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Am J Physiol Cell Physiol 294: C572-C578, 2008. First published December 5, 2007; doi:10.1152/ajpcell.00242.2007
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Opposing effects of coupled and uncoupled NOS activity on the Na+-K+ pump in cardiac myocytes

C. N. White,1 E. J. Hamilton,2 A. Garcia,1 D. Wang,1,2 K. K. M. Chia,1,2 G. A. Figtree,1,2 and H. H. Rasmussen1,2

1Department of Cardiology, Royal North Shore Hospital, Sydney, Australia; and 2Department of Medicine, University of Sydney, Sydney, Australia

Submitted 8 June 2007 ; accepted in final form 30 November 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Pharmacological delivery of nitric oxide (NO) stimulates the cardiac Na+-K+ pump. However, effects of NO synthesized by NO synthase (NOS) often differ from the effects of NO delivered pharmacologically. In addition, NOS can become "uncoupled" and preferentially synthesize O2·–, which often has opposing effects to NO. We tested the hypothesis that NOS-synthesized NO stimulates Na+-K+ pump activity, and uncoupling of NOS inhibits it. To image NO, we loaded isolated rabbit cardiac myocytes with 4,5-diaminofluorescein-2 diacetate (DAF-2 DA) and measured fluorescence with confocal microscopy. L-Arginine (L-Arg; 500 µmol/l) increased DAF-2 DA fluorescence by 51% compared with control (n = 8; P < 0.05). We used the whole cell patch-clamp technique to measure electrogenic Na+-K+ pump current (Ip). Mean Ip of 0.35 ± 0.03 pA/pF (n = 44) was increased to 0.48 ± 0.03 pA/pF (n = 7, P < 0.05) by 10 µmol/l L-Arg in pipette solutions. This increase was abolished by NOS inhibition with radicicol or by NO-activated guanylyl cyclase inhibition with 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one. We next examined the effect of uncoupling NOS using paraquat. Paraquat (1 mmol/l) induced a 51% increase in the fluorescence intensity of O2·–-sensitive dye dihydroethidium compared with control (n = 9; P < 0.05). To examine the functional effects of uncoupling, we measured Ip with 100 µmol/l paraquat included in patch pipette solutions. This decreased Ip to 0.28 ± 0.03 pA/pF (n = 12; P < 0.001). The paraquat-induced pump inhibition was abolished by superoxide dismutase (in pipette solutions). We conclude that NOS-mediated NO synthesis stimulates the Na+-K+ pump, whereas uncoupling of NOS causes O2·–-mediated pump inhibition.

myocyte; nitric oxide; superoxide; oxidative stress

WE HAVE PREVIOUSLY FOUND that nontargeted delivery of nitric oxide (NO) by the pharmacological NO donor sodium nitroprusside stimulates the Na+-K+ pump in ventricular myocytes (42). Since NO synthase (NOS) is expressed in cardiac myocytes, endogenously synthesized NO may be an important determinant of sarcolemmal Na+-K+ pump activity. However, this cannot be assumed: effects of NO can be highly compartmentalized in molecular microdomains (22), and the results obtained by using a pharmacological NO donor may be critically different from the effects of endogenously synthesized NO (12, 29).

NOS produces a mixture of NO and oxidant species with a composition that depends on cellular conditions, including oxidant stress, availability of cofactors, and the NOS substrate L-arginine (L-Arg) (12). When "uncoupled," NOS mediates synthesis of superoxide (O2·–) rather than NO. Superoxide and its derivatives, highly oxidizing reactive nitrogen (RNS) and oxygen species (ROS), can modify proteins, peptides, membrane lipids, and other cellular constituents. The balance between NO and O2·– synthesis can be critical for cell function since NO and O2·– derivatives can have opposing effects (3).

Although oxidative damage mediated by ROS/RNS has been implicated in the pathogenesis of many disease processes, including diseases of the heart (19), oxidative processes may also regulate normal cell function. Recent evidence suggests an important role for such "redox signaling" via modification of susceptible sulfhydryl groups on proteins (1, 7, 18). The Na+-K+ pump may be a candidate for oxidative modification because its subunits contain many sulfhydryl and other oxidizable residues (14). An effect of such modification on pump activity is supported by inhibition of the isolated enzymatic equivalent of the pump Na+-K+-ATPase by chemical oxidants (14), as well as the effect of chemical blocking of sulfhydryl groups on pump current in guinea pig ventricular myocytes (20). In the current study, we have examined whether endogenous NOS-mediated NO synthesis has an effect on the Na+-K+ pump similar to NO sourced from sodium nitroprusside. We also examined the effect on the pump of pharmacologically induced uncoupling of NOS from NO to O2·– synthesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cells. Cardiac myocytes were isolated from male White New Zealand rabbits. Details of anesthesia, excision of the heart, and cell isolation techniques have been described previously (11). The institutional review committee for animal research had approved the experimental protocols. The myocytes were used on the day of isolation only and were stored at room temperature in Krebs-Henseleit buffer solution until used.

Measurement of electrogenic Na+-K+ pump current. Myocytes were suspended in a tissue bath mounted on an inverted microscope for measurement of electrogenic Na+-K+ pump current (Ip). While we established the whole cell configuration, we perfused the bath with modified Tyrode's solution, which contained (in mmol/l) 140 NaCl, 5.6 KCl, 2.16 CaCl2, 1 MgCl2, 10 glucose, 0.44 NaH2PO4, and 10 HEPES. It was titrated to a pH of 7.40 at 35°C with NaOH. Within 2–3 min of establishing the whole cell configuration, we switched to a superfusate that was identical except that it was nominally Ca2+ free and contained 0.2 mmol/l CdCl2 and 2 mmol/l BaCl2. We replaced Na+-containing compounds with N-methyl-D-glucamine (NMG.Cl) in the superfusates in some experiments (21).

We used wide-tipped patch pipettes (4 µm) in all experiments. The pipette solutions contained (in mmol/l) 5 HEPES, 2 MgATP, 5 ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid; 70 potassium glutamate, 10 sodium glutamate, and 80 tetramethylammonium chloride (TMA.Cl), and they were titrated to a pH of 7.20 at 35°C with 1 mol/l KOH. We increased the Na+ concentration in pipette solutions to 80 mmol/l (osmotic balance was maintained by adjusting the concentration of TMA.Cl) in some experiments. Patch pipettes filled with these solutions had resistances of 0.8–1.1 M{Omega}. The series resistance after formation of the whole cell configuration was ≤2.8 M{Omega}, which satisfies criteria we have previously defined as essential for experimental validity (42).

Ip was identified at a holding potential of –40 mV as the difference between stable plateaus of holding current before and after Na+-K+ pump blockade with 100 µmol/l ouabain 10–15 min after the whole cell configuration was established. A stable current was identified when no drift could be identified on the digital display of the voltage clamp amplifier for at least 50 s. The plateaus were defined by the means of 10 samples with an electronic cursor taken at ~5-s intervals. Recordings were obtained by using the continuous single-electrode mode of Axoclamp 2A or 2B amplifiers supported by AxoTape and pCLAMP software (Axon Instruments, Foster City, CA). We report Ip normalized for membrane capacitance and hence cell size.

Imaging of intracellular NO and O2·– by fluorescent confocal microscopy. To detect NO production, we loaded myocytes with diacetylated 4,5-diaminofluorescein-2 (DAF-2 DA). This membrane-permeable dye is hydrolyzed intracellularly by cytosolic esterases releasing DAF-2, which is converted in the presence of NO into a fluorescent product, DAF-2 triazole (26). The DAF-2 DA was loaded at a concentration of 1 µmol/l in Krebs solution in the dark at 37°C for 30 min. The Krebs solution contained either L-Arg (500 µmol/l) or no L-Arg. To detect intracellular O2·–, we loaded isolated myocytes with dihydroethidium (DHE; 2 µmol/l) for 15 min in Krebs solution at 37°C in the dark. Cells were then exposed to paraquat (1 mmol/l for 10 min) or control solution. Myocytes were fixed in 2% paraformaldehyde on ice for 4 min. After a wash, they were mounted on poly-L-lysine-coated glass slides in Vectorshield. They were examined by laser-scanning fluorescent confocal microscopy (Nikon C1). The excitation wavelength was 488 nm and the emission wavelength was 530 nm in the case of DAF-2 DA fluorescence and was 488 nm/585 nm for DHE fluorescence. The fluorescence images were obtained by using constant settings of scanning speed, pinhole diameter, and voltage gain. Myocytes (≥6) representative of each experimental condition were selected randomly for measurement of fluorescence intensity (Photoshop, Adobe). Only myocytes with clear striations and rodlike shape were included in the analysis. The average of each experiment was normalized against its relevant control (DAF loaded, not exposed to L-Arg or DHE loaded, not exposed to paraquat). N refers to the number of individual experiments performed in ≥4 animals.

Chemicals and reagents. TMA.Cl and NMG.Cl were purum grade and were obtained from Fluka Chemicals. All other chemicals used in Tyrode's solutions were analytical grade and were obtained from BDH. Ouabain, CuZn SOD, radicicol, DAF-2 DA, and paraquat were obtained from Sigma Chemical (St Louis, MO). DHE was obtained from Invitrogen (Carlsbad, CA). 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ) was obtained from Calbiochem (La Jolla, CA), and 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) was obtained from Merck. ODQ and YC-1 were dissolved in dimethyl sulfoxide that was diluted to a final concentration of 0.037% or less in pipette solutions. Radicicol was dissolved in ethanol that was diluted to 0.08% in experimental solutions. Paraquat and SOD were dissolved directly in experimental solutions. Vectorshield was obtained from Vector Laboratories (Burlingame, CA).

Statistical analysis. Results are expressed as means ± SE. Student's t-test for unpaired data was used for statistical comparisons. We used Dunnett's test when the same control group was used for more than one comparison. P < 0.05 is regarded as significant in all comparisons. Student's t-test for paired data, with single-tail distribution, was used for analysis of differences in DAF-2-DA or DHE fluorescence intensity levels between experiments and controls.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
L-Arg stimulates Ip by augmenting myocyte NO levels. To examine the effect of L-Arg supplementation on NO levels, we loaded isolated myocytes with DAF-2 DA and examined them with laser-scanning confocal microscopy. We compared the fluorescence intensity of controls and myocytes exposed to 500 µmol/l L-Arg. Fluorescence images of a representative control myocyte and a myocyte exposed to L-Arg are shown in Fig. 1A (top). L-Arg supplementation resulted in augmentation of DAF fluorescence. Analysis of mean fluorescence intensity from control myocytes and myocytes exposed to L-Arg confirmed a significant increase in mean DAF fluorescence intensity in response to L-Arg, as shown in Fig. 1B.


Figure 1
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  		Fig. 1. A: fluorescent confocal micrographs demonstrating the effect of L-arginine (L-Arg; 500 µmol/l) supplementation on intracellular nitric oxide (NO) in myocytes loaded with the NO-sensitive dye 4,5-diaminofluorescein-2 diacetate (DAF-2 DA). Exposure to L-Arg augmented the fluorescence signal compared with control (n = 10; top). Radicicol had no effect on baseline fluorescence but abolished the increase in fluorescence induced by L-Arg (n = 4; bottom). B: mean DAF fluorescence intensity for myocytes supplemented with L-Arg in the presence and absence of radicicol. *Significant difference compared with control.

 
Since the molecular chaperone heat shock protein 90 (Hsp90) plays an important role in NOS activation, we examined the effect of exposing myocytes to 10 µmol/l of the Hsp90 inhibitor radicicol (30) for 15 min before incubation in solutions containing DAF and L-Arg. Fluorescence images of a representative control myocyte and a myocyte exposed to L-Arg preexposed to radicicol are shown in Fig. 1A. The mean fluorescence intensities are shown in Fig. 1B. Radicicol had no effect on baseline fluorescence. However, it abolished the increase in fluorescence induced by L-Arg.

To examine the effect of supplementing the intracellular compartment with L-Arg on sarcolemmal Na+-K+ pump activity, we patch clamped myocytes using pipettes that included 10 mmol/l Na+. This concentration was used in all experiments in the present study unless indicated otherwise. The pipette solutions were free of L-Arg, or they contained L-Arg in a concentration of 1, 10, 100, or 1,000 µmol/l. L-Arg was dissolved in water. A standard Na+-containing superfusate was used in the initial series of experiments. Fig. 2A shows the effect of Na+-K+ pump inhibition with ouabain on stable whole cell membrane currents of myocytes patch clamped by using a pipette solution that was free of L-Arg or contained 1,000 µmol/l L-Arg. The ouabain-induced shift in holding current (Ip) was larger for the myocyte patch clamped using the solution containing L-Arg than for control. The effect of increasing L-Arg concentration in pipette solution on mean Ip is shown in Fig. 2B. Inclusion of L-Arg in pipette solutions in concentrations of 10, 100, or 1,000 µmol/l induced a significant increase in Ip. We used L-Arg in a concentration of 10 µmol/l in all subsequent patch-clamp experiments unless otherwise specified.


Figure 2
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  		Fig. 2. A: the effect of L-Arg on Na+-K+ pump current (Ip) identified by the ouabain (Oua)-induced shift in whole cell holding current. Ip of the myocyte exposed to L-Arg (1,000 µmol/l) was 0.48 pA/pF, whereas Ip of the control myocyte was 0.37 pA/pF. Cm, membrane capacitance. B: effect of increasing L-Arg concentration in pipette solution on mean Ip. The numbers in parentheses indicate the number of experiments. *Significant difference compared with control.

 
The increase in Ip induced by L-Arg could be due to an increase in intrinsic Na+-K+ pump activity, or it could be indirectly due to enhanced influx of Na+ into myocytes and secondary pump stimulation. To distinguish between direct and indirect pump stimulation, we used patch pipette solutions that contained 10 mmol/l Na+ and 10 µmol/l L-Arg. The superfusate was nominally Na+-free to eliminate Na+ influx as a possible cause of Na+-K+ pump stimulation. Despite the lack of Na+ in the superfusate, the ouabain-induced shift in holding current was larger when the pipette solution contained L-Arg than control solution. The mean Ip for experiments using control pipette solutions and solutions containing L-Arg is shown in Fig. 3A. L-Arg induced a statistically significant increase in Ip. This indicates that the L-Arg-induced increase is not due to secondary pump stimulation arising from Na+ influx.


Figure 3
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  		Fig. 3. The effect of sodium-free superfusate on L-Arg-induced stimulation of the Na+-K+ pump. A: the mean Ip for experiments using control pipette solutions and solutions containing L-Arg. The superfusate was sodium free, whereas pipette solutions contained 10 mmol/l Na+. B: the effect of including 80 mM Na+ in pipette solutions on L-Arg-induced stimulation of the Na+-K+ pump. The mean Ip for experiments using control pipette solutions and solutions containing L-Arg. The numbers in parentheses indicate the number of experiments. *Significant difference compared with control.

 
To examine whether L-Arg enhances maximal Na+-K+ pump turnover, we used patch pipette solutions that included 80 mmol/l Na+. The solutions also included L-Arg, or they were L-Arg free. A Na+-containing superfusate was used. The mean Ip for experiments using control pipette solutions or solutions containing L-Arg is shown in Fig. 3B. L-Arg did not induce an increase in Ip, which suggests that it has no effect on maximal pump turnover.

To examine whether the increase in Ip induced by L-Arg is dependent on NOS, we included 10 µmol/l radicicol in pipette solutions. The effect of radicicol on the mean Ip of control myocytes or those voltage clamped by using pipette solutions containing L-Arg is shown in Fig. 4. Radicicol abolished the increase in Ip. We used ODQ to support a role of NO in mediating pump stimulation by L-Arg. Inclusion of 10 µmol/l of this competitive inhibitor of NO-activated guanylyl cyclase in pipette solutions abolished the L-Arg-induced pump stimulation. The effect of ODQ on mean Ip of control myocytes or myocytes voltage clamped by using pipette solutions containing L-Arg is shown in Fig. 4.


Figure 4
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  		Fig. 4. The role of NO synthase-dependent signaling in pump stimulation induced by L-Arg. Patch pipette solutions contained 10 µmol/l L-Arg as indicated. The effect of radicicol and 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ) on mean Ip and L-Arg-induced stimulation is shown. The numbers in parentheses indicate the number of experiments. *Significant difference.

 
In principle, perfusion of the intracellular compartment with pipette solution may deplete the NOS cofactor tetrahydrobiopterin (BH4) and have an effect on baseline pump currents. To examine whether supplementation of BH4 had an effect on Ip, we included it in solutions in a concentration of 10 µmol/l. The solutions did not contain L-Arg. Mean Ip (0.32 ± 0.03 pA/pF, n = 5) was not significantly different from mean Ip of control myocytes (0.35 ± 0.01 pA/pF, n = 44; 90% power to detect a 25% difference in mean Ip with an {alpha}-error level of 0.05).

Uncoupling NOS inhibits the Na+-K+ pump. We used paraquat to uncouple NOS. Paraquat shunts electrons from the heme domain of NOS to form the paraquat-free radical. This, in turn, reacts with molecular oxygen to form O2·–, effectively uncoupling NOS from NO to O2·– synthesis, i.e., preferentially synthesizing O2·– rather than NO (28). To ascertain that paraquat increases O2·– levels in the cardiac myocytes, we loaded myocytes with DHE. We compared fluorescence of controls and myocytes exposed to 1 mmol/l paraquat. Representative confocal fluorescent micrographs of a control myocyte and a myocyte exposed to paraquat are shown in Fig. 5A, and the mean fluorescence intensities are presented in Fig. 5B. Paraquat induced a significant increase in DHE fluorescence intensity compared with control.


Figure 5
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  		Fig. 5. A: confocal fluorescent micrographs of a control myocyte and a myocyte exposed to paraquat. B: mean dihydroethidium (DHE) fluorescence intensity. Paraquat increased DHE fluorescence intensity compared with control. *Significant difference compared with control (n = 9).

 
We next examined the effect of uncoupling of NOS on the Na+-K+ pump. Patch pipette solutions included 10 mmol/l Na+ and 10 µmol/l L-Arg. The superfusate contained Na+. Mean Ip of myocytes patch clamped by using pipette solutions containing 100 µmol/l paraquat or corresponding control pipette solutions is shown in Fig. 6. Paraquat induced a large decrease in Ip.


Figure 6
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  		Fig. 6. Effect of SOD and ebselen on paraquat-induced Na+-K+ pump inhibition. Histograms show the mean Ip of myocytes patch clamped by using pipette solutions containing 100 µmol/l paraquat or corresponding control pipette solutions. The numbers in parentheses indicate the number of experiments. *Significant difference.

 
To examine whether signaling downstream from NO is intact after exposure of myocytes to paraquat, we used pipette solutions that included paraquat and contained 10 µmol/l YC-1 or were free of YC-1. There was no L-Arg in the solutions. YC-1 directly stimulates NO-activated, soluble guanylyl cyclase (sGC) (15) and induces an increase in Ip that depends on activation of cGMP-dependent protein kinase (42). Despite the presence of paraquat, mean Ip measured by using pipette solutions that contained YC-1 was significantly larger than mean Ip measured by using solutions without YC-1 (0.43 ± 0.03 pA/pF, n = 8 vs. 0.29 ± 0.03 pA/pF, n = 6), which indicates that the downstream signaling cascade is intact.

To examine whether O2·– mediates the paraquat-induced decrease in Ip, we included 200 IU/ml SOD in patch pipette solutions. Results are shown in Fig. 6. SOD abolished the paraquat-induced decrease in mean Ip. We also examined the effect of the reactive oxidant scavenger ebselen. Ebselen was included in pipette solutions in a concentration of 10 µmol/l. Results are shown in Fig. 6. Ebselen caused a decrease in Ip in the absence of paraquat in the pipette solution. This is consistent with a previously reported decrease of Na+-K+-ATPase activity in vitro (4). Despite this, ebselen caused an increase in Ip in the presence of paraquat.


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Supplementing isolated cardiac myocytes with L-Arg, the key substrate for NOS, increased NO-sensitive DAF fluorescence and stimulated Na+-K+ pump activity. The involvement of NOS in L-Arg induced NO production, and pump stimulation was supported by their inhibition with the Hsp90-antagonist radicicol (Figs. 1 and 4). There was a plateau of pump stimulation at a concentration of 10 µmol/l L-Arg in the pipette solution. With perfusion of the intracellular compartment, the concentration in the pipette solution should approximate the concentration of freely exchangeable L-Arg in the myocyte. The similarity of the L-Arg dose dependency of pump stimulation and recombinant NOS activation (39) supports the role of NOS in the effect of L-Arg on Ip.

The increase in Ip reached a plateau at a concentration much lower than the concentration of L-Arg in intact cells [100–800 µmol/l in endothelial cells (8, 41)]. The simplest interpretation is that perfusion of the intracellular compartment with pipette solution containing little or no L-Arg depletes L-Arg to a level where its concentration becomes rate limiting for NOS-mediated NO synthesis. However, this interpretation is poorly compatible with the augmentation of NO-sensitive DAF fluorescence in intact myocytes. Separate cellular L-Arg pools that are not freely exchangeable may exist. Functionally, L-Arg contained in caveolae appears separate from L-Arg in the bulk phase of the cytosol, and colocalized L-Arg transporters and NOS-3 may be part of a functional unit (6, 41). This may explain the apparent "L-arginine paradox": supplementation of L-Arg in modest concentrations increases cellular NO synthesis despite an expected saturation of NOS at typical intracellular concentrations.

The L-Arg-induced Na+-K+ pump stimulation could be due to a direct effect of NO on the pump molecule or the lipid membrane they are embedded in. However, the effect of ODQ to abolish the increase in Ip induced by L-Arg (Fig. 4) strongly implicates downstream activation of sGC (17). We have previously found that activation of sGC using a pharmacological NO donor or YC-1 stimulates the Na+-K+ pump in cardiac myocytes (42) via downstream messengers that include cGMP-activated protein kinase. Whereas kinase-mediated phosphorylation has been implicated in Na+-K+ pump stimulation, phosphorylation sites on the pump molecule are poorly accessible (37), and the role of phosphorylation in pump regulation is uncertain (9). Phosphorylation of phospholemman, a membrane protein closely associated with the Na+-K+ pump, has been suggested to regulate the pump (36). However, phosphorylation of phospholemman is unlikely to mediate NO-dependent pump stimulation because the protein is not phosphorylated by PKG. The molecular mechanism by which L-Arg stimulates the Na+-K+ pump cannot be identified from our study.

Paraquat induced an increase in O2·–-sensitive DHE fluorescence and a decrease in Na+-K+ pump current. Because of its charge, O2·– has poor mobility in the lipid environment of the Na+-K+ pump molecule (25). It is converted to another, highly diffusible, oxidant species, H2O2, in a reaction catalyzed by SOD. However, H2O2 or its metabolites seem unlikely to mediate the paraquat-induced decrease in Ip because SOD abolished rather than enhanced the decrease (Fig. 6), implicating an alternative O2·–-dependent pathway in the paraquat-induced pump inhibition. O2·– can also react with NO to form the powerful oxidant peroxynitrite (ONOO–), which, in its conjugated acid form, OONOH, is highly membrane permeable (40) and may reach oxidizable groups on protein molecules embedded in the cell membrane, including Na+-K+ pump molecules. Ebselen can scavenge ONOO– (10) and should reverse the paraquat-induced decrease in pump current if it depends on ONOO–, as was found in the present study. However, since ebselen is not specific for ONOO– (16), a definite role for ONOO– cannot be identified from our study.

Whereas a specific compound is not identified, it is likely that a RNS/ROS derivative of O2·– mediates the effect of paraquat on Ip by inducing an oxidative change on target molecules, perhaps the Na+-K+ pump molecule itself. Thiol groups on cysteine residues are particularly susceptible to oxidation. Reactions they undergo include essentially irreversible sulfinic or sulfonic oxidation or, in the presence of glutathione, reversible S-glutathionylation, the most frequent form of oxidative thiol modification (27). The Na+-K+ pump's {alpha}- and β-subunits and the FXYD proteins closely associated with the pump all contain cysteine residues, and inhibition of isolated Na+-K+-ATPase exposed to oxidant stress in vitro has been attributed to oxidation of thiol groups (34). While the present study has not demonstrated oxidation of Na+-K+ pump subunits, it has shown that activity of the in situ Na+-K+ pump can be altered by oxidant stress sourced in the myocyte. Such an endogenous source of oxidant stress is a prerequisite for implicating oxidation of proteins in the regulation of their function (18).

Since the Na-K pump is a key determinant of excitation-contraction coupling, NOS-dependent pump stimulation may have important physiological implications. For example, stimulation of the Na+-K+ pump by NOS-derived NO may contribute to the previously demonstrated regulation of cardiac excitation-contraction coupling by NO (2, 35), and since membrane receptors are coupled to NOS (13, 29), the link between NOS activation and Na+-K+ pump stimulation shown in the present study may also provide an explanation for regulation of cardiac contractility by some hormones.

Our study may also have implications for cardiac pathophysiology, particularly heart failure. Raised levels of hormones regulating cardiovascular function, including angiotensin II (38), dysregulation of NO/ROS/RNS (31), and raised myocyte Na+ levels (32, 33) are key factors in the pathogenesis of the clinical syndrome of contractile abnormalities and cardiac arrhythmias in heart failure. The present study suggests that the interaction of these factors may be important for our understanding of the pathophysiology and treatment of the syndrome. Since angiotensin II activates NAD(P)H oxidase in cardiac myocytes (23), the O2·– derived from the oxidase may directly, or after amplification via oxidation of BH4 and NOS uncoupling (30), account for angiotensin II-induced sarcolemmal Na+-K+ pump inhibition we have reported previously (5, 24). Identification of steps in which an adverse interaction between cardiovascular hormones, NO/ROS/RNS, the Na+-K+ pump, and intracellular Na+ can be reversed may be important for the treatment of the electromechanical phenotype of heart failure.

In summary, we have shown that supplementation of the NOS substrate L-Arg induces an NO-mediated increase in Na+-K+ pump activity in cardiac myocytes, whereas the uncoupling of NOS causes O2·–-mediated pump inhibition. Such opposing effects of coupled and uncoupled NOS activity on the Na+-K+ pump in cardiac myocytes may be of particular importance in understanding the function of both the normal and the failing heart.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
The present study was supported by grants from the North Shore Heart Research Foundation and the National Health and Medical Research Council.


   ACKNOWLEDGMENTS
 
The authors acknowledge the facilities as well as scientific and technical assistance from staff in the Nanostructural Analysis Network Organisation Major National Research Facility at the Electron Microscopy Unit, the University of Sydney. In particular, the assistance from Ellie Kable and Professor Filip Braet was greatly appreciated.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. H. Rasmussen, Dept. of Cardiology, Royal North Shore Hospital, St. Leonards, NSW, 2065, Australia (e-mail: helger{at}med.usyd.edu.au)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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