AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 281: H2295-H2303, 2001;
0363-6135/01 $5.00
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Vol. 281, Issue 6, H2295-H2303, December 2001

SPECIAL TOPIC
Positive and negative effects of nitric oxide on Ca2+ sparks: influence of beta -adrenergic stimulation

Mark T. Ziolo, Hideki Katoh, and Donald M. Bers

Department of Physiology, Loyola University Medical Center, Maywood, Illinios 60153


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISUSSION
REFERENCES

Nitric oxide (NO) can have a positive or negative effect on cardiac contractility and the ryanodine receptor (RyR). This dual effect has been explained as being dependent on the concentration of NO. We find that cellular RyR response to NO is also dependent on the degree of beta -adrenergic stimulation, and thus the state of protein kinase A activation. Ca2+ spark frequency (CaSpF) in rat ventricular myocytes was used as an index of resting RyR activity. CaSpF response to beta -adrenergic stimulation was used as an index of protein kinase A activation. High concentration of isoproterenol, a beta -adrenergic agonist, caused a large increase in CaSpF; addition of NO (spermine NONOate, 300 µM) then caused a decrease in CaSpF. Low concentration of isoproterenol produced only a slight increase in CaSpF, but the same NO concentration now caused a large increase in CaSpF. A dual effect was also observed in twitch. Thus the net direction of the effects of NO on RyR activity and Ca2+ transients (directly or by alteration of sarcoplasmic reticulum Ca2+ load) can be reversed, depending on the ambient level of beta -adrenergic activation.

protein kinase A; ryanodine receptor; nitrosylation; excitation-contraction coupling


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISUSSION
REFERENCES

CONTRACTION OF CARDIAC MYOCYTES is regulated by a process termed excitation-contraction (EC) coupling (for review, see Ref. 3). Upon depolarization, L-type Ca2+ channels open, which causes an influx of Ca2+ (trigger Ca2+). This trigger Ca2+ then causes the release of Ca2+ from the sarcoplasmic reticulum (SR) in a process termed Ca2+-induced-Ca2+-release (CICR). Release of Ca2+ from the SR occurs through the SR Ca2+ release channel (ryanodine receptor, RyR). Ca2+ sparks are the fundamental unit of SR Ca2+ release (6). Ca2+ sparks can occur stochastically during diastole (independent of Ca2+ influx) (25), but the cellular Ca2+ transient is due to temporal and spatial summation of Ca2+ sparks synchronized by the trigger Ca2+.

An important physiological inotropic pathway is beta -adrenergic stimulation. This pathway has been well-characterized in cardiac myocytes. Binding to the beta -adrenergic receptor leads to activation of adenylate cyclase, increased cAMP levels, and activation of the cAMP-dependent protein kinase (PKA). PKA can phosphorylate various proteins involved in EC coupling, which results in a large increase in the Ca2+ transient and consequently contractility (e.g., 13). Phospholamban (PLB) phosphorylation by PKA stimulates SR Ca2+ uptake and increases the SR Ca2+ load (e.g., 3). This increase in SR Ca2+ load increases both the Ca2+ available for release as well as the fractional SR Ca2+ release during CICR (1). Another protein, which could be involved in the positive inotropic effect of beta -adrenergic stimulation, is the RyR. PKA activation leads to phosphorylation of RyR (11, 29) and does occur physiologically in intact rat ventricular myocytes (34). PKA phosphorylation of RyR can lead to an increase in the channel open probability (Po) (9), and this may be due to dissociation of FK-506 binding protein 12.6 from RyR (19). Thus, resting Ca2+ spark frequency (CaSpF) increases during beta -adrenergic stimulation due to PKA phosphorylation of RyR (to increase Po) and/or PLB (to increase SR Ca2+ load) (8, 36).

Nitric oxide (NO) is an important modulator of cardiac myocyte contractility (for review see, Ref. 15). NO can interact with proteins involved in EC coupling through two general signaling pathways: cGMP independent and cGMP dependent. NO is known to regulate the L-type Ca2+ channel (e.g., 5, 31) and the RyR (e.g., 35). Interestingly, NO can be a positive or negative inotropic agent. For example, it has been shown that NO can inhibit or stimulate the mammalian L-type Ca2+ current (ICa) in intact myocytes (5, 31). The negative effect of NO on ICa has been shown to occur through both signaling pathways: cGMP-dependent pathway (5, 31) and cGMP-independent pathway (nitrosylation) (12). The positive effect of NO on ICa has also been shown to occur through both pathways: cGMP-dependent pathway (21) or the cGMP-independent pathway (nitrosylation) (5).

This same dual effect (positive and negative) of NO has been observed on the RyR in lipid bilayer through the cGMP-independent pathway (nitrosylation) (28, 33, 35). No studies have investigated the effects of NO on RyR activity in intact cardiac myocytes yet. These different signaling pathways (cGMP dependent and cGMP independent) and effects on proteins involved in EC coupling (positive or negative inotropy) can also affect myocyte shortening. For example, the negative inotropic effect of NO has been shown to be via the cGMP-dependent pathway (e.g., 14, 37), as well as the cGMP-independent pathway (e.g., 23). Furthermore, the positive inotropic effect of NO on myocyte shortening has also been shown to be both cGMP dependent (e.g., 17, 20) and cGMP independent (e.g., 22, 24). Studies postulated that the dual effects of NO are due to the concentration of NO (e.g., 16, 20, 30). Thus low concentrations of NO (and small increase in cGMP levels) will have a positive inotropic effect via inhibition of cGMP-inhibited phosphodiesterase (16) or direct activation of adenylate cyclase (30). High concentrations of NO (and large increases in cGMP) will have a negative inotropic effect via activation of cGMP-dependent protein kinase (16, 30) and resultant inhibition of ICa and/or myofilament Ca2+ sensitivity (26). These studies did not find negative inotropic effects of NO via the cGMP-independent pathway. The effects of NO on cardiac myocyte function are complex due to the dynamics of its signaling pathways and numerous proteins it affects.

The purpose of this study was twofold: to examine the effects of NO on RyR activity in intact cardiac myocytes and to examine whether the degree of beta -adrenergic stimulation also determines the myocyte response to NO (at a constant concentration of NO). The hypothesis is that NO will effect RyR function in intact myocytes and that the degree of beta -adrenergic stimulation (and thus the state of PKA activation) will also determine the response of the myocyte to NO, either a positive (low level of PKA activation) or negative (high level of PKA activation) inotropic effect.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISUSSION
REFERENCES

Rat Cardiac Myocyte Isolation

Ventricular myocytes were isolated from rat hearts as previously described (2). Briefly, rats were anesthetized by intraperitoneal injection of pentobarbital sodium (100 mg/kg), and the heart from each rat was excised. The hearts were cleaned and flushed with nominally Ca2+-free Tyrode solution consisting of (in mM) 140 NaCl, 4 KCl, 1 MgCl2, 10 glucose, and 5 HEPES (pH = 7.4) and perfused using a Langendorff apparatus. Hearts were perfused with nominally Ca2+-free Tyrode solution for 5 min at 37°C. Perfusion was then switched to the same solution with 1 mg/ml collagenase (type I, Sigma; St. Louis, MO) and 0.16 mg/ml protease (Sigma) for 7 min, after which the heart was washed out with 50 ml of nominally Ca2+-free Tyrode. The heart was then taken down and the tissue minced, triturated, and then filtered. The cell suspension was rinsed and then stored in nominally Ca2+-free Tyrode. Cells were used within 4 h after isolation.

Dye Loading and Fluorescence Imaging

Myocytes were loaded with fluo-3 acetoxymethyl ester (AM) as previously described (25). Briefly, myocytes were loaded with fluo-3 AM (20 µM, Molecular Probes; Eugene, OR) for 20 min at room temperature. Excess dye was removed by washing out the dye with bath solution three times and allowing 30 min for intracellular deesterification of fluo-3 AM.

Fluorescence imaging was performed, as previously described (25), with a laser scanning confocal microscope (LSM 410, Carl Zeiss), coupled to an inverted microscope (Axiovert 100, Carl Zeiss) and equipped with a ×40 oil immersion objective (Plan-Neofluar, numerical aperature = 1.3; Carl Zeiss). Fluo-3 fluorescence was excited with the 488-nm line of an argon ion laser. Emitted fluorescence was measured at wavelengths >515 nm.

Image acquisition for the analysis of Ca2+ sparks and Ca2+ transients was made in the line-scan mode, with 512 pixels per line (0.25 µm per pixel) scanned at a sampling rate of 250 lines per second. Images were processed using IDL software (Research System; Boulder, CO) with intraceullar Ca2+ concentration ([Ca]i) calculated according to the formula [Ca]i = Kd · (F/Fo)/{(Kd/[Ca]i,rest) + 1 - (F/Fo)}, where Kd is the dissociation constant for fluo-3, F is the fluorescence intensity, Fo is the intensity at rest, and [Ca]i,rest is [Ca]i at rest (6). Fo was determined as the mean fluorescence intensity of the lowest 128 pixels along each line (512 pixels).

Ca2+ sparks were located visually. The fluorescence intensities of five adjacent pixels of an individual scan line, centered on the highest pixel value, were averaged and transformed to [Ca]i as described above. A local increase in fluorescence was counted as a spark when the peak amplitude exceeded 60 nM and the duration of half amplitude was at least 8 ms. The number of Ca2+ sparks counted per line scan image was normalized spatially (per µm3) and temporally (per s) as the spark frequency (CaSpF; sparks × pl-1 × s-1) (pl = picoliter). CaSpF was used as an index of RyR activity, and the CaSpF response to beta -adrenergic stimulation was used as an index of PKA activation. The Ca2+ transients evoked by electrical stimulation and caffeine application were derived from the changes in averaged fluorescence intensities along the scanned line and expressed as F/Fo.

Experimental Protocols

Myocytes were field stimulated via platinum electrodes (0.2 Hz). Cells were superfused with normal Tyrode (22-25°C), and when Ca2+ transients reached steady state, stimulation was stopped, and resting Ca2+ sparks were immediately measured. Sparks were continuously measured for 13.4 s in every protocol. Field stimulation was started again, and the solution was switched to normal Tyrode with isoproterenol (Iso), a beta -adrerengic agonist (0.01, 0.1, or 1 µM). These different concentrations of Iso were used to achieve different states of PKA activation. When cell response reached steady state (4 min), stimulation was stopped, and resting Ca2+ sparks were immediately measured (again for 13.4 s). Field stimulation was started again, and the solution was switched to normal Tyrode with Iso and spermine NONOate, a nitric oxide donor (300 µM). When cell response reached steady state (4 min), stimulation was stopped, and resting Ca2+ sparks were immediately measured for 13.4 s.

To examine the effect of NO on basal CaSpF, we increased extracellular Ca2+ to 2 mM to increase basal CaSpF so the changes in CaSpF with NO, if any, would be more evident. Once steady state was reached, stimulation was stopped, and resting Ca2+ sparks were immediately measured for 13.4 s. Field stimulation was started again, and the solution was switched to spermine NONOate (300 µM) for 4 min, stimulation was stopped, and resting Ca2+ sparks were immediately measured for 13.4 s. It should be noted that all these protocols ensure that SR Ca2+ stores are filled before observing CaSpF. Moreover, in rat ventricular myocytes there is no change in SR Ca2+ load expected during 13.4 s of rest [and only a modest rise in CaSpF (25)]. The SR Ca2+ content was estimated by rapid application of 10 mM caffeine dissolved in normal Tyrode.

Solutions

The Tyrode solution consisted of (in mM) 140 NaCl, 4 KCl, 1 MgCl2, 1 CaCl2, 10 glucose, and 5 HEPES (pH = 7.4). Iso (1 mM stock) and spermine NONOate were prepared fresh each day. Caffeine (10 mM) and spermine NONOate (300 µM) were dissolved directly in Tyrode solution. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 mM stock) was dissolved in DMSO. Equivalent concentration of DMSO was added to all solutions (<0.1%). All chemicals were purchased from Sigma except ODQ (Alexis; San Diego, CA).

Statistical Analysis

Results were expressed as means ± SE. Statistical significance was determined by repeated-measures ANOVA (followed by Newman-Keuls test). P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISUSSION
REFERENCES

Effect of NO on Ca2+ Spark Frequency After beta -Adrenergic Stimulation

We examined the effect of NO on resting CaSpF after beta -adrenergic stimulation. Figure 1 shows representative experiments performed on a single myocyte when NO had a positive effect on CaSpF. Line scan images obtained during control superfusion are shown in Fig. 1A, and there were few Ca2+ sparks. When the cell was superfused with Iso (0.01 µM), a beta -adrenergic agonist, there was a slight increase in the Ca2+ sparks (Fig. 1B). When the cell was superfused with Iso and spermine NONOate (300 µM), a NO donor (Iso + NO), there was a further increase in CaSpF (Fig. 1C).


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  		Fig. 1.   Nitric oxide (NO) increases Ca2+ spark frequency during low-level beta -adrenergic stimulation. Representative recordings of longitudinal line scan images of Ca2+ sparks (each image is a 2.2-s frame). Line plots of intracellular Ca2+ concentration ([Ca]i) taken from selected sites (white bars) are shown below images (width of 5 pixels). A: left and right, resting Ca2+ sparks in presence of control Tyrode solution (total of 1 spark for both images). Dotted lines, in line plot, represent threshold level of what is considered a spark. B: left and right, in presence of 0.01 µM isoproterenol (Iso), a beta -adrenergic agonist (total of 6 sparks for both images). C: left and right, in presence of Iso and 300 µM spermine NONOate (Iso + NO), a NO donor (total of 14 sparks for both images).

Figure 2 shows representative experiments performed on a single myocyte when NO had a negative effect on CaSpF. Figure 2 shows line scan images obtained during control superfusion where there were few Ca2+ sparks (A), with Iso (1 µM) (B) where CaSpF was greatly increased compared with Fig. 1B, and with Iso and spermine NONOate (300 µM) where CaSpF dramatically decreased (C). This is the opposite NO affect as was seen in Fig. 1C even though NO concentrations were the same.


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  		Fig. 2.   NO decreases Ca2+ spark frequency during high-level beta -adrenergic stimulation. Representative recordings of longitudinal line scan images of Ca2+ sparks (each image is a 2.2-s frame). Line plots of [Ca]i taken from selected sites (white bars) are shown below images (width of 5 pixels). A: left and right, resting Ca2+ sparks in presence of control Tyrode solution (total of 3 sparks for both images). Dotted lines, in line plot, represent threshold level of what is considered a spark. B: left and right, presence of 1 µM Iso, a beta -adrenergic agonist (total of 20 sparks for both images). C: left and right, presence of Iso and 300 µM spermine NONOate, a NO donor (total of 7 sparks for both images).

All cells were separated into two groups based on the response to NO (positive or negative effect on CaSpF). Pooled data (Fig. 3) show that there was no statistical difference between the two groups with respect to basal CaSpF (13 ± 6 vs. 17 ± 11 sparks · pl-1 · s-1, P = not significant). The cells that exhibited a positive NO effect were also the cells in which Iso had a small positive effect (CaSpF only increased to 22 ± 12 sparks · pl-1 · s-1 and Iso concentration was typically 0.01 µM). In contrast, when NO had a negative inotropic effect, the cell had already shown a large response to Iso to (194 ± 55 sparks · pl-1 · s-1, P < 0.05, and in this case Iso concentration was typically 1 µM).


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  		Fig. 3.   Differential effects of 300 µM spermine NONOate (Iso + NO) on Iso-stimulated Ca2+ spark frequency (CaSpF). A: positive effect of NO on CaSpF. B: negative effect of NO on CaSpF. Note: different response of CaSpF to Iso (A: typically [Iso] was 0.01 µM; B: typically [Iso] was 1 µM). Pooled data expressed as means ± SE. (*P < 0.05, ANOVA, n = 6-10 cells for each group). C: correlation between degree of Iso response (as CaSpF in Iso - CaSpF in control) and NO effect [as CaSpF in (Iso + NO) - CaSpF in Iso]. r2 = 0.87.

With the small response to Iso, superfusion with spermine NONOate produced a further fourfold increase of the Iso-stimulated CaSpF (from 22 to 87 ± 21 sparks · pl-1 · s-1, P < 0.05 compared with Iso alone). When there was a large response to Iso, superfusion with spermine NONOate (same concentration) decreased the Iso-stimulated CaSpF by -53% (from 194 to 91 ± 20 sparks · pl-1 · s-1, P < 0.05, compared with Iso alone). Thus, when there was a large response to Iso (i.e., a higher state of PKA activation), NO had a negative inotropic effect on CaSpF. When there was a small response to Iso (i.e., a low state of PKA activation), the same NO concentration had a positive inotropic effect on CaSpF (see Fig. 3C).

We also examined the effects of NO on basal CaSpF. As Fig. 4A shows, there was no effect of NO on basal CaSpF (41 ± 21 vs. 36 ± 23 sparks · pl-1 · s-1, P = not significant). In these experiments, we raised extracellular Ca2+ concentration from 1 to 2 mM to raise CaSpF and enhance our ability to detect NO-dependent changes in CaSpF. This accounts for the higher basal CaSpF (versus the Iso experiments). These results indicate that the effect of NO on CaSpF is dependent on the degree of beta -adrenergic stimulation and thus the state of PKA activation.


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  		Fig. 4.   A: effect of NO on basal CaSpF. Data are expressed as means ± SE (paired t-test, P = not significant, n = 6 cells). B: effect of Iso (0.01 µM), spermine NONOate (Iso + NO, 300 µM), and ODQ (Iso + NO + ODQ, 10 µM), a guanylate cyclase inhibitor, on CaSpF. Data are expressed as means ± SE (ANOVA, *P < 0.05 compared with Iso, n = 11 cells).

Guanylate Cyclase Involvement in NO-Induced Increase in beta -Adrenergic-Stimulated Ca2+ Sparks

Our laboratory (38) has preliminary data that the negative inotropic effect of NO (produced via iNOS) on CaSpF is independent of guanylate cyclase activation. For this study, we investigated whether the positive effect of NO on CaSpF was due to the activation of guanylate cyclase and thus increased production of cGMP. If the positive effect of NO on CaSpF is cGMP dependent, then inhibition of NO stimulation of guanylate cyclase should prevent this effect. Figure 4B shows that specific inhibition of NO-stimulated guanylate cyclase by ODQ (10 µM) did not prevent the NO-induced increase in CaSpF (after beta -adrenergic stimulation). These data suggest that the stimulatory effect of NO on CaSpF is independent of guanylate cyclase activation and cGMP but could be mediated by other actions (e.g., direct nitrosylation).

Effects of NO on Twitch Ca2+ Transients and SR Ca2+ Load After beta -Adrenergic Stimulation

In separate experiments, we examined the effects of NO superfusion in cardiac myocytes and its effect on twitch Ca2+ transient amplitude (Delta [Ca]i) and SR Ca2+ load. Myocytes were field stimulated at 0.2 Hz, and twitch Delta [Ca]i was measured under various conditions. SR Ca2+ load was also measured by rapid caffeine (10 mM) application. Figure 5 shows the pooled data for the effects of NO on Ca2+ transients after beta -adrenergic stimulation in cardiac myocytes. Figure 5A shows cells that exhibit a positive inotropic effect of NO. Iso caused an increase in the twitch Ca2+ transient compared with control (2.5 ± 0.4 vs. 1.7 ± 0.3 F/Fo, P < 0.05). When the cell was superfused with spermine NONOate (Iso + NO), there was a further increase in the twitch Ca2+ transient (4.0 ± 0.5 F/Fo, P < 0.05 compared with Iso alone).


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  		Fig. 5.   Differential effects of 300 µM spermine NONOate (Iso + NO) on Iso-stimulated twitch [Ca]i transients. A: pooled data for peak amplitude of twitch [Ca]i transients induced by field stimulation when NO further increased Iso-stimulated (0.01 or 0.1 µM) twitch transients. Data are are expressed as means ± SE (ANOVA, *P < 0.05 compared with control, **P < 0.05 compared with Iso, n = 9 cells). F, fluorescence intensity; Fo intensity at rest. B: pooled data for peak amplitude of twitch [Ca]i transients induced by field stimulation when NO decreased Iso-stimulated (0.1 or 1 µM) twitch transients. Data are expressed as means ± SE (ANOVA, *P < 0.05 compared with control and Iso + NO, n = 6 cells).

Figure 5B shows the pooled data for cells where NO had a negative inotropic effect. Iso caused an increase in the twitch Ca2+ transient compared with control (2.6 ± 0.5 vs. 2.1 ± 0.5 F/F0, P < 0.05). When the cell was superfused with spermine NONOate (Iso + NO), there was a decrease in the twitch Ca2+ transient (2.0 ± 0.4 F/F0, P < 0.05 compared with Iso alone). There was no effect of NO on basal twitch Delta [Ca]i (data not shown).

One of the main determinants of SR Ca2+ release is the Ca2+ load of the SR. We examined this by measuring caffeine-induced Ca2+ transients. Figure 6A shows the overall data of SR Ca2+ load when NO has a positive inotropic effect. As expected, Iso caused an increase in SR Ca2+ load compared with control (4.0 ± 0.4 vs. 3.4 ± 0.4 F/F0, P < 0.05). When the cell was superfused with spermine NONOate (Iso + NO), there was a further increase in SR Ca2+ load (4.6 ± 0.5 F/F0, P < 0.05 compared with Iso alone). Figure 6B shows the overall data of SR Ca2+ load when NO has a negative inotropic effect. As expected, Iso caused an increase in SR Ca2+ load compared with control (5.6 ± 0.8 vs. 5.0 ± 0.8 F/F0, P < 0.05). When the cell was superfused with spermine NONOate (Iso + NO), there was a decrease in SR Ca2+ load (4.7 ± 0.7 F/F0, P < 0.05 compared with Iso alone). There was no effect of NO on basal SR Ca2+ load (data not shown). These data suggest that a dual effect of NO on the whole cell Ca2+ transients in intact cardiac myocytes also occurs, and this effect may be due to the state of PKA activation because the same concentration of NO was used for the positive and negative effect.


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  		Fig. 6.   Differential effects of 300 µM spermine NONOate (Iso + NO) on Iso-stimulated, caffeine-induced Ca2+ transients. A: pooled sarcoplasmic reticulum (SR) Ca2+ load data when NO increased Iso-stimulated (0.01 or 0.1 µM) SR Ca2+ load. Data are expressed as means ± SE (ANOVA, *P < 0.05 compared with control, **P < 0.05 compared with Iso, n = 9 cells). B: pooled SR Ca2+ load data when NO decreased Iso-stimulated (0.1 or 1 µM) SR Ca2+ load. Data are expressed as means ± SE (ANOVA, *P < 0.05 compared with control and Iso + NO, n = 6 cells).


    DISUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISUSSION
REFERENCES

beta -Adrenergic stimulation leads to a positive inotropic effect through activation of PKA and phosphorylation of several proteins involved in EC coupling. One important protein in this pathway is the SR Ca2+ release channel RyR. Events mediated by RyR have been identified as Ca2+ sparks. Ca2+ sparks were used as an index of RyR activity. PKA activation leads to phosphorylation of RyR (11, 29), which can increase Po (9). PKA also phosphorylates PLB, which will lead to an increase in SR Ca2+ load (due to greater SR Ca2+-ATPase activity), and this can also increase RyR Po (18). This increased Po is seen as an increase in resting CaSpF (8, 36). Thus the frequency of Ca2+ sparks during beta -adrenergic stimulation was used as an index of PKA activation. That is, when there was a small increase in CaSpF, the functional state of PKA activation was considered to be low. When there was a large increase in CaSpF, the functional state of PKA activation was considered to be high. Different concentrations of Iso, a beta -adrenergic agonist, were used to generate different states of PKA activation. In most instances, the high Iso concentration caused a large increase in CaSpF, and the low Iso concentration caused a small increase in CaSpF. This is consistent with the higher Iso concentration activating more PKA and causing a greater beta -adrenergic response. Thus we believe CaSpF is a reliable, indirect measure of the functional state of PKA activation of the entire cell, not just localized to RyR.

NO is also a regulator of cardiac contractility (for review, see Ref. 15). Interestingly, it has been found that NO can have a positive inotropic effect, as well as a negative inotropic effect, on cardiac myocyte function (i.e., myocyte shortening, L-type Ca2+ current, and RyR). Studies have concluded that this dual effect of NO was due to the concentration of NO. That is, high concentrations of NO lead to a negative inotropic effect, whereas low concentrations lead to a positive inotropic effect (10, 16, 20, 30). These differing effects of NO are due to its signaling pathway. NO has two general pathways: cGMP dependent and cGMP independent. Studies have shown that both signaling pathways of NO can produce the positive and negative inotropic effects (see INTRODUCTION). Hence, the effect of NO on myocyte contractility is multifaceted and probably dependent on other factors, not just NO concentration. The object of this study was twofold: to determine whether NO regulation of RyR activity occurs in intact cardiac myocytes and to examine whether the state of PKA activation alters the myocyte response to NO. The RyR activity was selected to examine the effect of PKA activation on NO effects for two reasons: 1) RyR gating can be both stimulated or inhibited by NO in lipid bilayer studies (28, 33, 35), and 2) PKA activation state can be inferred from CaSpF.

PKA Activation and Response to NO

When there was a large increase in CaSpF in response to beta -adrenergic stimulation (1,392% increase from control), we infer that there was a high state of PKA activation. Perfusion with NO in this case attenuated the beta -adrenergic-stimulated CaSpF (53% decrease of Iso response) and had a negative inotropic effect (Figs. 2 and 3). When there was a small increase in CaSpF in response to beta -adrenergic stimulation (29% increase from control, i.e., a low state of PKA activation), NO caused a further increase in CaSpF (295% increase of Iso response) and a positive inotropic effect (Figs. 1 and 3). These data indicate that the state of PKA activation (1,392% vs. 29%) can be a crucial predisposing factor that can determine whether NO will be a negative or positive inotropic agent (-53% vs. +295%). There was no effect of NO on basal CaSpF (Fig. 4A). These data are in agreement with the majority of literature that shows NO does not effect basal cardiac function (e.g., 31). Thus the purpose of NO may be to regulate (or fine tune) the myocyte's response to beta -adrenergic stimulation (further enhance or attenuate). That is, when there is a high state of beta -adrenergic stimulation, NO will have a negative inotropic effect to prevent Ca2+ overload. When there is a low state of beta -adrenergic stimulation, NO will have a positive inotropic effect to enhance the beta -adrenergic response.

NO Effect on CaSpF: cGMP Independent or Dependent?

The effect of NO on RyR activity is thought to be mediated mainly by nitrosylation (cGMP independent). Preliminary results from our lab have shown that the negative inotropic effect of NO (produced via inducible NOS) on RyR activity was through the cGMP-independent pathway (38). Our results here with ODQ, a specific inhibitor of NO stimulation of guanylate cyclase (7), imply that the positive effect of NO on CaSpF is independent of guanylate cyclase and cGMP (Fig. 4B). Previous work has shown that ODQ is a specific inhibitor of guanylate cyclase in cardiac myocytes (ODQ selectively decreased cGMP levels in response to NO donors) (27). Because ODQ did not alter the effects of NO on beta -adrenergic-stimulated CaSpF, we conclude that the effect of NO to increase RyR activity is likely to be a direct effect via nitrosylation of a target protein. These data also agree with other studies that have shown NO can alter the gating activity of RyR incorporated into lipid bilayers via nitrosylation (28, 33, 35). Interestingly, two of these three studies found that nitrosylation of cardiac RyR led to increased probability of channel opening (28, 33), whereas the other found decreased probability (35). This may be due to different conditions in the bilayer, different redox states of RyR, different types of NO donors, different levels of NO produced, etc. In fact, Hart and Dulhunty (10) found a dual effect of NO (positive and negative), which was dependent on NO concentration (high vs. low), using RyR from skeletal muscle in lipid bilayers. Our data are consistent with the effect of NO on RyR activity being via the cGMP-independent pathway. Our data here suggest that there may also be different PKA phosphorylation states of RyR, which will respond differently to NO.

What is Cellular Target of NO to Alter Twitch Ca2+ Transients?

To understand mechanistically how NO alters twitch Ca2+ transients, one must consider the molecular targets of NO. A NO-induced change in beta -adrenergic-stimulated CaSpF (Fig. 3) and twitch Ca2+ transients (Fig. 5) could be due to changes in RyR, PLB, and/or L-type Ca2+ channel.

RyR. If the dominant effect of NO to increase twitch Ca2+ transients (Fig. 5A) were via a direct nitrosylation of RyR to increase Po, then the SR Ca2+ load would decrease. However, the NO-induced increase in twitch Ca2+ transients is associated with an increase in SR Ca2+ load (Fig. 6A). Additionally, if the effect of NO to decrease twitch Ca2+ transients (Fig. 5B) were via a direct nitrosylation of RyR to decrease Po, then the SR Ca2+ load would increase. However, the opposite effect occurred (Fig. 6B). Thus the primary effect of NO to alter twitch Ca2+ transients must be due to changes in SR Ca2+ uptake (seen as changes in SR Ca2+ load). This does not preclude direct alterations of RyR function by NO, but this may not be the primary pathway. When NO decreased CaSpF (Figs. 2 and 3B) and twitch Ca2+ transients (Fig. 5B), there was a high CaSpF and SR Ca2+ load (Fig. 6B) with Iso (indication of greater SR Ca2+ uptake via higher PKA activity). When NO increased CaSpF (Figs. 1 and 3A) and twitch Ca2+ transients (Fig. 5A), there was a low CaSpF and SR Ca2+ load with Iso (indicative of lower SR Ca2+ uptake via lower PKA activity). Therefore, it may be the PKA-regulated change in SR Ca2+ load (or phosphorylation of a PKA target) that dictates whether NO is inhibitory or stimulatory.

Although the mechanism of how NO altered SR Ca2+ load was not examined in this study, it could be speculated that NO-induced changes in phosphorylation levels of PLB and/or the L-type Ca2+ channel would lead to a change in SR Ca2+ load. We cannot distinguish whether this positive or negative effect of NO are via the same molecular target.

PLB and negative effect of NO? For example, we recently found that NO can decrease phosphorylation levels of PLB, in a cGMP-independent pathway, after a high degree of beta -adrenergic stimulation (27). Thus the negative effect of NO on CaSpF (Fig. 3B), twitch Ca2+ transients (Fig. 5B), and SR Ca2+ load (Fig. 6B) could simply be explained by NO-induced changes in SR Ca2+ load via modifications in PLB phosphorylation, as our previous study demonstrated.

L-type Ca2+ Channel and positive effect of NO? The positive effect of NO could be via stimulation of SR Ca2+ uptake by increasing ICa. We have shown here that the positive effect of NO on CaSpF is cGMP independent (Fig. 4B). Data in the literature suggest that the positive effect of NO on ICa is also cGMP independent (5), which is consistent with our data presented here. However, the negative effect of NO on ICa is cGMP dependent (5, 31), which does not match our data. Thus the positive effect of NO on CaSpF (Fig. 3A), twitch Ca2+ transients (Fig. 5A), and SR Ca2+ load (Fig. 6A) could be explained by NO-induced changes in SR Ca2+ load via modification of ICa. But these speculations will require further experimental tests.

In conclusion, we have shown that NO can alter (either as a positive or negative inotropic agent) beta -adrenergic-stimulated Ca2+ spark frequency, an index of RyR activity. This response may be a direct effect of NO on RyR or may also be dependent on the state of PKA-regulated change in SR Ca2+ load. Thus, not only the concentration of NO, but also the degree of beta -adrenergic stimulation is a determinant of the effect, positive or negative, of NO. This effect of NO on Ca2+ handling may also have clinical implications, because in human heart failure where beta -adrenergic signaling is altered (4), RyR is hyperphosphorylated (19) and NO production is increased (32). Thus NO effects may contribute to altered Ca2+ uptake and release events in heart failure.


    ACKNOWLEDGEMENTS

The authors thank Dr. Lars S. Maier and Dr. Thomas Shannon for helpful discussions and comments on the manuscript.


    FOOTNOTES

This study was supported by National Heart, Lung, and Blood Institute Grants HL-10122 (to M. T. Ziolo) HL-64098, and HL-64724 (to D. M. Bers).

Address for reprint requests and other correspondence: D. M. Bers, Dept. of Physiology, Loyola Univ. Medical Center, 2160 South First Ave., Maywood, IL 60153 (E-mail: dbers{at}lumc.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 1 June 2001; accepted in final form 23 August 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISUSSION
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Am J Physiol Heart Circ Physiol 281(6):H2295-H2303
0363-6135/01 $5.00 Copyright © 2001 the American Physiological Society


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