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Am J Physiol Cell Physiol 277: C461-C468, 1999;
0363-6143/99 $5.00
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Vol. 277, Issue 3, C461-C468, September 1999

Angiotensin regulates the selectivity of the Na+-K+ pump for intracellular Na+

Kerrie A. Buhagiar1,2, Peter S. Hansen1,2, David F. Gray1,2, Anastasia S. Mihailidou1, and Helge H. Rasmussen1,2

1 Department of Cardiology, Royal North Shore Hospital, St. Leonards 2065; and 2 University of Sydney, Sydney, New South Wales 2006, Australia


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Treatment of rabbits with angiotensin-converting enzyme (ACE) inhibitors increases the apparent affinity of the Na+-K+ pump for Na+. To explore the mechanism, we voltage clamped myocytes from control rabbits and rabbits treated with captopril with patch pipettes containing 10 mM Na+. When pipette solutions were K+ free, pump current (Ip) for myocytes from captopril-treated rabbits was nearly identical to that for myocytes from controls. However, treatment caused a significant increase in Ip measured with pipettes containing K+. A similar difference was observed when myocytes from rabbits treated with the ANG II receptor antagonist losartan and myocytes from controls were compared. Treatment-induced differences in Ip were eliminated by in vitro exposure to ANG II or phorbol 12-myristate 13-acetate or inclusion of the protein kinase C fragment composed of amino acids 530-558 in pipette solutions. Treatment with captopril had no effect on the voltage dependence of Ip. We conclude that ANG II regulates the pump's selectivity for intracellular Na+ at sites near the cytoplasmic surface. Protein kinase C is implicated in the messenger cascade.

cardiac myocytes; sodium; protein kinase C


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CYTOSOLIC LEVELS of Na+ in ventricular papillary muscle isolated from rabbits that have been treated with angiotensin-converting enzyme-inhibiting drugs (ACE inhibitors) are lower than levels in papillary muscle from untreated control rabbits (18). This is due to a treatment-induced enhancement of the activity of the sarcolemmal Na+-K+ pump. The enhanced pump activity can be demonstrated as an increase in electrogenic Na+-K+ pump current (Ip) in ventricular myocytes voltage clamped with patch pipettes containing Na+ in a concentration ([Na+]pip) that is near physiological intracellular levels. In contrast, treatment has no effect on Ip when [Na+]pip is high, indicating that there is no effect on maximal pump rate (18). These findings are likely to be important both for our understanding of the mechanism of action of ACE inhibitors and, by inference, for our understanding of the role of the renin-angiotensin system in cardiovascular homeostasis (17). In addition, the findings are of interest because they indicate that the Na+-K+ pump's apparent affinity for intracellular Na+ can be modulated in vivo.

Intracellular binding of three Na+ to the pump in each cycle is thought to occur at two negatively charged sites near the cytoplasmic surface and at an uncharged site located inside the membrane dielectric. Na+ competes with K+ for binding at the negatively charged sites, and because of the location of the sites near the cytoplasmic surface, this binding is voltage independent. In contrast, interaction with the uncharged site is highly selective for Na+ and dependent on membrane voltage (Vm) (16, 25, 26, 28).

The increase in the overall apparent affinity of the pump for intracellular Na+ induced by treatment with ACE inhibitors could be due to an increase in the affinity for Na+ relative to the affinity for K+ at the pump sites near the cytoplasmic surface. One would expect that such a change would be dependent on the presence of intracellular K+ and independent of Vm. The overall increase in apparent Na+ affinity could also be due to an increase in the intrinsic affinity for Na+ at the site inside the membrane dielectric. Binding of Na+ at this site is expected to be voltage dependent. A change in the affinity of the site for binding of Na+ is therefore expected to cause a change in the voltage dependence of steady-state pump activity if the binding can be a rate-limiting step in the pump cycle. It follows that an analysis of the effect of treatment with ACE inhibitors on the dependence of pump activity on intracellular K+ and Vm may provide insight into the mechanism for the treatment-induced increase in the apparent affinity of the pump for Na+. Such an analysis was performed in this study.


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

Treatment protocols. A group of male New Zealand White rabbits weighing 2.5-3.0 kg were given captopril in their drinking water for 8 days as described previously (18). Another group given captopril-free water served as controls. The effects of the treatment protocol on systemic blood pressure and serum renin activity have been described previously (18). A third group of rabbits were given losartan at 25 mg/kg body wt once daily by gavage. After completion of treatment protocols, rabbits were anesthetized with ketamine (50 mg/kg) and xylazine hydrochloride (20 mg/kg) given intramuscularly, and the heart was excised when deep anesthesia was achieved.

Measurement of Ip. Single ventricular myocytes were isolated as described previously (18). The isolation procedure typically lasted ~2 h. Unless indicated otherwise, cells were stored at room temperature in Krebs-Henseleit buffer (KHB) solution (17) until Ip was measured. Cells were used for experimentation on the day of isolation only, and Ip was typically measured within 2.5-8 h after cell isolation. In some experiments myocytes were preincubated for a period of 45 min at 35°C in KHB solution containing 10 nM ANG II or for 60 min in KHB solution containing 160 nM phorbol 12-myristate 13-acetate (PMA) before Ip was measured. For incubation with ANG II we used polypropylene test tubes to prevent the loss of ANG II by adhesion to glass. ANG II was dissolved in water, and PMA was dissolved in DMSO. The final concentration of DMSO was 0.01%. DMSO in this concentration has no effect on Ip (14).

Myocytes were suspended in a tissue bath mounted on an inverted microscope for measurement of Ip. The bath was perfused with modified Tyrode solution warmed to 35 ± 0.5°C. The solution contained (in mM) 140 NaCl, 5.6 KCl, 2.16 CaCl2, 1 MgCl2, 0.44 NaH2PO4, 10 glucose, and 10 HEPES. It was titrated with NaOH to a pH of 7.40 ± 0.01 at 35°C. This solution was used in all experiments until the whole cell configuration had been established and membrane capacitance had been measured. In most experiments we then switched to a superfusate that was identical except that it was nominally Ca2+ free and contained 0.2 mM CdCl2 and 2 mM BaCl2. In some experiments this superfusate was replaced with a Na+-free solution that contained (in mM) 14.56 KCl, 140 N-methyl-D-glucamine chloride, 0.44 KH2PO4, 10 HEPES, 1 MgCl2, 10 glucose, 2 BaCl2, and 0.2 CdCl2. The solution was titrated with HCl to a pH of 7.4 ± 0.01 at 35°C. In experiments designed to examine the effect on the apparent affinity for extracellular K+, the superfusate was similar to the Na+-containing, Ca2+-free superfusate described above except the K+ concentration was varied between 0 and 15 mM. The osmolality of these superfusates was maintained constant with tetramethylammonium (TMA) chloride.

Myocytes were patch clamped with wide-tipped (4-5 µm) pipettes made from borosilicate glass as described previously (31). Membrane currents were recorded by using the continuous single-electrode voltage-clamp mode of an Axoclamp-2A amplifier and AxoTape or pCLAMP software (Axon Instruments, Foster City, CA). Voltage-clamp protocols were generated with pCLAMP. Details of the experimental protocols used to measure membrane capacitance and Ip and details of the electronic recording system have been described previously (31).

For measurement of Ip at a fixed Vm of -40 mV we used pipette filling solutions that contained (in mM) 9 sodium glutamate, 1 NaH2PO4, 5 HEPES, 2 MgATP, 5 EGTA, and 0-140 KCl. The osmolality was maintained constant by the addition of 10-150 mM TMA chloride. The solutions were titrated with TMA hydroxide to a pH of 7.05 ± 0.01 at 35°C. In some experiments we included the protein kinase C (PKC) fragment composed of amino acids 530-558 (PKCF) in these solutions to activate PKC. PKCF was dissolved in a 0.05 M acetic acid stock solution for storage at -20°C and added to pipette solutions on the day of experimentation to a final concentration of 4 nM. Addition of the quantity of stock solution required to achieve this concentration did not alter the pH of pipette solutions.

For determination of the voltage dependence of Ip we used a filling solution that contained (in mM) 10 sodium glutamate, 1 KH2PO4, 5 HEPES, 5 EGTA, 2 MgATP, 60 TMA chloride, 20 tetraethylammonium chloride, 70 CsOH, and 50 aspartic acid. The solution was titrated with HCl to a pH of 7.05 ± 0.01 at 35°C. In experiments designed to determine the apparent affinity for extracellular K+, filling solutions contained (in mM) 80 sodium glutamate, 70 potassium glutamate, 1 KH2PO4, 5 HEPES, 5 EGTA, 2 MgATP, and 10 TMA chloride. The solution was titrated with KOH to a pH of 7.05 ± 0.01 at 35°C. Patch pipettes filled with the solutions used in this study had resistances of 0.8-1.1 MOmega .

Ip was usually identified as the shift in holding current induced by superfusion of 100 µM ouabain. We exposed myocytes to ouabain with the same latency of 10-12 min after the whole cell configuration had been established in all experiments to eliminate variability, which would arise if rundown of Ip were to occur. Ip was identified as the shift in holding current induced by switching from a K+-free to a K+-containing superfusate in one series of experiments. The shifts induced by ouabain or K+ were measured by sampling stable currents with an electronic cursor every 5-10 s five times before and five times after the onset of exposure, and the mean values of the samples were used to determine the effect of ouabain or K+. Unless specified otherwise, currents were normalized for membrane capacitance. When the voltage dependence of Ip was determined, we voltage clamped myocytes at -40 mV and applied 320-ms voltage steps to test Vm levels in 20-mV increments from -100 to +60 mV. Details of the experimental protocol used to determine the voltage dependence of Ip have been described previously (15).

Reagents and chemicals. TMA chloride was "purum" grade and purchased from Fluka. All other chemicals were analytical grade and purchased from BDH. ANG II, ouabain, and PKCF were purchased from Sigma, and PMA was purchased from Calbiochem. Captopril was donated by Bristol-Myers Squibb Pharmaceuticals, and losartan was donated by Merck.

Statistical analysis. Results are expressed as means ± SE. One-way ANOVA was used for statistical comparisons. Dunnett's test was used when the same control group was used in more than one comparison. An empirical equation describing the pipette K+ concentration ([K+]pip)-Ip relationship was fitted with nonlinear regression, and parameters were compared by Student's t-test for unpaired data. Ip-Vm relationships were compared by both linear regression and two-way ANOVA. P < 0.05 is regarded as significant in all comparisons.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Ip of right and left ventricular myocytes. We have previously found that treatment of rabbits with captopril for 8 days induces an increase in the Ip of myocytes isolated from the right ventricle (17, 18). In initial experiments in the present study, we wished to see if this observation can be extended to myocytes from the left ventricle. We isolated myocytes separately from the right and left ventricles of rabbits treated with captopril and from those of untreated controls. They were voltage clamped at -40 mV with pipettes containing K+ at a [K+]pip of 70 mM. The mean Ip values for right and left ventricular myocytes isolated from rabbits in the two groups are summarized in Fig. 1. Treatment with captopril caused a significant increase in Ip in both right and left ventricular myocytes. Ip and the effect of treatment were similar for right and left ventricular myocytes. We therefore used either right or left ventricular myocytes in all subsequent experiments in this study.


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  		Fig. 1.   Pump current (Ip) in left ventricular (LV) and right ventricular (RV) myocytes. Myocytes were isolated separately from LVs and RVs of control rabbits and rabbits treated with captopril. Pipette solutions contained 10 mM Na+ and 70 mM K+. Numbers of myocytes used are in parentheses. Treatment with captopril induced a significant increase in Ip values for myocytes from both LVs and RVs.

Dependence of Ip on [K+]pip. To determine if the effect of treatment with captopril on Ip is dependent on intracellular K+, we measured Ip with patch pipettes containing either 0, 35, 70, or 140 mM K+. Experiments were performed on 28 myocytes from 6 control rabbits and 41 myocytes from 11 rabbits treated with captopril. We used different [K+]pip values in experiments on myocytes from the same rabbit to reduce effects that might arise from interrabbit variability. Mean Ip values are shown in Fig. 2. Mean Ip values for myocytes from control rabbits and rabbits treated with captopril were similar when [K+]pip was 0 mM. In contrast, the mean Ip was significantly greater in myocytes from rabbits treated with captopril than in myocytes from controls when [K+]pip was 35, 70, or 140 mM.


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  		Fig. 2.   Effect of pipette K+ concentration ([K+]pip) on Ip. Ip was measured in myocytes from control rabbits, from rabbits treated with captopril or losartan, and from rabbits treated with captopril and subsequently exposed to ANG II in vitro. Median number of myocytes for each of the data points shown was 7 (range 5-15). Equation 1 was fitted to [K+]pip-Ip relationship for myocytes from control rabbits and from rabbits treated with captopril.

It appears from Fig. 2 that the inhibitory effect of [K+]pip is greater in myocytes from control rabbits than in myocytes from rabbits treated with captopril. To compare the [K+]pip values that cause half-maximal inhibition (K1/2 values), we fitted the [K+]pip-Ip relationships for myocytes from controls and from captopril-treated rabbits to an equation of the form
<IT>I</IT><SUB>p</SUB> = <IT>I</IT><SUP>max</SUP><SUB>p</SUB>&cjs0823;  [1 + ([K<SUP>+</SUP>]<SUB>pip</SUB>&cjs0823;  K<SUB>1&cjs0823;  2</SUB>)<SUP><IT>n</IT></SUP>] (1)
where Imaxp is the Ip measured at a [K+]pip of 0 mM. This equation is empirical and not intended to have mechanistic implications. The derived values for K1/2 were 55 ± 5 and 84 ± 8 mM for myocytes from control rabbits and myocytes from captopril-treated rabbits, respectively. The difference was statistically significant. Values for n were 1.7 ± 0.3 for myocytes from control and captopril-treated rabbits.

[K+]pip and effect of ANG II. Because treatment with captopril inhibits the synthesis of both ANG II and kinins (33), we examined if treatment of rabbits with the specific ANG II receptor antagonist losartan had an effect similar to that of treatment with captopril. We treated five rabbits with losartan. Because the method of administration differed from the protocol we used previously (17), we measured blood pressure by direct cannulation of an ear artery in anesthetized rabbits to ascertain the treatment effect. Satisfactory recordings were obtained for four rabbits. The mean systolic blood pressures measured before treatment was started and immediately before sacrifice were 97.5 ± 4.5 and 83.8 ± 1.9, respectively. The difference was statistically significant. Administration of placebo capsules not containing losartan had no effect on Ip (data not shown). The Ip values measured at [K+]pip values of 0, 35, 70, and 140 mM have been included in Fig. 2. The mean Ip of myocytes from rabbits treated with losartan and the mean Ip of myocytes from control rabbits were similar when [K+]pip was 0 mM. ANOVA indicated that mean Ip was significantly greater in myocytes from rabbits treated with losartan than in myocytes from controls when [K+]pip was 35, 70, or 140 mM. It should be noted that Ip values for myocytes from losartan-treated rabbits were similar to Ip values for myocytes from rabbits treated with captopril.

The similarity in the effect of treatment with captopril and ANG II receptor blockade on the [K+]pip-Ip relationship suggests that an effect of captopril on ANG II metabolism rather than on kinin metabolism induces the increase in Ip. To obtain independent support for this, we incubated myocytes from captopril-treated rabbits with 10 nM ANG II at 35°C as described previously (17). During the 45-min incubation period myocytes settled on the bottoms of the test tubes. The supernatant was then aspirated, and the cells were resuspended in ANG II-free solution and stored at room temperature until used for patch-clamp studies. The superfusates used for measurement of Ip were also ANG II free.

We measured Ip values for 26 myocytes isolated from rabbits treated with captopril and exposed to ANG II in vitro. The Ip values measured at [K+]pip values of 0, 35, 70, and 140 mM have been included in Fig. 2. The mean Ip for myocytes from the captopril-treated rabbits was significantly lower for myocytes exposed than for those not exposed to ANG II in vitro when [K+]pip was 35, 70, or 140 mM. In contrast, the mean Ip values for such myocytes were similar when [K+]pip was 0 mM. Figure 2 indicates that ANG II induced a reduction in Ip for myocytes from the captopril-treated rabbits to a level similar to that measured in myocytes that were isolated from the untreated controls and not subsequently exposed to ANG II.

Effect of PKC activation on Ip. Exposure of myocytes isolated from captopril-treated rabbits to the PKC activator PMA has an effect similar to the effect of ANG II (17). To examine if the effect of PMA is dependent on [K+]pip, we incubated myocytes from captopril-treated rabbits with 160 nM PMA for 60 min at 35°C as described previously (17). After the incubation we aspirated the supernatant and resuspended the cells in PMA-free solution at room temperature until they were used for patch-clamp experiments. The superfusates used for these experiments were also PMA free.

We measured Ip at a [K+]pip of 0 mM in nine myocytes that had been isolated from captopril-treated rabbits and subsequently exposed to PMA. The mean Ip was 0.88 ± 0.08 pA/pF. This was similar to the mean Ip for myocytes isolated from captopril-treated rabbits and not subsequently exposed to PMA (Fig. 2). Thus, as was the case for exposure to ANG II, exposure to PMA had no effect on Ip when [K+]pip was 0 mM. We also measured mean Ip at a [K+]pip of 70 mM. The mean Ip for seven myocytes isolated from captopril-treated rabbits and subsequently exposed to PMA was significantly lower than the mean Ip for similar myocytes not exposed to PMA (Fig. 3).


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  		Fig. 3.   Effect of PKC activation and ANG II on Ip. Myocytes were isolated from rabbits treated with captopril (capt) or losartan (los), and protein kinase C (PKC) was activated by incubation with phorbol 12-myristate 13-acetate (PMA) or by inclusion of PKC fragment composed of amino acids 530-538 (PKCF) in pipette solutions. A [K+]pip of 70 mM was used in all experiments. Number of myocytes in each group is in parentheses. * Significantly different vs. captopril treatment alone; ** significantly different vs. losartan treatment alone. Data in 1st, 2nd, and 5th bars from left, also shown in Fig. 2, have been included here to facilitate comparison.

PMA, although it is a rapid and potent activator of PKC, can also bind to receptors other than the PKC family of enzymes (32). We therefore performed a series of experiments in which myocytes from losartan-treated rabbits were dialyzed with pipette filling solution containing 4 nM PKCF to selectively activate PKC (19, 30). The mean Ip for seven myocytes measured at a [K+]pip of 0 mM was 0.96 ± 0.07 pA/pF. This is similar to the mean Ip shown in Fig. 2 for myocytes isolated from losartan-treated rabbits and not subsequently exposed to PKCF. We also examined the effect of PKCF at a [K+]pip of 70 mM. As shown in Fig. 3, the mean Ip was significantly lower than the mean Ip for myocytes isolated from losartan-treated rabbits and not exposed to PKCF. This [K+]pip dependence of the effect of PKCF on Ip is similar to the [K+]pip dependence we observed when we exposed myocytes isolated from rabbits treated with captopril to ANG II and to PMA.

Exposure to PMA and PKCF had an effect on Ip similar to the effect of ANG II. This suggests that the effect of ANG II on Ip is mediated via PKC. We performed additional experiments to obtain support for this. Myocytes were isolated from rabbits treated with captopril and then exposed to both ANG II and PKCF. We used a [K+]pip of 70 mM in these experiments. The mean Ip, shown in Fig. 3, was similar to the mean Ip for myocytes exposed to ANG II only. The absence of an additive effect of ANG II and PKCF is consistent with the involvement of the two peptides in the same messenger pathway.

Effect of captopril treatment on voltage dependence of Ip. The [K+]pip dependence of the effect of treatment with captopril suggests that the treatment alters the interaction of Na+ with pump sites near the cytoplasmic surface because it is at these sites that K+ competes with Na+ for binding. Any change in pump function at these sites should be independent of Vm. To examine the effect of captopril treatment on the pump's voltage dependence we determined the Ip-Vm relationships for myocytes from rabbits treated with captopril and for myocytes from controls.

Myocytes were patch clamped with pipettes containing a filling solution designed to eliminate time-dependent currents through ion channels (see MATERIALS AND METHODS for details). The voltage-clamp protocol and details of data analysis have been reported previously (15). To facilitate a comparison of the voltage dependence of the pump for myocytes from captopril-treated rabbits with that for myocytes from controls, we normalized Ip for each myocyte to its Ip recorded at 0 mV. Summaries of the normalized Ip values for six myocytes from captopril-treated rabbits and seven myocytes from controls are shown in Fig. 4A. The relationships were nearly linear and had a positive slope over the voltage range examined. There was no significant difference between the slopes of linear regression models fitted to the Ip-Vm relationships or between curves compared by using a two-way ANOVA.



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  		Fig. 4.   A: voltage (Vm) dependence of Na+-K+ pump current. Mean Ip values for myocytes isolated from control rabbits (open circle ) and rabbits treated with captopril () are shown. The Ip-Vm relationships were normalized to the Ip values recorded at 0 mV (Ip%) to facilitate comparison of their slopes. B: Vm dependence of Ip recorded in high-K+, Na+-free superfusates.

Release of Na+ and binding of K+ at extracellular sites are thought to be the most important steps generating the voltage dependence of the pump (24, 27), whereas binding of Na+ at intracellular sites only exhibits a modest voltage dependence (7, 16, 26). Variability between cells arising from the steps at extracellular pump sites might therefore make it difficult to detect a change in the voltage dependence of intracellular binding of Na+. We performed additional experiments using superfusates designed to eliminate the voltage dependence arising at extracellular pump sites.

The patch pipette filling solution and the voltage- clamp protocol were identical to those used for the experiments illustrated in Fig. 4A. However the superfusate used at the time Ip was measured was Na+ free and contained 15 mM K+ (see MATERIALS AND METHODS for details). The normalized Ip-Vm relationships for seven myocytes from captopril-treated rabbits and for eight myocytes from controls are summarized in Fig. 4B. The Ip-Vm relationships for myocytes from the two groups of rabbits were similar. We conclude that treatment with captopril does not alter voltage-dependent binding of Na+ at cytosolic Na+-K+ pump sites.

Effect of captopril treatment on the pump's apparent affinity for extracellular K+. To examine if the apparent affinity of the Na+-K+ pump for extracellular K+ is affected by treatment with captopril, we voltage clamped myocytes from rabbits treated with captopril with patch pipettes containing 80 mM Na+. This concentration was used to cause near-maximal pump activation to allow detection of small pump currents at low extracellular K+ concentrations. After stable holding currents were recorded at a holding potential of -40 mV, we inactivated the pump by switching to a K+-free superfusate. Ip was then identified as the shift in holding current induced by reexposure to solutions containing K+ in concentrations ranging from 0.5 to 15 mM in random order. We have previously established that such K+-induced shifts in holding currents are not contaminated by other K+-sensitive currents (15).

Each exposure to K+ was bracketed by exposure to K+-free superfusate, and the same nonzero K+ concentration was used for the first and the last exposures to detect if rundown of pump current had occurred. Figure 5 has been included to illustrate that K+-induced shifts in membrane currents were stable during long periods of recording. Figure 5 shows a tracing of the holding current of a myocyte exposed to K+-free and K+-containing superfusates over a period of >1 h. There was no evidence of rundown of the K+-activated current in this or 11 other experiments in the present study. We also found no evidence for rundown in 17 experiments in a previous study using a similar protocol (15).


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  		Fig. 5.   K+-induced shifts in holding current (Ih) during prolonged recording in a myocyte isolated from a rabbit treated with captopril. K+-induced shifts in Ih increased progressively with each increase in concentration ([K]o) over a period of ~1 h. The shift, measured with an electronic cursor, induced by the first exposure to 0.5 mM K+ (52 pA) was similar to shift induced by K+ in same concentration at completion of experimental protocol (54 pA). Experiment shown was not included in analysis of apparent K+ affinity of pump because different K+ concentrations were not used in random order.

Ip at each K+ concentration was normalized to the Ip recorded when superfusates contained 7 mM K+. The Hill equation was fitted to the relationship between the K+ concentration in the superfusate and the normalized Ip to derive K1/2. Details of data analysis have been reported previously (15). The mean K1/2 was 2.6 ± 0.2. This is similar to the value of 2.7 mM we have found previously for myocytes from control rabbits (15).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Modulation of apparent Na+ affinity. The increase in the apparent Na+ affinity of the sarcolemmal Na+-K+ pump induced by treatment with captopril (18) is not a unique mechanism by which activity of the pump is regulated. Both short-term in vitro and long-term in vivo interventions have previously been reported to alter the pump's apparent Na+ affinity. A change in the affinity with in vitro interventions has been reported for exposure of rat adipocytes and renal cells to insulin (8-10, 22), epidermal growth factor (10), and the alpha -adrenergic agonist oxymetazoline (1, 20) and for osmotically induced swelling or shrinkage of rabbit cardiac myocytes (31). Changes can also be induced in vivo by dietary cholesterol supplementation (15).

Although it is well established that the pump's apparent affinity for Na+ can be regulated, the mechanism for this regulation is poorly understood. To explore the mechanism for the captopril-induced changes, we examined the effect of [K+]pip and Vm on Ip at a fixed [Na+]pip of 10 mM. There was no effect of treatment with captopril when Na+ was the only monovalent cation expected to interact with intracellular pump sites. This suggests that a change in the intrinsic binding affinity for Na+ at the selective site within the membrane dielectric is unlikely. This conclusion is supported by the absence of an effect of treatment with captopril on the voltage dependence of Ip because interaction of Na+ with the pump at the selective pump site is expected to be voltage dependent (25, 26).

Effect of intracellular K+. The increase in Ip induced by treatment with captopril was dependent on [K+]pip (Fig. 2) and consistent with a captopril-induced decrease in inhibition of Ip by intracellular K+. The K1/2 values for Na+-K+-ATPase-rich membrane fragments (29) and Na+-K+-ATPase reconstituted into liposomes (6) have been reported to be ~10-20 and 40 mM, respectively. The inhibitory effect of K+ at cytoplasmic pump sites is highly dependent on experimental conditions (28), and meaningful comparisons of values for K1/2 between studies are difficult.

The ability of K+ to act as a competitive inhibitor of Na+ activation of the pump is reflected by the ratio of affinities for K+ and Na+ at intracellular sites (29). We used a fixed [Na+]pip of 10 mM, and the absence of an effect of treatment with captopril or losartan when pipette solutions were K+ free suggests that treatment alters the affinity for K+ rather than for Na+. However, a definite distinction between the effects of treatment on the apparent affinities at intracellular pump sites for Na+ and K+ based on a separate kinetic analysis for the interaction of the two ligands with intracellular pump sites cannot be made from the data.

The competition for Na+ and K+ exhibited by alpha 1-, alpha 2-, and alpha 3-isoforms of Na+-K+ ATPase obtained from different sources has been examined in a previous study (29). Identical experimental techniques were used to measure ATPase activity. The apparent affinity of K+ as a competitive inhibitor of Na+ binding was tissue rather than isoform specific. It was concluded that the primary structure of the alpha -isoform is not the sole determinant of intracellular cation selectivity, and it was speculated that a tissue-specific pump modulator regulates the inhibition of the pump by K+ at intracellular sites (29). The present study indicates that the inhibition within the same organ can be modulated by pharmacological intervention. An effect of treatment on a tissue-specific pump modulator might account for this.

PKC and the Na+-K+ pump. Isolated, purified Na+-K+-ATPase can be a substrate for PKC in vitro (2, 3, 5, 11, 21), and in situ pumps can be phosphorylated when PKC is activated by exposing a variety of intact cells to phorbol esters (5, 12, 13, 23). The functional significance of PKC-mediated pump phosphorylation has not been firmly established. Stimulation, inhibition, and a complete absence of any effect have been reported (see Refs. 4 and 12 for review). We found that the exposure of myocytes from rabbits treated with captopril to PMA had no effect on Ip when patch pipette solutions were K+ free. In contrast, a decrease in Ip was induced when pipettes contained 70 mM K+ (Fig. 3). These findings suggest that exposure of myocytes to PMA reduced the selectivity for Na+ relative to K+ at intracellular pump sites, a change that is expected to reduce the apparent affinity of the pump for Na+. Such K+ dependence of the effect of a phorbol ester on the pump has not been reported previously and has not been taken into account in previous studies. This is likely to have contributed to the controversy regarding the effects of phorbol ester-induced phosphorylation of the Na+-K+ pump.

Phorbol esters are convenient to use for activating PKC in intact cells because they are membrane permeable. We used one of these, PMA, in the present study because an extensive literature documenting the effect of phorbol esters on the Na+-K+ pump exists. However, it is widely recognized that they are not specific activators of PKC. The patch-clamp technique allowed the access of PKCF to the intracellular compartment in our study. This peptide is a highly specific activator of the PKC family of kinases (19), and the feasibility of the effective dialysis of it into patch-clamped myocytes has been demonstrated previously (30). The effect of PKCF was similar to that of PMA. This supports the conclusion, reached by previous studies using phorbol esters, that PKC can regulate the Na+-K+ pump in intact cells.

We have previously reported that an ANG II-induced decrease in Ip of myocytes from rabbits treated with captopril is abolished by inhibitors of PKC (17). Available inhibitors of PKC are not absolutely specific. However, the similar, nonadditive effects of ANG II and PKCF on Ip in the present study (Fig. 3) provide additional support for the involvement of PKC in the regulation of the Na+-K+ pump by ANG II. When one considers the evidence available from our studies and from studies by other groups, it is reasonable to think that an ANG II-induced protein kinase-dependent phosphorylation of the pump regulates competitive inhibition by K+ of Na+ binding to the pump at the sites near the cytoplasmic surface. Such a specific functional effect of a hormone-induced phosphorylation on the pump has not been implicated previously. Definitive proof for this scheme would require direct demonstration of changes in the phosphorylation of cytoplasmic Na+ binding sites accompanying the functional changes we demonstrated. Such a degree of resolution in the analysis of the pump's structure-function relationship is not possible at present.


    ACKNOWLEDGEMENTS

This study was supported by National Heart Foundation of Australia Grant G 96S 4589 and by the North Shore Heart Research Foundation.


    FOOTNOTES

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. §1734 solely to indicate this fact.

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

Received 24 September 1998; accepted in final form 1 June 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aperia, A., F. Ibarra, L.-B. Svensson, C. Klee, and P. Greengard. Calcineurin mediates alpha -adrenergic stimulation of Na+,K+-ATPase activity in renal tubule cells. Proc. Natl. Acad. Sci. USA 89: 7394-7397, 1992[Abstract/Free Full Text].

2.   Beguin, P., A. T. Beggah, A. V. Chibalin, P. Burgener-Kairuz, F. Jaisser, P. M. Mathews, B. C. Rossier, S. Cotecchia, and K. Geering. Phosphorylation of the Na,K-ATPase alpha -subunit by protein kinase A and C in vitro and in intact cells. J. Biol. Chem. 269: 24437-24445, 1994[Abstract/Free Full Text].

3.   Bertorello, A. M., A. Aperia, S. I. Walaas, A. C. Nairn, and P. Greengard. Phosphorylation of the catalytic subunit of Na+,K+-ATPase inhibits the activity of the enzyme. Proc. Natl. Acad. Sci. USA 88: 11359-11362, 1991[Abstract/Free Full Text].

4.   Bertorello, A. M., and A. I. Katz. Short-term regulation of renal Na-K-ATPase activity: physiological relevance and cellular mechanisms. Am. J. Physiol. 265 (Renal Fluid Electrolyte Physiol. 34): F743-F755, 1993[Abstract/Free Full Text].

5.   Chibalin, A. V., L. A. Vasilets, H. Hennekes, D. Pralong, and K. Geering. Phosphorylation of Na,K-ATPase alpha -subunits in microsomes and in homogenates of Xenopus oocytes resulting from the stimulation of protein kinase A and protein kinase C. J. Biol. Chem. 267: 22378-22384, 1992[Abstract/Free Full Text].

6.   Cornelius, F. Cis-allosteric effects of cytoplasmic Na+/K+ discrimination at varying pH. Low-affinity multisite inhibition of cytoplasmic K+ in reconstituted Na+/K+-ATPase engaged in uncoupled Na+-efflux. Biochim. Biophys. Acta 1108: 190-200, 1992[Medline].

7.   Cornelius, F. Hydrophobic ion interaction on Na+ activation and dephosphorylation of reconstituted Na+,K+-ATPase. Biochim. Biophys. Acta 1235: 183-196, 1995[Medline].

8.   Féraille, E., M. L. Carranza, B. Buffin-Meyer, M. Rousselot, A. Doucet, and H. Favre. Protein kinase C-dependent stimulation of Na+-K+-ATPase in rat proximal convoluted tubule. Am. J. Physiol. 268 (Cell Physiol. 37): C1277-C1283, 1995[Abstract/Free Full Text].

9.   Féraille, E., M. L. Carranza, M. Rousselot, and H. Favre. Insulin enhances sodium sensitivity of Na-K-ATPase in isolated rat proximal convoluted tubule. Am. J. Physiol. 267 (Renal Fluid Electrolyte Physiol. 36): F55-F62, 1994[Abstract/Free Full Text].

10.   Féraille, E., M. L. Carranza, M. Rousselot, and H. Favre. Modulation of Na+,K+-ATPase activity by a tyrosine phosphorylation process in rat proximal convoluted tubule. J. Physiol. (Lond.) 498: 99-108, 1997[Medline].

11.   Feschenko, M. S., and K. J. Sweadner. Conformation-dependent phosphorylation of Na,K-ATPase by protein kinase A and protein kinase C. J. Biol. Chem. 269: 30436-30444, 1994[Abstract/Free Full Text].

12.   Feschenko, M. S., and K. J. Sweadner. Phosphorylation of Na,K-ATPase by protein kinase C at Ser18 occurs in intact cells but does not result in direct inhibition of ATP hydrolysis. J. Biol. Chem. 272: 17726-17733, 1997[Abstract/Free Full Text].

13.   Fisone, G., G. L. Snyder, J. Fryckstedt, M. J. Caplan, A. Aperia, and P. Greengard. Na+,K+-ATPase in the choroid plexus. J. Biol. Chem. 270: 2427-2430, 1995[Abstract/Free Full Text].

14.   Gadsby, D. C., and M. Nakao. Steady-state current-voltage relationship of the Na/K pump in guinea pig ventricular myocytes. J. Gen. Physiol. 94: 511-537, 1989[Abstract/Free Full Text].

15.   Gray, D. F., P. S. Hansen, M. M. Doohan, L. C. Hool, and H. H. Rasmussen. Dietary cholesterol affects Na+-K+ pump function in rabbit cardiac myocytes. Am. J. Physiol. 272 (Heart Circ. Physiol. 41): H1680-H1689, 1997[Abstract/Free Full Text].

16.   Heyse, S., I. Wuddel, H.-J. Apell, and W. Stürmer. Partial reactions of the Na,K-ATPase: determination of rate constants. J. Gen. Physiol. 104: 197-240, 1994[Abstract/Free Full Text].

17.   Hool, L. C., D. F. Gray, B. G. Robinson, and H. H. Rasmussen. Angiotensin-converting enzyme inhibitors regulate the Na+-K+ pump via effects on angiotensin metabolism. Am. J. Physiol. 271 (Cell Physiol. 40): C172-C180, 1996[Abstract/Free Full Text].

18.   Hool, L. C., D. W. Whalley, M. M. Doohan, and H. H. Rasmussen. Angiotensin-converting enzyme inhibition, intracellular Na+, and Na+-K+ pumping in cardiac myocytes. Am. J. Physiol. 268 (Cell Physiol. 37): C366-C375, 1995[Abstract/Free Full Text].

19.   House, C., P. J. Robinson, and B. E. Kemp. A synthetic peptide analog of the putative substrate-binding motif activates protein kinase C. FEBS Lett. 249: 243-247, 1989[Medline].

20.   Ibarra, F., A. Aperia, L.-B. Svensson, A.-C. Eklöf, and P. Greengard. Bidirectional regulation of Na+,K+-ATPase activity by dopamine and an alpha -adrenergic agonist. Proc. Natl. Acad. Sci. USA 90: 21-24, 1993[Abstract/Free Full Text].

21.   Logvinenko, N. S., I. Dulubova, N. Fedosova, S. H. Larsson, A. C. Nairn, M. Esmann, P. Greengard, and A. Aperia. Phosphorylation by protein kinase C of serine-23 of the alpha -1 subunit of rat Na+,K+-ATPase affects its conformational equilibrium. Proc. Natl. Acad. Sci. USA 93: 9132-9137, 1996[Abstract/Free Full Text].

22.   Lytton, J. Insulin affects the sodium affinity of the rat adipocyte (Na+,K+)-ATPase. J. Biol. Chem. 260: 10075-10080, 1985[Abstract/Free Full Text].

23.   Middleton, J. P., W. A. Khan, G. Collinsworth, Y. A. Hannun, and R. M. Medford. Heterogeneity of protein kinase C-mediated rapid regulation of Na/K-ATPase in kidney epithelial cells. J. Biol. Chem. 268: 15958-15964, 1993[Abstract/Free Full Text].

24.   Nakao, M., and D. C. Gadsby. [Na] and [K] dependence of the Na/K pump current-voltage relationship in guinea pig ventricular myocytes. J. Gen. Physiol. 94: 539-565, 1989[Abstract/Free Full Text].

25.   Or, E., P. David, A. Shainskaya, D. M. Tal, and S. J. D. Karlish. Effects of competitive sodium-like antagonists on Na,K-ATPase suggest that cation occlusion from the cytoplasmic surface occurs in two steps. J. Biol. Chem. 268: 16929-16937, 1993[Abstract/Free Full Text].

26.   Or, E., R. Goldshleger, and S. J. D. Karlish. An effect of voltage on binding of Na+ at the cytoplasmic surface of the Na+-K+ pump. J. Biol. Chem. 271: 2470-2477, 1996[Abstract/Free Full Text].

27.   Sagar, A., and R. F. Rakowski. Access channel model for the voltage dependence of the forward-running Na+/K+ pump. J. Gen. Physiol. 103: 869-894, 1994[Abstract/Free Full Text].

28.   Schulz, S., and H.-J. Apell. Investigation of ion binding to the cytoplasmic binding sites of the Na,K-pump. Eur. Biophys. J. 23: 413-421, 1995[Medline].

29.   Therien, A. G., N. B. Nestor, W. J. Ball, and R. Blostein. Tissue-specific versus isoform-specific differences in cation activation kinetics of the Na,K-ATPase. J. Biol. Chem. 271: 7104-7112, 1996[Abstract/Free Full Text].

30.   Watson, C. L., and M. R. Gold. Modulation of Na+ current inactivation by stimulation of protein kinase C in cardiac cells. Circ. Res. 81: 380-386, 1997[Abstract/Free Full Text].

31.   Whalley, D. W., L. C. Hool, R. E. Ten Eick, and H. H. Rasmussen. Effect of osmotic swelling and shrinkage on Na+-K+ pump activity in mammalian cardiac myocytes. Am. J. Physiol. 265 (Cell Physiol. 34): C1201-C1210, 1993[Abstract/Free Full Text].

32.   Wilkinson, S. E., and T. J. Hallam. Protein kinase C: is its pivotal role in cellular activation overstated? Trends Pharmacol. Sci. 15: 53-57, 1994[Medline].

33.   Zusman, R. M. Eicosanoids: prostaglandins, thromboxane, and prostacyclin. In: The Heart and Cardiovascular System, edited by H. A. Fozzard, R. B. Jennings, E. Haber, A. M. Katz, and H. E. Morgan. New York: Raven, 1991, p. 1797-1815.

Am J Physiol Cell Physiol 277(3):C461-C468
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society


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