Voltage-dependent stimulation of the Na+-K+ pump by insulin in rabbit cardiac myocytes

Peter S. Hansen, Kerrie A. Buhagiar, David F. Gray, and Helge H. Rasmussen

Department of Cardiology, Royal North Shore Hospital, and Department of Medicine, University of Sydney, St. Leonards, New South Wales 2065, Australia


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Insulin enhances Na+-K+ pump activity in various noncardiac tissues. We examined whether insulin exposure in vitro regulates Na+-K+ pump function in rabbit ventricular myocytes. Pump current (Ip) was measured using the whole-cell patch-clamp technique at test potentials (Vms) from -100 to +60 mV. When the Na+ concentration in the patch pipette ([Na]pip) was 10 mM, insulin caused a Vm-dependent increase in Ip. The increase was ~70% when Vm was at near physiological diastolic potentials. This effect persisted after elimination of extracellular voltage-dependent steps and when K+ and K+-congeners were excluded from the patch pipettes. When [Na]pip was 80 mM, causing near-maximal pump stimulation, insulin had no effect, suggesting that it did not cause an increase in membrane pump density. Effects of tyrphostin A25, wortmannin, okadaic acid, or bisindolylmaleimide I in pipette solutions suggested that the insulin-induced increase in Ip involved activation of tyrosine kinase, phosphatidylinositol 3-kinase, and protein phosphatase 1, whereas protein phosphatase 2A and protein kinase C were not involved.

sodium-potassium-adenosinetriphosphatase; whole-cell voltage clamp; second messengers


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

STUDIES ON RAT ADIPOCYTES and isolated renal proximal convoluted tubule indicate that insulin stimulates the membrane Na+-K+ pump by increasing its sensitivity to intracellular Na+ (Nai), although there is no effect on maximal pump rate (5, 14, 16). Binding of Nai to the pump occurs at two relatively nonselective negatively charged sites at the cytoplasmic surface and at a third, highly selective, uncharged site located some distance inside the electrical field of the membrane (20, 21). Because Na+ competes with K+ for binding to the sites at the cytoplasmic surface, insulin might increase the overall sensitivity of the Na+-K+ pump to Na+ by increasing the Na+/K+ selectivity ratio. Detection of this should be dependent on intracellular K+ (Ki) and independent of membrane voltage. In contrast, because binding of Na+ to the third pump site is highly selective for Na+, an effect of insulin at these sites should be independent of Ki. However, because binding occurs within the membrane dielectric, such an effect should depend on membrane voltage.

The techniques used in the previous studies in adipocytes and renal tubule cells (5, 14, 16) do not allow selective experimental control of Ki and membrane voltage, and no information is available regarding the Ki and voltage dependence of the effect of insulin on the pump in these cells. We have used the whole-cell patch-clamp technique to study electrogenic Na+-K+ pump current (Ip) in single isolated cardiac myocytes. When wide-tipped patch pipettes are used, cardiac myocytes are small enough to allow dialysis of the intracellular compartment and accurate control of membrane voltage. However, because of a high pump density of cardiac myocytes, the Ip is large enough to be identified with high resolution (7). We found that insulin increases Ip when pipette solutions contain Na+ in a concentration near physiological intracellular levels. This effect is dependent on membrane voltage but is independent of Ki. Pump stimulation was abolished by including pharmacological blockers of the insulin tyrosine kinase receptor, phosphatidylinositol 3-kinase (PI 3-kinase) and serine/threonine protein phosphatase 1 (PP-1) in the patch pipette solution.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of single ventricular myocytes. Single ventricular myocytes were isolated from male New Zealand White rabbits as described previously (10). After isolation they were maintained at room temperature and used on the day of isolation only. Pump currents were always measured within 10 h of excising the heart.

Solutions. After isolation, myocytes were stored in Krebs-Henseleit buffer (KHB) solution containing the following (in mM): 130 NaCl, 4.8 KCl, 1.2 MgSO4, 1.2 NaH2PO4, 25 NaHCO3, 12.5 glucose, 0.5 CaCl2, and 1.0% BSA. The solution was bubbled with 5% CO2-95% O2 for 1 h before use to achieve a pH of 7.40 ± 0.05 at 35°C. Myocytes were transferred to a 350-µl tissue bath for patch-clamp studies. The bath was perfused at a rate of 5 ml/min with modified Tyrode solution warmed to 35°C. The solution contained the following (in mM): 140 NaCl, 5.6 KCl, 2.16 CaCl2, 0.44 NaH2PO4, 10 glucose, 1 MgCl2, and 10 HEPES. It was titrated with 1 M NaOH to a pH of 7.40 ± 0.01 at 35°C. This solution was used in all experiments until the whole-cell configuration was established and membrane capacitance had been measured. For measurement of Ip we switched to a solution that was identical to the solution used while the whole-cell configuration was established, except that it was nominally Ca2+-free and contained 0.2 mM CdCl2 to limit Na+-Ca2+ exchange. It also contained 2 mM BaCl2 to reduce membrane K+ conductance. Additional variations in the composition of superfusates are indicated in RESULTS.

Myocytes were voltage clamped with wide-tipped patch pipettes (4-5 µm) made as described previously (33). For the measurement of Ip at a fixed holding potential of -40 mV we filled pipettes with a solution containing the following (in mM): 70 potassium glutamate, 1 KH2PO4, 5 HEPES, 5 EGTA, 2 MgATP, and 90 sodium glutamate plus tetramethylammonium chloride (TMA-Cl). The solution was titrated with 1 M KOH to pH 7.05 ± 0.01 at 35°C. In experiments designed to examine the relationship between Ip and test potential (Vm) we blocked time-dependent K+ currents by including tetraethylammonium chloride (TEA-Cl) in pipette solutions and replacing potassium glutamate with CsCl. The solution contained the following (in mM): 10 sodium glutamate, 1 KH2PO4, 5 HEPES, 5 EGTA, 2 MgATP, 60 TMA-Cl, 20 TEA-Cl, 70 CsCl, and 50 aspartic acid. The solution was titrated with 1 M HCl to a pH of 7.05 ± 0.01 at 35°C. To examine the Ip-Vm relationship with a high pipette Na+ concentration ([Na]pip), the solution contained 80 mM sodium glutamate, 65 mM CsCl, and 45 mM aspartic acid. We omitted TMA-Cl in this solution to maintain osmotic balance. To examine the Ip-Vm relationship using K+- and K+-congener (Cs+)-free pipettes we eliminated K+- and Cs+-containing compounds. Osmotic balance was maintained by including TMA-Cl.

Insulin exposure protocols. In initial studies myocytes were exposed to insulin in the tissue bath both before and after we achieved the whole-cell configuration. The duration of this exposure was 15-40 min. Ca2+ was included in the superfusate during the initial period of exposure to insulin. We used insulin in a nominal concentration of 100 mU/ml. Because it is expected to adhere to glass we measured the concentration in the perfusion chamber. It was 20 ± 3 mU/ml (n = 12), a concentration reported to cause near-maximal pump stimulation (31). A second protocol was developed to examine the role of Ca2+ in insulin-induced pump stimulation and to allow blockade of intracellular messengers by drugs included in pipette solutions before cells were exposed to insulin. In these experiments insulin was only superfused for the period between establishment of the whole-cell configuration and measurement of Ip (10-12 min). The superfusate was Ca2+-free during that period.

Measurement of Ip. Ip was identified as the shift in holding current induced by 100 µM ouabain, a concentration known to completely block the Na+-K+ pump in rabbit ventricular myocytes (10). Membrane currents were recorded through the use of the continuous single-electrode voltage-clamp mode of an Axoclamp-2A amplifier and Axotape and 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 (10, 33). Ip is reported normalized for membrane capacitance unless specified otherwise.

Drugs and chemicals. "Purum" grade TMA-Cl was purchased from Fluka (Switzerland). All other chemicals were analytical grade and purchased from Sigma (St. Louis, MO). Tyrphostin A25, tyrphostin A63, wortmannin, bisindolylmaleimide I, sodium salt okadaic acid, and methyl ester okadaic acid were purchased from Calbiochem (La Jolla, CA). Sodium salt okadaic acid was dissolved in water. All other second-messenger inhibitors and their inactive controls were dissolved in 0.01-0.04% DMSO. Ouabain was purchased from Sigma.

Statistical analysis. Results are expressed as mean ± SE. Student's t-test for paired and unpaired data was used. Dunnett's test was used when the same control group was used for more than one comparison. P < 0.05 is regarded as significant in all comparisons. Slopes of Ip-Vm relationships were compared using linear regression. Nonlinear regression was used to fit the Hill equation to data.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of insulin on Ip. In an initial series of experiments we examined the effect on Ip when myocytes were exposed to 100 mU/ml insulin after they were transferred to the tissue bath. Insulin was included in the Ca2+-containing superfusate we used in all experiments in this study while we sought to establish the whole-cell configuration, and in the Ba2+-containing solution we used at the time Ip was measured. The latter solution was Ca2+-free and contained Cd2+. The duration of exposure to insulin before Ip was measured depended on the time required to achieve the whole-cell configuration. The range of exposure was 15-40 min. To reduce variability in Ip arising if rundown of pump activity were to occur, we always exposed myocytes to ouabain with the same latency of 10-12 min after the whole-cell configuration had been established. Myocytes were voltage clamped at -40 mV during this period and during the subsequent exposure to ouabain. We have previously published representative recordings of ouabain-induced shifts in holding currents (33). Mean Ip, measured using a [Na]pip of 10 mM, was 0.33 ± 0.01 pA/pF in 6 control myocytes and 0.43 ± 0.01 pA/pF in 13 myocytes exposed to insulin. The difference was statistically significant.

To examine whether insulin can stimulate the pump in a patch-clamped myocyte we included insulin only in the Ca2+-free, Ba2+- and Cd2+-containing superfusates used after the whole-cell configuration had been established. In these experiments myocytes were exposed to insulin for ~10-12 min. Control myocytes not exposed to insulin were maintained in the whole-cell configuration and superfused with Ca2+-free, Ba2+- and Cd2+-containing solution for the same period before Ip was measured. Mean Ip, measured using a [Na]pip of 10 mM, was 0.31 ± 0.01 pA/pF in six control myocytes and 0.41 ± 0.03 pA/pF in six myocytes exposed to insulin. The difference was statistically significant. This indicates that the messenger pathways linking the insulin receptor to the Na+-K+ pump can be activated within 10-12 min and that the pathways are functional despite the nominal absence of extracellular Ca2+ and buffering of cytosolic Ca2+ by the EGTA contained in pipette filling solutions.

Effect of insulin on voltage dependence of the Na+-K+ pump. To examine whether insulin affects the voltage dependence of Ip, we patch-clamped myocytes with the use of a [Na]pip of 10 mM. Pipette filling solutions included CsCl and TEA-Cl in these experiments. After we achieved the whole-cell configuration myocytes were superfused for 10-12 min with Ca2+-free solution that contained insulin or was insulin-free. The myocytes were voltage clamped at -40 mV during this time. We then applied voltage steps of 320-ms duration in 20-mV increments to test potentials ranging from -100 to +60 mV. Each test potential was bracketed by a return to the -40-mV holding potential for 2 s. The voltage-clamp protocol was applied three times before and three times after myocytes were exposed to 100 µM ouabain and averaged steady-state holding currents were determined. Figure 1A shows an example of currents recorded in a myocyte exposed to insulin. The criteria for identifying ouabain-induced shifts in currents at each test potential have been described previously (9). The Ip-Vm relationships for myocytes exposed to insulin and for control myocytes have been summarized in Fig. 1B. The Ip-Vm relationships for both groups of cells were nearly linear throughout the voltage range examined. Control myocytes had a clearly positive slope of the Ip-Vm relationship, whereas the slope appeared much less steep for myocytes exposed to insulin. The relationships were compared by linear regression. There was a statistically significant difference between their slopes. We conclude that insulin stimulates the Na+-K+ pump and reduces its voltage dependence when [Na]pip is 10 mM.


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Fig. 1.   A: holding currents pre- and post-ouabain exposure of a myocyte superfused with insulin-containing superfusates for ~10 min when Na+ concentration in the pipette ([Na]pip) was 10 mM. The ouabain-induced shift in currents was used to identify pump current (Ip) at each test potential (Vm). B: effect of insulin on Vm dependence of absolute Ip using [Na]pip of 10 mM. Ip-Vm relationships are averaged for 16 myocytes (5 rabbits) exposed to insulin-free superfusates (open circles) and 14 myocytes (5 rabbits) exposed to insulin-containing superfusates (filled circles).

We next examined whether insulin can be shown to stimulate Ip when [Na]pip is high. Pipette filling solutions were identical to those used in the experiments shown in Fig. 1 except that [Na]pip was increased to 80 mM, whereas the concentration of TMA-Cl was decreased by 60 mM and CsCl and aspartic acid by 5 mM each to maintain osmotic balance. The Ip-Vm relationships for myocytes exposed to insulin and for control myocytes have been summarized in Fig. 2. The mean levels of Ip for the two groups of cells were similar at all test potentials. This indicates that the voltage-dependent effect of insulin on Ip is eliminated when intracellular Na+ is at levels expected to nearly saturate the pump's binding sites.


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Fig. 2.   Effect of insulin on Vm dependence of absolute Ip using [Na]pip of 80 mM. Ip-Vm relationships are averaged for 8 myocytes (3 rabbits) exposed to insulin-free superfusates (open circles) and 7 myocytes (3 rabbits) exposed to insulin-containing superfusates (filled circles). Comparison with Fig. 1A indicates that increase in [Na]pip from 10 to 80 mM caused an approximately four- to sevenfold increase in mean Ip in control myocytes over the voltage range examined. There was no effect of insulin on Ip.

The interaction of Na+ and K+ with extracellular pump sites is the major contributor to the voltage dependence of Ip. This voltage dependence is eliminated if the Na+ concentration in the superfusate is low and the K+ concentration ([K]o) is maintained at or above physiological levels (see Ref. 25 for review). To examine whether the effect of insulin on the pump's voltage dependence is due to an effect at extracellular sites we determined the Ip-Vm relationship of myocytes through the use of a superfusate that was Ca2+-free and contained 1.5 mM Na+, 140 mM N-methyl-D-glucamine chloride (NMGC), 5.6 mM K+, and 0.2 mM Cd2+. The composition of the superfusate was adopted from a previous study on the voltage dependence of the Na+-K+ pump in cardiac myocytes (18). The pipette filling solution included 10 mM Na+ and 71 mM Cs+. Figure 3 shows the Ip-Vm relationship of control myocytes and myocytes exposed to insulin. Insulin stimulated Ip at negative test potentials, whereas it had no effect at positive potentials. The Ip-Vm relationships were compared by linear regression. There was a statistically significant difference between their slopes. We also normalized the Ip-Vm relationships in Fig. 3 to the Ip recorded at 0 mV (not shown) and compared the normalized relationships. There was a significant difference between their slopes. We conclude that a functional change in the interaction of Na+ and K+ with extracellular sites does not account for the effect of insulin on the pump's voltage dependence.


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Fig. 3.   Effect of insulin on Vm dependence of Ip when extracellular Na+ concentration ([Na]o) was 1.5 mM, extracellular K+ concentration ([K]o) 5.6 mM, and [Na]pip 10 mM. Ip-Vm relationships are averaged for 10 myocytes (3 rabbits) exposed to insulin-free superfusates (open circles) and 15 myocytes (3 rabbits) exposed to insulin-containing superfusates (filled circles).

Ki and effect of insulin on Ip. To examine the effect of Ki on the insulin-induced pump stimulation we used K+- and K+-congener (Cs+)-free pipette filling solutions. We maintained osmotic balance by replacing CsCl with TMA-Cl. The solution contained 10 mM Na+ and 20 mM TEA-Cl. The Ip-Vm relationship was determined in a Ca2+-free superfusate in which NaCl had been replaced with NMGC. [K]o was 15 mM. Figure 4 shows the Ip-Vm relationships for control myocytes and myocytes exposed to insulin. Insulin significantly stimulated Ip at negative test potentials and reduced the slope of the Ip-Vm relationship. The slope of the normalized Ip-Vm relationship (not shown) was also significantly altered by insulin.


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Fig. 4.   Effect of insulin on Vm dependence of Ip when pipette solution was K+- and K+-congener (Cs+)-free and contained 10 mM Na+. Superfusate composition eliminated an effect from extracellular voltage-dependent steps of the pump cycle. Ip-Vm relationships are averaged for 11 myocytes (3 rabbits) exposed to insulin-free superfusates (open circles) and 10 myocytes (3 rabbits) exposed to insulin-containing superfusates (filled circles).

Effect of insulin on the apparent K+ affinity. Because in vivo insulin deficiency is reported to affect the K+ affinity of the Na+-K+ pump (19), we next examined whether exposure of myocytes to insulin in vitro has an effect on the pump's sensitivity to extracellular K+. We used a [Na]pip of 80 rather than 10 mM in these experiments for two reasons. Insulin increases the turnover rate of the Na+-K+ pump when [Na]pip is 10 mM (Fig. 1B). The dependence of the pump's apparent K+ affinity on turnover rate (11) is therefore expected to induce a change in affinity even if insulin has no effect on the pump's interaction with extracellular K+. Use of a [Na]pip of 80 mM was also expected to facilitate detection of small pump currents when the [K]o was low.

After obtaining the whole-cell configuration we voltage clamped myocytes at -40 mV and switched to a superfusate that was Ca2+ free and contained 2 mM Ba2+, 0.2 mM Cd2+, and 5.6 mM K+. We maintained the myocyte in this solution for 10 min. If the holding currents were stable we then switched to a K+-free superfusate to inactivate the Na+-K+ pump. Ip was subsequently identified as the shift in holding current induced by reexposure to K+. We have previously shown that such a K+-induced shift in holding current is free from contamination by non-pump K+-sensitive currents (9). Each myocyte was exposed in random order to Ca2+-free superfusates with different K+ concentrations ranging from 0.5 to 15 mM. Each exposure to K+ was bracketed by exposure to the K+-free superfusate to ensure a return to the baseline holding current. To detect rundown of Ip we exposed eight randomly selected myocytes to the first [K]o used in the protocol and measured the K+-induced shift in holding current. There was no evidence for rundown in the time needed to complete the experimental protocol (25-32 min). An illustration of the experimental protocol and representative traces of holding currents have been published previously (9). We included insulin in all Ca2+-free superfusates used after the whole-cell configuration was established. The relationship between [K]o and Ip is shown in Fig. 5. We fitted the Hill equation to the data as described previously (9). The [K]o for half-maximal pump activation was 2.3 mM for control myocytes and 2.7 mM for myocytes exposed to insulin, whereas Hill coefficients were 1.17 and 1.28, respectively. There were no significant differences between these.


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Fig. 5.   Effect of [K]o on Ip in 12 myocytes (4 rabbits) exposed to insulin-free superfusates (open circles) or 7 myocytes (3 rabbits) exposed to insulin-containing superfusates (filled circles). Ip at each [K]o has been normalized to the current recorded when [K]o was 5.6 mM rather than to cell capacitance. This eliminates variability of data arising from cell capacitance determination.

Messenger pathway for the effect of insulin on Ip. Because all cellular effects of insulin are mediated by the insulin receptor tyrosine kinase (1), we first examined the effect of tyrosine kinase inhibition. We measured Ip at a fixed holding potential of -40 mV through the use of a Ca2+-free superfusate that included 140 mM Na+ and 5.6 mM K+. Pipette filling solutions included 10 mM Na+ and 71 mM K+. We inhibited tyrosine kinase by including 100 µM tyrphostin A25 in pipette filling solutions. Figure 6 summarizes mean Ip measured in myocytes through the use of drug- and solvent-free control filling solutions containing tyrphostin A25, its inactive analog tyrphostin A63, or DMSO used only to dissolve the drugs. Myocytes were either exposed or not to insulin. To allow time for dialysis of the compounds into the intracellular compartment we did not expose myocytes to insulin until the whole-cell configuration had been established for ~3-5 min. Figure 6 shows that DMSO and tyrphostin A63 had no effect on mean Ip, whereas tyrphostin A25 caused a small but statistically significant decrease in mean Ip compared with mean Ip of other cells not exposed to insulin. Tyrphostin A25 completely abolished the insulin-induced increase in mean Ip while this increase persisted in the presence of the inactive analog, tyrphostin A63.


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Fig. 6.   Effect of tyrosine kinase inhibition on response of Ip to insulin. Mean Ip from myocytes exposed to drug-free, tyrphostin A25- (100 µM), tyrphostin A63- (100 µM), or DMSO-containing (0.04%) pipette solutions and insulin-free superfusates are compared with mean Ip from myocytes exposed to drug-free, tyrphostin A25- (100 µM) or tyrphostin A63-containing (100 µM) pipette solutions and insulin-containing superfusate (con, control; ins, insulin). Numbers in parentheses indicate number of myocytes in each group. Experiments were obtained from three or more rabbits in each group.

Because the insulin receptor tyrosine kinase activates PI 3-kinase we next examined the effect of inhibition of PI 3-kinase on the insulin-induced increase in Ip. We included 1 µM wortmannin in patch pipette solutions and measured Ip in the presence or absence of insulin. The results are summarized in Fig. 7. Mean Ip of myocytes exposed to wortmannin was significantly lower than mean Ip of myocytes voltage clamped using wortmannin-free pipette solutions and wortmannin completely abolished the insulin-induced increase in mean Ip. The results summarized in Figs. 6 and 7 suggest that the increase in Ip induced by insulin is a specific insulin receptor-mediated effect.


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Fig. 7.   Effect of phosphatidylinositol 3-kinase inhibition on response of Ip to insulin. Mean Ips from myocytes exposed to drug-free or wortmannin-containing (wort, 1 µM) pipette solutions in insulin-free superfusates are compared with mean Ip from myocytes exposed to identical pipette solutions and insulin-containing superfusate (con, control; ins, insulin). Numbers in parentheses indicate number of myocytes in each group. Experiments were obtained from three rabbits in each group.

Because both protein kinase C (PKC) and serine/threonine phosphatases have been reported to mediate cellular effects of insulin, we also examined the effect of the PKC inhibitor bisindolylmaleimide I and the phosphatase inhibitor okadaic acid. In one series of experiments we included 1 µM bisindolylmaleimide I in pipette solutions. As shown in Fig. 8 this had no effect on basal Ip and it did not block the insulin-induced increase in mean Ip.


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Fig. 8.   Effect of protein kinase C inhibition on response of Ip to insulin. Mean Ip from myocytes exposed to drug-free or bisindolylmaleimide I-containing (B-I, 1 µM) pipette solutions in insulin-free superfusates are compared with mean Ip from myocytes exposed to identical pipette solutions and insulin-containing superfusate (con, control; ins, insulin). Numbers in parentheses indicate number of myocytes in each group. Experiments were obtained from three rabbits in each group.

To examine the effect of protein phosphatase inhibition we initially included 1 µM okadaic acid in pipette solutions. Okadaic acid in this concentration inhibits both PP-1 and serine/threonine protein phosphatase 2A (PP-2A). As shown in Fig. 9, 1 µM okadaic acid abolished the insulin-induced increase in mean Ip. The inactive okadaic acid analog, okadaic acid methyl ester, had no effect on Ip of control cells nor on the insulin-induced increase in Ip. Because PP-1 and PP-2A exhibit a differential sensitivity to okadaic acid we also examined the effect of including 10 nM okadaic acid in pipette solutions. This had no effect on the insulin-induced increase in mean Ip (Fig. 9).


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Fig. 9.   Effect of protein phosphatase 1 and 2A inhibition on response of Ip to insulin. Mean Ip from myocytes exposed to drug-free, okadaic acid sodium salt- (OA-N, 1 µM or 10 nM) or okadaic acid methyl ester-containing (OA-M, 1 µM) pipette solutions in insulin-free (con) superfusates are compared with mean Ips from myocytes exposed to identical pipette solutions and insulin-containing superfusate (ins). Numbers in parentheses indicate number of myocytes in each group. Experiments were obtained from three or more rabbits in each group.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The effect of insulin on the cardiac Na+-K+ pump has been examined previously by measuring ouabain-sensitive 86Rb+-uptake in ventricular slices or isolated myocytes. Some studies have reported that insulin stimulates 86Rb+ uptake (30, 32), whereas others have reported that insulin has no effect (4, 12). The membrane potential was not measured in any of the studies and depolarization induced by the tissue or cell isolation procedures or induced by the experimental solutions used to study 86Rb+ uptake cannot be ruled out. As illustrated by Fig. 1B, an effect of insulin on pump function might be difficult to demonstrate in depolarized cells. Conversely, if insulin does cause an increase in 86Rb+ uptake the increase does not necessarily indicate that insulin directly stimulates pump function. Insulin can enhance Na+-influx and cause an increase in Nai at least in noncardiac cells. The increase in Nai, in turn, stimulates pump function (27). Such indirect pump stimulation is difficult to rule out using the 86Rb+ uptake technique. In our study insulin-induced pump stimulation could be demonstrated even when the transmembrane electrochemical gradient for Na+ was directed outward (Figs. 3 and 4). This clearly indicates that the effect of insulin on the pump is not secondary to a rise in Nai.

Insulin and voltage dependence of the Na+-K+ pump. We are aware of only one previous study that has examined whether insulin-induced Na+-K+ pump stimulation is voltage dependent (6). Insulin-induced Na+-K+ pump stimulation was studied in rat renal tubular cells with a physiological membrane potential and in cells depolarized by exposure to 20 mM extracellular Rb+ or 3 mM Ba2+. It was concluded that the effect of insulin was voltage independent. The range of membrane voltage achieved in the experiments was not measured (6). However, the resting membrane potential of renal tubular cells is only approximately -50 to -60 mV and a substantial component of this is generated by the electrogenic Na+-K+ pump current (22). Interference with ion channel currents by extracellular Rb+ or Ba2+ should cause a shift in the membrane potential much smaller than the range from -100 to +60 mV used in this study and a voltage-dependent effect of insulin on pump activity is expected to be difficult to detect.

The voltage dependence of the insulin-induced pump stimulation we demonstrated has implications for the site of action of insulin on the pump. Release of Na+ and binding of K+ at extracellular sites are believed to be the major steps contributing to the voltage dependence in the Na+-K+ pump cycle (17, 23). The effect of insulin on the Ip-Vm relationship in our study persisted after voltage dependence arising at extracellular pump sites was expected to have been eliminated (Figs. 3 and 4), indicating that insulin influences some other voltage-dependent step in the pump cycle.

Studies on isolated Na+-K+ ATPase in noncellular experimental systems have indicated that binding of cytosolic Na+ is weakly voltage dependent, a finding consistent with binding within a shallow access channel (3, 8, 21, 28). The concentration of Na+ within such a channel should increase with a shift of Vm in a positive direction and this increase should stimulate pump activity. It follows that binding of Na+ to pump sites within the channel contributes to a positive slope of the Ip-Vm relationship.

The effect of insulin on the Ip-Vm relationship was demonstrated when we used a [Na]pip of 10 mM. According to an access channel model there is kinetic equivalence between ligand concentration and membrane voltage (26). The effect of insulin on the Ip-Vm relationship might therefore be eliminated when the intracellular Na+ concentration is at a level expected to saturate binding sites at all membrane voltages. In agreement with this expectation insulin had no effect on the Ip-Vm relationship when [Na]pip was increased to 80 mM (Fig. 2). Definitive support for an effect of insulin on binding of Na+ within an access channel might in principle be obtained by studying transient currents arising from charge movements in the Na+ limb of the Na+-K+ pump cycle in response to membrane voltage steps. However, transient currents arising from shallow internal access channels have not been reported (25), and are probably impossible to record without contamination by larger currents arising from interaction of Na+ with the pump in an external channel.

Mechanism for the effect of insulin on Ip. Insulin has been reported to induce translocation of a latent pool of Na+-K+ pumps to the cell membrane in some tissues (see Ref. 15 for example). However, the absence of an effect of insulin on Ip, measured when [Na]pip is expected to nearly saturate intracellular binding sites, in the present study suggests that insulin does not induce an increase in the membrane Na+-K+ pump density in cardiac myocytes. Similar conclusions have been reached from studies on rat skeletal muscle (2) and renal tubular cells (6). An effect of insulin on functional properties of preexisting pumps must be invoked to account for the increase in Ip in our study.

The intracellular messenger pathways linking the insulin receptor to effector molecules are incompletely understood, in part because available pharmacological blockers of the pathways are not absolutely specific (29). We used such blockers to examine whether the pharmacological profile of the effect of insulin in our study is similar to that reported in previous studies on cellular responses to insulin. The effect of insulin on Ip was blocked by an inhibitor of tyrosine kinase. We are not aware of evidence indicating that the Na+-K+ pump can be phosphorylated on tyrosine residues, and the effect of tyrosine kinase inhibition on Ip almost certainly reflects interruption of the upstream part of the messenger cascade only. The effect of insulin was also blocked by wortmannin. Wortmannin inhibits PI 3-kinase and mitogen-activated protein kinase (29). Because insulin-induced activation of mitogen-activated protein kinase has nuclear effects (29), it is unlikely to be involved in the short-latency response in our study. This suggests that PI 3-kinase is involved, a kinase implicated in most cellular responses to insulin. Bisindolylmaleimide I had no effect on the insulin-induced increase in Ip. This indicates that classical and novel PKC isoforms are not involved. However, the atypical isoform PKCzeta is relatively insensitive to pharmacological blockers (13), and we cannot rule out a role of PKCzeta . Unfortunately this isoform can only be blocked when PKC inhibitors are used in very high, nonspecific concentrations. The concentration-dependent effect of okadaic acid suggests that PP-1 may be involved in mediating the effect of insulin on Ip. Studies on rat myotubes have suggested that insulin causes activation of PP-1 and that PP-1 in turn dephosphorylates Na+-K+ pump alpha -subunits on serine/threonine residues (24). PP-1 may induce dephosphorylation of pump units in cardiac myocytes exposed to insulin in a similar manner. Our findings are consistent with a functional change in the binding site believed to be located within the membrane dielectric. However, this scheme is speculative, and currently available techniques do not allow the resolution in structure-function relationships required to obtain definitive support for it.


    ACKNOWLEDGEMENTS

This study was supported by the North Shore Heart Research Foundation. P. S. Hansen received a Postgraduate Medical Research Scholarship from the National Heart Foundation of Australia, K. A. Buhagiar was supported by National Heart Foundation of Australia Grant No. G96S4589, and D. F. Gray received a National Health & Medical Research Council Medical Postgraduate Research Scholarship.


    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, NSW 2065, Australia (E-mail: helger{at}mail.med.usyd.edu.au).

Received 3 May 1999; accepted in final form 18 October 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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