Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Heart failure wikipedia , lookup
Cardiac contractility modulation wikipedia , lookup
Rheumatic fever wikipedia , lookup
Electrocardiography wikipedia , lookup
Quantium Medical Cardiac Output wikipedia , lookup
Coronary artery disease wikipedia , lookup
Management of acute coronary syndrome wikipedia , lookup
Am J Physiol Heart Circ Physiol 287: H14 –H21, 2004; 10.1152/ajpheart.01235.2003. Invited Review: Point-Counterpoint Endocardial endothelium in the avascular frog heart: role for diffusion of NO in control of cardiac O2 consumption Alexandra Adler,1 Harer Huang,1 Zipping Wang,1 Joseph Conetta,2 Ellen Levee,2 Xiaoping Zhang,1 and Thomas H. Hintze1 1 Department of Physiology and 2Department of Comparative Medicine, New York Medical College, Valhalla, New York 10595 nitric oxide; Western blot analysis; Triton X-100; mitochondria; Griess reaction THE ENDOCARDIAL ENDOTHELIUM (EE) plays a vital role in the regulation of myocardial performance and electrical activity as recently reviewed by Brutsaert (3). Located between luminal blood and the underlying cardiac muscle, the EE synthesizes and releases nitric oxide (NO) in amounts sufficient to regulate myocardial contraction under basal conditions (4, 24). NO is also known to regulate O2 consumption under a variety of conditions (2, 12, 15, 22, 25). This regulation of myocardial metabolism may be one of NO’s most significant roles. We have proposed that endothelial NO synthase (eNOS), which is the most highly expressed isoform of NOS in vascular tissue under physiological conditions, contributes to the control of tissue O2 consumption by NO (16, 25). 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 Address for reprint requests and other correspondence: T. H. Hintze, Dept. of Physiology, New York Medical College, Valhalla, NY 10595. H14 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 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. 0363-6135/04 $5.00 Copyright © 2004 the American Physiological Society http://www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.2 on June 16, 2017 Adler, Alexandra, Harer Huang, Zipping Wang, Joseph Conetta, Ellen Levee, Xiaoping Zhang, and Thomas H. Hintze. Endocardial endothelium in the avascular frog heart: role for diffusion of NO in control of cardiac O2 consumption. Am J Physiol Heart Circ Physiol 287: H14 –H21, 2004; 10.1152/ajpheart.01235. 2003.—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-Nacetyl 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. Invited Review: Point-Counterpoint DIFFUSION OF NO TO MITOCHONDRIA 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. AJP-Heart Circ Physiol • VOL 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 䡠 HCl, pH 7.2, 1 mM EDTA, 10 mM dithiothreitol, 1 mg/ml PMSF, 100 g/ml leupeptin, 100 g/ml soybean trypsin inhibitor, and 20 g/ml aprotinin) for 15 s ⫻ 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 g 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 l of a 1% solution) and N-(1-naphthyl)ethylenediamine (50 l 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 287 • JULY 2004 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.2 on June 16, 2017 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. H15 Invited Review: Point-Counterpoint H16 DIFFUSION OF NO TO MITOCHONDRIA 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 Fig. 1. Inhibition of myocardial O2 consumption by bradykinin (10⫺4–10⫺8 mol/l) and carbachol (10⫺4–10⫺8 mol/l) in Xenopus. There was a significant, concentration-dependent decrease in myocardial O2 consumption at 37°C (Œ) 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. AJP-Heart Circ Physiol • VOL 287 • JULY 2004 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.2 on June 16, 2017 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 䡠min⫺1 䡠g⫺1 (n ⫽ 10)] compared with 37°C [459 ⫾ 45.9 nmol O2 䡠min⫺1 䡠g⫺1 (n ⫽ 7); P ⬎ 0.05]. These baselines were not affected by L-NAME [426 ⫾ 50.1, 303 ⫾ 39.8 nmol O2 䡠min⫺1 䡠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. Effect of carbachol on myocardial O2 consumption at 37°C and 25°C. Carbachol (10⫺8–10⫺4 mol/l) caused concentrationdependent decreases in myocardial O2 consumption (Fig. 1B) at 37°C (n ⫽ 6) (from 24.8 ⫾ 14.5 to 201.0 ⫾ 13.0 nmol/min) and at 25°C (n ⫽ 7) (from 26.8 ⫾ 7.1 to 72.7 ⫾ 11.0 nmol/min). The response to carbachol at 25°C was significantly less than at 37°C (P ⬍ 0.05). The addition of L-NAME significantly inhibited the response to carbachol at both temperatures. The response to carbachol with L-NAME was, however, not significantly different at the two temperatures. 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. Effect of Triton X-100 on myocardial O2 consumption. The addition of Triton X-100 (0.05%) caused a significant decrease Invited Review: Point-Counterpoint DIFFUSION OF NO TO MITOCHONDRIA H17 Fig. 2. Inhibition of myocardial O2 consumption by S-nitroso-Nacetyl penicillamine (SNAP; 10⫺4–10⫺7 mol/l) in Xenopus. There was a significant, concentration-dependent decrease in myocardial O2 consumption at 37°C (Œ) and 25°C (}) in response to SNAP. The decrease was significantly less at 25°C compared with 37°C. *P ⬍ 0.05, 25°C vs. 37°C. 15.3 to 112 ⫾ 14.0 nmol/min; without Triton X-100: from 22.1 ⫾ 8.3 to 218 ⫾ 16.3). Hematoxylin and eosin slides. Hematoxylin and eosin slides indicate that Triton X-100 removed the layer of endothelial Fig. 3. Addition of the detergent Triton X-100 abolished the regulation of O2 consumption by bradykinin (10⫺4–10⫺8 mol/l) and carbachol (10⫺4–10⫺8) at 37°C. A: in the presence of bradykinin, regulation of O2 consumption was significantly decreased by the addition of Triton X-100 (}) compared with the control (■). 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. AJP-Heart Circ Physiol • VOL 287 • JULY 2004 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.2 on June 16, 2017 in the control of O2 consumption by tissue from Xenopus hearts at 37°C in the presence of either BK or carbachol (Fig. 3, A and B). Triton X-100 altered O2 consumption in the presence of SNAP (10⫺7–10⫺4 mol/l) (with Triton X-100: from 50.2 ⫾ Invited Review: Point-Counterpoint H18 DIFFUSION OF NO TO MITOCHONDRIA cells around the lumen, the EE. Fig. 4, left, shows control tissue, whereas Fig. 4, right, shows tissue after perfusion with Triton X-100. The endothelial cells with the addition of Triton appear ripped, whereas the control cells are intact. Effect of Triton X-100 on eNOS protein. Levels of eNOS protein decreased in only some of the hearts that were perfused with Triton X-100 (Fig. 5). Although Triton X-100 ripped the EE, eNOS protein could still cling to underlying tissue. The eNOS would be inactive, however, because of the lack of an intact cell membrane. Effect of BK and Triton X-100 on nitrite production in heart tissue. Tissue perfused with Triton X-100 had significantly lower nitrite production in response to BK compared with control tissue (Fig. 6) (Griess reaction). L-NAME inhibited the production of nitrite in response to BK in the control tissue (BK: 81.4 ⫾ 23.7% change compared with control; BK ⫹ L-NAME: 23.7 ⫾ 19.2% change compared with control). L-NAME had no effect on the production of nitrite in response to BK in the tissue perfused with Triton X-100 (BK: 13.9 ⫾ 8.9% change compared with control; BK ⫹ L-NAME: 13.1 ⫾ 5.8% change compared with control). 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 DISCUSSION The results of the present study indicate that BK and carbachol significantly decrease O2 consumption in the myocardial Fig. 5. Levels of endothelial nitric oxide (NO) synthase (eNOS) protein was decreased only in some of the Triton X-100-perfused hearts. This suggests that eNOS protein still clings to the disrupted EE. N, normal. AJP-Heart Circ Physiol • VOL Fig. 6. Addition of the detergent Triton X-100 abolished the ability of bradykinin (10⫺4 M) to stimulate NO production (measured as nitrite) in frog heart tissue. Nitrite production in the normal tissue (solid bar) was nearly eight times higher than in the Triton X-100-perfused tissue (hatched bar). *P ⬍ 0.05, bradykinin vs. bradykinin ⫹ Triton X-100. 287 • JULY 2004 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.2 on June 16, 2017 Fig. 4. ⫻40 magnification of four sections: two control (left) and two Triton X-100 (right). The endocardial endothelium (EE) of the control hearts is intact, as indicated by the smooth outline of the tissue with regularly spaced, horizontally oriented nuclei. The EE of the tissue perfused with Triton X-100 appears ripped with nuclei and perhaps cell membranes remaining stuck to the underlying tissue. Invited Review: Point-Counterpoint DIFFUSION OF NO TO MITOCHONDRIA AJP-Heart Circ Physiol • VOL 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 ⫻40 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 perfor287 • JULY 2004 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.2 on June 16, 2017 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. H19 Invited Review: Point-Counterpoint H20 DIFFUSION OF NO TO MITOCHONDRIA 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 inoAJP-Heart Circ Physiol • VOL tropic 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 287 • JULY 2004 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.2 on June 16, 2017 mance 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. Invited Review: Point-Counterpoint DIFFUSION OF NO TO MITOCHONDRIA 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). REFERENCES 1. Adler A, Messina E, Sherman B, Wang Z, Huang H, Linke A, and Hintze TH. NAD(P)H oxidase generated superoxide anion accounts for the reduced control of myocardial oxygen consumption by NO in aged Fischer 344 rats. Am J Physiol Heart Circ Physiol 285: H1015–H1022, 2003. 1a.Balligand JL, Kelly RA, Marsden PA, Smith TW, and Michel T. Control of cardiac muscle cell function by endogenous nitric oxide signaling system. Proc Natl Acad Sci USA 90: 347–351, 1993. 2. Bernstein RD, Ochoa FY, Xu X, and Hintze TH. Function and production of nitric oxide in the coronary circulation of the conscious dog during exercise. Circ Res 79: 840 – 848, 1996. 3. Brutsaert DL. Cardiac endothelial-myocardial signaling: its role in cardiac growth, contractile performance and rhythmicity. Physiol Rev 83: 59 –115, 2003. 4. Brutsaert DL, Fransen P, Andries LJ, De Keulenaer GW, and Sys SU. Cardiac endothelium and myocardial function. Cardiovasc Res 38: 281– 290, 1998. 5. Cleeter MW, Cooper JM, Darley-Usmar VM, Moncada S, and Schapira AH. Reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, by nitric oxide. Implications for neurodegenerative diseases. FEBS Lett 345: 50 –54, 1994. 6. Clementi E, Brown GC, Foxwell N, and Moncada S. On the mechanism by which vascular endothelial cells regulate their oxygen consumption. Proc Natl Acad Sci USA 96: 1559 –1562, 1999. 7. Cottrell GA and Welsh ME. Effects of synthetic eledoisin and bradykinin on certain invertebrate preparations and the isolated frog heart. Nature 212: 838 – 839, 1966. 7a.Dagenais GR, Yusuf S, Bourassa MG, Yi Q, Bosch J, Lonn EM, Kouz S, and Grover J (HOPE Investigators). Effect of ramapril on coronary events in high-risk persons: results of the Heart Outcomes Prevention Evaluation Study. Circulation 104: 522–526, 2001. 8. Foxon GEH. Problems in the double circulation of vertebrates. Biol Rev 30: 196 –228, 1955. AJP-Heart Circ Physiol • VOL 9. Furchgott RF and Zawadski JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288: 373–376, 1980. 10. Ghiara P, Parente L, and Piomelli D. The cyclo-oxygenase pathway in the avascular heart of the frog, Rana esculenta L. Gen Pharmacol 15: 309 –313, 1984. 11. Gattuso A, Mazza R, Pellegrino D, and Tota B. Endocardial endothelium mediates luminal ACh-NO signaling in isolated frog heart. Am J Physiol Heart Circ Physiol 276: H633–H641, 1999. 12. Granger DL and Lehninger AL. Sites of inhibition of mitochondrial electron-transport in macrophage injured neoplastic cells. J Cell Biol 85: 527–535, 1982. 13. Grant RT and Regneir M. The comparative anatomy of the cardiac coronary vessels. Heart 13: 205–209, 1926. 14. Hermsmeyer K and Aprigliano O. Effects of chlorobutanol and bradykinin on myocardial excitation. Am J Physiol 230: 306 –310, 1976. 15. Hibbs JB, Taintor RR, Vavrin Z, and Rachlin EM. Nitric oxide: a cytotoxic activated macrophage effector molecule. Biochem Biophys Res Commun 157: 87–94, 1988. 15a.Lee TP, Kuo JF, and Greengard P. Role of muscarinic cholinergic receptors in the regulation for guanosine 3,5 cyclic monophosphate content in mammalian brain, heart muscle, and intestinal smooth muscle. Proc Natl Acad Sci USA 69: 3287–3289, 1972. 16. Litwin SE and Grossman W. Diastolic dysfunction as a cause of heart failure. J Am Coll Cardiol 22: 49A-55A, 1993. 16a.Loke KE, McConnell PI, Tuzman JM, Shesely EG, Smith CJ, Stackpole CJ, Thompson CI, Kaley G, Wolin MS, and Hintze TH. Endogenous endothelial nitric oxide synthase-derived nitric oxide is a physiological regulator of myocardial oxygen consumption. Circ Res 84: 840 – 845, 1999. 17. Loke KE, Messina EJ, Mital S, and Hintze TH. Impaired nitric oxide modulation of myocardial oxygen consumption in genetically cardiomyopathic hamsters. J Mol Cell Cardiol 32: 2299 –2306, 2000. 18. Loke KE, Messina EJ, Shesely EG, Kaley G, and Hintze TH. Potential role of eNOS in the therapeutic control of myocardial oxygen consumption by ACE inhibitors and amlodipine. Cardiovasc Res 49: 86 –93, 2001. 19. Miller VM and Vanhoutte PM. Prostaglandins but not nitric oxide are endothelium-derived relaxing factors in the trout aorta. Acta Pharmacol Sci 21: 871– 876, 2000. 20. Nobel Chronicles. 1936 Henry Dale (1875–1968), and Otto Loewi (1873– 1961). Lancet 353: 416, 1999. 20a.Paulus WJ, Vantrimpont PJ, and Shah AM. Acute effects of nitric oxide on left ventricular relaxation and diastolic distensibility in humans. Circulation 89: 2070 –2078, 1994. 21. Pittis M, Zhang X, Loke KE, Mital S, Kaley G, and Hintze TH. Canine coronary microvessel NO production regulates oxygen consumption in eNOS knockout mouse heart. J Mol Cell Cardiol 32: 1141–1146, 2000. 21a.Shah AM, Spurgeon HA, Sollot SJ, Talo A, and Lakatta E. 8-Bromo cGMP reduces the myofilament response to Ca in intact cardiac myocytes. Circ Res 74: 970 –978, 1994. 22. Shen W, Xu X, Ochoa M, Zhao G, Wolin MS, and Hintze TH. Role of nitric oxide in the regulation of oxygen consumption in conscious dogs. Circ Res 75: 1086 –1095, 1994. 23. Staley NA and Benson ES. The ultrastructure of frog ventricular cardiac muscle and its relationship to mechanism of excitation-contraction coupling. J Cell Biol 38: 99 –114, 1968. 24. Sys SU, Pellegrino D, Mazza R, Gattuso A, Andries LJ, and Tota B. Endocardial endothelium in the avascular heart of the frog: morphology and role of nitric oxide. J Exp Biol 200: 3109 –3118, 1997. 25. Trochu JN, Bouhour JB, Kaley G, and Hintze TH. Role of endothelium-derived nitric oxide in the regulation of cardiac oxygen metabolism: implications in health and disease. Circ Res 87: 1108 –1117, 2000. 26. Zhang XP, Xie X, Nasjletti A, Xu X, Wolin MS, and Hintze TH. ACE inhibitors promote nitric oxide accumulation to modulate myocardial oxygen consumption. Circulation 95: 176 –182, 1997. 27. Zhang XP, Scicli GA, Xu X, Nasjletti A, and Hintze TH. Role of endothelial kinin formation in the control of nitric oxide production in canine coronary microvessels. Hypertension 30: 1105–1111, 1997. 287 • JULY 2004 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.2 on June 16, 2017 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. H21