Na+ influx and Na+-K+ pump activation during short-term exposure of cardiac myocytes to aldosterone

Anastasia S. Mihailidou1, Kerrie A. Buhagiar1,2, and Helge H. Rasmussen1,2

Department of Cardiology, 1 Royal North Shore Hospital, and 2 University of Sydney, Sydney, New South Wales 2065, Australia

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

To examine the effect of aldosterone on sarcolemmal Na+ transport, we measured ouabain-sensitive electrogenic Na+-K+ pump current (Ip) in voltage-clamped ventricular myocytes and intracellular Na+ activity (aiNa) in right ventricular papillary muscles. Aldosterone (10 nM) induced an increase in both Ip and the rate of rise of aiNa during Na+-K+ pump blockade with the fast-acting cardiac steroid dihydroouabain. The aldosterone-induced increase in Ip and rate of rise of aiNa was eliminated by bumetanide, suggesting that aldosterone activates Na+ influx through the Na+-K+-2Cl- cotransporter. To obtain independent support for this, the Na+, K+, and Cl- concentrations in the superfusate and solution of pipettes used to voltage clamp myocytes were set at levels designed to abolish the inward electrochemical driving force for the Na+-K+-2Cl- cotransporter. This eliminated the aldosterone-induced increase in Ip. We conclude that in vitro exposure of cardiac myocytes to aldosterone activates the Na+-K+-2Cl- cotransporter to enhance Na+ influx and stimulate the Na+-K+ pump.

ion transport; intracellular sodium; sodium-potassium-two chloride cotransport; mineralocorticoid receptor; cell membrane

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE KIDNEY IS regarded as the classical target organ for aldosterone. However, aldosterone can also bind with high affinity to other organs, including the heart in rat (23), rabbit (18), and humans (17). A functional role for aldosterone in the heart has been suggested by a study demonstrating aldosterone-induced cellular uptake of Na+ in isolated rat cardiac myocytes maintained under tissue-culture conditions. This, in turn, activates synthesis of alpha 1- and beta 1-subunits of the sarcolemmal Na+-K+ pump. These effects occur with a considerable delay and can only be demonstrated ~6-12 h after the onset of exposure (15). In several noncardiac tissues, aldosterone has been shown to have effects on membrane Na+ transport that develop with a much shorter latency.

Aldosterone has regulatory effects on the plasmalemmal Na+/H+ exchanger of amphibian (22) and canine kidney cells (27) with a latency of 10-20 min and of human lymphocytes with a latency of 1-2 min (28, 29). The effects on intracellular Na+ levels were not reported in the study on kidney cells. However, in the lymphocytes, exposure to aldosterone eliminated a spontaneous decline in the intracellular Na+ concentration that occurred in control cells. Aldosterone has also been reported to stimulate Na+-K+ pump-mediated 86Rb+ uptake in renal cortical collecting tubules isolated from rats (10). This effect was evident within the first 30 min of exposure, and it was concluded that the aldosterone-induced pump stimulation precedes any induction of synthesis of new pumps.

Although it is well established that aldosterone can regulate membrane Na+ transport in cardiac cells with long latency via a genomic effect (15), it is not known if effects of aldosterone on sarcolemmal Na+ transport can also occur with short latency. An effect with short latency is of interest because it would imply that aldosterone can modify functional properties of preexisting sarcolemmal transport mechanisms. We have therefore examined early effects of aldosterone on regulation of intracellular Na+ in cardiac myocytes. We used the whole cell patch-clamp technique to measure electrogenic Na+-K+ pump current (Ip) in ventricular myocytes, and we used the ion-sensitive microelectrode technique to measure intracellular pH (pHi) and intracellular Na+ activity (aiNa) in papillary muscles.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Male New Zealand White rabbits were used for the study. They 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 myocytes from either ventricle were isolated as described previously (14). They were stored at room temperature in modified Tyrode solution until used for experimentation. The solution contained (in mM) 140 NaCl, 5.6 KCl, 2.16 CaCl2, 0.44 NaH2PO4, 10 glucose, 1.0 MgCl2, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). The solution also contained 16 mg/l gentamicin to inhibit bacterial growth and 0.5% bovine serum albumin. It was titrated to a pH of 7.40 ± 0.01 at 35°C with 1 M NaOH. The Na+ concentration in the final titrated solution was 146 mM. The solution used subsequently when Ip was to be measured was of an identical composition in most experiments except that the bovine serum albumin was omitted. These solutions were warmed to 35°C. When indicated, additional superfusates used were designed to alter transmembrane electrochemical driving gradients.

The myocytes were voltage clamped using wide-tipped (4-5 µm) patch pipettes manufactured as described previously (14). Three different filling solutions were used. In an initial series of experiments, they contained (in mM) 70 potassium glutamate, 1 KH2PO4, 5 HEPES, 5 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 2 MgATP, 10 sodium glutamate, and 80 tetramethylammonium chloride (TMA-Cl). The solutions used in a second series of experiments were identical except that they were Na+ free and contained 90 mM TMA-Cl. The composition of a third filling solution was designed to eliminate the inward driving gradient for the Na+-K+-2Cl- cotransporter. The Cl- concentration in those solutions ([Cl-]pip) was increased to 150 mM by substituting the 70 mM potassium glutamate used in the initial series of experiments with 70 mM KCl. All solutions were titrated to a pH 7.2 at 22°C with KOH. The patch pipettes had resistances of 0.9-1.1 MOmega when filled with these solutions.

A 3 M KCl agar bridge to a Ag-AgCl reference electrode was used to minimize junction potentials. Off-set potentials were nulled as described previously (14). Myocytes were voltage clamped using the continuous single-electrode voltage-clamp mode of an Axoclamp 2A amplifier supported by AxoTape or pCLAMP software (Axon Instruments, Foster City, CA). Ip was identified as the shift in holding current induced by 50 µM ouabain. We have previously shown that ouabain in this concentration causes nearly complete inhibition on the Na+-K+ pump in rabbit cardiac myocytes under a variety of experimental conditions (13, 14). Reported currents are normalized for membrane capacitance. Details of the experimental setup and of the experimental protocols used to measure membrane capacitance and Ip have been described previously (30).

Measurement of aiNa and pHi. Conventional voltage-sensitive microelectrodes were made from filamented borosilicate glass tubing and had resistances of 11-22 MOmega when filled with 3 M KCl. Ion-sensitive microelectrodes, based on liquid sensors, were manufactured from unfilamented borosilicate glass tubing as described previously (30). Na+-sensitive microelectrodes were calibrated using the reciprocal dilutions method (26). H+-sensitive microelectrodes were calibrated in solutions containing 150 mM KCl, 10 mM NaCl, and 10 mM HEPES. The solutions were titrated to a pH of 6.5 or 7.5 with 1 M NaOH to determine the slope. The slopes of our Na+- and H+-sensitive electrodes and the selectivity of the Na+-sensitive electrodes against Ca2+ have been reported previously (14). Calibration of electrodes was performed after impalements in all experiments. In a subset of experiments, we calibrated the electrodes both before and after impalements. There was no difference in their performance.

Reagents and chemicals. TMA-Cl was "purum" grade and was purchased from Fluka. All other chemicals were "analytical" grade and were purchased from BDH. Ouabain and aldosterone (Sigma Chemical, St. Louis, MO) were added to superfusates from a stock solution of ethanol. The final concentration of ethanol in superfusates was <0.1%. The aldosterone receptor in rabbit heart has a dissociation constant of 0.25 nM (18). Unless indicated otherwise, we used aldosterone in a concentration of 10 nM to facilitate fast binding to the receptor. Dihydroouabain (DHO) and potassium canrenoate (Sigma Chemical) were dissolved directly in superfusates. Bumetanide was dissolved in dimethyl sulfoxide (DMSO; BDH). The final concentration of DMSO in the superfusate was 0.5%. In agreement with a previous study on cardiac myocytes (12), DMSO in this concentration had no effect on Ip. Tetrodotoxin (TTX; Sigma Chemical) was dissolved in modified Tyrode solution to achieve a final concentration of 10 µM.

Statistical analysis. Results are expressed as means ± SE. Statistical comparisons were made using unpaired Student's t-test, one-way analysis of variance followed by a Tukey's test, and repeated measures analysis of variance. Differences were regarded as statistically significant when P < 0.05.

    RESULTS
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Abstract
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Materials & Methods
Results
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References

Effect of aldosterone on aiNa. In the first series of experiments, we measured aiNa before and immediately after exposure of isolated papillary muscles to aldosterone. Papillary muscles were initially superfused with aldosterone-free Tyrode solution. We identified stable, satisfactory recordings of both voltage-sensitive and ion-sensitive electrodes according to previously published criteria (14). We then switched to a superfusate that contained 10 nM aldosterone. We did not observe any detectable change in aiNa over the initial 10-min period after the onset of superfusion of aldosterone. We next examined the rate of rise in aiNa on blockade of Na+ extrusion via the Na+-K+ pump. Control papillary muscles and papillary muscles exposed to aldosterone were superfused with 500 µM DHO. We exposed the tissue to aldosterone for ~20 min to ensure binding before we superfused DHO. Figure 1 shows electrode recordings during superfusion of DHO in a representative experiment. The papillary muscle used in this experiment was not exposed to aldosterone. The aiNa recorded immediately before exposure to DHO was 9.9 ± 0.7 mM in seven papillary muscles exposed to aldosterone while aiNa in seven controls was 7.7 ± 0.7 mM. The difference was statistically significant. Figure 2 summarizes the DHO-induced rise in aiNa in control papillary musles and in papillary muscles exposed to aldosterone. The rate of increase in aiNa upon exposure to DHO was significantly greater for papillary muscles exposed to aldosterone than for controls.


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Fig. 1.   Measurement of steady-state intracellular Na+ activity (aiNa) and rate of increase in aiNa during Na+-K+ pump inhibition. Traces show potentials of voltage-sensitive (Vm) and Na+-sensitive (VNa) microelectrodes and subtracted difference potential (Vdiff) during superfusion of a papillary muscle from a control rabbit with dihydroouabain (DHO)-free solution and solution containing 500 µM DHO.


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Fig. 2.   Mean increase in aiNa during inhibition of Na+ efflux with DHO in 7 control papillary muscles (open circle ), 7 papillary muscles superfused with aldosterone alone (bullet ), and 6 papillary muscles superfused with both aldosterone and bumetanide (down-triangle). Increase in aiNa was significantly greater for papillary muscles exposed to aldosterone than for controls. Bumetanide significantly inhibited effect of aldosterone on rate of increase in aiNa (repeated measures analysis of variance).

Effects of aldosterone on pHi. Because aldosterone has been reported to activate the Na+/H+ exchanger in noncardiac tissue with a short latency, we examined whether exposure to aldosterone can alter pHi. When stable recordings with voltage- and pH-sensitive electrodes were achieved, we switched from an aldosterone-free superfusate to a superfusate containing aldosterone. To facilitate detection of an early change, we used aldosterone in a concentration of 100 nM in these experiments rather than the 10 nM used in all other experiments in the study. Figure 3 shows a recording of pHi during superfusion of aldosterone. We could not detect any change in pHi during ~15 min of exposure to aldosterone of this or four other papillary muscles. In an independent series of experiments, we examined the effect on pHi of a longer duration of exposure to aldosterone. Six papillary muscles were exposed to 100 nM aldosterone before we impaled microelectrodes. By the time pHi was determined, they had been exposed to aldosterone for 47-60 min. The mean pHi was 6.83 ± 0.09. Mean pHi in six controls was 6.95 ± 0.03. The difference was not statistically significant. These results suggest that aldosterone does not activate the Na+/H+ exchanger in cardiac tissue.


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Fig. 3.   Effect of acute exposure to aldosterone on electrode potentials during measurement of intracellular pH. Traces show membrane potential (Vm) and difference potential (Vdiff) between voltage-sensitive and pH-sensitive electrodes before and after superfusion of 100 nM aldosterone.

Effect of aldosterone on Ip. To examine the effect of aldosterone on Ip, we superfused isolated myocytes with control Tyrode solution or solution containing 10 nM aldosterone. The myocytes were patch clamped using pipettes containing 10 mM Na+, a concentration near physiological intracellular levels. The Na+, K+, and Cl- concentrations of pipette solutions and superfusates are summarized in Table 1, condition A. When the whole cell configuration had been established, we measured the membrane capacitance. We then voltage clamped the myocytes at -40 mV and switched the superfusate to Tyrode solution containing 1 mM Ba2+. Superfusion of Ba2+ induced an inward shift in membrane current. When a new stable holding current was recorded, we identified Ip as the additional shift induced by 50 µM ouabain. Typical recordings of holding currents in experiments using similar experimental protocols have been published previously (14, 30). Myocytes were exposed to aldosterone for 27-90 min or exposed to aldosterone-free Tyrode solution. The duration of exposure to aldosterone was dependent on the time it took to achieve the whole cell configuration. Mean Ip of myocytes exposed to aldosterone and mean Ip of control myocytes are included in Fig. 4. Aldosterone induced a significant increase in Ip.

                              
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Table 1.   Transmembrane ionic gradients during the different experimental conditions and calculated free energy


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Fig. 4.   Effect of transsarcolemmal Na+ and Cl- gradients on aldosterone-induced pump stimulation. Pipette Na+ concentration was 10 mM in all experiments, and transmembrane K+ gradient was maintained constant. Subscripts o and i indicate concentrations in superfusate and pipette solutions, respectively. Open bars show mean Na+-K+ pump current (Ip) in control myocytes, and solid bars show mean Ip of myocytes exposed to 10 nM aldosterone. Numbers in parentheses indicate number of myocytes studied for each experiment. Statistically significant differences between Ip of myocytes exposed to aldosterone and Ip of corresponding control myocytes are indicated by asterisks.

We performed additional experiments using Na+-free patch pipette filling solutions. Myocytes were exposed to aldosterone for 20-60 min or exposed to aldosterone-free solution. In agreement with previous studies (14, 21, 30), a small ouabain-sensitive current could be identified in control myocytes when patch pipette filling solutions were Na+ free. However, mean Ip of myocytes exposed to aldosterone was substantially larger. The mean Ip values from the two groups of myocytes are included in Fig. 5. The difference between them was statistically significant.


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Fig. 5.   Effect of Na+ channel blockade, Na+-K+-2Cl- cotransport inhibition, and aldosterone receptor blockade on aldosterone-induced pump stimulation. Pipette solutions were Na+ free, and myocytes were superfused with control solutions or solutions containing either 10 nM aldosterone (Ald) only or aldosterone combined with 10 µM tetrodotoxin (TTX) or 10 µM bumetanide (Bumet) or 1 µM potassium canrenoate (K-can). Numbers in parentheses indicate number of myocytes studied. Asterisks indicate statistically significant differences between mean Ip values (analysis of variance).

We designed a separate series of experiments to examine if brief exposure to aldosterone can stimulate Ip. We used Na+-free solutions in these experiments, since it appears that the stimulatory effect of aldosterone on Ip is more marked when pipette filling solutions are Na+ free than when they contain 10 mM Na+. The whole cell configuration was established before the myocytes were exposed to 10 nM aldosterone, and we measured Ip 5-10 min after superfusion of aldosterone was started. The time elapsed from when the whole cell configuration was established until we measured Ip was similar for control myocytes and myocytes exposed to aldosterone. Mean Ip of 12 myocytes exposed to aldosterone was 0.09 ± 0.03 pA/pF. This was not significantly larger than the mean Ip of seven myocytes not exposed to aldosterone (0.05 ± 0.01 pA/pF).

Transmembrane ionic gradients, aldosterone, and Ip. Aldosterone induced an increase in the rate of rise in aiNa during exposure to DHO. This suggests that the increase in Ip of myocytes exposed to aldosterone may be due to enhanced transmembrane Na+ influx and secondary pump stimulation by a rise in the intracellular Na+ concentration. To examine this possibility, we manipulated the transmembrane electrochemical gradient for Na+ in patch-clamped myocytes. We voltage clamped myocytes at 0 mV using pipettes with filling solutions that contained 10 mM Na+. The whole cell configuration was established in the Tyrode solution that contained 146 mM Na+. For measurement of Ip, we then switched to a superfusate in which the Na+ concentration had been reduced to 16 mM by isosmotic substitution of NaCl with TMA-Cl. The Na+, K+, and Cl- concentrations of pipette solutions and superfusates at the time we measured Ip are summarized in Table 1, condition B. In initial experiments, myocytes died within a few minutes of switching to the low-Na+ superfusate. This problem was eliminated when we used a low-Na+ superfusate that was nominally Ca2+ free. Myocytes were exposed to aldosterone for 21-108 min or exposed to aldosterone-free solution. The dependence of the aldosterone-induced increase in Ip on the transsarcolemmal electrochemical gradient for Na+ is illustrated in Fig. 4. It is apparent that an aldosterone-induced increase in Ip is converted to a decrease when the transmembrane gradient for Na+ is reduced. This, in turn, indicates that the aldosterone-induced increase in Ip is due to enhanced transsarcolemmal Na+ influx rather than a direct stimulatory effect of aldosterone on the pump.

Aldosterone-induced Na+ influx could occur through voltage-sensitive Na+ channels, through the Na+/H+ exchanger, or through the Na+-K+-2Cl- cotransporter. Because aldosterone did not shift pHi in an alkaline direction, the Na+/H+ exchanger is unlikely to be involved. To examine if Na+ channels are involved, we examined the effect of TTX on the aldosterone-induced increase in Ip. We patch clamped myocytes exposed to aldosterone for at least 20 min using Na+-free pipette filling solutions. After we had established the whole cell configuration, we switched to a superfusate that contained 10 µM TTX. To facilitate binding to Na+ channels, we then activated the channels by applying six depolarizing pulses to +20 mV for 20 ms from a holding potential of -80 mV. The interval between each depolarization was 2 s. We then measured Ip at a holding potential of -40 mV. Mean Ip of myocytes exposed to both TTX and aldosterone was similar to mean Ip of myocytes exposed to aldosterone only (Fig. 5).

Because the Na+-K+-2Cl- cotransporter is a mechanism for Na+ uptake in cardiac myocytes that potentially has a large transport capacity (9, 16), we examined whether eliminating the driving gradients for the cotransporter could abolish the aldosterone-induced increase in Ip. The Na+, K+, and Cl- concentrations of pipette solutions and superfusates at the time Ip was measured are included in Table 1, condition C. The free energy (Gnet) expected to be associated with Na+-K+-2Cl- cotransport under these and the two other conditions used in measurement of Ip are also included in Table 1. We calculated Gnet according to Aickin and Brading (1), assuming that activity coefficients are identical in the cytosol and patch pipette filling solutions and that complete control of intracellular ionic concentrations by patch clamping were achieved. The myocytes were voltage clamped at -40 mV at the time Ip was measured. Myocytes were exposed to aldosterone for 5-27 min or exposed to aldosterone-free solution. The mean Ip values have been included in Fig. 4. Figure 4 and the values for Gnet in Table 1 illustrate how the aldosterone-induced increase in Ip is eliminated when the net inward driving force for the Na+-K+-2Cl- cotransporter is eliminated.

Effect of bumetanide on Ip and aiNa. The effect of increasing the Cl- concentration in the patch pipette filling solution on the aldosterone-induced increase in Ip suggests that aldosterone induces an increase in transsarcolemmal Na+ influx through the Na+-K+-2Cl- cotransporter. We examined the effect of bumetanide to obtain independent support for this. We voltage clamped myocytes at -40 mV using Na+-free patch pipettes. They were superfused with Tyrode solution containing 10 nM aldosterone and 10 µM bumetanide for 10-70 min before we measured Ip. The effect of bumetanide on the aldosterone-induced increase in Ip is illustrated in Fig. 5. Bumetanide blocked the aldosterone-induced increase in Ip.

We examined the effect of bumetanide on Ip in the absence of aldosterone in an independent series of control experiments. We exposed seven myocytes to 10 µM bumetanide. The myocytes were voltage clamped at -40 mV with patch pipettes containing 10 mM Na+. Mean Ip was 0.29 ± 0.03 pA/pF. This is similar to the mean Ip of 0.32 ± 0.03 pA/pF measured under experimental conditions that were identical except that the myocytes were not exposed to bumetanide. We conclude that 10 µM bumetanide does not directly inhibit the Na+-K+ pump.

We also examined whether bumetanide could abolish the effect of aldosterone on the increase in aiNa after pump blockade with DHO. We exposed six papillary muscles to a superfusate that contained both 10 µM bumetanide and 10 nM aldosterone. When stable impalements had been established, we switched to a superfusate that contained DHO. The time course of aiNa upon exposure to DHO was virtually superimposable on the time course for control papillary muscles (Fig. 2). Bumetanide alone had no effect on the increase in aiNa on exposure to DHO.

Effect of potassium canrenoate on Ip. We performed experiments to examine the effect of blockade of the mineralocorticoid receptor on the aldosterone-induced increase in Ip. We superfused myocytes with Tyrode solution containing 10 nM aldosterone and 1 µM potassium canrenoate. They were voltage clamped at -40 mV with Na+-free patch pipettes. The mean Ip, measured after 21- to 45-min exposure to aldosterone and potassium canrenoate, has been included in Fig. 5. Mean Ip was significantly smaller than mean Ip of myocytes exposed to aldosterone only. We conclude that potassium canrenoate blocked the effect of aldosterone on Ip. We examined the effect of potassium canrenoate on Ip in the absence of aldosterone in an independent series of control experiments. We exposed myocytes to 1 µM potassium canrenoate. They were voltage clamped at -40 mV with patch pipettes containing 10 mM Na+. Mean Ip of five myocytes was 0.38 ± 0.06 pA/pF. This is similar to the mean Ip of 0.32 ± 0.03 pA/pF measured under experimental conditions that were identical except that the myocytes were not exposed to potassium canrenoate (included in Fig. 4). We conclude that the effect of 1 µM potassium canrenoate to abolish the aldosterone-induced increase in Ip is not due to direct inhibition of the Na+-K+ pump by potassium canrenoate.

    DISCUSSION
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Abstract
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Materials & Methods
Results
Discussion
References

Rationale for experimental model. We used ion-sensitive microelectrodes to demonstrate that aldosterone enhances Na+ influx in isolated papillary muscles. The whole cell patch-clamp technique was then used to explore the mechanism for this effect in single cardiac myocytes. When this technique is used to study the Na+-K+ pump, it is usually a key objective to achieve good control of intracellular ionic concentrations. Although such control to a large extent can be achieved in cardiac myocytes (11, 21), measured pump currents may exhibit some deviation from the current predicted by the Na+ concentration in pipette solutions because a concentration gradient exists between the tip of the patch pipette and the cytoplasm (20) or between the cytoplasm and a diffusion-restricted subsarcolemmal space (5).

When transsarcolemmal Na+ fluxes are large relative to our ability to control the Na+ concentration at intracellular pump sites, the relationship between pipette Na+ concentration and Ip does not accurately reflect Na+ activation of the pump (5, 20). Experimentally, the errors arising from this problem can be minimized by reducing non-pump transsarcolemmal Na+ fluxes with appropriate design of experimental solutions and voltage-clamp protocol (11, 21). The ouabain-sensitive current recorded when using Na+-free patch pipette filling solutions gives an indication of the degree of control of intracellular Na+ that can be achieved. Nakao and Gadsby (21) reported that this current amounts to ~0.04 pA/pF, a value that is only ~3.5% of the current recorded at saturating levels of pipette Na+ concentration. They demonstrated that the current was largely due to electrogenic pumping of Na+ leaked into cells through the sarcolemma. An increase in this leak would be expected to induce an increase in the measured Ip (20). When using Na+-free patch pipettes, we measured an Ip of 0.05 ± 0.01 pA/pF in control myocytes. Exposure to aldosterone induced a large increase in Ip. It is unlikely that a direct effect of aldosterone on the pump induced this increase in Ip, since steady-state aiNa was significantly increased rather than decreased after aldosterone superfusion. In addition, the aldosterone-induced increase in Ip was converted to a decrease when the transmembrane gradient for Na+ was reduced (Fig. 4). Induction of transsarcolemmal influx of Na+ and the development of a concentration gradient between intracellular pump sites and the patch pipette filling solution offers the simplest explanation for the increase in Ip recorded with exposure to aldosterone.

Mechanism for aldosterone-induced Na+ influx. Transsarcolemmal Na+ influx could occur through Na+ channels, through the Na+/H+ exchanger, or through the Na+-K+-2Cl- cotransporter. The Na+ channels are expected to be inactivated at the holding potentials we used (-40 or 0 mV), and exposure of myocytes to TTX had no effect on the aldosterone-induced increase in Ip. In addition, the aldosterone-induced increase in Ip was converted to a decrease when the [Cl-]pip was increased without any change in the transsarcolemmal electrochemical gradient for Na+ (Fig. 4). These findings indicate that an effect on passive entry of Na+ through an ion channel cannot be involved in the aldosterone-induced increase in Ip.

The Na+/H+ exchanger in cardiac cells is expected to be largely inactivated at the level of pH we used in patch pipette solutions (19). In agreement with this, the pHi in intact papillary muscles in this study was ~0.2-0.3 units lower than the pH of pipette solutions. An ~0.3 alkaline shift of pHi has been reported to be induced by aldosterone in amphibian kidney cells (22). A similar activation of the exchanger could mediate transsarcolemmal Na+ influx in cardiac myocytes. However, we found no evidence for an aldosterone-induced shift in pHi to support this. Experimental manipulation of transsarcolemmal ionic gradients in patch-clamped myocytes also indicate that entry of Na+ through the Na+/H+ exchanger is not involved in the aldosterone-induced increase in Ip. In experiments performed using a pipette Na+ concentration of 10 mM, the aldosterone-induced increase in Ip was converted to a decrease when extracellular Na+ concentration was low or [Cl-]pip was high (Fig. 4). This conversion occurred despite the thermodynamic driving force for Na+ via the Na+/H+ exchanger being inwardly directed in all experiments.

It is apparent from Table 1 that the thermodynamic driving force for Na+ via the Na+-K+-2Cl- cotransporter is outwardly directed when the extracellular Na+ concentration was low. An aldosterone-induced activation of the cotransporter may have reduced the intracellular Na+ concentration. This, in turn, could account for the aldosterone-induced decrease in Ip (Fig. 4). In experiments performed using a high [Cl-]pip, Na+-K+-2Cl- cotransport is expected to be reduced because intracellular Cl- inhibits the cotransporter, an effect which is independent of the direction of the net driving force (2). The calculated net driving force was outwardly directed but small. It is uncertain if this small driving force can account for the aldosterone-induced decrease in Ip. In this regard, it is important to note that Gnet was calculated assuming that intracellular and extracellular activity coefficients are identical, an assumption that cannot be verified. In addition, one might speculate that an aldosterone-activated cotransporter is not perfectly ion selective and that the calculated driving force for transport therefore is not completely accurate. Regardless of the mechanism, the conversion from an aldosterone-induced increase in Ip to a decrease when using a high [Cl-]pip (Fig. 4) is consistent with activation of Na+-K+-2Cl- cotransport.

We used bumetanide to obtain independent support for the conclusion that aldosterone activates the Na+-K+-2Cl- cotransporter. Bumetanide in the concentration we used appears to be specific for the cotransporter in rabbit cardiac myocytes (6, 7). Bumetanide abolished both the aldosterone-induced increase in Ip of isolated myocytes (Fig. 5) and the aldosterone-induced increase in the rate of rise of aiNa during pump inhibition with DHO (Fig. 2). This supports the conclusion that aldosterone activates the Na+-K+-2Cl- cotransporter in rabbit heart. It is interesting to note that bumetanide alone had no inhibitory effect on the increase in aiNa or on Ip of patch-clamped myocytes. This suggests that, unless activated, the cotransporter does not contribute to Na+ uptake under the in vitro conditions we used.

Aldosterone receptors and the Na+-K+-2Cl- cotransporter. A two-step model for the effect of aldosterone on cells has been proposed (28). According to this model, aldosterone binds to both a receptor on the surface of cell membranes and to an intracellular receptor. Binding to the surface receptor is characterized by a short latency (minutes) of the response and by a lack of sensitivity to canrenone. The receptor has been linked to changes in electrolyte transport. In contrast, the response to binding to the intracellular receptor occurs with long latency (hours) and is blocked by canrenone. A genomic effect of binding to this receptor is usually invoked. We found that Na+-K+-2Cl- cotransport was activated with a relatively short latency, but we were not able to demonstrate activation within the few minutes of latency reported for activation of Na+/H+ exchange in human lymphocytes (28, 29). In our study, the effect of aldosterone within the first few minutes could only be studied if we achieved the whole cell configuration before exposure was started. Intracellular Ca2+ is expected to be clamped at a low level by the EGTA in the pipette filling solution. This may have prevented detection of a response to aldosterone, since release of Ca2+ from intracellular stores and changes in cytosolic levels of Ca2+ are thought to be involved in the messenger cascade for nongenomic effects (24). The classical mineralocorticoid receptor blocker potassium canrenoate abolished the aldosterone-induced increase in Ip (Fig. 5). Within the framework of the two-step model for aldosterone effects, this suggests an involvement of the intracellular receptor, although the latency of the response in our study implies a nongenomic mechanism. A detailed exploration of this possibility was beyond the scope of the study.

Functional significance of aldosterone-induced Na+-K+-2Cl- cotransport. The presence of aldosterone receptors in the heart (17, 18, 23) suggests that aldosterone plays a role in regulation of cardiac function. However, this role is poorly understood at present (3). Our study has demonstrated that aldosterone enhances influx of Na+ through the cotransporter. This may reduce extrusion of Ca2+ through the Na+/Ca2+ exchanger and hence enhance contractility. In support of these speculations, a positive inotropic effect of aldosterone on isolated cat papillary muscles has been reported (25). Diastolic properties of cardiac muscle might also be altered by Na+-K+-2Cl- cotransport activation. Activation of the cotransporter increases cardiac myocyte volume, and this may decrease cardiac compliance and hence impair ventricular filling (8). The sarcolemmal Na+-K+-2Cl- cotransporter is inhibited by atrial natriuretic factor (8) and, as demonstrated in this study, activated by aldosterone. Levels of both hormones are elevated in heart failure (4, 31). If the effects demonstrated in vitro also occur in vivo, one might speculate that an adverse effect of aldosterone may be compensated for by release of atrial natriuretic factor. Such interaction would have important therapeutic implications.

    ACKNOWLEDGEMENTS

This study was supported by National Heart Foundation of Australia Grant G93S3842 and by the North Shore Heart Research Foundation.

    FOOTNOTES

Address for reprint requests: H. H. Rasmussen, Dept. of Cardiology, Royal North Shore Hospital, Pacific Highway, St. Leonards, Sydney, NSW 2065, Australia.

Received 31 March 1997; accepted in final form 26 September 1997.

    REFERENCES
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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