) (from 24.0 ± 5.4 to 77.7 ± 12.7 nmol/min). The response to BK at 25°C was significantly less than that at 37°C (P < 0.05). The addition of L-NAME significantly inhibited the response to BK at both temperatures. The response to BK with L-NAME was, however, not significantly different at the two temperatures.
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Effect of SNAP on myocardial O2 consumption at 37°C and 25°C. SNAP (10–7–10–4 mol/l) caused concentration-dependent decreases in myocardial O2 consumption at (Fig. 2) 37°C (n = 7) (from 22.1 ± 82.9 to 218 ± 16.3 nmol/min) and at 25°C (n = 3) (from 9.49 ± 2.6 to 89.5 ± 8.4 nmol/min). L-NAME did not significantly alter the response to SNAP at any temperature.
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The results of the present study indicate that BK and carbachol significantly decrease O2 consumption in the myocardial tissues of female Xenopus frogs at 37°C. The abolition of these responses after the addition of L-NAME, an NOS inhibitor, indicates a role for NO in the regulation of mitochondrial respiration in the heart of frogs. When O2 consumption was examined at 25°C, the response to BK and carbachol was significantly decreased. Importantly, the regulation of O2 consumption in response to BK and carbachol was nearly abolished in tissue perfused with Triton X-100, a detergent that strips away the EE. Removal of the EE was confirmed with the use of hematoxylin and eosin slides of tissue in the presence and absence of Triton X-100. Western blot analysis indicated that eNOS protein levels were variably decreased by Triton X-100. Activity of eNOS (and NO production), however, was significantly reduced in Triton X-100-perfused tissue, suggesting its singular role in NO production in the frog heart. The effect of SNAP was not altered by the addition of Triton X-100, suggesting that the underlying myocytes were still functional and that the EE is the sole source of NO in the frog heart. Thus NO must diffuse from the EE to other parts of the heart (i.e., myocytes) to regulate myocardial O2 consumption.
Our data indicate that pretreatment with L-NAME had no effect on baseline O2 consumption in the frog hearts. We have interpreted this previously to suggest that the basal release of NO is small in the absence of an agonist that stimulates NO production in our preparation. Our data also suggest that the ability of endogenous NO to regulate O2 consumption was greatly reduced in tissues studied at 25°C compared with those studied at 37°C. Such a reduction leads to reduced ability of BK, for example, to regulate myocardial mitochondrial O2 consumption. The effect of the exogenous NO donor SNAP was significantly decreased at 25°C as well, demonstrating that the tissue is less sensitive to NO at a lower temperature. The role of temperature is of interest in amphibians because they are cold-blooded and their enzyme activity varies over a range of temperatures, the Q10. The reduction in NO bioactivity at 25°C may be dependent upon the competitive relationship between NO and O2 for cytochrome c oxidase. At 25°C, biochemical work by the heart is decreased, intramitochondrial PO2 is higher (5, 6, 25), and NO does not compete as well for the binding site of O2 as it would at a higher temperature (i.e., high O2 consumption). Therefore, the effect of BK on the control of O2 consumption at 25°C is predictably less than it would be at 37°C, as we have shown.
One of the most exciting findings in this study was that the control of O2 consumption by endogenous NO was nearly abolished in tissue from hearts perfused with Triton X-100. The addition of Triton X-100 significantly decreased the ability of BK or carbachol to regulate O2 consumption. Triton X-100 has previously been studied and shown to affect cardiac function and to destroy EE cells (24). This finding suggests that the EE may be the sole source of NO in the frog heart and that when it is removed, control of O2 consumption by BK and carbachol is severely limited. Examination of hematoxylin and eosin slides verified the absence of viable endothelial cells in hearts perfused with Triton X-100. The EE of the control tissue appeared intact while the EE of the perfused tissue with Triton was disrupted. To better understand our findings, Western blot analysis for eNOS and nitrite, the hydration product of NO, production studies were performed. Although levels of eNOS protein were variably reduced, production of nitrite (an indicator of eNOS activity) was significantly reduced in the Triton X-100-perfused tissue. From these findings, it seems likely that NO was produced solely in the EE and that NO diffuses from the EE to myocytes to regulate myocardial O2 consumption.
BK, the B2 kinin receptor agonist, stimulates endogenous NO production through the activation of eNOS. This production is blocked in the presence of L-NAME. BK has previously been studied in frog hearts, and it has been shown that there are no BK receptors present in frog myocardial cells (14). Therefore, BK cannot modulate NO production in frog myocytes. These receptors may, however, exist in other parts of the heart, particularly the EE (7). BK caused a significantly smaller reduction in myocardial O2 consumption in hearts perfused with Triton X-100 compared with control hearts, suggesting that an intact EE is vital for the activity of BK.
Carbachol, a muscarinic receptor agonist that stimulates endogenous NO production through the activation of eNOS, was used as well. Carbachol also caused a significantly smaller reduction in myocardial O2 consumption in hearts previously perfused with Triton X-100. These results again support the conclusion that the EE is the sole source of NOS and NO and that NO must therefore diffuse to other tissues in the heart to regulate O2 consumption. The decreased effect of carbachol or BK in Triton-perfused tissue is due to decrease eNOS activity.
Because there are no BK receptors on frog cardiac myocytes, the use of BK after Triton X-100 does not directly address the possibility that frog myocytes make NO. However, this is not the case with carbachol, whose actions, like those of ACh (Vagusstoff), on the heart were originally described by Loewi in 1903 (20). Furthermore, Gattuso et al. (11) have shown more recently that ACh regulates cardiac contraction through NO production.
In our study, the exogenous NO donor SNAP reduced tissue O2 consumption to a similar degree in the control and Triton X-100-perfused heart tissue. The loss of the ability of BK and carbachol to reduce O2 consumption is therefore not a result of an impairment of the ability of NO to modulate tissue O2 consumption (i.e., altered mitochondrial sensitivity to NO) but rather a reduction in biologically active NO. This is due to the loss of the EE in the Triton X-100-perfused heart tissue.
Hematoxylin and eosin slides provided qualitative evidence for removal of the EE after treatment with Triton X-100. The slides, examined at x40 magnification, showed the physical effect of Triton X-100 on EE cells. In control hearts, the EE appears as a smooth layer of cells lining the lumen and covering the sinusoids. This smooth endothelial layer appears distorted in the heart perfused with Triton X-100. Although the nuclei remain, they are clumped and not located in intact cells.
In our study, with Western blot analysis, we found that tissue perfused with Triton X-100 did not, on average, significantly lower eNOS levels. It seems likely that although the EE is destroyed, eNOS protein, like the cell nuclei, may still cling to remaining tissue. These conclusions were reinforced in studies using the Griess reaction to measure nitrite production in the buffer. Nitrite production was decreased by 85.4% in the Triton X-100-perfused tissue compared with the control tissue. This suggests that, whereas eNOS protein may still cling to remaining cells in the lumen of the frog heart, it is inactive after treatment with Triton X-100. Our data suggest that intact endocardial endothelial cells are necessary for eNOS activity. Therefore, when the EE is distorted and cells are no longer intact, eNOS is inactive, and NO production does not occur. The lack of effect by BK in the Triton X-100-perfused tissue also supports the concept that the EE is the only NO source in the frog heart.
In the present study, we have clearly shown that the stimulation of endogenous NO production failed to reduce tissue O2 consumption in the presence of Triton X-100. Previous work performed in frogs has suggested that they are ideal models in which to study the EE. It is known that NOS is only found in the EE and that the EE is important to the myocardial performance of mammals (24). The frog ventricle is highly trabeculated: it has a much higher endothelial surface-to-myocardial volume ratio than higher vertebrates. Unlike the hearts of mammals, the frog heart has no coronary circulation and can therefore be used to study the interaction between the EE and the myocardium without interference (13). NO produced by the EE modulates intramyocardial cGMP levels and contractility and therefore acts as a short-distance messenger (bidirectional) between EE cells and myocytes, which are separated only by a thin layer of extra cellular material (13).
The findings of the present study support our previous work indicating that BK activates eNOS to regulate cardiac O2 consumption and that this does not occur in the eNOS knockout mouse heart (16, 18). More importantly, we demonstrated previously the transfer of NO from dog coronary microvessels coincubated with eNOS knockout mouse heart regulates tissue O2 consumption (21). Those studies and the current one support the concept that NO can diffuse over a distance to regulate myocardial O2 consumption (25), just as NO can diffuse from a single layer of endothelial cells to regulate vascular tone (9).
We have used the frog heart to explore the possibility that NO can diffuse from one cell to another to regulate myocardial O2 consumption. Control of O2 consumption was found to be temperature dependent in the frog heart. We have also shown that the EE in the frog heart produces enough NO to regulate myocardial O2 consumption (21). Triton X-100 abolished the ability of an agent that stimulates NO production to regulate O2 consumption in the cardiac tissue, suggesting that eNOS is present only in the EE cells. Hematoxylin and eosin slides confirmed the ability of Triton X-100 to destroy the EE cells. Although levels of eNOS protein were variable between control and Triton X-100-perfused tissue, NO production was reduced in the Triton X-100-treated tissue, suggesting that eNOS cannot function without an intact EE and that the EE is the sole eNOS source in the frog heart. This suggests that NO must diffuse from the EE to other parts of the heart, such as myocytes, and in this way, regulate O2 consumption. The avascular frog heart is a unique model in which the roles of the EE and NO as a messenger can be studied without the confounding effects of the vascular endothelium or the presence of myocyte NOS.
POINT-COUNTERPOINT: ROLE OF NO IN THE HEART: CONTROL OF BEATING OR BREATHING
The study by Paulus and Bronzwaer proposes that the reduction in systolic time, the early onset of diastole, and increased compliance caused by NO in cardiac myocytes result in reduced systolic tension or pressure development and can be misconstrued as a reduction in cardiac contractility. This occurs whether NO is produced by endothelial cells subsequent to stimulation by substance P or is given as an NO donor. Furthermore, the source of NO may be either inducible NOS or eNOS and would in the main have a beneficial effect on cardiac contraction. Coupled with a reduction in vascular tone and aortic impedance, the reduction in systolic function may also lead to increased ejection, even in a failing heart. In the presence of adrenergic stimulation, the reduction in systolic time would cause a reduced systolic contraction and appear as a negative inotropic effect. If the NO were from an endogenous source, then an NOS inhibitor would appear to increase inotropic state and increase systolic tension development, an observation made in both experimental animals and in humans with heart failure. This interesting view of the literature by Paulus and Bronzwaer provides an alternative explanation for the paradoxical conclusion that what is otherwise a physiological important substance at low concentrations is an etiologic agent in the development of cardiac dysfunction at the same concentrations. By redefining the mechanism of reduced cardiac contraction by NO from a negative inotropic effect to an early onset of cardiac relaxation, at the same inotropic state, the onerous implications of reduced contraction to the development of disease are lessened. It should be remembered that catecholamines enhance the velocity of cardiac cell relaxation by a cAMP-associated mechanism, and this is considered of substantial physiological significance at high heart rates and to maintain filling during cardiac dysfunction. However, catecholamines are notoriously O2 wasting and the early relaxation caused by NO may serve to reduce this O2 wasting effect, which is especially important in the diseased heart.
The mechanism by which NO causes an early onset of cardiac relaxation is most probably an increase in cGMP and phosphorylation of troponin I (TNI) (21a). This phosphorylation of TNI initiates relaxation regardless of the intracellular calcium levels, thus at any given inotropic state. A similar although undefined process encompassing the interaction of cAMP and ACh on cardiac contraction was discussed by Greengard (15a) as the "Yin-Yang" hypothesis of cardiac contractions as early as the 1970s. The theory proposed by Paulus and Bronzwaer maybe an explanation for those early findings. The signaling mechanisms involved are not the focus of the discussion provided rather, the focus is on basic and clinical implications of this mechanism.
These can be summarized as follows: 1) the phosphorylation of TNI by cGMP results in an early onset of cardiac relaxation abbreviating isometric contraction without affecting the velocity of contraction; 2) the phosphorylation of TNI results in increased distensibility; 3) the interaction of this mechanism with catecholamines results in a reduction of cardiac isovolumic contraction without a change in inotropic state; 4) the inhibition of catecholamine-induced contraction is O2 sparing; and 5) coupled with a reduction in impedance, stroke volume can be maintained or enhanced without increasing O2 consumption even in the presence of catecholamines and cardiac failure. Importantly, diastolic dysfunction is thought to be an early hallmark of heart failure (16), perhaps reduced NO production is partially responsible. These are important consequences with substantial implications for our understanding of the role of NO in the genesis and treatment of cardiovascular disease.
In relation to our study, we found it most interesting that infusion of substance P even into the human heart resulted in abbreviation of systole and increased compliance (16). The source of NO was attributed to the endothelial cells. Furthermore, the mechanism of action of angiotensin-converting enzyme inhibitors, perhaps the most useful drugs in the treatment of heart failure in particular and cardiovascular disease in general (7a), was at least in part attributed to this mechanism of action and a kinin-mediated release of NO from the endothelium. With the use of the same reasoning, the effects of L-NMMA on the failing heart of patients in the presence of high adrenergic tone could also be explained by interfering with the effects of the production of NO from the endothelium on the myocardium. Thus the conclusions by Paulus and Bronswaer support our contention that endothelium-derived NO may diffuse to the adjacent cardiac cells to modulate cardiac contractile function in this case or mitochondrial function as discussed in our studies. This by no means indicates that the only source of NO in the normal or diseased heart is the endothelium. Several studies have proposed focal concentrations of NO based on immunohistochemical localization of NOS isoforms in cardiac myocytes (1a). The question to be addressed is not the potential of myocytes to make NO but the concentration gradient over which NO is diffusing. If endothelial eNOS is downregulated and cardiac myocyte NOS, whatever the isoform, is upregulated then the cardiac isoform may the primary contributor. When isoform swithching occurs in the disease process is an area in need of some perspective and for future studies. Our hypothesis that NO regulates cardiac myocyte O2 consumption by regulating mitochondrial function (25) would be complimentary with the proposal that NO regulates cardiac contractile function associated consumption of O2 even in the face of O2 wasting effects of excess catecholamines.
GRANTS
This study was supported by National Heart, Lung, and Blood Institute Grants PO-1 HL-41023, HL-60142, and HL-61290 (to T. H. Hintze) and American Heart Association Grant 02-35493T (to X. Zhang).
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FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
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  A. Robinet, H. Millart, F. Oszust, W. Hornebeck, and G. Bellon Binding of elastin peptides to S-Gal protects the heart against ischemia/reperfusion injury by triggering the RISK pathway FASEB J, July 1, 2007; 21(9): 1968 - 1978. [Abstract] [Full Text] [PDF]
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) and 25°C (
) in response to bradykinin (A) and carbachol (B). The decrease was significantly less at 25°C compared with 37°C. *P < 0.05, 25°C vs. 37°C.
). B: in the presence of carbachol, regulation of O2 consumption was significantly decreased by the addition of Triton X-100 compared with control. *P < 0.05 with Triton X-100.
