Mechanisms of Na+-K+ pump regulation in cardiac myocytes during hyposmolar swelling

N. L. Bewick1, C. Fernandes1,2, A. D. Pitt1, H. H. Rasmussen1,2, and D. W. Whalley1,2

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have previously demonstrated that the sarcolemmal Na+-K+ pump current (Ip) in cardiac myocytes is stimulated by cell swelling induced by exposure to hyposmolar solutions. However, the underlying mechanism has not been examined. Because cell swelling activates stretch-sensitive ion channels and intracellular messenger pathways, we examined their role in mediating Ip stimulation during exposure of rabbit ventricular myocytes to a hyposmolar solution. Ip was measured by the whole cell patch-clamp technique. Swelling-induced pump stimulation altered the voltage dependence of Ip. Pump stimulation persisted in the absence of extracellular Na+ and under conditions designed to minimize changes in intracellular Ca2+, excluding an indirect influence on Ip mediated via fluxes through stretch-activated channels. Pump stimulation was protein kinase C independent. The tyrosine kinase inhibitor tyrphostin A25, the phosphatidylinositol 3-kinase inhibitor LY-294002, and the protein phosphatase-1 and -2A inhibitor okadaic acid abolished Ip stimulation. Our findings suggest that swelling-induced pump stimulation involves the activation of tyrosine kinase, phosphatidylinositol 3-kinase, and a serine/threonine protein phosphatase. Activation of this messenger cascade may cause activation by the dephosphorylation of pump units.

osmolarity; tyrosine kinase; ion transport; phosphatases


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE SWELLING OF CARDIAC myocytes induced by exposure to hyposmolar superfusates stimulates the activity of the membrane Na+-K+ pump (27, 32). However, the cellular mechanisms involved in the transduction of the osmotic stress into pump stimulation have not been investigated. The swelling of myocytes activates stretch-sensitive ion channels, including channels permeable to Na+ and Ca2+ (3, 23). Pump stimulation could therefore be secondary to an influx of extracellular Na+ or Ca2+, resulting in a rise in cytosolic Na+ or Ca2+ concentrations, known determinants of pump activity (33). Cell swelling is also known to rapidly activate a variety of intracellular messengers, including protein tyrosine kinase and serine(threonine) protein kinase (24, 26; see Ref. 25 for review). Serine(threonine) protein kinases can induce the phosphorylation of the Na+-K+ pump, whereas tyrosine kinases are involved in inducing the dephosphorylation of pump molecules via a mechanism involving activation of protein serine/threonine phosphatases (21). Because the activation of kinases and phosphatases can regulate the Na+-K+ pump (2, 7), increased pump activity during exposure of cardiac myocytes to hyposmolar superfusates could be mediated by protein kinase- and/or protein phosphatase-dependent pathways.

Understanding the mechanism by which the Na+-K+ pump is regulated by osmotic stress is desirable because the pump plays a role in the maintenance of cell volume under physiological conditions and during pathophysiological states, including acute myocardial ischemia and reperfusion. Using the whole cell patch-clamp technique, we studied Na+-K+ pump current (Ip) in ventricular myocytes exposed to hyposmolar superfusates. We found that swelling-induced Na+-K+ pump stimulation is independent of the influx of extracellular Na+ and Ca2+. However, stimulation was dependent on tyrosine kinase and involved the activation of an okadaic acid-inhibitable protein phosphatase. Pump stimulation induced by exposure to hyposmolar superfusates was rapidly reversible on reexposure to an isosmolar superfusate. This reversal of pump stimulation could be blocked by inhibition of protein kinase. We propose that the Na+-K+ pump is regulated by cell volume via steps which involve the phosphorylation and dephosphorylation of the pump molecule by kinases and phosphatases, respectively.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cells and solutions. Single ventricular myocytes were isolated from male New Zealand White rabbits weighing 2.5-3.5 kg as described previously (32). They were stored at room temperature until used. For measurement of Ip, myocytes were suspended in superfusates flowing through a tissue bath mounted on an inverted microscope. The superfusate used initially in all experiments contained (in mM) 140 NaCl, 5.6 KCl, 2.16 CaCl2, 0.44 NaH2PO4, 10 glucose, 10 HEPES, and 1 MgCl2. The solution was titrated to pH 7.4 ± 0.01 at 35°C with 1 M NaOH. Hyposmolar and isosmolar Ca2+-free modified Tyrode solutions, used subsequently when Ip was measured, contained (in mM) 105 NaCl, 5.6 KCl, 0.44 NaH2PO4, 10 glucose, 10 HEPES, 2 MgCl2, 0.2 CdCl2, and 2 BaCl2. BaCl2 was added to block K+ channels, and CdCl2 was added to block the Na+/Ca2+ exchanger. The solutions were titrated to pH 7.4 ± 0.01 at 35°C with 1 M NaOH. The isosmolar solution contained 70 mM sucrose, whereas the hyposmolar solutions were sucrose free. We measured solution osmolarity with a vapor pressure osmometer (model 5500 osmometer; Wescor, Logan, UT). The osmolarities of the hyposmolar and isosmolar solutions were 240 ± 3 and 305 ± 4 mosM, respectively (mean ± SD). Superfusates used at the time Ip was measured were warmed to 35 ± 0.5°C. For measurements of Ip at a fixed membrane voltage (Vm) of -40 mV, the pipette filling solution contained (in mM) 70 potassium glutamate, 1 KH2PO4, 5 HEPES, 5 EGTA, and 2 MgATP. Unless otherwise indicated, the solution also contained 10 mM sodium glutamate and 80 mM tetramethylammonium chloride (TMA-Cl). It was titrated to pH 7.05 ± 0.01 at 35°C with 1 M KOH. In experiments designed to examine the Ip-Vm relationship, the filling solution of the patch pipettes contained (in mM) 10 sodium glutamate, 1 KH2PO4, 5 HEPES, 5 EGTA, 2 MgATP, 60 TMA-Cl, 20 tetraethylammonium chloride, 70 CsOH, and 50 aspartic acid. The solution was titrated to a pH of 7.05 ± 0.01 at 35°C with HCl. The resistances of pipettes filled with either solution were 0.8-1.0 MOmega . The offset potential was nulled by placing both the ground electrode and patch pipette in the pipette filling solution (20). We used the computer program JPCalc (P. Barry, Univ. of New South Wales) to estimate junction potentials between the ground electrode and superfusates. The differences in these junction potentials were <0.3 mV between the different superfusates used. They are not corrected for when Ip is reported.

Measurement of membrane currents. We measured Ip in freshly isolated cells 2-9 h after excision of the heart. Ip was defined as the shift in holding current induced by 100 µM ouabain (Sigma Chemical, St. Louis, MO). For each cell Ip was measured during the superfusion of either isosmolar or hyposmolar Ca2+-free Tyrode solutions. 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). The Ip values reported were normalized for membrane capacitance as described previously (32). For measurement of the Ip-Vm relationship, we superfused myocytes with Ca2+-free solutions having a composition identical to that of solutions used to measure Ip at the fixed Vm of -40 mV with the exception that we added 1 mM anthracene-9-carboxylic acid (9-AC; Sigma Chemical) to inhibit stretch-activated Cl- channels (12).

Reagents. In some experiments tyrphostin A25 or its inactive analog, tyrphostin A63, okadaic acid or its inactive analog, okadaic acid methyl ester, or staurosporine was added to the patch pipette. All were purchased from Calbiochem (La Jolla, CA). Tyrphostin A25 and A63 were dissolved in DMSO. The final concentration of DMSO was 0.04%. Staurosporine and okadaic acid methyl ester were also dissolved in DMSO; the final concentrations of DMSO were 0.005 and 0.2%, respectively. In experiments designed to examine the Ip-Vm relationship, 9-AC was dissolved in DMSO with a final concentration of 0.1%. It has previously been shown that DMSO in concentrations higher than those used in the present study have no effect on the steady-state holding current or Ip in isolated cardiac myocytes (19). LY-294002, purchased from Calbiochem, was dissolved in ethanol and added to the superfusates on the day of experimentation. The final ethanol concentration in the superfusates was 0.03%. Ethanol at this concentration has previously been shown in control experiments to have no effect on basal Na+-K+ pump activity (11).

Statistical analysis. Results are expressed as means ± SE unless otherwise indicated. Comparisons are made by using Student's t-test for unpaired observations. Statistical comparisons based on the same control experiments were made with Dunnett's test. Comparisons between the voltage dependence curves were made by two-way ANOVA for the interaction of voltage and osmolarity. Differences between means were regarded as statistically significant at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Role of Ca2+ influx in stimulation of Ip by cell swelling. We have previously shown that exposure of rabbit cardiac myocytes to hyposmolar solutions stimulates Ip when the patch pipette Na+ concentration ([Na]pip) is near physiological intracellular levels (32). In contrast, Sasaki et al. (27) did not report a significant enhancement of the Ip values for patch-clamped guinea pig cardiac myocytes under similar experimental conditions ([Na]pip 10 mM). Our experiments were performed in the presence of extracellular Ca2+, whereas the experiments of Sasaki et al. (27) were performed with superfusates that were nominally Ca2+ free. This raises the possibility that the pump stimulation induced by exposure to hyposmolar superfusates may be dependent on the influx of Ca2+ via stretch-activated channels. Because it cannot be ensured that the dialysis of the pipette solution completely changes intracellular Ca2+ levels, such an influx might cause changes in the intracellular concentrations of this ion. We performed experiments to examine this possibility. The pipette solution we used included 10 mM Na+, and, to buffer intracellular Ca2+, 5 mM EGTA. The whole cell configuration was first established while myocytes were being superfused with isosmolar Ca2+-containing Tyrode solution. We then switched to a superfusate that contained 105 mM Na+ and that was isosmolar (310 mosM) or hyposmolar (240 mosM). These solutions were nominally Ca2+ free. The Ca2+-free Tyrode solution was superfused for 3 min before we measured Ip. We measured Ip values for nine cells superfused with isosmolar solution and for seven cells superfused with hyposmolar solution. Representative recordings of shifts in holding currents during the measurement of Ip have been published previously (32). The means of the Ip values were 0.25 ± 0.03 pA/pF in isosmolar superfusates and 0.58 ± 0.06 pA/pF in hyposmolar superfusates. The difference in mean Ip values was statistically significant.

We previously found that swelling-induced stimulation of Ip, measured by using Ca2+-containing superfusates, only occurred when [Na]pip was near physiological levels of intracellular Na+; there was no effect when [Na]pip was high (32). We wished to examine whether a similar [Na]pip dependence of the pump stimulation was apparent when Ca2+-free superfusates were used. We therefore performed experiments using an [Na]pip of 80 mM, a concentration that produces near-maximal pump activity. The mean Ip was 1.68 ± 0.12 pA/pF (n = 12) in cells exposed to isosmolar superfusate and 1.79 ± 0.16 pA/pF (n = 12) in cells exposed to hyposmolar superfusate. This difference was not statistically significant. The results of experiments performed at [Na]pip values of 10 and 80 mM are summarized in Fig. 1. Taken together, the data in Fig. 1 indicate that the [Na]pip-dependent stimulatory effect of the exposure of myocytes to hyposmolar superfusates on Ip is not dependent on the influx of extracellular Ca2+.


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Fig. 1.   Effect of osmotic swelling on sarcolemmal Na+-K+ pump current (Ip). Superfusate was isosmolar (Iso) or hyposmolar (Hypo), and patch pipette Na+ concentration ([Na+]pip) was either 10 or 80 mM. Ip has been normalized for cell capacitance. No. in parentheses is no. of experiments in each group.

Role of Na+ influx in pump stimulation. Cell swelling and mechanical stretch open nonselective stretch-activated cation channels (23). An influx of Na+ through such channels might stimulate pump activity in the present study by increasing the Na+ concentration at intracellular binding sites in a restricted subsarcolemmal space to levels above [Na]pip. To examine this possibility, we measured Ip while myocytes were being superfused with Na+-free isosmolar or hyposmolar superfusates. Both superfusates were Ca2+ free. We replaced Na+ in the superfusates with 105 mM N-methyl-D-glucamine. Isosmolar superfusates contained 70 mM sucrose, whereas hyposmolar solutions were sucrose free. [Na]pip was 10 mM. In the absence of extracellular Na+, the mean Ip of eight myocytes superfused with hyposmolar solutions was 0.59 ± 0.04 pA/pF, whereas the mean Ip of six myocytes superfused with isosmolar solutions was 0.31 ± 0.03 pA/pF. The difference was statistically significant (P < 0.001). This indicates that the stimulatory effect of cell swelling on Ip is not due to an increased Na+ influx. A direct effect of cell swelling on the Na+-K+ pump is suggested.

Effect of cell swelling on pump Ip-Vm relationship. The experiments described above indicate that myocyte swelling stimulates the Na+-K+ pump at a fixed membrane voltage (Vm) of -40 mV. Because pump function is voltage dependent, we examined if stimulation also occurs at other voltages. After establishing the whole cell configuration, we switched to a Ca2+-free superfusate and voltage-clamped myocytes at a holding potential of -40 mV. We then applied 320-ms voltage steps to test potentials (Vm) from -100 to +60 mV in 20-mV increments. The command voltage was returned to -40 mV for 2 s between each step to a test potential. To derive Ip at each test potential, the membrane currents recorded during the superfusion of ouabain were subtracted from the membrane currents at corresponding voltages before the superfusion of ouabain. Details of the protocol and representative current traces have been described previously (10). Figure 2A summarizes the Ip-Vm relationships for six myocytes superfused with isosmolar solution and six myocytes superfused with hyposmolar solution. Pump stimulation induced by cell swelling was most prominent at the most negative values of Vm and decreased with a shift of Vm toward positive levels. Ip values measured in these experiments using a [Na]pip of 10 mM are larger during the superfusion of both isosmolar and hyposmolar solutions than those measured in experiments performed at a constant Vm of -40 mV with 10 mM [Na]pip (see Fig. 1). This difference can be attributed to differences in the compositions of the pipette solutions used in the two experimental protocols. In experiments examining the Ip-Vm relationship, we used a pipette solution containing Cs+ as a substitute for K+ to abolish time- and voltage-dependent K+ conductances. We have previously shown that the inhibitory effect of intracellular Cs+ on Ip is significantly less than the inhibitory effect of intracellular K+. Such a reduction in inhibitory effect is expected to cause an increase in Ip (14). The Ip-Vm relationships have been normalized to the Ip recorded at 0 mV in Fig. 2B to facilitate the comparison of their slopes. The Ip-Vm relationship in isosmolar solutions was nearly linear and had a positive slope throughout the entire range of test potentials. The Ip-Vm relationship for six myocytes superfused with hyposmolar solution appeared less steep at negative potentials than that for myocytes superfused with isosmolar solutions. It had a negative slope at potentials more positive than -40 mV. The difference in these curves was statistically significant (P < 0.001; 2-way ANOVA). We conclude that pump stimulation induced by myocyte swelling is voltage dependent.


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Fig. 2.   A: voltage dependence of Ip values for myocytes superfused with isosmolar (open circle ) and hyposmolar superfusates (). Ip-membrane voltage (Vm) relationships of 6 myocytes in each group have been summarized by plotting mean ± SE Ip measured at each test potential. B: normalized voltage dependence of Ip. Plots were obtained by first normalizing Ip-Vm relationship for each myocyte to Ip recorded at 0 mV and then averaging normalized Ip values at each test potential.

Effect of kinase inhibition on swelling-induced pump stimulation. Both mechanical deformation of the cardiac sarcolemmal membrane and osmotic swelling in a hyposmolar solution activate protein kinase C (PKC) (24). Because PKC and other kinases, in turn, modulate Na+-K+ pump activity in a variety of noncardiac tissues (2, 7), we examined the effect of the potent but nonselective protein kinase inhibitor staurosporine and the effect of bisindolylmaleimide I, an inhibitor believed to be specific for PKC. We added 10 nM staurosporine to the patch pipette solution. [Na]pip was 10 mM in all experiments. After the whole cell configuration had been established, we waited for 5 min to allow intracellular dialysis of the staurosporine before myocytes were exposed to isosmolar or hyposmolar superfusate, and then Ip was determined. The mean Ip of seven cells exposed to isosmolar solution was 0.24 ± 0.02 pA/pF, and the mean Ip of six cells exposed to hyposmolar solution was 0.40 ± 0.04 pA/pF. The difference was statistically significant. This indicates that nonselective kinase inhibition does not prevent the stimulation of Ip induced by cell swelling. A similar result was obtained when we used 1 µM bisindolylmaleimide I.

Effect of tyrosine kinase inhibition on pump stimulation in hyposmolar solutions. Swelling and mechanical stretch of cardiac myocytes rapidly activate tyrosine kinase (24, 26). Because activation of the insulin receptor tyrosine kinase has an effect on the Ip-Vm relationship (13) similar to the effect of swelling found in this study, we examined the effect of the tyrosine kinase inhibitor tyrphostin A25. In the first series of experiments, we examined the effect of tyrphostin A25 on pump stimulation induced by myocyte swelling at a fixed Vm of -40 mV. We included 100 µM tyrphostin A25 in the patch pipette solution. [Na]pip was 10 mM. After the whole cell configuration was established, myocytes were superfused with either isosmolar or hyposmolar Ca2+-free solutions and Ip was measured. The mean Ip of six myocytes superfused with isosmolar solution was 0.26 ± 0.03 pA/pF. This is similar to the mean Ip measured under similar conditions with tyrphostin A25-free pipette solutions (Fig. 1), suggesting that there is no tonic effect of tyrosine kinase on the Ip of myocytes in isosmolar solutions. When tyrphostin A25 was included in pipette solutions, the mean Ip of six myocytes exposed to hyposmolar solutions was 0.29 ± 0.04 pA/pF. This is similar to mean Ip values of the myocytes exposed to isosmolar solutions, indicating that tyrphostin A25 completely blocked the pump stimulation induced by cell swelling. To exclude a nonspecific effect of tyrphostin A25 on the Na+-K+ pump, experiments were repeated with 100 µM tyrphostin A63, the inactive analog of tyrphostin A25, in the patch pipette. In the presence of tyrphostin A63, the mean Ip of five myocytes superfused with isosmolar solution and the mean Ip of five myocytes superfused with hyposmolar solutions were 0.20 ± 0.03 and 0.48 ± 0.07 pA/pF, respectively. The difference was statistically significant. Results obtained with tyrphostin A25 and A63 in pipette solutions have been summarized in Fig. 3A. We conclude that the tyrosine kinase inhibitor tyrphostin A25 can abolish the pump stimulation induced by cell swelling and that this is a specific effect of the drug.


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Fig. 3.   A: effect of tyrosine kinase and phosphatidylinositol 3-kinase (PI3K) inhibition. Tyrosine kinase inhibitor tyrphostin A25 (Tyr A25) and its inactive analog, tyrphostin A63 (Tyr A63), were included in patch pipette solutions. In a separate group of experiments PI3K inhibitor LY-294002 was included in superfusates. Inhibition of tyrosine kinase or PI3K abolished swelling-induced pump stimulation. No. in parentheses is no. of experiments in each group. B: effect of tyrphostin A25 on Ip-Vm relationship during exposure to isosmolar (open circle ) and hyposmolar () solutions.

In a second series of experiments, we examined the effect of tyrphostin A25 on the Ip-Vm relationship. Normalized Ip-Vm relationships obtained from six myocytes exposed to isosmolar solutions and six myocytes exposed to hyposmolar solutions have been summarized in Fig. 3B. Their slopes were similar to the slopes of Ip-Vm relationships for myocytes superfused with isosmolar solution and voltage clamped with tyrphostin A25-free patch pipettes (Fig. 2B). Tyrphostin A25 completely eliminated the effect of the hyposmolar solution on the slope of the Ip-Vm relationship. We also determined the Ip-Vm relationship with the inactive analog tyrphostin A63 included in pipette solutions. The shift in the Ip-Vm relationship induced by exposure to hyposmolar solutions (Fig. 2B) was unaffected by tyrphostin A63 (data not shown).

Role of phosphatidylinositol 3-kinase in mediating the stimulation of Ip. Activation of phosphatidylinositol 3-kinase (PI3K) is an essential step in the transduction of cellular signals mediated via tyrosine kinase-dependent processes (4, 15, 31). We investigated the role of PI3K in the stimulation of Ip by hyposmolar solutions. Myocytes were superfused before and after establishment of the whole cell configuration with solutions containing the specific PI3K inhibitor LY-294002 at a concentration of 5 µM. The mean Ip of six myocytes superfused with LY-294002-containing isosmolar solutions was 0.29 ± 0.02 pA/pF, and the mean Ip of six myocytes superfused with LY-294002-containing hyposmolar solutions was 0.27 ± 0.02 pA/pF. The difference was not statistically significant. We have included the results of experiments performed with LY-294002 in Fig. 3A. Because PI3K is strongly linked to tyrosine kinase-mediated messenger pathways, the abolition of swelling-induced pump stimulation by LY-294002 strongly supports the conclusion derived from experiments using tyrphostin A25, i.e., that pump stimulation is mediated by the activation of tyrosine kinase.

Role of serine/threonine protein phosphatase in Ip stimulation. Tyrosine kinase has been demonstrated to activate serine/threonine protein phosphatase 1 (PP1) (6). PP1 and another protein phosphatase, PP2A, may play a role in the regulation of the Na+-K+ pump in the kidney (16). We examined the effect of okadaic acid, an inhibitor of both PP1 and PP2A, on the stimulation of Ip by hyposmolar solutions. In initial experiments we added 1 µM okadaic acid to the patch pipette solution to completely inhibit both PP1 and PP2A (5). We measured Ip in isosmolar and hyposmolar superfusates. The mean Ip in six myocytes measured during exposure to isosmolar superfusate was 0.33 ± 0.03 pA/pF, whereas the mean Ip in eight myocytes measured during exposure to hyposmolar superfusate was 0.37 ± 0.05 pA/pF. This difference was not statistically significant. This suggests that Na+-K+ pump stimulation during exposure to hyposmolar solutions involves the activation of PP1 and/or PP2A. To exclude a nonspecific effect of okadaic acid on the pump, we repeated the experiments with the inactive analog, okadaic acid methyl ester (1 µM) in pipette solutions. The mean Ip in seven myocytes measured during exposure to isosmolar superfusate was 0.33 ± 0.02 pA/pF, whereas the mean Ip of seven myocytes exposed to hyposmolar superfusates was 0.49 ± 0.03 pA/pF. The difference was statistically significant.

In multiple noncardiac tissues, 10 nM okadaic acid has been shown to inhibit PP2A almost completely but to have no effect on PP1 (5). This differential sensitivity of phosphatases to okadaic acid has also been assumed to exist in cardiac myocytes (17). We used this presumed differential sensitivity to examine which of the phosphatases might be involved in the Na+-K+ pump stimulation induced by exposure to hyposmolar solutions. We added 10 nM okadaic acid to patch pipette solutions and measured Ip in isosmolar and hyposmolar solutions. The mean Ip in eight myocytes measured during exposure to isosmolar solutions was 0.28 ± 0.03 pA/pF, whereas the mean Ip in nine myocytes measured during exposure to hyposmolar solutions was 0.51 ± 0.02 pA/pF. The difference was statistically significant. The results of experiments performed with okadaic acid are summarized in Fig. 4. Figure 4 illustrates that swelling-induced Na+-K+ pump stimulation can be eliminated by the inhibition of protein phosphatases. The dependence of this inhibition on the concentration of okadaic acid suggests that PP1 rather than PP2A is involved.


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Fig. 4.   Effect of serine/threonine phosphatase inhibition on swelling-induced Ip stimulation. Experiments were performed during exposure to isosmolar or hyposmolar solution in presence of 1 µM or 10 nM okadaic acid. No. in parentheses is no. of experiments in each group.

Reversibility of swelling-induced pump stimulation. We have previously found that changes in the intracellular Na+ activity of intact papillary muscles induced by exposure to hyposmolar superfusates are rapidly reversible (32). We performed a series of experiments to examine if this is reflected by reversible changes in the Ip of patch-clamped myocytes. After establishing the whole cell configuration, we superfused isosmolar Ca2+-free solution for 3 min. We then switched to a hyposmolar Ca2+-free superfusate to induce cell swelling and pump stimulation. After 3 min we switched back to the isosmolar superfusate for 5 min and measured Ip. Figure 5 illustrates the changes in cell size during the experimental protocol. Exposure to the hyposmolar superfusate induced a myocyte swelling that was completely reversible on switching back to the isosmolar superfusate for 5 min. Ip was determined after switching back to isosmolar superfusate at the end of the 5-min period. To control for a possible time-dependent Na+-K+ pump run-down, control myocytes were superfused with isosmolar solution for 11 min after the whole cell configuration was established before Ip was determined. The protocols for exposure to superfusates and measurement of Ip are illustrated in Fig. 6A. In eight myocytes briefly exposed to hyposmolar solution, the mean Ip measured after switching back to isosmolar solution was 0.33 ± 0.02 pA/pF. The mean Ip of nine myocytes exposed to isosmolar solution only was 0.27 ± 0.03 pA/pF. The difference was not statistically significant. These results suggest that stimulation of Ip by cell swelling is rapidly reversible with restoration of extracellular osmolarity and cell size.


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Fig. 5.   Reversibility of osmotically induced cell swelling in patch-clamped cardiac myocyte. Shown is a photomicrograph of a patch-clamped myocyte during sequence of exposure to isosmolar (A) and hyposmolar solution (B) and during reexposure to isosmolar solution (C). Calibration bars indicate myocyte width under basal isosmolar conditions.



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Fig. 6.   Reversibility of osmotically induced Ip stimulation. A: protocol used to examine role of protein kinase C in reversing swelling-induced Ip stimulation. B: effect of staurosporine (Stauro) on reversibility of swelling-induced Ip stimulation. Open bars and solid bars, results of protocol without and with staurosporine, respectively, in patch pipette. Hypo, hyposmolar. No. in parentheses is no. of experiments in each group.

To explore the role of kinases in restoring Ip to control levels, we included 10 nM staurosporine in all pipette solutions. The experimental protocols were otherwise identical to those illustrated in Fig. 6A. The mean Ip of eight myocytes transiently exposed to the hyposmolar superfusate was 0.44 ± 0.03 pA/pF, whereas the mean Ip of seven myocytes exposed to isosmolar solution was 0.24 ± 0.02 pA/pF. The difference was statistically significant. The experiments examining the effect of transient swelling on Ip are summarized in Fig. 6B. Figure 6B illustrates that pump stimulation is rapidly reversible and that the reversibility can be blocked by a potent but nonspecific kinase inhibitor. A separate series of experiments performed with bisindolylmaleimide I in patch pipette solutions gave results similar to those with staurosporine, implicating PKC in the mechanism allowing the rapid reversal of Ip on the reversal of cell swelling.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Role of transmembrane ion fluxes during myocyte swelling. We have previously reported that Ip in rabbit cardiac myocytes is stimulated by exposure to hyposmolar superfusates (32). This effect was observed when [Na]pip was near the physiological level of intracellular Na+ concentration, whereas there was no effect when [Na]pip was high. We concluded that the stimulation was due to an increase in the apparent affinity of the pump for intracellular Na+ rather than to an influx of extracellular Na+ and secondary pump stimulation. In the present study, we have directly ruled out any effect of Na+ influx during myocyte swelling by demonstrating that swelling activates Ip even in the absence of extracellular Na+.

A study of patch-clamped guinea pig ventricular myocytes has found that exposure to hyposmolar superfusates had no consistent effect on Ip when [Na]pip was near physiological intracellular levels, whereas it increased Ip when [Na]pip was high (27). Because these findings were not compatible with our previously published results, we considered differences in experimental protocols that might account for the discrepancy. The Ip of guinea pig myocytes was measured by using superfusates that were nominally Ca2+ free and by using pipette solutions containing 10 mM EGTA to buffer cytosolic Ca2+ (27), whereas we used Ca2+-containing superfusates and 5 mM EGTA in pipette solutions in our earlier study (32). In the present study we measured Ip in nominally Ca2+-free superfusates and we used 5 mM EGTA in pipette solutions. As in our previous study, exposure to hyposmolar superfusates stimulated Ip when [Na]pip was low but had no effect when [Na]pip was high. We conclude that transmembrane fluxes of Ca2+ are not involved in the mechanism activating the pumps of myocytes exposed to hyposmolar solutions and that differences in extracellular Ca2+ and intracellular Ca2+ buffering do not account for the discrepancy between guinea pig and rabbit myocytes in the [Na]pip dependence of the pump response to swelling.

Site of effect of myocyte swelling on the Na+-K+ pump. Exposure of myocytes to hyposmolar solutions shifted the Ip-Vm relationship in a negative direction (Fig. 2B), suggesting that myocyte swelling affects an electrogenic step in the pump cycle. Voltage dependence is thought to arise, at least in part, from the interaction of the transported ligands with the pump in access channels within the electrical field of the membrane (see Ref. 22 for review). At extracellular sites, the interaction of Na+ and K+ with the pump within access channels contributes to the slope of the Ip-Vm relationship in a positive and a negative direction, respectively. Experimentally, the voltage dependence of the pump arising from the interaction of Na+ with extracellular sites can be eliminated by using Na+-free superfusates. Unless the K+ concentration is very low, the voltage dependence arising from the interaction of K+ with extracellular sites is also eliminated in such Na+-free solutions (22). In the present study, swelling-induced pump stimulation persisted when myocytes were superfused with Na+-free superfusates containing 5.6 mM K+, suggesting that it is not one of the voltage-dependent steps at the extracellular sites that is stimulated by cell swelling.

The voltage dependence of the Na+-K+ pump cycle is also thought to arise at the intracellular sites for Na+ binding. Two Na+ are believed to bind in a voltage-independent manner to sites near the cytoplasmic surface. At these sites, Na+ competes with K+ for binding. Voltage dependence appears to arise from the highly selective binding of a third Na+ in an access channel within the membrane dielectric. At positive or negative membrane voltages, the concentration of Na+ within the channel is expected to increase or decrease, respectively. These changes in Na+ concentrations will cause an increase in Ip, with a shift of Vm in a positive direction. It is conceivable that cell swelling alters the depth of the Na+ access channel. A decrease in depth might account for the shift in the Ip-Vm relationship in the negative direction, as shown in Fig. 2. The absence of an effect of cell swelling on Ip when [Na]pip was 80 mM supports this speculation because the binding of Na+ to intracellular sites is expected to be independent of access channel depth when intracellular Na+ concentration is high (22).

Messenger cascade linking cell volume to pump activity. We examined the effect of including staurosporine and bisindolylmaleimide I in the pipette solution because a previous study had indicated that the mechanical stretch of cardiac myocytes activates PKC (24). The drugs did not abolish the increase in Ip induced by cell swelling. This might be taken to indicate that PKC does not regulate sarcolemmal Na+-K+ pump activity. However, it should be noted that total PKC activity, rather than the activity of specific isoforms, was measured in the study of the effect of mechanical stretch of myocytes (24). Because the effects of PKC are believed to be isoform specific (see Ref. 18 for review), it is reasonable to think that the isoforms activated by stretch have no effect on Ip. Previous studies have indicated that PKC is activated during osmotically induced cell shrinkage (29). We found that staurosporine and bisindolylmaleimide I prevented the return of Ip to control values after reexposure of the osmotically swollen myocyte to the isosmolar solution (Fig. 6B). These findings suggest that the PKC isoform(s) activated by cell shrinkage regulates the Na+-K+ pump in cardiac tissue.

Tyrosine kinase has been demonstrated to be activated within seconds when cardiac myocytes and intestinal epithelial cells are exposed to osmotic swelling and mechanical stretch (24, 30). Such activation has been implicated as an essential step in the activation of volume-sensitive ion conductances and membrane transport pathways (28, 30). Because tyrphostin A25 and LY-294002 abolished pump stimulation induced by cell swelling in the present study, a messenger pathway involving tyrosine kinase is implicated. Tyrosine kinase-dependent pathways have been reported to stimulate the pump in previous studies (8, 21). Activation of receptor tyrosine kinase by insulin and epidermal growth factor thus stimulates Na+-K+-ATPase activity in rat proximal convoluted tubules (8) and in cultured rat skeletal muscle cells (21). As in the present study, stimulation only occurred at low intracellular Na+ levels and had no effect at high Na+ levels. Finally, in support of the role of tyrosine kinase in pump activation by cell swelling, it should be noted that the insulin exposure of cardiac myocytes has an effect on the Ip-Vm relationship (13) virtually identical to the effect of swelling in the present study (Fig. 2B).

The differential effect of 10 nM and 1 µM okadaic acid suggests that PP1 rather than PP2A is involved in the messenger cascade linking cell swelling to Na+-K+ pump stimulation. Similar findings for the activation of the pump in cultured rat skeletal muscle by insulin have been reported (21). PP1 induced the dephosphorylation of pump molecules, and it was suggested that this dephosphorylation caused pump activation. It is reasonable to think that a similar mechanism is involved in the activation of the pump by cell swelling in the present study. However, this cannot be firmly concluded because we did not directly measure the phosphorylation of pump molecules. Figure 6 indicates that pump stimulation develops within 3 min of the onset of swelling. If this stimulation is mediated by dephosphorylation, we can conclude that messenger mechanisms involving the activation of tyrosine kinase, PI3K, and protein phosphatase in intact cardiac myocytes allow the dephosphorylation of pump molecules within this time span. Our model, however, does not allow a detailed kinetic analysis of the presumed pump dephosphorylation.

Swelling-induced pump stimulation was rapidly reversible, as indicated by Fig. 6. The reversal was blocked by kinase inhibition. Previous studies have indicated that isolated Na+-K+-ATPase can be directly phosphorylated by protein kinase A and PKC (1, 9). Direct phosphorylation of Na+-K+ pump molecules by a protein kinase would offer the simplest explanation for the reversal of pump stimulation on the return to a normal cell volume, although we cannot rule out the possibility that the phosphorylation of intermediate pump-regulatory proteins is involved. Taken together, the effect of the sequential exposure of cardiac myocytes to solutions of different osmolarities, in combination with the use of inhibitors of phosphatases and kinases, supports the suggestion that Na+-K+ pump activity in intact cells can be rapidly regulated by a balance between the effects of kinases and phosphatases on the pump molecules. The experimental model developed in the present study offers a convenient tool for the study of these processes.


    ACKNOWLEDGEMENTS

This study was supported by a grant from 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: D. Whalley, Cardiology Dept., Royal North Shore Hospital, Pacific Highway, St. Leonards, NSW 2065, Australia.

Received 13 August 1998; accepted in final form 1 February 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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