Anti-adrenergic effects of nitric oxide donor SIN-1 in rat cardiac myocytes

Miroslav O. Stojanovic1, Mark T. Ziolo1,3, Gordon M. Wahler3, and Beata M. Wolska1,2

1 Department of Physiology and Biophysics, and 2 Section of Cardiology, Department of Medicine, College of Medicine, University of Illinois at Chicago, Chicago 60612; and 3 Department of Physiology, Midwestern University, Downers Grove, Illinois 60515


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We studied how the nitric oxide (NO · ) donor 3-morpholinosydnonimine (SIN-1) alters the response to beta -adrenergic stimulation in cardiac rat myocytes. We found that SIN-1 decreases the positive inotropic effect of isoproterenol (Iso) and decreases the extent of both cell shortening and Ca2+ transient. These effects of SIN-1 were associated with an increased intracellular concentration of cGMP, a decreased intracellular concentration of cAMP, and a reduction in the levels of phosphorylation of phospholamban (PLB) and troponin I (TnI). The guanylyl cyclase inhibitor 1H-8-bromo-1,2,4-oxadiazolo (3,4-d)benz(b)(1,4)oxazin-1-one (ODQ) was not able to prevent the SIN-1-induced reduction of phosphorylation levels of PLB and TnI. However, the effects of SIN-1 were abolished in the presence of superoxide dismutase (SOD) or SOD and catalase. These data suggest that, in the presence of Iso, NO-related congeners, rather than NO · , are responsible for SIN-1 effects. Our results provide new insights into the mechanism by which SIN-1 alters the positive inotropic effects of beta -adrenergic stimulation.

3-morpholino-sydnonimine; phospholamban; troponin I; beta -adrenergic stimulation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ALTHOUGH THERE HAVE BEEN many studies on the physiological and pathological roles of nitric oxide (NO · ) in the regulation of heart function, the intracellular signaling pathways are not fully understood. There are controversial reports showing positive, negative, or biphasic inotropic effects of NO ·  on the heart (5, 13, 16, 22, 23). Some of these discrepancies may be due to variations among species, differences in the concentrations of NO ·  donors used, or to differences in the conditions under which the experiments were performed, such as the presence or absence of beta -adrenergic agonists (1, 4, 30). Interpretation of the data on the effect of NO ·  may also be related to complex effects depending on reactions of NO ·  and superoxides (O<SUP><IT>·−</IT></SUP><SUB><IT>2</IT></SUB>).

In experiments reported here, we treated rat heart myocytes with 3-morpholinosydnonimine (SIN-1) to determine mechanisms by which NO ·  and peroxynitrites affect the response to beta -adrenergic stimulation. SIN-1, which is the active metabolite of the vasodilatory drug molsidomine, alters the positive inotropic effect of beta -agonist stimulation and also generates superoxide in addition to NO ·  (7, 26). The superoxide reacts with NO · , generating a potent oxidizing product, peroxynitrite (ONOO-)


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Peroxynitrite reacts with many biological substances and has been shown to cause several pathological conditions (2, 3, 31). Moreover, in the presence of superoxide dismutase (SOD), formation of hydrogen peroxide (H2O2) is favored, which has been shown to stimulate the cytosolic or soluble form of guanylate cyclase (9).

Our results suggest that, in single rat cardiac myocytes, SIN-1 alters the positive inotropic effect of the beta -agonist isoproterenol (Iso) by altering phosphorylation levels of the sarcoplasmic reticulum protein phospholamban (PLB) and the myofilament protein troponin I (TnI). These effects of SIN-1 were associated with an increased intracellular concentration of cGMP and a decreased intracellular concentration of cAMP.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Myocyte isolation. Left ventricular myocytes were isolated from rat hearts by means of a technique previously described by Wolska et al. (32). After isolation, cells were resuspended in fresh control solution (in mmol/l: 0.2 CaCl2, 133.5 NaCl, 4 KCl, 1.2 NaH2PO4, 1.2 MgSO4, 10 HEPES, and 11 glucose) if the cells were used for contraction experiments or in the Na-HEPES phosphate-free buffer (in mmol/l: 4.8 KCl, 1.2 MgSO4, 132 NaCl, 10 HEPES, 2.5 Na-pyruvate, 10 glucose, and 0.2 CaCl2) if the cells were used for phosphorylation experiments (33). The cells were stored in control solution or in the Na+-HEPES phosphate-free buffer with 500 µmol/l Ca2+ at room temperature (22-23°C) until used.

Measurement of intracellular free Ca2+ transients and cell shortening. Fura 2 fluorescence and shortening of cells were monitored simultaneously, as described in detail elsewhere (32, 33). SIN-1, 3-morpholinoiminoacetonitrile (SIN-1C), and Iso were dissolved in H2O. All stock solutions were prepared freshly and added to the superfusion solution just before use.

Labeling of mouse myocytes with 32P. Experiments aimed at determining the level of protein phosphorylation in myocyte preparations were done as described by Wolska et al. (33) by use of a protocol modified from that described by Gupta et al. (10). Myocytes were incubated in 1.0 mmol/l Ca2+-Na-HEPES phosphate-free buffer with 0.5 mCi [32P]orthophosphate for 30 min at room temperature. Thereafter, cells were washed twice with the Na-HEPES phosphate-free solution with 1 mmol/l Ca2+. The myocyte suspensions (150 µl) were mixed with 150 µl of the Na-HEPES phosphate-free buffer with 1 mmol/l Ca2+ with or without added drugs, as described in the figure legends. After 2 min, the reaction was stopped by adding 150 µl of SDS stop solution (in mmol/l: 1 dithiothreitol, 30 Tris · HCl, and 3 EDTA; 6% SDS, 15% glycerol, and a trace of bromphenol blue). The samples were mixed well and stored at -20°C. Before analysis by SDS-PAGE, the samples were boiled for 10 min to convert the high molecular weight of PLB into its low-molecular-weight form. The 1H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one (ODQ) stock solution was prepared in DMSO. An equivalent amount of DMSO was added to all control samples. In experiments in which ODQ was used cells were preincubated with these drugs for 10 min.

Determination of protein phosphorylation. Gel electrophoresis was performed using a linear 5-20% polyacrylamide gradient gel as previously described (33). An aliquot of cells containing 50 µg of protein, as determined using the Lowry method (17), was applied to each lane. The SDS-PAGE gels were scanned using Personal Densitometer SI (Molecular Dynamics), vacuum dried, and placed in Storage Phosphor Screen Cassettes (Kodak) for overnight exposure. The cassettes were scanned in a Molecular Dynamics Storm 840 PhosphorImager. The ImageQuant software from Molecular Dynamics was used for data processing. Phosphorimage and densitometric data were adjusted for background and used for calculations. Data were normalized to the maximum values. Myofilament proteins were identified by comigration with known standards. PLB was identified using a monoclonal antibody.

Determination of cGMP and cAMP levels. Cyclic nucleotide measurements were performed on isolated myocytes by use of a modification of the method of George et al. (8). In this procedure, sample aliquots from myocyte preparations were resuspended in 1.5 ml of bath solution (control or drug). After 10 min, the myocyte samples were centrifuged at 14,000 g for 45 s, the supernatant was aspirated, and the cells were resuspended in 600 µl of cold 65% ethanol. The ethanol-myocyte solution was sonicated and then centrifuged at 14,000 g for 20 min at 4°C. The supernatants were aspirated and placed into separate microcentrifuge tubes, the ethanol was evaporated, and the remaining residue was stored at -20°C. The pellets were resuspended in 1% SDS for measurement of total protein by use of the bichinchoninic acid protein assay (Pierce, Rockford, IL). For measurement of cGMP levels, the frozen residue was first reconstituted with 50 mmol/l acetate buffer, and the cGMP content was determined by radioimmunoassay (RIA) with a commercially available kit (Biomedical Technologies, Stoughton, MA). The cAMP content was also determined by RIA with a commercially available kit (Amersham Pharmacia Biotech, Piscataway, NJ). We have previously used these methods to measure cAMP and cGMP levels in cardiac myocytes (35).

Statistical analysis. Data are presented as means ± SE. The significance of differences between the means was evaluated using appropriate analysis of variance (ANOVA) and the Student-Newman-Keul test for multiple comparisons. Values with P < 0.05 were considered statistically significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of SIN-1 and Iso on cell shortening and Ca2+ transients. We studied the effects of the NO ·  donor SIN-1 on twitch contractions and on fura 2 fluorescence ratios as a measure of intracellular free Ca2+ in single cardiac myocytes, isolated from rat hearts. Measurements were made before and after stimulation with the beta -adrenergic agonist Iso. A representative example of the effects of 3-min perfusion with 1 µM Iso alone on the Ca2+ transient is shown in Fig. 1A and the effects on cell shortening during twitch contraction in Fig. 1B. Three-minute perfusion with Iso resulted in a significant steady-state increase in cell shortening (to 237 ± 9% of control; n = 6) and an increase in the amplitude of the Ca2+ transient (to 157 ± 15% of control; n = 6). A representative example of the effects of 3-min perfusion with a combination of 1 µM Iso and 200 µM SIN-1 on the Ca2+ transient is shown in Fig. 1C and on those on cell shortening in Fig. 1D. When the cells were perfused for 3 min with 1 µM Iso and 200 µM SIN-1, cell shortening increased only to 167 ± 17% of control (n = 6), and the amplitude of the Ca2+ transient was increased to 124 ± 15% of control (n = 6). The reduced response to Iso was also observed when the cells were perfused for 5 or 7 min with addition of 200 µM SIN-1. Figure 2 summarizes the effects of 1 µM Iso in the presence and absence of 200 µM SIN-1 on the peak amplitude of the Ca2+ transient and amplitude of twitch contraction at three different time points (Fig. 2A) and on changes in time to 50 and 75% of relaxation of cell shortening after 3 min perfusion with Iso or a combination of Iso and SIN-1 (Fig. 2B).


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Fig. 1.   Effects of 200 µM 3-morpholinosydnonimine (SIN-1) on Ca2+ transients (A and C) and on myocyte shortening (B and D) in the presence of 1 µM isoproterenol (Iso). The recordings shown were performed in 2 separate cells in control conditions and after 3 min of perfusion with Iso or a combination of Iso and SIN-1.



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Fig. 2.   Summary of the effects of 1 µM Iso in the presence and absence of 200 µM SIN-1 on the peak amplitude of the Ca2+ transient and amplitude of twitch contraction at different time points (A) and on changes in time to 50 and 75% of relaxation of cell shortening after 3-min perfusion with Iso or a combination of Iso and SIN-1 (B). Data are presented as means ± SE; n = 6. *Significant difference between Iso and combination of Iso and SIN-1.

Effects of SIN-1 and Iso on protein phosphorylation. There are a number of mechanisms that could account for the altered Ca2+ transient and shortening of cells in the presence of SIN-1. One possibility is that SIN-1 may alter phosphorylation levels of proteins involved in excitation-contraction coupling. To determine this possibility directly, we measured phosphorylation of myocyte proteins under control conditions and after treatment with 1) 0.1 µM Iso; 2) 0.1 µM Iso + 200 µM SIN-1; 3) 0.1 µM Iso + 200 µM SIN-1C; 4) 0.5 µM Iso; 5) 0.5 µM Iso + 200 µM SIN-1; and 6) 0.5 µM Iso + 200 µM SIN-1C (Fig. 3A). SIN-1C, the final product of SIN-1 oxidation and which does not release NO · , was used as a negative control. Treatment with Iso resulted in a significant increase in 32P incorporation into PLB, cTnI, and C protein. Maximal phosphorylation of PLB and TnI (taken as 100%) was observed at 0.5 µM Iso. The level of phosphorylation of light chain 2 (LC2) was not altered by 0.5 µM Iso. SIN-1 (200 µM) or SIN-1C (200 µM) did not alter the basal levels of phosphorylation of PLB, cTnI, or LC2. However, 200 µM SIN-1 resulted in a significant decrease in the levels of phosphorylation of PLB and cTnI caused by Iso. There was no significant change in the levels of phosphorylation of LC2 or C protein. Addition of SIN-1C (200 µM) did not significantly reduce the phosphorylation of cTnI and PLB induced by Iso. Figure 3B summarizes the effects of buffer with vehicle alone (control), 0.5 µM Iso, 0.5 µM Iso plus 200 µM SIN-1, and 0.5 µM Iso plus 200 µM SIN-1C on the PLB, cTnI, and LC2 phosphorylation.


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Fig. 3.   Effects of SIN-1 and 3-morpholinoiminoacetonitrile (SIN-1C) on protein phosphorylation in basal conditions and in the presence of different concentrations of Iso. A: representative phosphorimage of proteins from 32P-labeled myocytes separated on a linear 5-20% gradient SDS-PAGE. Lane A, 0.1 µM Iso; lane B, 0.5 µM Iso; lane C, 0.1 µM Iso and 200 µM SIN-1; lane D, 0.5 µM Iso and 200 µM SIN-1; lane E, 0.1 µM Iso and 200 µM SIN-1C; lane F, 0.5 µM Iso and 200 µM SIN-1C; lane G, 200 µM SIN-1; lane H, 200 µM SIN-1C; lane I, buffer. B: summary of the effects of 200 µM SIN-1, 200 µM SIN-1C, and buffer with vehicle alone (control) on phosphorylation of cyclic troponin I (cTnI), phospholamban (PLB), and light chain 2 (LC2) in the presence of 0.5 µM Iso. Data are presented as means ± SE; n >=  6. *Significant difference from treatment with Iso.

Effects of SIN-1 on protein phosphorylation in the presence of ODQ. To test whether soluble guanylyl cyclase is involved in the effect of SIN-1 on the protein phosphorylation induced by Iso, we performed a series of experiments in the presence of 10 µM ODQ, a selective inhibitor of soluble guanylyl cyclase. Figure 4 shows a SDS-PAGE analysis (Fig. 4A) and phosphorimage (Fig. 4B) of proteins from isolated cardiac myocytes in the presence of 10 µM ODQ with and without 0.5 µM Iso and 200 µM SIN-1. Even in the presence of ODQ, SIN-1 significantly decreased the level of phosphorylation of cTnI and PLB caused by Iso. The summary of the phosphorylation levels of cTnI and PLB relative to the level of phosphorylation in the presence of 10 µM ODQ and 0.5 µM Iso is presented in Fig. 4C.


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Fig. 4.   Effects of 200 µM SIN-1 on Iso-dependent protein phosphorylation in the presence of 10 µM 1H-8-bromo-1,2, 4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin- 1-one (ODQ). Representative linear 5-20% gradient SDS-PAGE of cardiac myocyte proteins (A) and its phosphorimage (B). C-prot., C protein. Different treatments are indicated below the phosphroimage. C: summary of the effects of SIN-1 on protein phosphorylation in the presence of ODQ. Data are presented as means ± SE; n = 3. *Significant difference from treatment with Iso and ODQ.

SOD and catalase diminished the inhibitory effects of SIN-1 on Iso-induced protein phosphorylation.

To investigate whether NO ·  or NO-related congeners are involved in alteration of PLB and cTnI phosphorylation, we repeated the experiments in the presence of 50 U/ml SOD and 300 U/ml catalase. We did not see any significant changes in PLB and cTnI phosphorylation between myocytes treated with Iso-SOD-catalase and Iso-SOD-catalase-SIN-1 (data not shown). The presence of SOD and catalase prevented SIN-1 from decreasing phosphorylation of PLB and cTnI, indicating that these effects are related to NOx. The presence of SOD-catalase also increased removal of H2O2. To test whether H2O2 is involved in the effects of SIN-1 on Iso-induced protein phosphorylation, we performed our experiments with SOD alone. Figure 5 shows that the presence of SOD alone was sufficient to prevent SIN-1 from decreasing phosphorylation of PLB and cTnI. These data suggest that H2O2 is not involved in the effect of SIN-1 on protein phosphorylation in the presence of Iso and that NOx rather than NO ·  is responsible for SIN-1 effects.


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Fig. 5.   Effects of 200 µM SIN-1 on protein phosphorylation in the presence of 0.5 µM Iso with or without 50 U/ml superoxide dismutase (SOD). Data are presented as means ± SE; n = 3. *Significant difference from treatment with Iso and SOD; #significant difference from Iso, SOD, and SIN-1.

Effects of SIN-1 on shortening and Ca2+ transient in the presence of SOD. As shown in Fig. 5, the presence of SOD alone prevented SIN-1 from decreasing PLB and cTnI phosphorylation. We also wanted to know whether preventing changes in phosphorylation of PLB and cTnI would be sufficient to reverse functional changes in myocyte shortening and Ca2+ transients. We compared cell shortening and Ca2+ transients in myocytes exposed to the following conditions: 1) 35 U/ml SOD; 2) 35 U/ml SOD and 1 µM Iso; or 3) 35 U/ml SOD, 1 µM Iso, and 200 µM SIN-1. In the presence of SOD, SIN-1 did not significantly reduce the extent of shortening or the Ca2+ transient that were increased by Iso (Fig. 6). These data suggest that alteration in PLB and cTnI phosphorylation may be major mechanisms responsible for SIN-1 effects on shortening and the Ca2+ transient.


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Fig. 6.   Summary of the effects of 200 µM SIN-1 on shortening and Ca2+ transients in the presence of 1 µM Iso and 35 U/ml SOD. Data are presented as means ± SE; n >=  6.

Effects of SIN-1 on intracellular levels of cAMP and cGMP. To test the extent to which alterations in Ca2+ transient, cell shortening, and levels of protein phosphorylation are due to altered activities of cAMP- and cGMP-dependent kinases, we measured the levels of cAMP and cGMP in isolated rat ventricular myocytes with different treatments. Treatment of myocytes with Iso resulted in significant increases in the intracellular level of cAMP and no change in the level of cGMP. In the presence of Iso, addition of SIN-1 resulted in a significant increase in cGMP and a decrease in cAMP. Data from different treatments are summarized in Table 1.

                              
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Table 1.   Different treatments of myocytes


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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To the best of our knowledge, our results are the first to show directly that, in the presence of beta -adrenergic stimulation, SIN-1 alters phosphorylation of proteins involved in excitation-contraction coupling, specifically PLB and TnI. Important questions are 1) what the mechanism is by which NO is able to alter phosphorylation of regulatory proteins PLB and TnI; and 2) how alterations in phosphorylation levels of PLB and TnI can explain the results of functional studies with isolated cells.

The effect of NO on cardiac contractility may be biphasic, depending on the concentration of the NO ·  donor used (22). In the present study, we focused on the mechanisms responsible for altered cardiomyocyte contractility by high concentrations of SIN-1 in the presence of the beta -agonist Iso. Incubation of rat cardiac myocytes with SIN-1 resulted in an increased cGMP concentration in both the presence and absence of Iso. This first set of data suggests that the guanylyl cyclase and/or cGMP-stimulated or -inhibited phosphodiesterases (PDEs) may be involved in the observed effects of SIN-1. In addition to an increase in cGMP levels, SIN-1 caused a decrease in the concentration of cAMP, which suggests stimulation of PDEII and increased degradation of cAMP. However, after blocking soluble guanylyl cyclase by ODQ we found that, although cGMP level was now unchanged by SIN-1, SIN-1 still reduced the level of phosphorylation of PLB and TnI. These data indicate a cGMP-independent pathway for alterations in phosphorylation and are consistent with recently published results of Sandirasegarane and Diamond (25). They have shown that, in the presence of Iso, high concentrations of the NO donors S-nitroso-N-penicillamine and DEA resulted in marked attenuation of Iso-mediated increase in cell shortening, in both the presence and absence of ODQ. Moreover, Weiss et al. (29) found that, in isolated Langendorff-perfused rat hearts, effects of intracoronary perfusion of nitroprusside were unrelated to an increase in cGMP- or cGMP-dependent changes in cAMP.

Another important finding on the mechanism of SIN-1 action comes from our experiments with catalase and SOD. Our data show that the effects of SIN-1 on phosphorylation of PLB and TnI, as well as on blunting the Ca2+ transient and myocyte shortening, are related to NOx, and H2O2 is not involved in its action.

How can alterations in phosphorylation levels of PLB and TnI explain the results of functional studies with isolated cells? PLB is an inhibitory protein of the SR Ca2+ pump in its unphosphorylated state, and phosphorylation or ablation releases this inhibition (6, 14, 18). Reduction in PLB phosphorylation, as was observed in our experiments, would result in a reduction in Ca2+ transients and decrease contractility. At the same time, we found lower levels of TnI phosphorylation, which would result in increased myofilament Ca2+ sensitivity (24, 27). Thus TnI phosphorylation has an opposite effect on contractility compared with reduced phosphorylation of PLB. In isolated single myocytes, we found decreased contractility, which suggests that reduction in PLB phosphorylation is more important in determining effects of SIN-1 on contractility than on the increase myofilament sensitivity to Ca2+. Reduced levels of phosphorylation of PLB and TnI by SIN-1 in the presence of Iso would also result in the smaller effect on the kinetics of relaxation of cell shortening, as was observed in our experiments.

Although our experiments strongly suggest that effects of SIN-1 on rat cardiac myocyte contractility and levels of protein phosphorylation are largely cGMP independent, the cGMP-dependent mechanism of SIN-1 that has been previously postulated by us (28) and others (5) cannot be completely excluded and may play a significant role in different experimental conditions. For example, in frog, rat, and guinea pig ventricular myocytes, SIN-1 had no effect on ICa in the basal state (20, 21, 28). However, depending on the dose, SIN-1 had a stimulatory or inhibitory effect on ICa previously stimulated by forskolin, cAMP, or Iso (21, 28). In guinea pig and rat myocytes, it has previously been found that the inhibitory effect of SIN-1 on Ca2+ current was PDE independent (21, 28). In rat Langendorff-perfused hearts, Klabunde et al. (15) found that the effect of the NO inhibitor N-methyl-L-arginine on myocardial contractility was through an alteration in cGMP level, which led to an increase in the PDE activity and to a decreased cAMP level. Decreased concentration of cAMP would be expected to result in decreased activity of protein kinase A (PKA) and reduced phosphorylation of both cTnI and PLB. However, in our experiments, the decreased concentration of cAMP was probably not due to changes in cGMP-dependent PDE activity. Another possible mechanism by which SIN-1 reduced cAMP levels and PKA activity is its direct or indirect effect on phosphatase activity. It is important to note that Yokoyama et al. (34) reported that tumor necrosis factor-alpha , which is known to be elevated in heart failure and to work, at least in part, through a NO ·  pathway, caused a reduction in the phosphorylation levels of TnI and PLB with no change in phosphorylation of LC2 by a concentration-dependent activation of phosphatase type 2A and no effect on type 1 phosphatase activity; both phosphorylated PLB and TnI are excellent substrates for phosphatase type 2A (19).

In summary, the results of the present study demonstrate that, in rat cardiac myocytes and during beta -adrenergic stimulation, the negative inotropic effect of the exogenous NO ·  donor SIN-1 in cardiac myocytes is associated with a reduction in the peak amplitude of the Ca2+ transient and phosphorylation levels of TnI and PLB. These effects depend on both cAMP- and cGMP-dependent protein kinases. The results presented in this paper add important new insights into our understanding of pathophysiological conditions such as human heart failure in which the NO ·  signaling pathway is altered (11, 12).


    ACKNOWLEDGEMENTS

This study was supported by National Heart, Lung, and Blood Institute Grants R29 HL-58591 (B. M. Wolska) and T32 HL-07692 (M. O. Stojanovic and M. T. Ziolo). B. M. Wolska was supported by an Established Investigator Grant of the American Heart Association. G. M. Wahler was supported by a grant from the American Heart Association of Metropolitan Chicago and from funds provided by the Research Affairs Office of Midwestern University.


    FOOTNOTES

Address for reprint requests and other correspondence: B. M. Wolska, Section of Cardiology, Dept. of Medicine (M/C 787), Univ. of Illinois at Chicago, 840 S. Wood St. Chicago, IL 60612 (E-mail: bwolska{at}uic.edu).

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 19 December 2000; accepted in final form 26 February 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

1.   Balligand, JL, Kelly RA, Marsden PA, Smith TW, and Michel T. Control of cardiac muscle cell function by an endogenous nitric oxide signaling system. Proc Natl Acad Sci USA 90: 347-351, 1993[Abstract].

2.   Ballinger, SW, Patterson C, Yan CN, Doan R, Burow DL, Young CG, Yakes FM, Van Houten B, Ballinger CA, Freeman BA, and Runge MS. Hydrogen peroxide- and peroxynitrite-induced mitochondrial DNA damage and dysfunction in vascular endothelial and smooth muscle cells. Circ Res 86: 960-966, 2000[Abstract/Free Full Text].

3.   Beckman, JS, Beckman TW, Chen J, Marshall PA, and Freeman BA. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci USA 87: 1620-1624, 1990[Abstract].

4.   Brady, AJB, Warren JB, Poolewilson PA, Williams TJ, and Harding SE. Nitric oxide attenuates cardiac myocyte contraction. Am J Physiol Heart Circ Physiol 265: H176-H182, 1993[Abstract/Free Full Text].

5.   Chesnais, JM, Fischmeister R, and Mery PF. Positive and negative inotropic effects of NO donors in atrial and ventricular fibres of the frog heart. J Physiol (Lond) 518: 449-461, 1999[Abstract/Free Full Text].

6.   Colyer, J, and Wang JH. Dependence of cardiac sarcoplasmic reticulum calcium pump activity on the phosphorylation status of phospholamban. J Biol Chem 266: 17486-17493, 1991[Abstract/Free Full Text].

7.   Feelisch, M, Ostrowski J, and Noack E. On the mechanism of NO release from sydnonimines. J Cardiovasc Pharmacol 14, Suppl 11: S13-S22, 1989[ISI][Medline].

8.   George, EE, Romano FD, and Dobson JG, Jr. Adenosine and acetylocholine reduce isoproterenol-induced protein phosphorylation of rat myocytes. J Mol Cell Cardiol 23: 749-764, 1991[ISI][Medline].

9.   Gergel, D, Misik V, Ondrias K, and Cederbaum AI. Increased cytotoxicity of 3-morpholinosydnonimine to HepG2 cells in the presence of superoxide dismutase. Role of hydrogen peroxide and iron. J Biol Chem 270: 20922-20929, 1995[Free Full Text].

10.   Gupta, RC, Neumann J, Boknik P, and Watanabe AM. M2-specific muscarinic cholinergic receptor-mediated inhibition of cardiac regulatory protein phosphorylation. Am J Physiol Heart Circ Physiol 266: H1138-H1144, 1994[Abstract/Free Full Text].

11.   Hare, JM, Givertz MM, Creager MA, and Colucci WS. Increased sensitivity to nitric oxide synthase inhibition in patients with heart failure: potentiation of beta -adrenergic inotropic responsiveness. Circulation 97: 161-166, 1998[Abstract/Free Full Text].

12.   Haywood, GA, Tsao PS, vonderLeyen HE, Mann MJ, Kelling PJ, Trindade PT, Lewis NP, Byrne CD, Rickenbacher PR, Bishopric NH, Cooke JP, McKenna WJ, and Fowler MB. Expression of inducible nitric oxide synthase in human heart failure. Circulation 93: 1087-1094, 1996[Abstract/Free Full Text].

13.   Ito, N, Bartunek J, Spitzer KW, and Lorell BH. Effects of the nitric oxide donor sodium nitroprusside on intracellular pH and contraction in hypertrophied myocytes. Circulation 95: 2303-2311, 1997[Abstract/Free Full Text].

14.   James, P, Inui M, Tada M, Chiesi M, and Carafoli E. Nature and site of phospholamban regulation of the Ca pump of sarcoplasmic reticulum. Nature 342: 90-92, 1989[ISI][Medline].

15.   Klabunde, RE, Kimber ND, Kuk JE, Helgren MC, and Forstermann U. NG-methyl-L-arginine decreases contractility, cGMP and cAMP in isoproterenol-stimulated rat hearts in vitro. Eur J Pharmacol 223: 1-7, 1992[ISI][Medline].

16.   Kojda, G, Kottenberg K, Nix P, Schluter KD, Piper HM, and Noack E. Low increase in cGMP induced by organic nitrates and nitrovasodilators improves contractile response of rat ventricular myocytes. Circ Res 78: 91-101, 1996[Abstract/Free Full Text].

17.   Lowry, DH, Rosebrough NJ, Farr AL, and Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 193: 165-175, 1951.

18.   Luo, WS, Grupp IL, Harrer J, Ponniah S, Grupp G, Duffy JJ, Doetschman T, and Kranias EG. Targeted ablation of the phospholamban gene is associated with markedly enhanced myocardial contractility and loss of beta -agonist stimulation. Circ Res 75: 401-409, 1994[Abstract].

19.   MacDougall, LK, Jones LR, and Cohen P. Identification of the major protein phosphatases in mammalian cardiac muscle which dephosphorylate phospholamban. Eur J Biochem 196: 725-734, 1991[Abstract].

20.   Mery, PF, Lohmann SM, Walter U, and Fischmeister R. Ca2+ current is regulated by cyclic GMP-dependent protein kinase in mammalian cardiac myocytes. Proc Natl Acad Sci USA 88: 1197-1201, 1991[Abstract].

21.   Mery, PF, Pavoine C, Belhassen L, Pecker F, and Fischmeister R. Nitric oxide regulates cardiac Ca2+ current---involvement of cGMP-inhibited and cGMP-stimulated phosphodiesterases through guanylyl cyclase activation. J Biol Chem 268: 26286-26295, 1993[Abstract/Free Full Text].

22.   Mohan, P, Brutsaert DL, Paulus WJ, and Sys SU. Myocardial contractile response to nitric oxide and cGMP. Circulation 93: 1223-1229, 1996[Abstract/Free Full Text].

23.   Paolocci, N, Ekelund UE, Isoda T, Ozaki M, Vandegaer K, Georgakopoulos D, Harrison RW, Kass DA, and Hare JM. cGMP-independent inotropic effects of nitric oxide and peroxynitrite donors: potential role for nitrosylation. Am J Physiol Heart Circ Physiol 279: H1982-H1988, 2000[Abstract/Free Full Text].

24.   Robertson, SP, Johnson JD, Holroyde MJ, Kranias EG, Potter JD, and Solaro RJ. The effect of troponin I phosphorylation on the Ca2+-binding properties of the Ca2+-regulatory site of bovine cardiac troponin. J Biol Chem 257: 260-263, 1982[Free Full Text].

25.   Sandirasegarane, L, and Diamond J. The nitric oxide donors, SNAP and DEA/NO, exert a negative inotropic effect in rat cardiomyocytes which is independent of cyclic GMP elevation. J Mol Cell Cardiol 31: 799-808, 1999[ISI][Medline].

26.   Singh, RJ, Hogg N, Joseph J, Konorev E, and Kalyanaraman B. The peroxynitrite generator, SIN-1, becomes a nitric oxide donor in the presence of electron acceptors. Arch Biochem Biophys 361: 331-339, 1999[ISI][Medline].

27.   Strang, KT, Sweitzer NK, Greaser ML, and Moss RL. beta -Adrenergic receptor stimulation increases unloaded shortening velocity of skinned single ventricular myocytes from rats. Circ Res 74: 542-549, 1994[Abstract].

28.   Wahler, GM, and Dollinger SJ. Nitric oxide donor SIN-1 inhibits mammalian cardiac calcium current through cGMP-dependent protein kinase. Am J Physiol Cell Physiol 37: C45-C54, 1995[ISI].

29.   Weiss, HR, Sadoff JD, Scholz PM, and Klabunde RE. Nitric oxide reduces myocardial contractility in isoproterenol-stimulated rat hearts by a mechanism independent of cyclic GMP or cyclic AMP. Pharmacology 55: 202-210, 1997[ISI][Medline].

30.   Weyrich, AS, Ma XL, Buerke M, Murohara T, Armstead VE, Lefer AM, Nicolas JM, Thomas AP, Lefer DJ, and Vinten-Johansen J. Physiological concentrations of nitric oxide do not elicit an acute negative inotropic effect in unstimulated cardiac muscle. Circ Res 75: 692-700, 1994[Abstract].

31.   White, CR, Brock TA, Chang LY, Crapo J, Briscoe P, Ku D, Bradley WA, Gianturco SH, Gore J, and Freeman BA. Superoxide and peroxynitrite in atherosclerosis. Proc Natl Acad Sci USA 91: 1044-1048, 1994[Abstract].

32.   Wolska, BM, Kitada Y, Palmiter KA, Westfall MV, Johnson MD, and Solaro RJ. CGP-48506 increases contractility of ventricular myocytes and myofilaments by effects on actin-myosin reaction. Am J Physiol Heart Circ Physiol 39: H24-H32, 1996[ISI].

33.   Wolska, BM, Stojanovic MO, Luo WS, Kranias EG, and Solaro RJ. Effect of ablation of phospholamban on dynamics of cardiac myocyte contraction and intracellular Ca2+. Am J Physiol Cell Physiol 40: C391-C397, 1996[ISI].

34.   Yokoyama, T, Arai M, Sekiguchi K, Tanaka T, Kanda T, Suzuki T, and Nagai R. Tumor necrosis factor-alpha decreases the phosphorylation levels of phospholamban and troponin I in spontaneously beating rat neonatal cardiac myocytes. J Mol Cell Cardiol 31: 261-273, 1999[ISI][Medline].

35.   Ziolo, MT, Dollinger SJ, and Wahler GM. Myocytes isolated from rejecting transplanted rat hearts exhibit reduced basal shortening which is reversible by aminoguanidine. J Mol Cell Cardiol 30: 1009-1017, 1998[ISI][Medline].


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