AJP - Heart Journal of Neurophysiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 279: H1982-H1988, 2000;
0363-6135/00 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (51)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Paolocci, N.
Right arrow Articles by Hare, J. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Paolocci, N.
Right arrow Articles by Hare, J. M.
Vol. 279, Issue 4, H1982-H1988, October 2000

cGMP-independent inotropic effects of nitric oxide and peroxynitrite donors: potential role for nitrosylation

Nazareno Paolocci, Ulf E. G. Ekelund, Takayoshi Isoda, Michitaka Ozaki, Koenraad Vandegaer, Dimitrios Georgakopoulos, Robert W. Harrison, David A. Kass, and Joshua M. Hare

Division of Cardiology, Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, Maryland 21287-6568


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Nitric oxide (NO) has concentration-dependent biphasic myocardial contractile effects. We tested the hypothesis, in isolated rat hearts, that NO cardiostimulation is primarily non-cGMP dependent. Infusion of 3-morpholinosydnonimine (SIN-1, 10-5 M), which may participate in S-nitrosylation (S-NO) via peroxynitrite formation, increased the rate of left ventricular pressure rise (+dP/dt; 19 ± 4%, P < 0.001, n = 11) without increasing effluent cGMP or cAMP. Superoxide dismutase (SOD; 150 U/ml) blocked SIN-1 cardiostimulation and led to cGMP elaboration. Sodium nitroprusside (10-10-10-7 M), an iron nitrosyl compound, did not augment +dP/dt but increased cGMP approximately eightfold (P < 0.001), whereas diethylamine/NO (DEA/NO; 10-7 M), a spontaneous NO· donor, increased +dP/dt (5 ± 2%, P < 0.05, n = 6) without augmenting cGMP. SIN-1 and DEA/NO +dP/dt increase persisted despite guanylyl cyclase inhibition with 1H-(1,2,4)oxadiazolo-(4,3,-a)quinoxalin-1-one (10-5 M, P < 0.05 for both donors), suggesting a cGMP-independent mechanism. Glutathione (5 × 10-4 M, n = 15) prevented SIN-1 cardiostimulation, suggesting S-NO formation. SIN-1 also produced SOD-inhibitable cardiostimulation in vivo in mice. Thus peroxynitrite and NO donors can stimulate myocardial contractility independently of guanylyl cyclase activation, suggesting a role for S-NO reactions in NO/peroxynitrite-positive inotropic effects in intact hearts.

myocardial contractility; 3-morpholinosydnonimine; cyclic nucleotides; superoxide dismutase; glutathione; 1H-(1,2,4) oxadiazolo-(4,3,-a)quinoxalin-1-one; guanosine 3',5'-cyclic monophosphate


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE MECHANISM(S) by which nitric oxide (NO) influences myocardial contractility remains controversial (17). At least two biochemical mechanisms may be relevant to NO signaling in the heart: activation of heme-containing proteins and nitrosylation (35). First, NO activates soluble guanylyl cyclase by binding to its heme moiety, leading to the production of cGMP (13,25). Second, NO, a free radical, may also react with sulfhydryl moieties on either low molecular compounds or proteins (34, 37, 44, 45). Protein nitrosylation has been shown in vitro to activate various proteins involved in the regulation of myocardial contractility. Most prominently, NO may activate the L-type calcium channel (3) and the ryanodine receptor (46).

NO influences contractility in a concentration-dependent biphasic manner. In vitro studies (5, 19, 23, 41) indicate that lower concentrations of NO may be cardiostimulatory, whereas higher concentrations become cardiodepressant. In vivo, organic nitrate NO donors have no effect or a weak stimulatory effect (29). With regard to the chemical signaling pathways responsible, cGMP has been shown in some studies to mediate biphasic effects (19, 41). On the other hand, direct protein nitrosylation that is cGMP independent could potentially augment contractility via increases in Ca2+ cycling (3, 46).

The purpose of this study was to test the hypothesis that NO donors well characterized to participate in nitrosylation reactions (24) would have a positive inotropic effect independent of cGMP activation in both isolated hearts and in vivo. To further clarify the determinants of this reaction, we tested the effect of agents that inhibit guanylyl cyclase activity [e.g., 1-H-(1,2,4)oxadiazolo-(4,3,-a)quinoxalin-one (ODQ)] or modify nitrosylation [e.g., superoxide dismutase (SOD) and the reduced form of glutathione (GSH)] on this inotropic response.


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

Reagents. 3-Morpholinosydnonimine-HCl (SIN-1), diethylenetiamine pentaacetic acid (DTPA), Cu/Zn SOD, and GSH were purchased from Sigma Chemicals (St. Louis, MO). SIN-1 was prepared immediately before use as a 10 mM stock solution. ODQ was purchased from Tocris Cookson (Ballwin, MO) and was dissolved in 10% ethanol (0.1% final concentration in the heart), which did not alter contractility per se (data not shown). Sodium nitroprusside (SNP) was from Elkins-Sinn (Cherry Hill, NJ). Diethylamine/NO (DEA/NO) complex was a generous gift from Dr. D. A. Wink (Radiation Biology Branch, National Institutes of Health, Bethesda, MD). DEA/NO was dissolved and diluted in ice-cold 10 mM NaOH to prevent NO release until addition to the heart perfusate. NaOH alone did not alter contractility (data not shown).

Isolated heart preparation. Hearts were rapidly excised from male Wistar rats (n = 77) premedicated with 1,000 U im heparin and retrogradely perfused with oxygenated perfusion buffer at 37°C (15). A polyvinyl chloride balloon attached to a polyethylene-190 tubing balloon (Clay Adams-Becton-Dickinson, Parsipanny, NJ) was placed through the left atrium and mitral valve into the left ventricle. The balloon was filled with saline to achieve a maximum isovolumic developed pressure, which typically occurred at an end-diastolic pressure of 10-15 mmHg. Hearts were perfused at a constant flow by a peristaltic pump, initially titrated to achieve a coronary perfusion pressure of 80 mmHg. Constant flow was used to avoid confounding alterations in contractility due to the Gregg effect (11). The perfusate contained (in mmol/l) 144 sodium, 5 potassium, 1.5 calcium, 17.5 bicarbonate, 1.2 magnesium, and 134 chloride, along with 5 µg/ml lidocaine. This was equilibrated with a gas mixture of 95% O2-5% CO2, resulting in a perfusate pH of 7.4. Finally, the metal-chelating agent DTPA (100 µM) was added (made from a stock solution of 1 mM in water) to prevent lipid oxidation (43). The hearts were placed in a heated bath at 37°C and paced at 300 beats/min. Left ventricular (LV) pressure, the rate of change of LV pressure (dP/dt), and the mean coronary perfusion pressure were measured continuously (Gould, Cleveland, OH) and digitized at 1,000 Hz. The animal protocol was approved by the Johns Hopkins University School of Medicine Animal Care and Use Committee.

Drug protocol. In preliminary experiments, SIN-1 was observed to have long-lasting effects on myocardial contraction. Accordingly, the effects of 1, 10, and 100 µM SIN-1 (final concentrations within the coronary circulation) were tested in separate experiments. In all experiments, steady-state conditions were established over a 15-min period. The SIN-1 solution was then infused (Harvard Instruments) through a sidearm of the aortic cannula at 1% of the coronary flow rate. The effect of SIN-1 (10 µM for 15 min) was also evaluated in a subset of experiments omitting DTPA in the perfusate (n = 5).

Potential mechanisms for the effects of SIN-1 were probed by the following experiments: 1) To prevent SIN-1 decomposition to ONOO-, Cu/Zn SOD (150 IU/ml) was infused 5 min before and continued during SIN-1 administration (for 15 min). 2) To test whether a cGMP-dependent mechanism contributed to inotropic effects of SIN-1, ODQ (10-5 M) was infused for 15 min and continued during SIN-1 (10-5 M) coinfusion. 3) In additional hearts, experiments were conducted with coinfusion of GSH (5 × 10-4 M), which served as a "competing" thiol to block myocardial nitrosylation-based reactions. 4) To contrast NO donors with nitrosylating effects versus those without nitrosylating effects (20), SNP (an iron nitrosyl) was infused (10-10-10-7 M) to test for effects on myocardial contractility and cyclic nucleotide levels (cGMP and cAMP) in the coronary sinus drainage. SNP (10-8-10-7 M) was also coinfused with ODQ (10-5 M) to confirm the activity of this agent to block cGMP elaboration. 5) Finally, to verify whether other NO donors can enhance myocardial contractility, we infused DEA/NO (10-7 M), a spontaneous NO· donor in aqueous solutions, alone and after ODQ.

Cyclic nucleotide assays. In additional experiments, effluent from perfused hearts was collected to determine the concentration of cAMP and cGMP in response to SIN-1 (10-5 M) alone, SNP (10-10-10-7 M) alone, SIN-1 in the presence of SOD (150 U, n = 6) and ODQ (10-5 M, n = 9), SNP in presence of ODQ (n = 6), and DEA/NO (10-7 M, n = 6) alone. Samples (10 mL) were immediately frozen in liquid nitrogen. Assays were performed using an enzyme immunoassay (Biotrak, Amersham Pharmacia Biotech, Piscataway, NJ) using lyopholized samples. Cyclic nucleotide concentrations are expressed in picomolars per minute per gram of heart tissue. The assay has a sensitivity of detection of 2 fM.

Murine in vivo experiments. Male C57BL/6 mice (n = 25, 12-18 wk old, 20-35 g body wt; Jackson Laboratories) were used. Animals were housed under diurnal lighting conditions and allowed food and tap water ad libitum. Animal treatment and care was provided in accordance with institutional guidelines, and the protocol was approved by the Animal Care and Use Committee of the Johns Hopkins University.

Mice were anesthetized and ventilated as described (8). A combined micromanometer-conductance catheter (9) (model SPR-719, Millar Instruments, Houston, TX) was placed retrograde into the left ventricle in open-chest animals. Infusions were administered via the right jugular vein cannulated with a 30-gauge needle. The recorded volume signal from the conductance catheter requires calibration for absolute volume (offset) and stroke volume (gain). Absolute volume was derived using a saline wash in technique (1). Stroke volume calibration was derived from cardiac output obtained from direct measurements of aortic flow, obtained using a flow probe (AT01RB, Transonic Systems, Ithaca, NY) placed around the aorta, and the flow per minute was recorded (AT106, Transonic Systems) via a lateral thoracotomy. Pressure, volume, and flow signals were digitized at 1,000 Hz, stored to disk, and analyzed with custom software.

Statistical analysis. Data are presented as means ± SE. Concentration-effect responses were analyzed by two-way ANOVA tests using an identification term for each individual experiment and the Student-Newman-Keuls post hoc test (42). For experiments comparing single dose effects, paired t-tests were applied. A P value <0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of SIN-1 on myocardial contractility and cyclic nucleotide concentrations. The baseline conditions of isolated hearts are shown in Table 1. Figure 1 depicts the concentration-dependent effects of SIN-1 on myocardial contractility. The peak rate of LV pressure rise (peak +dP/dt) rose by 9 ± 4% (P < 0.05, n = 5) at 1 µM and 19 ± 4% (P < 0.001, n = 11) at 10 µM but was unchanged (n = 4) at 100 µM. In separate experiments where DPTA was omitted from the perfusate, SIN-1 augmented +dP/dt to a similar degree (data not shown). Coronary perfusion pressure decreased by 12 ± 3% (P = 0.002) at 10 µM but not at 1 µM. Furthermore, SIN-1 (10 µM) decreased LV end-diastolic pressure (LVEDP) from 13 ± 2 to 8 ± 2 mmHg (P = 0.004 vs. baseline) and shortened the time constant of relaxation from 28 ± 2 to 20 ± 2 mmHg (P = 0.005 vs. baseline). The positive inotropic effect of SIN-1 was not accompanied by changes in effluent concentrations of cGMP or cAMP (Fig. 1 and Table 2).

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Baseline conditions in isolated rat hearts



View larger version (20K):
[in this window]
[in a new window]
  		Fig. 1.   Effect of 3-morpholinosydnonimine (SIN-1), with and without superoxide dismutase (SOD), on myocardial contractility and cGMP production. Retrograde perfused rat hearts were administered SIN-1 alone or SIN-1 after pretreatment with SOD at the indicated molar concentrations. The percentage of increase in peak rate of left ventricular pressure rise (peak +dP/dt) from baseline (A) and the increase in cGMP concentration in coronary sinus drainage (B) is depicted. Data represent means ± SE. *P < 0.05 vs. baseline.


                              
View this table:
[in this window]
[in a new window]
  		Table 2.   Cyclic nucleotide levels in control and SIN-1-treated hearts

Redox modulation of SIN-1-positive inotropic effect. To assess whether SIN-1 augmentation of contractility depended on a balance of superoxide and NO (i.e., peroxynitrite formation), SOD was coinfused with SIN-1. SOD, which had no effect on +dP/dt alone, fully prevented the SIN-1-positive inotropic effect (3,056 ± 164 and 3,100 ± 171 mmHg/s, SOD vs. SOD + SIN-1, respectively, n = 17). Moreover, in the presence of SOD, SIN-1 now modestly augmented effluent cGMP (Fig. 1).

Impact of GSH on SIN-1 inotropic effects. To test whether administration of a competing thiol could block the SIN-1-positive inotropic response, we coinfused the low-molecular-weight thiol GSH (5 × 10-4 M). GSH alone did not affect +dP/dt but fully prevented SIN-1 inotropy (2,511 ± 144 and 2,393 ± 126 mmHg/s, GSH vs. GSH + SIN-1, respectively, n = 15).

Impact of ODQ on SIN-1-positive inotropic effects and cGMP perfusate levels. To further assess the impact of guanylyl cyclase on SIN-1-positive inotropic responses, we performed an additional series of experiments in which SIN-1 (10-5 M) was administered after 15 min of ODQ (10-5 M). As shown in Fig. 2, ODQ alone significantly increased +dP/dt from 2,507 ± 152 to 2,731 ± 199 mmHg/s (9 ± 3%, P < 0.05 vs. baseline, n = 9), and SIN-1 further augmented +dP/dt to 2,898 ± 171 mmHg/s (16 ± 3%, P < 0.05 vs. baseline and ODQ alone). ODQ lowered cGMP levels in the perfusate by 40 ± 15% (P < 0.05 vs. control, n = 5), and SIN-1 did not change cGMP concentrations further (P < 0.05 vs. control; not significant vs. ODQ alone).


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 2.   Effect of 1-H-(1,2,4)oxadiazolo-(4,3,-a)quinoxalin-one (ODQ) on the inotropic and cGMP response to SIN-1. The percentage of increase in peak +dP/dt from baseline (A; n = 9) and the absolute change in cGMP perfusate level (B; n = 5) is depicted. Data represent means ± SE. *P < 0.05 vs. baseline; dagger P < 0.05 vs. ODQ alone.

Effects of SNP on myocardial contractility and cyclic nucleotide concentrations. SNP infused over 10-10-10-7 M did not alter peak +dP/dt (n = 5, Fig. 3). Similar to the effects of SIN-1, SNP decreased coronary perfusion pressure, with a maximal decrease of 15 ± 2% (P < 0.0001) at 10-7 M, and decreased LVEDP by 18 ± 3% (P < 0.0005). In marked contrast to SIN-1, SNP profoundly increased effluent cGMP concentrations (~8-fold, P < 0.002, n = 3; Fig. 3) but did not change cAMP levels (data not shown). In the presence of ODQ, the increase in cGMP was completely prevented (Fig. 3).


View larger version (20K):
[in this window]
[in a new window]
  		Fig. 3.   Effect of sodium nitroprusside (SNP), with and without ODQ, on myocardial contractility and cGMP perfusate levels. A: dose-response curve to SNP alone (n = 5) and with ODQ (n = 6). B: increase in cGMP levels from baseline with SNP alone (n = 3) and SNP + ODQ (n = 5). Data represent means ± SE. *P < 0.05 vs. baseline.

Effects of DEA/NO on myocardial contractility and cyclic nucleotide concentrations. To assess whether the SIN-1-positive inotropic effect was dependent on peroxynitrite, we infused another spontaneous NO donor, DEA/NO. As shown in Fig. 4, DEA/NO (10-7 M for 15 min) augmented +dP/dt by 5 ± 2% (n = 6, P < 0.05). This inotropic effect was not accompanied by any changes in cGMP in the heart perfusate (P = not significant, n = 6). Moreover, this augmentation also persisted in the presence of ODQ (10-5 M for 15 min). ODQ alone augmented +dP/dt by 9 ± 2% (P < 0.05 from baseline, n = 10), and the subsequent addition of DEA/NO further increased myocardial contractility (+14 ± 3% from baseline, P < 0.005, n = 10).


View larger version (25K):
[in this window]
[in a new window]
  		Fig. 4.   Effect of diethylamine/nitric oxide (DEA/NO) alone (n = 6) and in combination with ODQ (n = 10) on myocardial contractility. Data represent means ± SE. *P < 0.05 vs. baseline; dagger P < 0.05 vs. ODQ alone.

In vivo effects of SIN-1. To test the relevance of these findings in vivo, we infused SIN-1 with and without SOD pretreatment to anesthetized C57BL/6 mice instrumented with a combined pressure-volume catheter. Baseline conditions are depicted in Table 3. Graded infusion of SIN-1 (80, 160, and 320 µg · kg-1 · min-1, ~9-36 µM, n = 18) caused a positive inotropic response. Ventricular elastance, the slope of the end-systolic pressure-volume relation, exhibited marked increases in slope from 18.7 ± 4.2 to 29.7 ± 10.5 mmHg/µl, maximal at 320 µg · kg-1 · min-1 (P < 0.04; Fig. 5). This positive inotropic effect was reflected in multiple indexes of myocardial contractility, namely the preload recruitable stroke work and the dP/dt corrected for pressure. In the presence of 30 IU Cu/Zn SOD (n = 6), the positive inotropic effect of SIN-1 was abolished or converted to a negative inotropic effect (Table 3).

                              
View this table:
[in this window]
[in a new window]
  		Table 3.   Hemodynamic effects of SIN-1 and SIN-1 in combination with Cu/Zn SOD in mice



View larger version (41K):
[in this window]
[in a new window]
  		Fig. 5.   Representative pressure-volume data generated with transient occlusion of the inferior vena cava. The pressure-volume loops before (solid loops) and after SIN-1 infusion (dashed loops, 160 µg · kg-1 · min-1) are shown. The solid line indicates slope of the end-systolic pressure-volume relationship obtained by the nonlinear regression of the end-systolic pressure volume points. The pressure-volume relationship shifted to the left, indicating a positive inotropic effect.

SIN-1 in the presence or absence of SOD did not change preload, as measured by LVEDP (8 ± 1 vs. 8 ± 1 mmHg, respectively), LV end-diastolic volume (19 ± 2 vs. 21 ± 2 µl, respectively), or heart rate (Table 3). On the other hand, SIN-1 exerted a vasodilator action: arterial elastance (afterload) decreased 17 ± 5% from the control value of 14.4 ± 6.4 mmHg/µl (P = 0.02). This effect was not different in the presence of SOD. Thus SIN-1 had both a positive inotropic and vasodilator effect in mice.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we demonstrate in both isolated crystalloid perfused hearts and in vivo that the NO/peroxynitrite donor SIN-1 has positive inotropic effects independent of cGMP or cAMP formation. In contrast, SNP, which potently stimulated cGMP formation, did not augment myocardial contraction. The SIN-1-positive inotropic effect could be abrogated by SOD, which also led to increased cGMP. The SIN-1 response persisted in the presence of guanylyl cyclase inhibition but was blocked by GSH, supporting a role for a thiol-nitrosylation-dependent mechanism of action. Finally, a spontaneous donor of NO·, DEA/NO, also had a positive inotropic effect independent of cGMP release.

The conditions under which NO stimulates or inhibits myocardial contractility remain controversial. Initial studies focused on the contractile impact of cGMP. NO has been reproducibly demonstrated in vivo to have a negative inotropic effect in the presence of beta -adrenergic stimulation (14, 16, 18), likely mediated by cGMP formation (32, 33). Positive inotropic effects of NO donors in the absence of beta -adrenergic stimulation have also been described (29). With regard to cGMP signaling, this nucleotide appears to have a biphasic effect on basal myocardial contractility, with low concentrations being cardiostimulatory (27). Whereas diverse metabolic (21, 39) and physiological (12) effects have been attributed to the NO synthase 3 isoform found in many cardiac cellular constituents, including myocytes, recent investigations (10, 40) have questioned the obligatory role of NO synthase 3 in myocardial contractile regulation.

Recently, the cardiac effects of NO that are cGMP independent are increasingly being appreciated in both amphibian (5) and mammalian (41) systems. One mechanism that could contribute to cardiac stimulation is nitrosylation of the L-type calcium channel (3) and the ryanodine receptor (46), which has been shown to activate these calcium-cycling proteins in vitro.

In the present study, we infused different NO donors to isolated rat hearts and measured the contractile and cyclic nucleotide responses. The NO donor SIN-1 releases equimolar NO and O2-·, which react to form ONOO-, in a reaction six times faster than SOD scavenging at physiological ionic strength (6, 24, 30, 36). ONOO- is an effective nitrosating species (24) and has been previously used in experiments regarding activation of the L-type calcium channel (3) and neutrophil-endothelium interactions in myocardial ischemia-reperfusion (26). SIN-1 stimulated myocardial contraction without altering the concentration of either cGMP or cAMP in the heart effluent. The iron nitrosyl NO donor SNP, which potently stimulated cGMP formation, did not increase contractility. Supporting this cGMP independence, SIN-1 inotropic responses were maintained in the presence of the soluble guanylyl cyclase inhibitor ODQ. In constrast, the response of SIN-1 could be converted to one resembling SNP (increasing cGMP without influencing contractility) by pretreatment with SOD. SOD would be anticipated to quench superoxide and prevent ONOO- formation. Finally, DEA/NO, which spontaneously releases NO· in aqueous solution (7), also stimulated myocardial contractility in the presence of ODQ, suggesting that other NO donors can similarly enhance contractility in a cGMP-independent manner. Thus different chemical signaling of NO/peroxynitrite donors produces different inotropic effects. The redox dependence of these reactions points out a factor that may not have been controlled for in previous studies.

In contrast to the divergence in inotropic effects, both SIN-1 and SNP had positive lusitropic effects, reducing LVEDP, the time constant of relaxation (tau ), or both. With regard to SNP, this response is most likely due to cGMP elevation, consistent with numerous in vitro (23, 33) and in vivo studies (28). The SIN-1-positive lusitropic effect, in the absence of cGMP elevation, is likely due to the same mechanism responsible for the positive inotropic effect (i.e., a Ca2+-mediated action).

Several recent studies have examined NO-positive inotropic responses in vitro. Vila-Petroff et al. (41) demonstrated in rat myocytes that NO donors at low concentrations stimulated myocyte contractile amplitude in association with an increase in calcium transients as well as cAMP production. Higher concentrations of NO donors inhibited contractile amplitude in a cGMP-dependent manner. Chesnais and colleagues reported similar observations in frog myocytes and also described that positive inotropic responses to NO donors (5) or peroxynitrite (4) could be inhibited or converted to negative inotropic responses by SOD. An important regulatory link between NO synthase and superoxide has been suggested by the colocalization of NO synthase 3 and SOD in myocytes (2). Taken together, these studies support a redox-sensitive, non-cGMP dependent mechanism for NO-related positive inotropy. The present study extends these findings to a whole heart and in vivo preparations and raises the issue that these effects are due to nitrosylation-related mechanisms.

The physiological roles of nitrosylation-based reactions are being increasingly appreciated. Thiol-nitrosylation reactions have been shown to confer NO-like vasodilator activity to albumin (37) and other low-molecular-weight thiols (31), enhance tissue plasminogen activator function (34), participate in NO entry into cells (47), maintain balance between blood vessel tone and tissue oxygen requirements (38), and contribute to the regulation of caspases (22). The present findings extend in vitro observations that the L-type Ca2+ channel and the ryanodine receptor undergo thiol-nitrosylation leading to increased calcium cycling to the level of myocardial contractility.

Given that our infusions were performed in a crystalloid-perfused preparation, we sought to confirm the SIN-1 effects in vivo. Using mice, we confirmed that SIN-1 stimulates myocardial contraction and that this positive inotropic effect could be prevented by pretreatment with SOD. Thus these reactions are relevant in the intact cardiovascular system.

Two issues warrant mention. First, guanylyl cyclase inhibition with ODQ produced a positive inotropic effect, consistent with basal suppression of contractility by cGMP. Second, in the presence of ODQ, the SIN-1 inotropic effect was of less magnitude than that of SIN-1 alone (see Figs. 1 and 2). On the other hand, the NO donor DEA/NO produced similar responses (5% increases in +dP/dt) alone and after ODQ administration (see Fig. 4). These findings suggest that there may be important differences between the cross-talk between peroxynitrite versus NO· and cGMP signaling in the regulation of myocardial contractility.

This study is limited by the lack of direct biochemical measurement of protein nitrosylation. Endogenous nitrosylation of ryanodine receptors purified from dog myocytes has been demonstrated by Xu et al. (46). Future work is aimed at directly correlating NO effects on myocardial contractility with biochemical measures of protein nitrosylation. In addition, we did not measure intracellular cyclic nucleotide concentration changes in response to various stimuli. The cyclic nucleotide changes observed in the effluent of perfused hearts may not totally reflect intracellular events (e.g., SNP increases and ODQ decreases cGMP levels). Nevertheless, the changes observed are qualitatively those expected and are consistent with the intracellular observations previously reported (41).

In summary, the present data demonstrate positive inotropic effects of NO donors that are independent of cyclic nucleotide production. Conversely, NO donors that increase cGMP production do not increase myocardial contractility. Blockade of the response by GSH support the idea that this effect involves a nitrosylation reaction. The present findings may have broad implications for the regulation of a wide number of processes based on calcium cycling.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grant K08-HL-03238 and a Grant-in-Aid from the American Heart Association (to J. M. Hare) and National Heart, Lung, and Blood Institute Grant HL-47511 (to D. A. Kass). N. Paolocci was supported by Universita' di Perugia, and U. E. G. Ekelund was supported by a grant from the Swedish Foundation for International Cooperation in Research and Higher Education.


    FOOTNOTES

Address for reprint requests and other correspondence: J. M. Hare, Johns Hopkins Hosp., Cardiology Div., 600 N. Wolfe St., Carnegie 568, Baltimore, MD 21287-6568 (E-mail: jhare{at}mail.jhmi.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 13 September 1999; accepted in final form 17 April 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Baan, J, Van der Velde ET, de Brun HG, Smeenk GJ, Koops J, van Dijk AD, Temmerman D, Senden J, and Buis B. Continuous measurement of left ventricular volume in animals and humans by conductance catheter. Circulation 70: 812-823, 1994[Abstract/Free Full Text].

2.   Brahmajothi, MV, and Campbell DL. Heterogeneous basal expression of nitric oxide synthase and superoxide dismutase isoforms in mammalian heart: implications for mechanisms governing indirect and direct nitric oxide-related effects. Circ Res 85: 575-587, 1999[Abstract/Free Full Text].

3.   Campbell, DL, Stamler JS, and Strauss HC. Redox modulation of L-type calcium channels in ferret ventricular myocytes. Dual mechanism regulation by nitric oxide and S-nitrosothiols. J Gen Physiol 108: 277-293, 1996[Abstract/Free Full Text].

4.   Chesnais, JM, Fischmeister R, and Mery PF. Peroxynitrite is a positive inotropic agent in atrial and ventricular fibres of the frog heart. J Physiol (Lond) 521: 375-388, 1999[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.   Cudd, A, and Fridovitch I. Electrostatic interactions in the reaction mechanism of bovine erythrocyte superoxide dismutase. J Biol Chem 257: 11443-11447, 1982[Abstract/Free Full Text].

7.   Feelisch, M, and Stamler JS. Donors of nitrogen oxides. In: Methods in Nitric Oxide Research, edited by Feelisch M, and Stamler JS.. Chichester: Wiley, 1996, p. 71-115.

8.   Georgakopoulos, D, Christe ME, Giewat M, Seidman CM, Seidman JG, and Kass DA. The pathogenesis of familial hypertrophic cardiomyopathy: early and evolving effects from an alpha -cardiac myosin heavy chain missense mutation. Nat Med 5: 327-330, 1999[ISI][Medline].

9.   Georgakopoulos, D, Mitzner WA, Chen C, Byrne BJ, Millar HD, Hare JM, and Kass DA. In vivo murine left ventricular pressure-volume relations by miniaturized conductance micromanometry. Am J Physiol Heart Circ Physiol 274: H1416-H1422, 1998[Abstract/Free Full Text].

10.   Godecke, A, Decking UK, Ding Z, Hirchenhain J, Bidmon HJ, Godecke S, and Schrader J. Coronary hemodynamics in endothelial NO synthase knockout mice. Circ Res 82: 186-194, 1998[Abstract/Free Full Text].

11.   Goto, Y, Slinker BK, and LeWinter MM. Effect of coronary hyperemia on Emax and oxygen consumption in blood-perfused rabbit hearts. Circ Res 68: 482-492, 1991[Abstract/Free Full Text].

12.   Han, X, Kubota I, Feron O, Opel DJ, Arstall MA, Zhao Y, Huang P, Fishman MC, Michel T, and Kelly RA. Muscarinic cholinergic regulation of cardiac myocyte ICa-L is absent in mice with targeted disruption of endothelial nitric oxide synthase. Proc Natl Acad Sci USA 95: 6510-6515, 1998[Abstract/Free Full Text].

13.   Hare, JM, and Colucci WS. Role of nitric oxide in the regulation of myocardial function. Prog Cardiovasc Dis 38: 155-166, 1995[ISI][Medline].

14.   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].

15.   Hare, JM, Kim B, Flavahan NA, Ricker KM, Peng X, Colman L, Weiss RG, and Kass DA. Pertussis toxin-sensitive G proteins influence nitric oxide synthase III activity and protein levels in rat heart. J Clin Invest 101: 1424-1431, 1998[ISI][Medline].

16.   Hare, JM, Loh E, Creager MA, and Colucci WS. Nitric oxide inhibits the contractile response to beta -adrenergic stimulation in humans with left ventricular dysfunction. Circulation 92: 2198-2203, 1995[Abstract/Free Full Text].

17.   Hare, JM, and Stamler JS. NOS: modulator, not mediator of cardiac performance. Nat Med 5: 273-274, 1999[ISI][Medline].

18.   Keaney, JF, Jr, Hare JM, Balligand JL, Loscalzo J, Smith TW, and Colucci WS. Inhibition of nitric oxide synthase augments myocardial contractile responses to beta -adrenergic stimulation. Am J Physiol Heart Circ Physiol 271: H2646-H2652, 1996[Abstract/Free Full Text].

19.   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].

20.   Lipton, SA, Choi YB, Pan ZH, Lei SZ, Chen HSV, Sucher NJ, Loscalzo J, Singel DJ, and Stamler JS. A redox-based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and related nitroso-compounds. Nature 364: 626-632, 1993[Medline].

21.   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[Abstract/Free Full Text].

22.   Mannick, JB, Hausladen A, Liu L, Hess DT, Zeng M, Miao QX, Kane LS, Gow AJ, and Stamler JS. Fas-induced caspase denitrosylation. Science 284: 651-654, 1999[Abstract/Free Full Text].

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

24.   Mohr, S, Stamler JS, and Brune B. Mechanism of covalent modification of glyceraldehyde-3-phosphate dehydrogenase at its active site thiol by nitric oxide, peroxynitrite and related nitrosating agents. FEBS Lett 348: 223-227, 1994[ISI][Medline].

25.   Moncada, SA. The L-arginine-nitric oxide pathway. N Engl J Med 329: 2002-2012, 1993[Free Full Text].

26.   Nossuli, TO, Hayward R, Jensen D, Scalia R, and Lefer AM. Mechanisms of cardioprotection by peroxynitrite in myocardial ischemia and reperfusion injury. Am J Physiol Heart Circ Physiol 275: H509-H519, 1998[Abstract/Free Full Text].

27.   Ohba, M, and Kawata H. Biphasic nature of inotropic action of nitric oxide donor NOC7 in guinea-pig ventricular trabeculae. Jpn J Physiol 49: 389-394, 1999[ISI][Medline].

28.   Paulus, WJ, Vantrimpont PJ, and Shah AM. Acute effects of nitric oxide on left ventricular relaxation and diastolic distensibility in humans. Assessment by bicoronary sodium nitroprusside infusion. Circulation 89: 2070-2078, 1994[Abstract/Free Full Text].

29.   Preckel, B, Kojda G, Schlack W, Ebel D, Kottenberg K, Noack E, and Thamer V. Inotropic effects of glyceryl trinitrate and spontaneous NO donors in the dog heart. Circulation 96: 2675-2682, 1997[Abstract/Free Full Text].

30.   Rigo, A, and Viglino P. Effect of ionic strength on the activity of bovine superoxide dismutase. FEBS Lett 50: 86-88, 1975[ISI][Medline].

31.   Scharfstein, JS, Keaney JF, Jr, Slivka A, Welch GN, Vita J, Stamler JS, and Loscalzo J. In vivo transfer of nitric oxide between a plasma protein-bound reservoir and low molecular weight thiols. J Clin Invest 94: 1432-1439, 1994.

32.   Shah, AM, Lewis MJ, and Henderson AH. Effects of 8-bromo-cyclic GMP on contraction and on inotropic response of ferret cardiac muscle. J Mol Cell Cardiol 23: 55-64, 1991[ISI][Medline].

33.   Shah, AM, Spurgeon HA, Sollott SJ, Talo A, and Lakatta EG. 8-Bromo-cGMP reduces the myofilament response to Ca2+ in intact cardiac myocytes. Circ Res 74: 970-978, 1994[Abstract/Free Full Text].

34.   Simon, DI, Mullins ME, Jia L, Gaston B, Singel DJ, and Stamler JS. Polynitrosylated proteins: characterization, bioactivity, and functional consequences. Proc Natl Acad Sci USA 93: 4736-4741, 1996[Abstract/Free Full Text].

35.   Stamler, JS. Redox signaling: nitrosylation and related target interactions of nitric oxide. Cell 78: 931-936, 1994[ISI][Medline].

36.   Stamler, JS, and Feelisch M. Biochemistry of nitric oxide and redox-related species. In: Methods in Nitric Oxide Research, edited by Feelisch M, and Stamler JS.. Chichester: Wiley, 1996, p. 19-28.

37.   Stamler, JS, Jaraki O, Osborne J, Simon DI, Keaney JF, Jr, Vita J, Singel D, Valeri CR, and Loscalzo J. Nitric oxide circulates in mammalian plasma primarily as an S-nitroso adduct of serum albumin. Proc Natl Acad Sci USA 89: 7674-7677, 1992[Abstract/Free Full Text].

38.   Stamler, JS, Jia L, Eu JP, McMahon TJ, Demchenko IT, Bonaventura J, Gernert K, and Piantadosi CA. Blood flow regulation by S-nitrosohemoglobin in the physiological oxygen gradient. Nature 276: 2034-2037, 1997.

39.   Tada, H, Thompson CI, Recchia FA, Loke KE, Ochoa M, Smith CJ, Shesely EG, Kaley G, and Hintze TH. Myocardial glucose uptake is regulated by nitric oxide via endothelial nitric oxide synthase in langendorff mouse heart. Circ Res 86: 270-274, 2000[Abstract/Free Full Text].

40.   Vandecasteele, G, Eschenhagen T, Scholz H, Stein B, Verde I, and Fischmeister R. Muscarinic and beta -adrenergic regulation of heart rate, force of contraction and Ca2+ current is preserved in mice lacking endothelial nitric oxide synthase. Nat Med 5: 331-334, 1999[ISI][Medline].

41.   Vila-Petroff, MG, Younes A, Egan J, Lakatta EG, and Sollott SJ. Activation of distinct cAMP-dependent and cGMP-dependent pathways by nitric oxide in cardiac myocytes. Circ Res 84: 1020-1031, 1999[Abstract/Free Full Text].

42.   Wallenstein, S, Zucker C, and Fleiss J. Some statistical methods useful in circulation research. Circ Res 47: 1-9, 1980[Abstract/Free Full Text].

43.   White, R, Crow JH, Spear N, Thomas S, Patel RP, Green I, Beckman JS, and Darley-Usmar VM. Making and working with peroxynitrite. In: Methods in Molecular Biology: Nitric Oxide Protocols, edited by Titheradge MA.. Totowa, NJ: Humana, 1998, p. 215-230.

44.   Wink, DA, Cook JA, Kim SY, Vodovotz Y, Pacelli R, Krishna MC, Russo A, Mitchell JB, Jourd'heuil D, Miles AM, and Grisham MB. Superoxide modulates the oxidation and nitrosation of thiols by nitric oxide-derived reactive intermediates. J Biol Chem 272: 11147-11151, 1997[Abstract/Free Full Text].

45.   Wink, DA, and Mitchell JB. Chemical biology of nitric oxide: insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radic Biol Med 25: 434-456, 1998[ISI][Medline].

46.   Xu, L, Eu JP, Meissner G, and Stamler JS. Activation of the cardiac calcium release channel (ryanodine receptor) by poly-S-nitrosylation. Science 279: 234-237, 1998[Abstract/Free Full Text].

47.   Zai, A, Rudd MA, Scribner AW, and Loscalzo J. Cell-surface protein disulfide isomerase catalyzes transnitrosation and regulates intracellular transfer of nitric oxide. J Clin Invest 103: 393-399, 1999[ISI][Medline].

Am J Physiol Heart Circ Physiol 279(4):H1982-H1988
0363-6135/00 $5.00 Copyright © 2000 the American Physiological Society


This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
M. J. Kohr, H. Wang, D. G. Wheeler, M. Velayutham, J. L. Zweier, and M. T. Ziolo
Targeting of phospholamban by peroxynitrite decreases {beta}-adrenergic stimulation in cardiomyocytes
Cardiovasc Res, January 15, 2008; 77(2): 353 - 361.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
R. Yang and L. A. Barouch
Leptin Signaling and Obesity: Cardiovascular Consequences
Circ. Res., September 14, 2007; 101(6): 545 - 559.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
T. Dai, Y. Tian, C. G. Tocchetti, T. Katori, A. M. Murphy, D. A. Kass, N. Paolocci, and W. D. Gao
Nitroxyl increases force development in rat cardiac muscle
J. Physiol., May 1, 2007; 580(3): 951 - 960.
[Abstract] [Full Text] [PDF]

Home page
 HypertensionHome page
E. Takimoto and D. A. Kass
Role of Oxidative Stress in Cardiac Hypertrophy and Remodeling
Hypertension, February 1, 2007; 49(2): 241 - 248.
[Full Text] [PDF]

Home page
 CirculationHome page
J. M. Zimmet and J. M. Hare
Nitroso-Redox Interactions in the Cardiovascular System
Circulation, October 3, 2006; 114(14): 1531 - 1544.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
A. S. Jung, H. Kubo, R. Wilson, S. R. Houser, and K. B. Margulies
Modulation of contractility by myocyte-derived arginase in normal and hypertrophied feline myocardium
Am J Physiol Heart Circ Physiol, May 1, 2006; 290(5): H1756 - H1762.
[Abstract] [Full Text] [PDF]

Home page
 Exp. Biol. Med.Home page
E. B. Manukhina, H. F. Downey, and R. T. Mallet
Role of nitric oxide in cardiovascular adaptation to intermittent hypoxia.
Experimental Biology and Medicine, April 1, 2006; 231(4): 343 - 365.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
M. Mongillo, C. G. Tocchetti, A. Terrin, V. Lissandron, Y.-F. Cheung, W. R. Dostmann, T. Pozzan, D. A. Kass, N. Paolocci, M. D. Houslay, et al.
Compartmentalized Phosphodiesterase-2 Activity Blunts {beta}-Adrenergic Cardiac Inotropy via an NO/cGMP-Dependent Pathway
Circ. Res., February 3, 2006; 98(2): 226 - 234.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
E. Nozik-Grayck, E. J. Whalen, J. S. Stamler, T. J. McMahon, P. Chitano, and C. A. Piantadosi
S-nitrosoglutathione inhibits {alpha}1-adrenergic receptor-mediated vasoconstriction and ligand binding in pulmonary artery
Am J Physiol Lung Cell Mol Physiol, January 1, 2006; 290(1): L136 - L143.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
X.-W. Yu, M.-Y. G Liu, R. H Kennedy, and S. J Liu
Both cGMP and peroxynitrite mediate chronic interleukin-6-induced negative inotropy in adult rat ventricular myocytes
J. Physiol., July 15, 2005; 566(2): 341 - 353.
[Abstract] [Full Text] [PDF]

Home page
 Sci Aging Knowl EnvironHome page
S. V. Y. Raju, L. A. Barouch, and J. M. Hare
Nitric Oxide and Oxidative Stress in Cardiovascular Aging
Sci. Aging Knowl. Environ., May 25, 2005; 2005(21): re4 - re4.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
C. Penna, G. Alloatti, S. Cappello, D. Gattullo, G. Berta, B. Mognetti, G. Losano, and P. Pagliaro
Platelet-activating factor induces cardioprotection in isolated rat heart akin to ischemic preconditioning: role of phosphoinositide 3-kinase and protein kinase C activation
Am J Physiol Heart Circ Physiol, May 1, 2005; 288(5): H2512 - H2520.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
H. C. Champion, D. Georgakopoulos, E. Takimoto, T. Isoda, Y. Wang, and D. A. Kass
Modulation of In Vivo Cardiac Function by Myocyte-Specific Nitric Oxide Synthase-3
Circ. Res., March 19, 2004; 94(5): 657 - 663.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
D. A. Wink, K. M. Miranda, T. Katori, D. Mancardi, D. D. Thomas, L. Ridnour, M. G. Espey, M. Feelisch, C. A. Colton, J. M. Fukuto, et al.
Orthogonal properties of the redox siblings nitroxyl and nitric oxide in the cardiovascular system: a novel redox paradigm
Am J Physiol Heart Circ Physiol, December 1, 2003; 285(6): H2264 - H2276.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
W. F. Saavedra, N. Paolocci, M. E. St. John, M. W. Skaf, G. C. Stewart, J.-S. Xie, R. W. Harrison, J. Zeichner, D. Mudrick, E. Marban, et al.
Imbalance Between Xanthine Oxidase and Nitric Oxide Synthase Signaling Pathways Underlies Mechanoenergetic Uncoupling in the Failing Heart
Circ. Res., February 22, 2002; 90(3): 297 - 304.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
M. T. Ziolo, H. Katoh, and D. M. Bers
Positive and negative effects of nitric oxide on Ca2+ sparks: influence of beta -adrenergic stimulation
Am J Physiol Heart Circ Physiol, December 1, 2001; 281(6): H2295 - H2303.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
Y. K. Zhu, X. D. Liu, M. C. Skold, T. Umino, H. Wang, D. J. Romberger, J. R. Spurzem, T. Kohyama, F.-Q. Wen, and S. I. Rennard
Cytokine Inhibition of Fibroblast-Induced Gel Contraction Is Mediated by PGE2 and NO Acting Through Separate Parallel Pathways
Am. J. Respir. Cell Mol. Biol., August 1, 2001; 25(2): 245 - 253.