The nitric oxide donor sodium nitroprusside stimulates the Na+–K+ pump in isolated rabbit cardiac myocytes
1 Department of Cardiology, Royal North Shore Hospital, St Leonards, Sydney, NSW 2065, Australia
2 Department of Cardiology, Gosford Hospital, Gosford, NSW 2250, Australia
3 Department of Medicine, University of Sydney, NSW 2006, Australia
4 School of Chemistry, University of Sydney, NSW 2006, Australia
Abstract
Nitric oxide (NO) affects the membrane Na+–K+ pump in a tissue-dependent manner. Stimulation of intrinsic pump activity, stimulation secondary to NO-induced Na+ influx into cells or inhibition has been reported. We used the whole-cell patch clamp technique to measure electrogenic Na+–K+ pump current (Ip) in rabbit ventricular myocytes. Myocytes were voltage clamped with wide-tipped patch pipettes to achieve optimal perfusion of the intracellular compartment, and Ip was identified as the shift in holding current induced by 100 μM ouabain. The NO donor sodium nitroprusside (SNP) in concentrations of 1, 10, 50 or 100 μM induced a significant increase in Ip when the intracellular compartment was perfused with pipette solutions containing 10 mM Na+, a concentration near physiological levels. SNP had no effect when the pump was near-maximally activated by 80 mM Na+ in pipette solutions. Stimulation persisted in the absence of extracellular Na+, indicating its independence of transmembrane Na+ influx. The SNP-induced pump stimulation was abolished by inhibition of soluble guanylyl cyclase (sGC) with 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one, by inhibition of protein kinase G (PKG) with KT-5823 or by inhibition of protein phosphatase with okadaic acid. Inclusion of the non-hydrolysable cGMP analogue 8pCPT-cGMP, activated recombinant PKG or the sGC-activator YC-1 in patch pipette filling solutions reproduced the SNP-induced pump stimulation. Pump stimulation induced by YC-1 was dependent on the Na+ concentration but not the K+ concentration in pipette filling solutions, suggesting an altered sensitivity of the Na+–K+ pump to intracellular Na+.
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Introduction
The simple diatomic molecule nitric oxide (NO) participates in, or modulates, messenger pathways that regulate a variety of cellular functions in all mammalian organs. Its production is mediated by NO synthase (NOS) within endothelial and parenchymal cells. NO may also be supplied exogenously by pharmacological donors frequently used in the treatment of cardiovascular diseases. In cardiac myocytes, NO has a prominent role in the regulation of Ca2+ handling. It modifies Ca2+ influx via L-type sarcolemmal Ca2+ channels, it regulates the sarcoplasmic reticulum (SR) Ca2+ release channel and it may inhibit Ca2+-ATPase-mediated SR reuptake of Ca2+ (Hare, 2003). Since sarcolemmal Na+–Ca2+ exchange is a key determinant of cardiac myocyte Ca2+ content, an effect of NO on the sarcolemmal Na+–K+ pump ultimately should affect Ca2+ handling because the pump maintains the electrochemical gradient for Na+. Previous studies in various non-cardiac tissues have reported that NO does regulate the Na+–K+ pump. However, results are conflicting.
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Na+–K+ ATPase activity in isolated membrane fragments, measured with saturating ligand concentrations, was reduced after exposure of opossum kidney cells to the NO donor sodium nitroprusside (SNP; Liang & Knox, 2000). Sodium nitroprusside and other NO donors induced similar inhibition of Na+–K+ ATPase activity in permeabilized tissue slices from the renal medulla (McKee et al. 1994), choroid plexus (Ellis et al. 2000) and ciliary processes (Ellis et al. 2001). However, the NO donor spermine NONOate, had no effect on Na+–K+ pump activity in the thick ascending limb from rat kidney. Activity was measured at high saturating or physiological rate-limiting levels of intracellular Na+ (Ortiz et al. 2001). Varela et al. (2004) reported that an inhibitory effect of NO donors on the pump activity in the thick ascending limb is time dependent. SNP induced Na+–K+ pump stimulation in rabbit aorta (Gupta et al. 1994) and human corpus cavernosum smooth muscle (Gupta et al. 1995). Pump stimulation in the aorta was thought to be secondary to Na+–H+ exchange-mediated influx of Na+ and an increase in its intracellular concentration. The mechanism for stimulation in corpus cavernosum smooth muscle was not determined.
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Tissue-specific differences in Na+–K+ pump regulation, differences in methodology used to study the Na+–K+ pump and variable delivery of NO and its bioactive derivatives by the donor compounds may have contributed to the conflicting results between studies. We have used the whole-cell patch clamp technique to examine the effect of NO on the sarcolemmal Na+–K+ pump in isolated rabbit ventricular myocytes. Provided wide-tipped patch pipettes are used, the technique allows control of the ligands of the Na+–K+ pump at intra- and extracellular sites and control of membrane voltage. It also allows the intracellular delivery of membrane-impermeable drugs and compounds dissolved in patch pipette solutions, including compounds of large molecular size. Electrogenic Na+–K+ pump current (Ip), arising from the 3Na+:2K+ exchange ratio, can be identified as the shift in holding current induced by blocking the pump with ouabain. We show that SNP induces an increase in Ip. To further characterize the mechanism, we examine the dependence of Na+–K+ pump stimulation on intracellular Na+ and K+. To avoid non-specific effects of pharmacological NO donors in these studies, we used the synthetic benzyl indazole derivative YC-1 (Russwurm & Koesling, 2002) to selectively activate soluble guanylyl cyclase (sGC), a downstream target molecule for NO.
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Methods
Cells
Single ventricular myocytes isolated from male New Zealand White rabbits were used. The rabbits, weighing 2.8–3.5 kg, were anaesthetized with 50 mg kg–1 ketamine and 20 mg kg–1 xylazine hydrochloride given intramuscularly. The heart was excised when deep anaesthesia was assured as indicated by the absence of a corneal reflex and response to deep pressure between the metatarsal bones. Details of techniques used to isolate ventricular myocytes have been previously described (Hool et al. 1995). The institutional review committee for animal research had approved experimental protocols. The myocytes were used on the day of isolation only. They were stored at room temperature in Krebs–Henseleit buffer solution until used for patch-clamp studies.
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Solutions
Myocytes were suspended in a tissue bath mounted on an inverted microscope for experimentation. While we established the whole-cell configuration, the bath was perfused with modified Tyrode solution, which contained (mM): NaCl, 140; KCl, 5.6; CaCl2, 2.16; MgCl2, 1; glucose, 10; NaH2PO4, 1; sodium glutamate, 9; and Hepes, 10. It was titrated to a pH of 7.40 ± 0.01 at 35°C with NaOH. For measurement of Ip, we switched to a superfusate that usually was identical except that it was nominally Ca2+ free and contained 0.2 mM CdCl2 and 2 mM BaCl2. In some experiments we modified this solution by replacing Na+-containing compounds with N-methyl-D-glucamine chloride (NMG-Cl; Hansen et al. 2000). The K+ concentration in the superfusate was 5.6 mM in all experiments unless otherwise indicated.
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For most experiments, wide-tipped patch pipettes (4–5 μm diameter) were filled with solutions containing (mM): Hepes, 5; MgATP, 2; EGTA, 5; potassium glutamate, 0–140; and sodium glutamate, 10. Osmotic balance was maintained with 150–10 mM tetramethylammonium chloride (TMA-Cl). We eliminated Na+-containing compounds in pipette solutions in some experiments (replaced with TMA-Cl) while we increased the Na+ concentration to 80 mM in others (osmotic balance was maintained by adjusting the concentration of TMA-Cl). The dependence of Ip on the intracellular Na+ concentration in the absence of intracellular K+ was examined using pipette solutions that included 0–80 mM sodium glutamate. The osmotic balance was maintained with TMA-Cl. All pipette solutions were titrated to a pH of 7.05 ± 0.01 at 35°C with 1 mM TMA-OH.
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Wide-tipped patch pipettes were used to optimize control of the concentration of the intracellular ligands for the Na+–K+ pump. The patch pipettes had initial resistances of 0.8–1.1 M when filling solutions included K+ at the concentration of 70 mM used in most experiments. Results were independent of series resistance in the whole-cell configuration only if levels were 2.8 M; the levels in accepted experiments ranged from 1.6 to 2.8 M. Since K+ has a higher conductivity than the other ions in pipette solutions (Hille, 1992) the series resistance was higher when we used K+-free pipette filling solutions. Control of the concentration of intracellular pump ligands, as indicated by independence of the measured pump currents of the series resistance, was achieved at levels 4.0 M.
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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 μM ouabain. Na+–K+ pump inhibition is saturated when rabbit ventricular myocytes are exposed to ouabain at this concentration (Drewnowska & Baumgarten, 1991; Hool et al. 1995). A stable current plateau 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 acquired with an electronic cursor at 5 s intervals. Recordings were obtained using the continuous single-electrode mode of Axoclamp 2A or 2B amplifiers supported by AxoTape and pCLAMP software (Axon Instruments, Foster City, CA, USA). We report Ip normalized for membrane capacitance and hence cell size.
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We used ouabain to inhibit the pump in this study because alternative, faster acting, cardiac steroids are less potent and would have to be used at a higher concentration to assure complete Na+–K+ pump inhibition. Any compound at a high concentration may have effects on non-pump membrane currents. We did not attempt to achieve wash-off of ouabain because, even when short-acting cardiac steroids are used, their effects on inotropy and intracellular Na+ in cardiac tissue remain for > 30 min (Lee et al. 1980; Boyett et al. 1986). In agreement with this, Na+–K+ pump currents of ventricular myocytes (Mogul et al. 1990) do not return to control levels within a time frame that assures drift-free recordings.
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Chemicals and reagents
TMA-Cl and NMG-Cl were purum grade and were obtained from Fluka Chemicals (Switzerland). All other chemicals used in Tyrode solutions were analytical grade and were obtained from BDH (Australia). Ouabain, SNP and bovine Cu,Zn superoxide dismutase were obtained from Sigma-Aldrich (St Louis, MO, USA) and YC-1, 1H-[1,2,4]Oxadiazole[4,3-a]quinoxalin-1-one (ODQ), KT-5823, okadaic acid, methyl okadaic acid, 8-pCPT-cGMP and recombinant bovine cGMP-activated protein kinase (PKG) were supplied by Calbiochem (La Jolla, CA, USA).
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Statistical analysis
Results are expressed as means ± S.E.M. Student's t test for unpaired data is used for statistical comparisons. We used Dunnett's test when the same control group was used for more than one comparison and a Mann–Whitney rank sum test for unequal sample sizes when data could not be assumed to be normally distributed. P < 0.05 is regarded as significant in all comparisons.
Results
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Effect of sodium nitroprusside on pump current
To examine whether the NO donor SNP has an effect on the Na+–K+ pump we measured Ip of control myocytes and of myocytes exposed to 50 μM SNP. Patch pipette solutions contained 10 mM Na+, a concentration near physiological intracellular levels in rabbit ventricular myocytes (Hool et al. 1995). The concentration of K+ in pipette solutions was 70 mM. The superfusate included 150 mM Na+. We maintained control myocytes as well as myocytes exposed to SNP in the whole-cell configuration for 10–12 min before exposing them to ouabain. Figure 1A shows traces of holding currents recorded from a control myocyte and a myocyte exposed to SNP. Ouabain induced a larger shift in holding current of the myocyte exposed to SNP than of the control myocyte.
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Patch pipette filling solutions and superfusates included 10 and 150 mM Na+ for experiments shown in A, 0 and 150 mM for B and 10 and 0 mM for experiments shown in C. The traces show holding currents before and after exposure to ouabain (Oua). The membrane capacitance, Cm (in pF) is indicated to facilitate comparisons.
Additional experiments were performed to examine whether SNP induces an increase in Ip due to an increase in transsarcolemmal Na+ influx. Such influx could cause secondary stimulation of the pump due to an increase in the intracellular Na+ concentration. In one set of experiments we patch clamped myocytes using pipette filling solutions that were nominally Na+ free. The superfusate contained Na+. Figure 1B shows traces of holding currents of a control myocyte and of a myocyte exposed to SNP. Ouabain-induced shifts in holding currents were barely detectable for either myocyte. Similar results were obtained for two additional myocytes exposed to SNP. If SNP enhances transsarcolemmal Na+ influx it does not cause an increase in the intracellular Na+ concentration that can be detected as an increase in Ip.
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To obtain independent support for the conclusion that the SNP-induced increase in Na+–K+ pump activity is not due to enhanced Na+ influx we used patch pipette solutions that contained 10 mM Na+ and a superfusate that was Na+ free. This eliminates any possible SNP-induced Na+ influx. Figure 1C shows holding currents of a control myocyte and of a myocyte exposed to SNP. Ouabain induced a larger shift in the holding current of the myocyte exposed to SNP than of the control myocyte. The mean levels of Ip in experiments performed using Na+-containing patch pipettes and superfusates that contained Na+ or were Na+ free are summarized in Fig. 2. Mean Ip recorded using control, SNP-free superfusates appeared higher in the presence than in the absence of extracellular Na+. We attribute this to an allosteric effect of extracellular Na+ which accelerates conversion of the enzyme from the low-affinity E2 state to the high-Na+-affinity E1 state and enhances the apparent affinity for Na+ at cytosolic Na+–K+ pump sites (Buhagiar et al. 2004). However, a statistically significant increase in Ip with exposure to SNP persisted in the absence of extracellular Na+.
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The Na+ concentration in patch pipette filling solutions was 10 mM in all experiments. Mean (± S.E.M.) Ip values recorded in a superfusate that included 150 mM Na+ (A) or was Na+ free (B) are shown. The extracellular Na+ concentration ([Na+]o) is indicated in the figure. The number of experiments is indicated in parentheses. *P < 0.05.
To examine the relationship between the concentration of SNP and Ip we exposed myocytes to SNP in concentrations ranging from 1 to 1000 μM. Patch pipettes and superfusates contained 10 mM Na+ and 70 mM K+. The superfusate contained 150 mM Na+. Results are shown in Fig. 3. SNP at concentrations of 1, 10, 50 and 100 μM induced a statistically significant increase in Ip. However, there was no significant stimulation relative to control myocytes when the concentration was increased further to 1000 μM, i.e. there was not a simple concentration-dependent effect of SNP. Since SNP at high concentrations has oxidant effects in vitro that can be prevented by superoxide dismutase (Jaworski et al. 2001) we included 200 μM cytosolic Cu,Zn superoxide dismutase (SOD) in pipette filling solutions and exposed myocytes 1000 μM SNP. Mean Ip has been included in Fig. 3. Superoxide dismutase restored SNP-induced stimulation of Ip.
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The Na+ concentration in patch pipette filling solutions was 10 mM. The superfusate contained 150 mM Na+ and 5.6 mM K+. Superoxide dismutase (SOD) was included in the patch pipette solution as indicated. Mean (± S.E.M.) Ip values at different concentrations of SNP are indicated. The numbers of experiments are indicated in parenthesis. Asterisk indicates a statistically significant SNP-induced increase in Ip.
Intracellular messengers linking NO to Na+–K+ pump stimulation
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Soluble guanylyl cyclase is the classical target molecule mediating cellular effects of NO. However, since some effects of NO are independent of sGC, we measured Ip while blocking sGC and its downstream messengers. Patch pipettes contained 10 mM Na+ and 70 mM K+. The superfusate contained 150 mM Na+ and we used SNP at a concentration of 50 μM. We included 10 μM of the NO-competitive antagonist ODQ in patch pipette solutions to block access of NO to sGC and so block stimulation of cGMP synthesis. Figure 4 shows that ODQ abolished the SNP-induced pump stimulation.
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Myocytes were superfused with control solutions or solutions containing SNP. Patch pipettes contained control solutions or solutions containing inhibitors of soluble guanylyl cyclase (ODQ), protein kinase G (KT-5823) or protein phosphatase (okadaic acid, OA). Numbers in parentheses indicate the number of myocytes studied with each set of conditions. * Significant difference between mean (± S.E.M.) Ip values of myocytes exposed or not exposed to SNP; # significant difference between Ip of myocytes exposed to SNP and patch clamped using control pipette filling solutions and solutions containing ODQ, KT-5823 or OA. Results of experiments using an SNP-free superfusate and ODQ, KT-5823 or OA in pipette solutions are also shown.
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The inhibition of the SNP-induced pump stimulation by ODQ implicates protein kinase G (PKG) in the downstream messenger pathways that mediate the stimulation. However, since cGMP can directly activate effector proteins independent of phosphorylation (Dimmeler et al. 1999), we examined the effect of inhibiting PKG by including 0.5 μM KT-5823 in patch pipette solutions. KT-5823 abolished the SNP-induced pump stimulation, implicating a role for PKG in the stimulation. If the pump stimulation is due to PKG directly phosphorylating Na+–K+ pump molecules, inhibition of phosphatase-mediated dephosphorylation might enhance the effect of SNP. To examine this we included 0.1 μM okadaic acid in patch pipette solutions. Figure 4 shows that phosphatase inhibition abolished, rather than enhanced, the SNP-induced pump stimulation.
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Figure 3 shows that there is not a simple concentration-dependent effect of the NO donor SNP on Ip. To examine the effect of activating messengers downstream from the NO-activated sGC we activated PKG by including a cGMP analogue in patch pipette solutions. We used the non-hydrolysable analogue 8-pCPT-cGMP to prevent its rapid intracellular breakdown. Figure 5 shows that 8-pCPT-cGMP induced a concentration-dependent increase in Ip. Since 8-pCPT-cGMP in high concentrations might activate protein kinase A (PKA), we examined the effect of adding 0.5 μM of H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride) to pipette solutions containing 1 mM 8-pCPT-cGMP. The mean Ip recorded in such experiments is included in Fig. 5. The 8-pCPT-cGMP-induced increase in Ip persisted after PKA blockade.
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Myocytes were exposed to control superfusates, and the compounds indicated in the figure were included in patch pipette solutions. Numbers in parentheses indicate the number of myocytes for each set of experimental conditions. Asterisk indicates a significant difference (P < 0.05) between Ip values of myocytes patch clamped using control patch pipette filling solutions and solutions containing 8-pCPT-cGMP, TEA. The protein kinase A inhibitor H-89 did not prevent the activation of Ip induced by the highest concentration of 8-pCPT-cGMP,TEA. # Significant difference (P < 0.05) between Ip values of myocytes patch clamped using filling solutions containing 20 μM 8-pCPT-cGMP, TEA with or without recombinant bovine protein kinase G (PKG).
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To avoid non-specific effects of high concentrations of 8-pCPT-cGMP we also examined the effect of including 400 U ml–1 recombinant bovine PKG in the patch pipette solution (White et al. 2000). To activate PKG we included 20 μM 8-pCPT-cGMP in the solution. Figure 5 shows that 20 μM 8-pCPT-cGMP in patch pipette solutions had no effect on Ip in the absence of recombinant PKG. However, the combination of 20 μM 8-pCPT-cGMP and recombinant PKG in pipette solutions induced a large increase in Ip.
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Activation of sGC with YC-1
Figure 5 shows that there is a clear concentration-dependent effect of an analogue of the product of sGC, cGMP, while Fig. 3 shows that the relationship between the NO donor SNP and Ip is much less predictable. In addition, activation of sGC by exposure to SNP causes a smaller increase in Ip than activation of messenger pathways downstream from sGC. This suggests that there are non-specific effects of SNP. To avoid this while still inducing activation via the classical target molecule for NO, sGC, we included 1 μM YC-1 in patch pipette solutions. The solutions included 10 mM Na+ and 70 mM K+. Superfusates included 5.6 mM K+ and they contained 150 mM Na+ or they were Na+ free. Figure 6A shows that ouabain induced a shift in holding current in the presence or absence of extracellular Na+. All experiments using the same protocols are summarized in Fig. 6B. YC-1 induced a large, statistically significant increase in Ip that was independent of the presence or absence of extracellular Na+.
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Inclusion of 1 μM YC-1 in pipette solutions is indicated in the figure. A shows shifts in holding current induced by ouabain (Oua) recorded using Na+ at a concentration ([Na+]pip) of 10 mM Na+ in patch pipette solutions. The Na+ concentration in superfusates ([Na+]o) was 150 mM (upper traces) or 0 mM (lower traces). B summarizes mean (± S.E.M.) Ip values recorded in Na+-containing and Na+-free superfusates. C shows ouabain-induced shifts in holding current recorded using 80 mM Na+ in patch pipette solutions, recorded in Na+-containing superfusates. D summarizes mean Ip values recorded using 80 mM Na+ in pipette solutions. Cm indicates membrane capacitance in pF. *P < 0.05.
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The Na+–K+ pump stimulation induced by YC-1 might be due to an increase in maximal turnover of the pump or due to an increase in its sensitivity to intracellular Na+. An increase in maximal turnover should be reflected by an increase in Ip recorded when intracellular pump sites are nearly saturated by a high concentration of Na+. We patch clamped myocytes using 80 mM Na+ and 70 mM K+ in pipette solutions. The superfusate included 150 and 5.6 mM K+. Figure 6C shows examples of ouabain-induced shifts in holding currents of a control myocyte and a myocyte exposed to YC-1. The ouabain-induced shifts in currents were much larger than those recorded using 10 mM Na+ in pipette solutions (Fig. 6A) and they were similar for a myocyte exposed or not exposed to YC-1. All experiments performed using 80 mM Na+ in the pipette solution are summarized in Fig. 6D. There was no significant difference between the mean Ip of control myocytes and that of myocytes exposed to YC-1, suggesting that YC-1 does not stimulate maximal pump turnover.
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To ascertain that YC-1 activates the same messenger pathways as those implicated for SNP-induced Na+–K+ pump stimulation we examined the effects of ODQ, KT-5823 or okadaic acid in patch pipette solutions. The pipette solutions included 10 mM Na+ and 70 mM K+. Figure 7 shows that ODQ, KT-5823 and okadaic acid abolished the YC-1-induced Na+–K+ pump stimulation, i.e. the pattern of inhibition by blockers of implicated messenger pathways was identical to that shown for SNP in Fig. 4.
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Patch pipettes contained control solutions or solutions that contained YC-1 and inhibitors of soluble guanylyl cyclase (ODQ), protein kinase G (KT-5823) or protein phosphatase (okadaic acid, OA) as indicated. The Na+ concentrations in pipette solutions and superfusates were 10 and 150 mM, respectively. Numbers in parentheses indicate the number of myocytes studied with each set of conditions. * Significant difference (*P < 0.05) between mean (± S.E.M.) Ip values of myocytes perfused or not perfused with pipette solutions containing YC-1; # significant difference (*P < 0.05) between mean Ip values of myocytes perfused with pipette solutions containing YC-1 alone and solutions containing both YC-1 and ODQ, KT-5823 or OA. Results of control experiments using ODQ, KT-5823 or OA alone are shown in Fig. 4.
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Dependence of Na+–K+ pump stimulation on intracellular Na+ and K+
The dependence of pump stimulation on a non-saturating concentration of intracellular Na+ suggests that YC-1 causes a change in the sensitivity of the pump to intracellular Na+. Since Na+ binds at two sites on the cytosolic side of the pump in competition with K+ and at a third site selective for Na+, we examined the dependence of pump stimulation induced by YC-1 on the intracellular K+ concentration. We voltage clamped myocytes using patch pipettes containing 10 mM Na+ and K+ at a concentration ([K]pip) ranging from 0 to 140 mM. Superfusates included 150 mM Na+ and 5.6 mM K+. Figure 8 shows a summary of Ip of control myocytes and of myocytes patch clamped using 1 μM YC-1 in pipette filling solutions. As expected, there was a [K]pip-dependent decrease in Ip. YC-1 induced a significant increase in Ip in the presence or absence of K+ in patch pipette filling solutions.
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Ip was measured in myocytes voltage clamped using control pipette filling solutions () or solutions containing YC-1 (). The pipette filling solutions contained Na+ at a concentration of 10 mM and K+ at a concentration ([K+]pip) ranging from 0 to 140 mM. The numbers of experiments for each set of conditions are indicated in parentheses. Mean (± S.E.M.) Ip values for myocytes perfused with pipette solutions containing YC-1 were significantly (*P < 0.05) higher than for myocytes perfused with control solutions at all levels of [K]pip.
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Since YC-1 induces Na+–K+ pump stimulation at a non-saturating concentration of intracellular Na+ only and since stimulation can occur in the nominal absence of intracellular K+, we examined whether YC-1 alters the sensitivity of the pump to intracellular Na+ in the absence of intracellular K+. We patch clamped myocytes using pipette solutions that were K+ free and included Na+ at concentrations ranging from 0 to 80 mM. To eliminate binding of K+ at extracellular sites as a rate-limiting step at high pipette Na+ concentration we used a K+ concentration of 15 mM in the superfusate in these experiments. The Na+ concentration in the superfusate was 150 mM. Figure 9A shows the ouabain-induced shift in holding current of a control myocyte patch clamped using a pipette solution that contained 80 mM Na+. The ouabain-induced shift in current appeared much larger than the shift recorded using K+-containing pipette filling solutions with the same Na+ concentration (Fig. 6). Figure 9B summarizes experiments performed using different Na+ concentrations in pipette solutions.
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Ip was measured in myocytes voltage clamped using control pipette filling solutions () or solutions containing YC-1 (). The pipette filling solutions contained Na+ at concentrations ranging from 0 to 80 mM. They were nominally K+ free. The numbers of experiments for each set of conditions are indicated in parentheses. Mean (± S.E.M.) Ip values for myocytes perfused with pipette solutions containing YC-1 were significantly higher than for myocytes perfused with control solutions when the pipette Na+ concentration was at rate-limiting levels.
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For the purpose of assigning a value to the intracellular Na+ concentration that induces half-maximal pump activation we fitted the Hill equation to the data. We derived values of 24.0 mM for control myocytes and 14.1 mM for myocytes perfused with pipette solutions containing YC-1. Since Ip is not a direct measure of Na+ binding, the fit is without mechanistic implications and is therefore not shown. Mean Ip is similar for control myocytes and for myocytes exposed to YC-1 when [Na+]pip is 0 or 80 mM. However, YC-1 seemed to induce an increase in Ip when [Na]pip was at the rate-limiting concentrations of 5, 10 or 20 mM. A Mann–Whitney test indicated that YC-1 induces a significant increase in Ip when data obtained at these three Na+ concentrations are considered in combination.
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Discussion
Previous studies have reported inhibition (McKee et al. 1994; Liang & Knox, 2000; Ellis et al. 2000, 2001; Varela et al. 2004), the absence of any effect (Ortiz et al. 2001) and stimulation (Gupta et al. 1994, 1995) of the membrane Na+–K+ pump when various tissues were exposed to NO donor compounds. Stimulation was secondary to enhanced Na+ influx rather than due to a direct effect on the Na+–K+ pump in vascular smooth muscle (Gupta et al. 1994). In our study, SNP stimulated intrinsic pump activity, independent of enhanced transsarcolemmal Na+ influx. The discrepancy between our results and previous studies may be organ dependent (Therien & Blostein, 2000). However, use of pharmacological NO donor compounds may also have contributed to inconsistent results. The pathways leading to formation of NO from the compounds and derivative side reactions are exquisitely sensitive to the local oxygen tension, and the amount of bio-active intermediate and end products that form during metabolism and degradation may exceed the amount of NO (Ignarro et al. 2002). The net response may arise from a balance between, at times opposing, effects of compounds generated. NO and its derivatives can directly inhibit isolated Na+–K+ ATPase, in part via effects on membrane fluidity (Muriel & Sandoval, 2000), and the derivative peroxynitrite inhibits Na+–K+ ATPase activity in renal cells exposed to NO donors. The inhibition is abolished when the peroxynitrite precursor, superoxide, is eliminated with SOD (Varela et al. 2004). In our study, SNP induced Na+–K+ pump stimulation at all but the highest concentration used, and stimulation at the high SNP concentration was restored by SOD.
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Activation and inhibition of messengers implicated in NO-mediated pathways
While SNP consistently induced Na+–K+ pump stimulation, there was not a simple concentration -dependent increase in Ip and there was considerable variability of Ip with exposure to SNP (Fig. 3). To avoid ambiguities of pharmacological NO donors we used YC-1 in patch pipette solutions to selectively activate sGC, a downstream target molecule for NO. Since a scaffold protein directs sGC to a membrane association in close proximity to nitric oxide synthase (Russwurm & Koesling, 2002), YC-1 activates sGC at sites of endogenous NO synthesis (Russwurm et al. 2002). Such activation may better reflect physiological signalling than exposure of cells to SNP because effects of YC-1 should be largely restricted to the microdomains of synthesis and breakdown of NO. YC-1 reproduced the SNP-induced stimulation of Ip. This, in combination with the effect of selective inhibition of sGC with ODQ (Russwurm et al. 2002) in pipette solutions (see Figs 4 and 7), strongly implicates sGC in Na+–K+ pump stimulation. The effect of cGMP synthetized on activation of sGC is usually mediated by PKG. However, it may also be due to a cGMP-mediated increase in cAMP and activation of PKA, it may be due to activation of PKA by cGMP in high concentrations or due to a direct effect of cGMP on effector proteins (White, 1999). The persistence of pump stimulation when H-89 is included in patch pipette solutions rules out involvement of PKA.
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Since KT-5823 has imperfect selectivity for PKG we included recombinant PKG in the pipette solution and activated it with 20 μM 8-pCPT-cGMP. PKG reproduced effects of SNP and YC-1. Of note, 20 μM 8-pCPT-cGMP in the absence of PKG in pipette solutions had no effect on Ip. While a difference in sensitivity to activation of endogenous and recombinant PKG may account for this, PKG endogenous to the myocyte may have been exposed to 8-pCPT-cGMP in a concentration lower than 20 μM because 8-pCPT-cGMP is highly membrane permeable. Rate-limiting diffusion through the patch pipette tip can result in an intracellular concentration in voltage clamped cardiac myocytes of membrane-permeable compounds that is lower than the concentration in pipette solutions (Mathias et al. 1990).
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Taken together, the data indicate that PKG mediates Na+–K+ pump stimulation induced by SNP and YC-1. There are few established molecular targets for PKG (Moreno et al. 2001). Phosphorylation of the pump molecule or of the closely associated FXYD1 protein implicated in pump regulation (Cornelius et al. 2001) is unlikely because FXYD1 is not reported to be phosphorylated by PKG and the pump itself is not easily phosphorylated by PKG (Fotis et al. 1999). In addition, if Na+–K+ pump stimulation were mediated by phosphorylation of the pump molecule or the FXYD1 protein, inhibition of protein phosphatases with okadaic acid should enhance rather than abolish stimulation. Protein phosphatases are often implicated in PKG-mediated signalling (White, 1999; Moreno et al. 2001), and phosphatase-mediated dephosphorylation of FXYD1 has been reported (Neumann et al. 1999). PKG can mediate phosphorylation of a regulatory protein for protein phosphatase 1 in smooth muscle myocytes. However, the phosphorylation inhibits rather than activates phosphatase activity (Tokui et al. 1996) and cannot account for our results. The available information cannot identify the okadaic acid-sensitive step in our study.
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Effect of NO on functional properties of the Na+–K+ pump
Interpretation of the YC-1- and SNP-induced change in the sensitivity of the Na+–K+ pump to intracellular Na+ depends on the assumption that Na+ concentrations in the cytosol and pipette solutions are similar. Concentration gradients could exist between the pipette solution and the intracellular bulk phase (Mathias et al. 1990) or between the bulk phase and a diffusion-restricted subsarcolemmal space (Despa & Bers, 2003; Silverman et al. 2003). Transsarcolemmal influx should maintain intracellular Na+ at a level high enough to support an easily detectable Na+–K+ pump current when Na+-free pipette solutions are used, unless the intracellular concentration largely reflects the concentration in the patch pipettes (Mathias et al. 1990; Silverman et al. 2003). Nakao & Gadsby (1989) used wide-tipped patch pipettes to optimize control of intracellular Na+. When Ip was recorded in Na+-containing superfusates with Na+-free patch pipettes it was 3.5% of the Ip recorded with pipettes containing 50 mM Na+. This fraction was reduced to 0.8% when superfusates were Na+ free to eliminate transsarcolemmal Na+ influx. The Ip we recorded with Na+-free patch pipettes was 0.3% of the Ip expected when pipettes contained 50 mM Na+ (extrapolated from Fig. 9B). This low level was achieved in Na+-containing superfusates, and suggests that a diffusion-restricted subsarcolemmal space does not give rise to substantial Na+ concentration gradients. In support of this we have reported that a ouabain-sensitive current is easily detectable when the Na+ concentration in pipette solutions is as low as 0.1 mM (Hansen et al. 2002). We conclude that the Na+ concentration at cytosolic pump sites approximates that in pipette solutions in our experiments.
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Na+–K+ pump regulation can depend on intracellular K+ (Buhagiar et al. 1999, 2004), which is consistent with an effect on the backward E1 + 2K+ E2(K+)2 reaction (Buhagiar et al. 2004) where E1 is the unphosphorylated conformation of the enzyme with a high affinity for cytoplasmic NA+ ions and E2 is the alternative unphosphorylated conformation which is stabilised by cytoplasmic K+ ions. However, such a mechanism cannot account for the pump stimulation in this study because stimulation could be demonstrated in the absence of intracellular K+ (Figs 8 and 9). A change in the apparent Na+ affinity might account for pump stimulation. A change in the affinity could be due to a change in intrinsic Na+ binding to the E1 conformation of pump molecules or due to an apparent change in affinity from a change in the rate of reactions determining availability of the E1 conformation (Apell & Karlish, 2001). Mechanistic details cannot be determined from this study.
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The relative increase in Ip induced by YC-1 was larger at 5.6 than at 15 mM extracellular K+ (Figs 8 and 9). The higher concentration must stimulate the pump. Any additional stimulation may then be less evident if subsequent steps, independent of K+, become rate limiting. Effects of binding of K+ to the E2 conformation should be small because binding is nearly saturated at 5.6 mM. Nor can an increase in the rate of the K+-stimulated dephosphorylation reaction account for stimulation because the reaction is not rate limiting (Kane et al. 1998; Kong & Clarke, 2004). However, allosteric binding of K+ stimulates rate-limiting (Kong & Clarke, 2004) deocclusion of K+ and the E2 E1 conformational transition (Forbush, 1987). We considered the stimulation this may mediate. We modified a mathematical model of the Na+–K+ pump cycle (Kong & Clarke, 2004) to take into account allosteric effects of extracellular K+ (see Appendix for details). Simulations indicated that one might expect a 28% increase in pump rate when extracellular K+ is increased from 5.6 to 15 mM. A quantitative interpretation of the simulations is not justified because most parameters in the model were derived from presteady-state kinetic measurements on purified Na+–K+ ATPase from kidney while our experimental data were obtained in intact cardiac cells. However, the simulations are consistent with the qualitative conclusion that the difference in extracellular K+ concentration may affect the relative size of pump stimulation induced by YC-1. This highlights how details of experimental conditions may affect analysis of Na+–K+ pump regulation.
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Appendix
To examine allosteric effects of K+ we simulated steady-state Na+–K+ pump activity under the experimental conditions used here (extracellular Na+ concentration 150 or 140 mM, extracellular K+ concentration 5.6 or 15 mM, intracellular Na+ concentration 10 mM, intracellular K+ concentration 0 mM and intracellular ATP concentration 2 mM). We modified eqn (5) of a previously published model (Kong & Clarke, 2004) by changing the dissociation constants for the activation of enzyme dephosphorylation by the two transported K+ ions, KoK1 and KoK2. They were estimated at 2.8 and 4.3 mM (Gray et al. 1997). In addition, the equation for the observed rate constant for the E2 E1 transition, kobs1, was modified to include an allosteric effect of external K+. The new equation was:
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We refer to Table 1 of Kong & Clarke (2004) for the values and meanings of the parameters already included in the model.
We made the assumption that the binding of Na+ and K+ to the allosteric sites is non-competitive and that the effects of the two ions are simply additive. KoK represents the dissociation constant of the external allosteric K+ ions from the E2 conformation of the enzyme, irrespective of whether Na+ and/or ATP are simultaneously bound to allosteric sites. It has been set to the same value as KoN, the dissociation constant for allosteric external Na+ ions, i.e. 31 mM, in accord with the results of Forbush (1987) that the affinity of the enzyme for various allosteric ions is not substantially different. ke1, kf1, kg1 and kh1 represent rate constants for the E2 E1 transition (with up to two K+ ions initially bound to E2 at transport sites) when the allosteric sites are saturated by K+ alone (ke1), by K+ and Na+ (kf1), by K+ and ATP (kg1) and by K+, Na+ and ATP (kh1). In the absence of ATP, Na+ and K+ have similar effects in accelerating the E2 E1 transition (Forbush, 1987). Thus, the values of ke1 and kf1 have been chosen to be equal to kb1, the rate constant for the transition at which only allosteric external Na+ ions are bound, i.e. 0.8 s–1.
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External K+ causes approximately 16% greater acceleration of the E2 E1 transition than external Na+ in the presence of ATP (Forbush, 1987). Accordingly, since kd1, the rate constant for the transition when the enzyme is saturated by allosteric Na+ and ATP, has been determined to have a value of approximately 70 s–1 (Lüpfert et al. 2001), the value of kg1 was taken as 81 s–1. When the allosteric sites are saturated by ATP, Na+ and K+, no experimental data are presently available on the value of the rate constant, i.e. kh1. For the purpose of the simulation we assumed the effects of Na+ and K+ are additive, i.e. kh1 = kd1 + kg1 = 151 s–1.
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The simulations were carried out using the commercially available program Berkeley Madonna 7.0 via the variable step size Rosenbrock integration method for stiff systems of differential equations. The simulations yield the time course of the concentration of each enzyme intermediate, as well as the concentration of inorganic phosphate, from which the turnover number of the enzyme can be calculated. The simulations show that the turnover number of the enzyme is predicted to be 28 s–1 in the presence of 5.6 mM external K+ and 36 s–1 in the presence of 15 mM external K+.
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2 Department of Cardiology, Gosford Hospital, Gosford, NSW 2250, Australia
3 Department of Medicine, University of Sydney, NSW 2006, Australia
4 School of Chemistry, University of Sydney, NSW 2006, Australia
Abstract
Nitric oxide (NO) affects the membrane Na+–K+ pump in a tissue-dependent manner. Stimulation of intrinsic pump activity, stimulation secondary to NO-induced Na+ influx into cells or inhibition has been reported. We used the whole-cell patch clamp technique to measure electrogenic Na+–K+ pump current (Ip) in rabbit ventricular myocytes. Myocytes were voltage clamped with wide-tipped patch pipettes to achieve optimal perfusion of the intracellular compartment, and Ip was identified as the shift in holding current induced by 100 μM ouabain. The NO donor sodium nitroprusside (SNP) in concentrations of 1, 10, 50 or 100 μM induced a significant increase in Ip when the intracellular compartment was perfused with pipette solutions containing 10 mM Na+, a concentration near physiological levels. SNP had no effect when the pump was near-maximally activated by 80 mM Na+ in pipette solutions. Stimulation persisted in the absence of extracellular Na+, indicating its independence of transmembrane Na+ influx. The SNP-induced pump stimulation was abolished by inhibition of soluble guanylyl cyclase (sGC) with 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one, by inhibition of protein kinase G (PKG) with KT-5823 or by inhibition of protein phosphatase with okadaic acid. Inclusion of the non-hydrolysable cGMP analogue 8pCPT-cGMP, activated recombinant PKG or the sGC-activator YC-1 in patch pipette filling solutions reproduced the SNP-induced pump stimulation. Pump stimulation induced by YC-1 was dependent on the Na+ concentration but not the K+ concentration in pipette filling solutions, suggesting an altered sensitivity of the Na+–K+ pump to intracellular Na+.
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Introduction
The simple diatomic molecule nitric oxide (NO) participates in, or modulates, messenger pathways that regulate a variety of cellular functions in all mammalian organs. Its production is mediated by NO synthase (NOS) within endothelial and parenchymal cells. NO may also be supplied exogenously by pharmacological donors frequently used in the treatment of cardiovascular diseases. In cardiac myocytes, NO has a prominent role in the regulation of Ca2+ handling. It modifies Ca2+ influx via L-type sarcolemmal Ca2+ channels, it regulates the sarcoplasmic reticulum (SR) Ca2+ release channel and it may inhibit Ca2+-ATPase-mediated SR reuptake of Ca2+ (Hare, 2003). Since sarcolemmal Na+–Ca2+ exchange is a key determinant of cardiac myocyte Ca2+ content, an effect of NO on the sarcolemmal Na+–K+ pump ultimately should affect Ca2+ handling because the pump maintains the electrochemical gradient for Na+. Previous studies in various non-cardiac tissues have reported that NO does regulate the Na+–K+ pump. However, results are conflicting.
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Na+–K+ ATPase activity in isolated membrane fragments, measured with saturating ligand concentrations, was reduced after exposure of opossum kidney cells to the NO donor sodium nitroprusside (SNP; Liang & Knox, 2000). Sodium nitroprusside and other NO donors induced similar inhibition of Na+–K+ ATPase activity in permeabilized tissue slices from the renal medulla (McKee et al. 1994), choroid plexus (Ellis et al. 2000) and ciliary processes (Ellis et al. 2001). However, the NO donor spermine NONOate, had no effect on Na+–K+ pump activity in the thick ascending limb from rat kidney. Activity was measured at high saturating or physiological rate-limiting levels of intracellular Na+ (Ortiz et al. 2001). Varela et al. (2004) reported that an inhibitory effect of NO donors on the pump activity in the thick ascending limb is time dependent. SNP induced Na+–K+ pump stimulation in rabbit aorta (Gupta et al. 1994) and human corpus cavernosum smooth muscle (Gupta et al. 1995). Pump stimulation in the aorta was thought to be secondary to Na+–H+ exchange-mediated influx of Na+ and an increase in its intracellular concentration. The mechanism for stimulation in corpus cavernosum smooth muscle was not determined.
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Tissue-specific differences in Na+–K+ pump regulation, differences in methodology used to study the Na+–K+ pump and variable delivery of NO and its bioactive derivatives by the donor compounds may have contributed to the conflicting results between studies. We have used the whole-cell patch clamp technique to examine the effect of NO on the sarcolemmal Na+–K+ pump in isolated rabbit ventricular myocytes. Provided wide-tipped patch pipettes are used, the technique allows control of the ligands of the Na+–K+ pump at intra- and extracellular sites and control of membrane voltage. It also allows the intracellular delivery of membrane-impermeable drugs and compounds dissolved in patch pipette solutions, including compounds of large molecular size. Electrogenic Na+–K+ pump current (Ip), arising from the 3Na+:2K+ exchange ratio, can be identified as the shift in holding current induced by blocking the pump with ouabain. We show that SNP induces an increase in Ip. To further characterize the mechanism, we examine the dependence of Na+–K+ pump stimulation on intracellular Na+ and K+. To avoid non-specific effects of pharmacological NO donors in these studies, we used the synthetic benzyl indazole derivative YC-1 (Russwurm & Koesling, 2002) to selectively activate soluble guanylyl cyclase (sGC), a downstream target molecule for NO.
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Methods
Cells
Single ventricular myocytes isolated from male New Zealand White rabbits were used. The rabbits, weighing 2.8–3.5 kg, were anaesthetized with 50 mg kg–1 ketamine and 20 mg kg–1 xylazine hydrochloride given intramuscularly. The heart was excised when deep anaesthesia was assured as indicated by the absence of a corneal reflex and response to deep pressure between the metatarsal bones. Details of techniques used to isolate ventricular myocytes have been previously described (Hool et al. 1995). The institutional review committee for animal research had approved experimental protocols. The myocytes were used on the day of isolation only. They were stored at room temperature in Krebs–Henseleit buffer solution until used for patch-clamp studies.
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Solutions
Myocytes were suspended in a tissue bath mounted on an inverted microscope for experimentation. While we established the whole-cell configuration, the bath was perfused with modified Tyrode solution, which contained (mM): NaCl, 140; KCl, 5.6; CaCl2, 2.16; MgCl2, 1; glucose, 10; NaH2PO4, 1; sodium glutamate, 9; and Hepes, 10. It was titrated to a pH of 7.40 ± 0.01 at 35°C with NaOH. For measurement of Ip, we switched to a superfusate that usually was identical except that it was nominally Ca2+ free and contained 0.2 mM CdCl2 and 2 mM BaCl2. In some experiments we modified this solution by replacing Na+-containing compounds with N-methyl-D-glucamine chloride (NMG-Cl; Hansen et al. 2000). The K+ concentration in the superfusate was 5.6 mM in all experiments unless otherwise indicated.
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For most experiments, wide-tipped patch pipettes (4–5 μm diameter) were filled with solutions containing (mM): Hepes, 5; MgATP, 2; EGTA, 5; potassium glutamate, 0–140; and sodium glutamate, 10. Osmotic balance was maintained with 150–10 mM tetramethylammonium chloride (TMA-Cl). We eliminated Na+-containing compounds in pipette solutions in some experiments (replaced with TMA-Cl) while we increased the Na+ concentration to 80 mM in others (osmotic balance was maintained by adjusting the concentration of TMA-Cl). The dependence of Ip on the intracellular Na+ concentration in the absence of intracellular K+ was examined using pipette solutions that included 0–80 mM sodium glutamate. The osmotic balance was maintained with TMA-Cl. All pipette solutions were titrated to a pH of 7.05 ± 0.01 at 35°C with 1 mM TMA-OH.
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Wide-tipped patch pipettes were used to optimize control of the concentration of the intracellular ligands for the Na+–K+ pump. The patch pipettes had initial resistances of 0.8–1.1 M when filling solutions included K+ at the concentration of 70 mM used in most experiments. Results were independent of series resistance in the whole-cell configuration only if levels were 2.8 M; the levels in accepted experiments ranged from 1.6 to 2.8 M. Since K+ has a higher conductivity than the other ions in pipette solutions (Hille, 1992) the series resistance was higher when we used K+-free pipette filling solutions. Control of the concentration of intracellular pump ligands, as indicated by independence of the measured pump currents of the series resistance, was achieved at levels 4.0 M.
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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 μM ouabain. Na+–K+ pump inhibition is saturated when rabbit ventricular myocytes are exposed to ouabain at this concentration (Drewnowska & Baumgarten, 1991; Hool et al. 1995). A stable current plateau 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 acquired with an electronic cursor at 5 s intervals. Recordings were obtained using the continuous single-electrode mode of Axoclamp 2A or 2B amplifiers supported by AxoTape and pCLAMP software (Axon Instruments, Foster City, CA, USA). We report Ip normalized for membrane capacitance and hence cell size.
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We used ouabain to inhibit the pump in this study because alternative, faster acting, cardiac steroids are less potent and would have to be used at a higher concentration to assure complete Na+–K+ pump inhibition. Any compound at a high concentration may have effects on non-pump membrane currents. We did not attempt to achieve wash-off of ouabain because, even when short-acting cardiac steroids are used, their effects on inotropy and intracellular Na+ in cardiac tissue remain for > 30 min (Lee et al. 1980; Boyett et al. 1986). In agreement with this, Na+–K+ pump currents of ventricular myocytes (Mogul et al. 1990) do not return to control levels within a time frame that assures drift-free recordings.
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Chemicals and reagents
TMA-Cl and NMG-Cl were purum grade and were obtained from Fluka Chemicals (Switzerland). All other chemicals used in Tyrode solutions were analytical grade and were obtained from BDH (Australia). Ouabain, SNP and bovine Cu,Zn superoxide dismutase were obtained from Sigma-Aldrich (St Louis, MO, USA) and YC-1, 1H-[1,2,4]Oxadiazole[4,3-a]quinoxalin-1-one (ODQ), KT-5823, okadaic acid, methyl okadaic acid, 8-pCPT-cGMP and recombinant bovine cGMP-activated protein kinase (PKG) were supplied by Calbiochem (La Jolla, CA, USA).
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Statistical analysis
Results are expressed as means ± S.E.M. Student's t test for unpaired data is used for statistical comparisons. We used Dunnett's test when the same control group was used for more than one comparison and a Mann–Whitney rank sum test for unequal sample sizes when data could not be assumed to be normally distributed. P < 0.05 is regarded as significant in all comparisons.
Results
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Effect of sodium nitroprusside on pump current
To examine whether the NO donor SNP has an effect on the Na+–K+ pump we measured Ip of control myocytes and of myocytes exposed to 50 μM SNP. Patch pipette solutions contained 10 mM Na+, a concentration near physiological intracellular levels in rabbit ventricular myocytes (Hool et al. 1995). The concentration of K+ in pipette solutions was 70 mM. The superfusate included 150 mM Na+. We maintained control myocytes as well as myocytes exposed to SNP in the whole-cell configuration for 10–12 min before exposing them to ouabain. Figure 1A shows traces of holding currents recorded from a control myocyte and a myocyte exposed to SNP. Ouabain induced a larger shift in holding current of the myocyte exposed to SNP than of the control myocyte.
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Patch pipette filling solutions and superfusates included 10 and 150 mM Na+ for experiments shown in A, 0 and 150 mM for B and 10 and 0 mM for experiments shown in C. The traces show holding currents before and after exposure to ouabain (Oua). The membrane capacitance, Cm (in pF) is indicated to facilitate comparisons.
Additional experiments were performed to examine whether SNP induces an increase in Ip due to an increase in transsarcolemmal Na+ influx. Such influx could cause secondary stimulation of the pump due to an increase in the intracellular Na+ concentration. In one set of experiments we patch clamped myocytes using pipette filling solutions that were nominally Na+ free. The superfusate contained Na+. Figure 1B shows traces of holding currents of a control myocyte and of a myocyte exposed to SNP. Ouabain-induced shifts in holding currents were barely detectable for either myocyte. Similar results were obtained for two additional myocytes exposed to SNP. If SNP enhances transsarcolemmal Na+ influx it does not cause an increase in the intracellular Na+ concentration that can be detected as an increase in Ip.
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To obtain independent support for the conclusion that the SNP-induced increase in Na+–K+ pump activity is not due to enhanced Na+ influx we used patch pipette solutions that contained 10 mM Na+ and a superfusate that was Na+ free. This eliminates any possible SNP-induced Na+ influx. Figure 1C shows holding currents of a control myocyte and of a myocyte exposed to SNP. Ouabain induced a larger shift in the holding current of the myocyte exposed to SNP than of the control myocyte. The mean levels of Ip in experiments performed using Na+-containing patch pipettes and superfusates that contained Na+ or were Na+ free are summarized in Fig. 2. Mean Ip recorded using control, SNP-free superfusates appeared higher in the presence than in the absence of extracellular Na+. We attribute this to an allosteric effect of extracellular Na+ which accelerates conversion of the enzyme from the low-affinity E2 state to the high-Na+-affinity E1 state and enhances the apparent affinity for Na+ at cytosolic Na+–K+ pump sites (Buhagiar et al. 2004). However, a statistically significant increase in Ip with exposure to SNP persisted in the absence of extracellular Na+.
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The Na+ concentration in patch pipette filling solutions was 10 mM in all experiments. Mean (± S.E.M.) Ip values recorded in a superfusate that included 150 mM Na+ (A) or was Na+ free (B) are shown. The extracellular Na+ concentration ([Na+]o) is indicated in the figure. The number of experiments is indicated in parentheses. *P < 0.05.
To examine the relationship between the concentration of SNP and Ip we exposed myocytes to SNP in concentrations ranging from 1 to 1000 μM. Patch pipettes and superfusates contained 10 mM Na+ and 70 mM K+. The superfusate contained 150 mM Na+. Results are shown in Fig. 3. SNP at concentrations of 1, 10, 50 and 100 μM induced a statistically significant increase in Ip. However, there was no significant stimulation relative to control myocytes when the concentration was increased further to 1000 μM, i.e. there was not a simple concentration-dependent effect of SNP. Since SNP at high concentrations has oxidant effects in vitro that can be prevented by superoxide dismutase (Jaworski et al. 2001) we included 200 μM cytosolic Cu,Zn superoxide dismutase (SOD) in pipette filling solutions and exposed myocytes 1000 μM SNP. Mean Ip has been included in Fig. 3. Superoxide dismutase restored SNP-induced stimulation of Ip.
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The Na+ concentration in patch pipette filling solutions was 10 mM. The superfusate contained 150 mM Na+ and 5.6 mM K+. Superoxide dismutase (SOD) was included in the patch pipette solution as indicated. Mean (± S.E.M.) Ip values at different concentrations of SNP are indicated. The numbers of experiments are indicated in parenthesis. Asterisk indicates a statistically significant SNP-induced increase in Ip.
Intracellular messengers linking NO to Na+–K+ pump stimulation
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Soluble guanylyl cyclase is the classical target molecule mediating cellular effects of NO. However, since some effects of NO are independent of sGC, we measured Ip while blocking sGC and its downstream messengers. Patch pipettes contained 10 mM Na+ and 70 mM K+. The superfusate contained 150 mM Na+ and we used SNP at a concentration of 50 μM. We included 10 μM of the NO-competitive antagonist ODQ in patch pipette solutions to block access of NO to sGC and so block stimulation of cGMP synthesis. Figure 4 shows that ODQ abolished the SNP-induced pump stimulation.
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Myocytes were superfused with control solutions or solutions containing SNP. Patch pipettes contained control solutions or solutions containing inhibitors of soluble guanylyl cyclase (ODQ), protein kinase G (KT-5823) or protein phosphatase (okadaic acid, OA). Numbers in parentheses indicate the number of myocytes studied with each set of conditions. * Significant difference between mean (± S.E.M.) Ip values of myocytes exposed or not exposed to SNP; # significant difference between Ip of myocytes exposed to SNP and patch clamped using control pipette filling solutions and solutions containing ODQ, KT-5823 or OA. Results of experiments using an SNP-free superfusate and ODQ, KT-5823 or OA in pipette solutions are also shown.
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The inhibition of the SNP-induced pump stimulation by ODQ implicates protein kinase G (PKG) in the downstream messenger pathways that mediate the stimulation. However, since cGMP can directly activate effector proteins independent of phosphorylation (Dimmeler et al. 1999), we examined the effect of inhibiting PKG by including 0.5 μM KT-5823 in patch pipette solutions. KT-5823 abolished the SNP-induced pump stimulation, implicating a role for PKG in the stimulation. If the pump stimulation is due to PKG directly phosphorylating Na+–K+ pump molecules, inhibition of phosphatase-mediated dephosphorylation might enhance the effect of SNP. To examine this we included 0.1 μM okadaic acid in patch pipette solutions. Figure 4 shows that phosphatase inhibition abolished, rather than enhanced, the SNP-induced pump stimulation.
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Figure 3 shows that there is not a simple concentration-dependent effect of the NO donor SNP on Ip. To examine the effect of activating messengers downstream from the NO-activated sGC we activated PKG by including a cGMP analogue in patch pipette solutions. We used the non-hydrolysable analogue 8-pCPT-cGMP to prevent its rapid intracellular breakdown. Figure 5 shows that 8-pCPT-cGMP induced a concentration-dependent increase in Ip. Since 8-pCPT-cGMP in high concentrations might activate protein kinase A (PKA), we examined the effect of adding 0.5 μM of H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride) to pipette solutions containing 1 mM 8-pCPT-cGMP. The mean Ip recorded in such experiments is included in Fig. 5. The 8-pCPT-cGMP-induced increase in Ip persisted after PKA blockade.
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Myocytes were exposed to control superfusates, and the compounds indicated in the figure were included in patch pipette solutions. Numbers in parentheses indicate the number of myocytes for each set of experimental conditions. Asterisk indicates a significant difference (P < 0.05) between Ip values of myocytes patch clamped using control patch pipette filling solutions and solutions containing 8-pCPT-cGMP, TEA. The protein kinase A inhibitor H-89 did not prevent the activation of Ip induced by the highest concentration of 8-pCPT-cGMP,TEA. # Significant difference (P < 0.05) between Ip values of myocytes patch clamped using filling solutions containing 20 μM 8-pCPT-cGMP, TEA with or without recombinant bovine protein kinase G (PKG).
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To avoid non-specific effects of high concentrations of 8-pCPT-cGMP we also examined the effect of including 400 U ml–1 recombinant bovine PKG in the patch pipette solution (White et al. 2000). To activate PKG we included 20 μM 8-pCPT-cGMP in the solution. Figure 5 shows that 20 μM 8-pCPT-cGMP in patch pipette solutions had no effect on Ip in the absence of recombinant PKG. However, the combination of 20 μM 8-pCPT-cGMP and recombinant PKG in pipette solutions induced a large increase in Ip.
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Activation of sGC with YC-1
Figure 5 shows that there is a clear concentration-dependent effect of an analogue of the product of sGC, cGMP, while Fig. 3 shows that the relationship between the NO donor SNP and Ip is much less predictable. In addition, activation of sGC by exposure to SNP causes a smaller increase in Ip than activation of messenger pathways downstream from sGC. This suggests that there are non-specific effects of SNP. To avoid this while still inducing activation via the classical target molecule for NO, sGC, we included 1 μM YC-1 in patch pipette solutions. The solutions included 10 mM Na+ and 70 mM K+. Superfusates included 5.6 mM K+ and they contained 150 mM Na+ or they were Na+ free. Figure 6A shows that ouabain induced a shift in holding current in the presence or absence of extracellular Na+. All experiments using the same protocols are summarized in Fig. 6B. YC-1 induced a large, statistically significant increase in Ip that was independent of the presence or absence of extracellular Na+.
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Inclusion of 1 μM YC-1 in pipette solutions is indicated in the figure. A shows shifts in holding current induced by ouabain (Oua) recorded using Na+ at a concentration ([Na+]pip) of 10 mM Na+ in patch pipette solutions. The Na+ concentration in superfusates ([Na+]o) was 150 mM (upper traces) or 0 mM (lower traces). B summarizes mean (± S.E.M.) Ip values recorded in Na+-containing and Na+-free superfusates. C shows ouabain-induced shifts in holding current recorded using 80 mM Na+ in patch pipette solutions, recorded in Na+-containing superfusates. D summarizes mean Ip values recorded using 80 mM Na+ in pipette solutions. Cm indicates membrane capacitance in pF. *P < 0.05.
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The Na+–K+ pump stimulation induced by YC-1 might be due to an increase in maximal turnover of the pump or due to an increase in its sensitivity to intracellular Na+. An increase in maximal turnover should be reflected by an increase in Ip recorded when intracellular pump sites are nearly saturated by a high concentration of Na+. We patch clamped myocytes using 80 mM Na+ and 70 mM K+ in pipette solutions. The superfusate included 150 and 5.6 mM K+. Figure 6C shows examples of ouabain-induced shifts in holding currents of a control myocyte and a myocyte exposed to YC-1. The ouabain-induced shifts in currents were much larger than those recorded using 10 mM Na+ in pipette solutions (Fig. 6A) and they were similar for a myocyte exposed or not exposed to YC-1. All experiments performed using 80 mM Na+ in the pipette solution are summarized in Fig. 6D. There was no significant difference between the mean Ip of control myocytes and that of myocytes exposed to YC-1, suggesting that YC-1 does not stimulate maximal pump turnover.
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To ascertain that YC-1 activates the same messenger pathways as those implicated for SNP-induced Na+–K+ pump stimulation we examined the effects of ODQ, KT-5823 or okadaic acid in patch pipette solutions. The pipette solutions included 10 mM Na+ and 70 mM K+. Figure 7 shows that ODQ, KT-5823 and okadaic acid abolished the YC-1-induced Na+–K+ pump stimulation, i.e. the pattern of inhibition by blockers of implicated messenger pathways was identical to that shown for SNP in Fig. 4.
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Patch pipettes contained control solutions or solutions that contained YC-1 and inhibitors of soluble guanylyl cyclase (ODQ), protein kinase G (KT-5823) or protein phosphatase (okadaic acid, OA) as indicated. The Na+ concentrations in pipette solutions and superfusates were 10 and 150 mM, respectively. Numbers in parentheses indicate the number of myocytes studied with each set of conditions. * Significant difference (*P < 0.05) between mean (± S.E.M.) Ip values of myocytes perfused or not perfused with pipette solutions containing YC-1; # significant difference (*P < 0.05) between mean Ip values of myocytes perfused with pipette solutions containing YC-1 alone and solutions containing both YC-1 and ODQ, KT-5823 or OA. Results of control experiments using ODQ, KT-5823 or OA alone are shown in Fig. 4.
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Dependence of Na+–K+ pump stimulation on intracellular Na+ and K+
The dependence of pump stimulation on a non-saturating concentration of intracellular Na+ suggests that YC-1 causes a change in the sensitivity of the pump to intracellular Na+. Since Na+ binds at two sites on the cytosolic side of the pump in competition with K+ and at a third site selective for Na+, we examined the dependence of pump stimulation induced by YC-1 on the intracellular K+ concentration. We voltage clamped myocytes using patch pipettes containing 10 mM Na+ and K+ at a concentration ([K]pip) ranging from 0 to 140 mM. Superfusates included 150 mM Na+ and 5.6 mM K+. Figure 8 shows a summary of Ip of control myocytes and of myocytes patch clamped using 1 μM YC-1 in pipette filling solutions. As expected, there was a [K]pip-dependent decrease in Ip. YC-1 induced a significant increase in Ip in the presence or absence of K+ in patch pipette filling solutions.
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Ip was measured in myocytes voltage clamped using control pipette filling solutions () or solutions containing YC-1 (). The pipette filling solutions contained Na+ at a concentration of 10 mM and K+ at a concentration ([K+]pip) ranging from 0 to 140 mM. The numbers of experiments for each set of conditions are indicated in parentheses. Mean (± S.E.M.) Ip values for myocytes perfused with pipette solutions containing YC-1 were significantly (*P < 0.05) higher than for myocytes perfused with control solutions at all levels of [K]pip.
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Since YC-1 induces Na+–K+ pump stimulation at a non-saturating concentration of intracellular Na+ only and since stimulation can occur in the nominal absence of intracellular K+, we examined whether YC-1 alters the sensitivity of the pump to intracellular Na+ in the absence of intracellular K+. We patch clamped myocytes using pipette solutions that were K+ free and included Na+ at concentrations ranging from 0 to 80 mM. To eliminate binding of K+ at extracellular sites as a rate-limiting step at high pipette Na+ concentration we used a K+ concentration of 15 mM in the superfusate in these experiments. The Na+ concentration in the superfusate was 150 mM. Figure 9A shows the ouabain-induced shift in holding current of a control myocyte patch clamped using a pipette solution that contained 80 mM Na+. The ouabain-induced shift in current appeared much larger than the shift recorded using K+-containing pipette filling solutions with the same Na+ concentration (Fig. 6). Figure 9B summarizes experiments performed using different Na+ concentrations in pipette solutions.
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Ip was measured in myocytes voltage clamped using control pipette filling solutions () or solutions containing YC-1 (). The pipette filling solutions contained Na+ at concentrations ranging from 0 to 80 mM. They were nominally K+ free. The numbers of experiments for each set of conditions are indicated in parentheses. Mean (± S.E.M.) Ip values for myocytes perfused with pipette solutions containing YC-1 were significantly higher than for myocytes perfused with control solutions when the pipette Na+ concentration was at rate-limiting levels.
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For the purpose of assigning a value to the intracellular Na+ concentration that induces half-maximal pump activation we fitted the Hill equation to the data. We derived values of 24.0 mM for control myocytes and 14.1 mM for myocytes perfused with pipette solutions containing YC-1. Since Ip is not a direct measure of Na+ binding, the fit is without mechanistic implications and is therefore not shown. Mean Ip is similar for control myocytes and for myocytes exposed to YC-1 when [Na+]pip is 0 or 80 mM. However, YC-1 seemed to induce an increase in Ip when [Na]pip was at the rate-limiting concentrations of 5, 10 or 20 mM. A Mann–Whitney test indicated that YC-1 induces a significant increase in Ip when data obtained at these three Na+ concentrations are considered in combination.
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Discussion
Previous studies have reported inhibition (McKee et al. 1994; Liang & Knox, 2000; Ellis et al. 2000, 2001; Varela et al. 2004), the absence of any effect (Ortiz et al. 2001) and stimulation (Gupta et al. 1994, 1995) of the membrane Na+–K+ pump when various tissues were exposed to NO donor compounds. Stimulation was secondary to enhanced Na+ influx rather than due to a direct effect on the Na+–K+ pump in vascular smooth muscle (Gupta et al. 1994). In our study, SNP stimulated intrinsic pump activity, independent of enhanced transsarcolemmal Na+ influx. The discrepancy between our results and previous studies may be organ dependent (Therien & Blostein, 2000). However, use of pharmacological NO donor compounds may also have contributed to inconsistent results. The pathways leading to formation of NO from the compounds and derivative side reactions are exquisitely sensitive to the local oxygen tension, and the amount of bio-active intermediate and end products that form during metabolism and degradation may exceed the amount of NO (Ignarro et al. 2002). The net response may arise from a balance between, at times opposing, effects of compounds generated. NO and its derivatives can directly inhibit isolated Na+–K+ ATPase, in part via effects on membrane fluidity (Muriel & Sandoval, 2000), and the derivative peroxynitrite inhibits Na+–K+ ATPase activity in renal cells exposed to NO donors. The inhibition is abolished when the peroxynitrite precursor, superoxide, is eliminated with SOD (Varela et al. 2004). In our study, SNP induced Na+–K+ pump stimulation at all but the highest concentration used, and stimulation at the high SNP concentration was restored by SOD.
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Activation and inhibition of messengers implicated in NO-mediated pathways
While SNP consistently induced Na+–K+ pump stimulation, there was not a simple concentration -dependent increase in Ip and there was considerable variability of Ip with exposure to SNP (Fig. 3). To avoid ambiguities of pharmacological NO donors we used YC-1 in patch pipette solutions to selectively activate sGC, a downstream target molecule for NO. Since a scaffold protein directs sGC to a membrane association in close proximity to nitric oxide synthase (Russwurm & Koesling, 2002), YC-1 activates sGC at sites of endogenous NO synthesis (Russwurm et al. 2002). Such activation may better reflect physiological signalling than exposure of cells to SNP because effects of YC-1 should be largely restricted to the microdomains of synthesis and breakdown of NO. YC-1 reproduced the SNP-induced stimulation of Ip. This, in combination with the effect of selective inhibition of sGC with ODQ (Russwurm et al. 2002) in pipette solutions (see Figs 4 and 7), strongly implicates sGC in Na+–K+ pump stimulation. The effect of cGMP synthetized on activation of sGC is usually mediated by PKG. However, it may also be due to a cGMP-mediated increase in cAMP and activation of PKA, it may be due to activation of PKA by cGMP in high concentrations or due to a direct effect of cGMP on effector proteins (White, 1999). The persistence of pump stimulation when H-89 is included in patch pipette solutions rules out involvement of PKA.
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Since KT-5823 has imperfect selectivity for PKG we included recombinant PKG in the pipette solution and activated it with 20 μM 8-pCPT-cGMP. PKG reproduced effects of SNP and YC-1. Of note, 20 μM 8-pCPT-cGMP in the absence of PKG in pipette solutions had no effect on Ip. While a difference in sensitivity to activation of endogenous and recombinant PKG may account for this, PKG endogenous to the myocyte may have been exposed to 8-pCPT-cGMP in a concentration lower than 20 μM because 8-pCPT-cGMP is highly membrane permeable. Rate-limiting diffusion through the patch pipette tip can result in an intracellular concentration in voltage clamped cardiac myocytes of membrane-permeable compounds that is lower than the concentration in pipette solutions (Mathias et al. 1990).
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Taken together, the data indicate that PKG mediates Na+–K+ pump stimulation induced by SNP and YC-1. There are few established molecular targets for PKG (Moreno et al. 2001). Phosphorylation of the pump molecule or of the closely associated FXYD1 protein implicated in pump regulation (Cornelius et al. 2001) is unlikely because FXYD1 is not reported to be phosphorylated by PKG and the pump itself is not easily phosphorylated by PKG (Fotis et al. 1999). In addition, if Na+–K+ pump stimulation were mediated by phosphorylation of the pump molecule or the FXYD1 protein, inhibition of protein phosphatases with okadaic acid should enhance rather than abolish stimulation. Protein phosphatases are often implicated in PKG-mediated signalling (White, 1999; Moreno et al. 2001), and phosphatase-mediated dephosphorylation of FXYD1 has been reported (Neumann et al. 1999). PKG can mediate phosphorylation of a regulatory protein for protein phosphatase 1 in smooth muscle myocytes. However, the phosphorylation inhibits rather than activates phosphatase activity (Tokui et al. 1996) and cannot account for our results. The available information cannot identify the okadaic acid-sensitive step in our study.
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Effect of NO on functional properties of the Na+–K+ pump
Interpretation of the YC-1- and SNP-induced change in the sensitivity of the Na+–K+ pump to intracellular Na+ depends on the assumption that Na+ concentrations in the cytosol and pipette solutions are similar. Concentration gradients could exist between the pipette solution and the intracellular bulk phase (Mathias et al. 1990) or between the bulk phase and a diffusion-restricted subsarcolemmal space (Despa & Bers, 2003; Silverman et al. 2003). Transsarcolemmal influx should maintain intracellular Na+ at a level high enough to support an easily detectable Na+–K+ pump current when Na+-free pipette solutions are used, unless the intracellular concentration largely reflects the concentration in the patch pipettes (Mathias et al. 1990; Silverman et al. 2003). Nakao & Gadsby (1989) used wide-tipped patch pipettes to optimize control of intracellular Na+. When Ip was recorded in Na+-containing superfusates with Na+-free patch pipettes it was 3.5% of the Ip recorded with pipettes containing 50 mM Na+. This fraction was reduced to 0.8% when superfusates were Na+ free to eliminate transsarcolemmal Na+ influx. The Ip we recorded with Na+-free patch pipettes was 0.3% of the Ip expected when pipettes contained 50 mM Na+ (extrapolated from Fig. 9B). This low level was achieved in Na+-containing superfusates, and suggests that a diffusion-restricted subsarcolemmal space does not give rise to substantial Na+ concentration gradients. In support of this we have reported that a ouabain-sensitive current is easily detectable when the Na+ concentration in pipette solutions is as low as 0.1 mM (Hansen et al. 2002). We conclude that the Na+ concentration at cytosolic pump sites approximates that in pipette solutions in our experiments.
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Na+–K+ pump regulation can depend on intracellular K+ (Buhagiar et al. 1999, 2004), which is consistent with an effect on the backward E1 + 2K+ E2(K+)2 reaction (Buhagiar et al. 2004) where E1 is the unphosphorylated conformation of the enzyme with a high affinity for cytoplasmic NA+ ions and E2 is the alternative unphosphorylated conformation which is stabilised by cytoplasmic K+ ions. However, such a mechanism cannot account for the pump stimulation in this study because stimulation could be demonstrated in the absence of intracellular K+ (Figs 8 and 9). A change in the apparent Na+ affinity might account for pump stimulation. A change in the affinity could be due to a change in intrinsic Na+ binding to the E1 conformation of pump molecules or due to an apparent change in affinity from a change in the rate of reactions determining availability of the E1 conformation (Apell & Karlish, 2001). Mechanistic details cannot be determined from this study.
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The relative increase in Ip induced by YC-1 was larger at 5.6 than at 15 mM extracellular K+ (Figs 8 and 9). The higher concentration must stimulate the pump. Any additional stimulation may then be less evident if subsequent steps, independent of K+, become rate limiting. Effects of binding of K+ to the E2 conformation should be small because binding is nearly saturated at 5.6 mM. Nor can an increase in the rate of the K+-stimulated dephosphorylation reaction account for stimulation because the reaction is not rate limiting (Kane et al. 1998; Kong & Clarke, 2004). However, allosteric binding of K+ stimulates rate-limiting (Kong & Clarke, 2004) deocclusion of K+ and the E2 E1 conformational transition (Forbush, 1987). We considered the stimulation this may mediate. We modified a mathematical model of the Na+–K+ pump cycle (Kong & Clarke, 2004) to take into account allosteric effects of extracellular K+ (see Appendix for details). Simulations indicated that one might expect a 28% increase in pump rate when extracellular K+ is increased from 5.6 to 15 mM. A quantitative interpretation of the simulations is not justified because most parameters in the model were derived from presteady-state kinetic measurements on purified Na+–K+ ATPase from kidney while our experimental data were obtained in intact cardiac cells. However, the simulations are consistent with the qualitative conclusion that the difference in extracellular K+ concentration may affect the relative size of pump stimulation induced by YC-1. This highlights how details of experimental conditions may affect analysis of Na+–K+ pump regulation.
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Appendix
To examine allosteric effects of K+ we simulated steady-state Na+–K+ pump activity under the experimental conditions used here (extracellular Na+ concentration 150 or 140 mM, extracellular K+ concentration 5.6 or 15 mM, intracellular Na+ concentration 10 mM, intracellular K+ concentration 0 mM and intracellular ATP concentration 2 mM). We modified eqn (5) of a previously published model (Kong & Clarke, 2004) by changing the dissociation constants for the activation of enzyme dephosphorylation by the two transported K+ ions, KoK1 and KoK2. They were estimated at 2.8 and 4.3 mM (Gray et al. 1997). In addition, the equation for the observed rate constant for the E2 E1 transition, kobs1, was modified to include an allosteric effect of external K+. The new equation was:
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We refer to Table 1 of Kong & Clarke (2004) for the values and meanings of the parameters already included in the model.
We made the assumption that the binding of Na+ and K+ to the allosteric sites is non-competitive and that the effects of the two ions are simply additive. KoK represents the dissociation constant of the external allosteric K+ ions from the E2 conformation of the enzyme, irrespective of whether Na+ and/or ATP are simultaneously bound to allosteric sites. It has been set to the same value as KoN, the dissociation constant for allosteric external Na+ ions, i.e. 31 mM, in accord with the results of Forbush (1987) that the affinity of the enzyme for various allosteric ions is not substantially different. ke1, kf1, kg1 and kh1 represent rate constants for the E2 E1 transition (with up to two K+ ions initially bound to E2 at transport sites) when the allosteric sites are saturated by K+ alone (ke1), by K+ and Na+ (kf1), by K+ and ATP (kg1) and by K+, Na+ and ATP (kh1). In the absence of ATP, Na+ and K+ have similar effects in accelerating the E2 E1 transition (Forbush, 1987). Thus, the values of ke1 and kf1 have been chosen to be equal to kb1, the rate constant for the transition at which only allosteric external Na+ ions are bound, i.e. 0.8 s–1.
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External K+ causes approximately 16% greater acceleration of the E2 E1 transition than external Na+ in the presence of ATP (Forbush, 1987). Accordingly, since kd1, the rate constant for the transition when the enzyme is saturated by allosteric Na+ and ATP, has been determined to have a value of approximately 70 s–1 (Lüpfert et al. 2001), the value of kg1 was taken as 81 s–1. When the allosteric sites are saturated by ATP, Na+ and K+, no experimental data are presently available on the value of the rate constant, i.e. kh1. For the purpose of the simulation we assumed the effects of Na+ and K+ are additive, i.e. kh1 = kd1 + kg1 = 151 s–1.
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The simulations were carried out using the commercially available program Berkeley Madonna 7.0 via the variable step size Rosenbrock integration method for stiff systems of differential equations. The simulations yield the time course of the concentration of each enzyme intermediate, as well as the concentration of inorganic phosphate, from which the turnover number of the enzyme can be calculated. The simulations show that the turnover number of the enzyme is predicted to be 28 s–1 in the presence of 5.6 mM external K+ and 36 s–1 in the presence of 15 mM external K+.
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