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Sarcolemmal cation channels and exchangers modify the increase in intracellular calcium in cardiomyocytes on inhibiting Na⁺-K⁺-ATPase

Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, and Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada

Submitted 3 January 2007 ; accepted in final form 21 February 2007

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Although inhibition of the sarcolemmal (SL) Na⁺-K⁺-ATPase is known to cause an increase in the intracellular concentration of Ca²⁺ ([Ca²⁺]_i) by stimulating the SL Na⁺/Ca²⁺ exchanger (NCX), the involvement of other SL sites in inducing this increase in [Ca²⁺]_i is not fully understood. Isolated rat cardiomyocytes were treated with or without different agents that modify Ca²⁺ movements by affecting various SL sites and were then exposed to ouabain. Ouabain was observed to increase the basal levels of both [Ca²⁺]_i and intracellular Na⁺ concentration ([Na⁺]_i) as well as to augment the KCl-induced increases in both [Ca²⁺]_i and [Na⁺]_i in a concentration-dependent manner. The ouabain-induced changes in [Na⁺]_i and [Ca²⁺]_i were attenuated by treatment with inhibitors of SL Na⁺/H⁺ exchanger and SL Na⁺ channels. Both the ouabain-induced increase in basal [Ca²⁺]_i and augmentation of the KCl response were markedly decreased when cardiomyocytes were exposed to 0–10 mM Na⁺. Inhibitors of SL NCX depressed but decreasing extracellular Na⁺ from 105–35 mM augmented the ouabain-induced increase in basal [Ca²⁺]_i and the KCl response. Not only was the increase in [Ca²⁺]_i by ouabain dependent on the extracellular Ca²⁺ concentration, but it was also attenuated by inhibitors of SL L-type Ca²⁺ channels and store-operated Ca²⁺ channels (SOC). Unlike the SL L-type Ca²⁺-channel blocker, the blockers of SL Na⁺ channel and SL SOC, when used in combination with SL NCX inhibitor, showed additive effects in reducing the ouabain-induced increase in basal [Ca²⁺]_i. These results support the view that in addition to SL NCX, SL L-type Ca²⁺ channels and SL SOC may be involved in raising [Ca²⁺]_i on inhibition of the SL Na⁺-K⁺-ATPase by ouabain. Furthermore, both SL Na⁺/H⁺ exchanger and Na⁺ channels play a critical role in the ouabain-induced Ca²⁺ increase in cardiomyocytes.

sodium-calcium exchanger; sodium-hydrogen exchanger; sarcolemmal Ca²⁺ transport

The critical role of NCX in the increase in [Ca²⁺]_i due to Na⁺-K⁺-ATPase inhibition has been demonstrated in cardiomyocytes isolated from NCX-knockout mice (31). Although various investigators (10, 44, 46) have also indicated the participation of other Ca²⁺-regulatory sites of both SL and sarcoplasmic reticulum (SR) membranes in the increase of [Ca²⁺]_i due to the inhibition of Na⁺-K⁺-ATPase, the results are controversial. In this regard, Santana et al. (37) have reported the influx of Ca²⁺ through cardiac Na⁺ channels after the inhibition of Na⁺-K⁺-ATPase in cardiomyocytes (slip-mode conductance), whereas Le Grand et al. (19) have shown an increase in L-type and T-type Ca²⁺ currents after the inhibition of Na⁺-K⁺-ATPase. Although some investigators have failed to observe the effect of cardiac glycosides on slip-mode conductance (28) and SL Na⁺ channels (1), others have reported a reduction in Ca²⁺ current after the inhibition of SL Na⁺-K⁺-ATPase (22). Likewise, some studies (32) have shown the specific binding of cardiac glycosides to the SR membrane and the activation of ryanodine receptors in the cardiac SR vesicles, whereas others (1) have denied the participation of SR Ca²⁺ stores in the ouabain-mediated increase in [Ca²⁺]_i. On the other hand, no information is available in the literature regarding the involvement of SL sites such as the SL Na⁺/H⁺ exchanger (NHE) and store-operated Ca²⁺ channels (SOC), which have been shown to regulate the [Ca²⁺]_i in cardiomyocytes (15, 34), in Ca²⁺ mobilization due to the inhibition of Na⁺-K⁺-ATPase.

Therefore, it appears that the roles of various Ca²⁺-regulatory sites of the SL and SR membranes in the action of cardiac glycosides in the heart are not clearly and fully understood. It should be pointed out that most investigators have targeted a single site at which to define the action of cardiac glycosides in Ca²⁺ mobilization in their studies, and thus a comprehensive investigation involving various SL and SR Ca²⁺-regulatory sites under the same experimental conditions is lacking.

The present study was focused on examining the role of different cation channels and exchangers in the regulation of [Ca²⁺]_i in both quiescent and KCl-depolarized cardiomyocytes on inhibiting Na⁺-K⁺-ATPase activity with ouabain, a known inhibitor of Na⁺-K⁺-ATPase (27). This isolated multicellular cardiomyocyte preparation has been employed previously (30, 33–36, 47) to examine the mechanisms of ATP-, low Na⁺-, phosphatidic acid-, NHE inhibition-, and -adrenoceptor stimulation-mediated increase in [Ca²⁺]_i. To confirm whether the effects of different concentrations of ouabain on [Ca²⁺]_i are associated with inhibition of Na⁺-K⁺-ATPase and an increase in [Na⁺]_i, Na⁺-K⁺-ATPase activity was measured in isolated SL vesicles and the fluorescence due to changes in [Na⁺]_i was measured in cardiomyocytes in the absence and presence of ouabain. To investigate the participation of Na⁺ in increasing the [Ca²⁺]_i due to ouabain, alterations in [Ca²⁺]_i were studied on exposing cardiomyocytes to NHE and Na⁺-channel inhibitors. Furthermore, some experiments were carried out in the absence of extracellular Na⁺ to obtain an understanding of the dependency of ouabain-mediated Ca²⁺ mobilization on Na⁺. The involvement of NCX in mediating the ouabain-induced increase in [Ca²⁺]_i under our experimental conditions was established by treating cardiomyocytes with NCX inhibitors. To gain understanding of the involvement of other SL sites in ouabain-induced Ca²⁺-mobilization, cardiomyocytes were treated with blockers of L-type Ca²⁺ channels, SL SOC, and SL Ca²⁺ pump before exposure to ouabain.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The use of animals in this study and all protocols were approved by the University of Manitoba Animal Care Committee in accordance with the standards of the Canadian Council on Animal Care.

Isolation of cardiomyocytes. Ventricular myocytes were isolated as described previously (36). In brief, male Sprague-Dawley rats (250–300 g) were anesthetized with a mixture of ketamine (90 mg/kg) and xylazine (9 mg/kg). The hearts were quickly excised, mounted on the Langendorff apparatus, perfused for 5 min with Ca²⁺-free buffer containing (in mM) 90 NaCl, 10 KCl, 1.2 KH₂PO₄, 5 MgSO₄, 15 NaHCO₃, 30 taurine, and 20 glucose, and gassed with 95% O₂-5% CO₂ at 37°C, pH 7.4. These hearts were then switched to the same perfusion medium containing 0.04% collagenase, 0.1% BSA, and 50 µM CaCl₂. At the end of a 25-min recirculation period, the hearts were removed from the cannula. The ventricles were cut into small pieces and were subjected to another 15-min digestion in a fresh collagenase solution containing 1% BSA gassed with a mixture of 95% O₂-5% CO₂ in a shaking water bath at 37°C. The ventricular fragments were triturated gently (twice per minute) with a plastic pipette. The cells from three to four harvests were combined and were filtered through a 200-µm nylon mesh. The cardiomyocytes were resuspended for 5 min in buffers containing gradually increasing extracellular Ca²⁺ (250, 500, and 750 µM) to a final concentration of 1 mM. The cell viability in all experimental groups was determined by using the trypan blue (Sigma-Aldrich, Oakville, ON, Canada) exclusion method. The unstained, stained, and total numbers of cells were counted by Neubauer chamber. The final cell suspension had 80–85% viable quiescent cardiomyocytes; 3–5% of cardiomyocytes were observed to beat spontaneously.

Spectrofluorometric measurement of [Ca²⁺]_i. Freshly isolated cardiomyocytes were incubated with 5 µM fura 2-AM for 40 min in a buffer (pH 7.4) containing (in mM) 90 NaCl, 10 KCl, 1.2 KH₂PO₄, 5 MgSO₄, 15 NaHCO₃, 30 taurine, 20 glucose, 1 CaCl₂, and 1% BSA. The cells were washed twice with the same solution to remove any extracellular dye. The final cell number in the cuvette was adjusted to 0.3 x 10⁶ cells/ml for all experimental groups. Alterations in the fluorescence intensity were monitored by a SLM DMX-1100 dual-wavelength spectrofluorometer (SLM Instruments, Urbana, IL) adjusted to an excitation wavelength of 340/380 nm, emission wavelength of 510 nm, integration time of 0.95 s, and resolution time of 1.0 s. The [Ca²⁺]_i level was calculated as described previously (33, 35, 36). In all experiments, treatment with different concentrations of ouabain was performed for 10 min inside the cuvette containing fura 2-loaded cells. No photobleaching of fluorometric recording was observed during this time period.

Treatments with different pharmacological agents for the modulation of [Ca²⁺]_i were performed by incubating the fura 2-loaded cells in the buffer containing the desired concentration of pharmacological agents for 10 min before the measurement of fluorescence in the presence and absence of ouabain. The concentrations of different pharmacological interventions used in the present study were selected on the basis of our previous experience with these agents (33, 34, 36). For examining the effect of different concentrations of Na⁺ on ouabain-mediated alterations in [Ca²⁺]_i, cardiomyocytes were suspended in Krebs-Henseleit buffer (pH 7.4) containing 70, 35, 10, and 0 mM extracellular Na⁺ for 10 min at room temperature, and the osmolarity of the solution was maintained by adding choline chloride as described earlier (30). Note that no change in cell viability was observed by treatment with various pharmacological interventions under incubation conditions. The increase in [Ca²⁺]_i at peak value due to KCl (30 mM), a depolarizing agent, was calculated as the net increase above the basal value in each experiment. The difference between the responses in the absence and presence of ouabain treatment was taken as the ouabain-induced increase in [Ca²⁺]_i.

Measurement of [Ca²⁺]_i in single cells by confocal microscopy. The measurement of [Ca²⁺]_i in single cardiomyocytes was measured by confocal microscopy. The cells were cultured overnight on glass-bottom culture dishes (MatTek, Ashland, MA) and then were loaded with 10 µM fluo 3-AM for 30 min. After two washes with buffer containing 1 mM Ca²⁺, the cells were scanned in time series. Alterations in the fluorescence intensity of fluo 3 were monitored by a confocal microscope (Nikon Eclipse TE2000-U; Nikon, Mississauga, ON, Canada) using a 488 nm argon laser, and the data were analyzed with the EZ C1 program (48).

Spectrofluorometric measurement of [Na⁺]_i. Freshly isolated cardiomyocytes were incubated with 10 µM SBFI-AM for 40 min. The changes in SBFI fluorescence intensity were monitored with a SLM DMX-1100 dual-wavelength spectrofluorometer adjusted to an excitation wavelength of 340/380 nm, emission wavelength of 510 nm, integration time of 0.95 s, and resolution time of 1.0 s. Treatments with different pharmacological agents for the modulation of [Na⁺]_i were performed by incubating the SBFI-loaded cells in the buffer containing the desired concentration of pharmacological agents for 10 min before the measurement of fluorescence in the presence and absence of ouabain.

Preparation of cardiac SL membrane and measurement of ATPase activities. To examine the effect of ouabain on Na⁺-K⁺-ATPase, SL was isolated from control hearts and the Na⁺-K⁺-ATPase activity was measured as described previously (41) with some modification. Briefly, 10 µg of SL vesicle were preincubated at 37°C with (in mM) 1.0 EGTA·Tris (pH 7.4), 5 NaN₃, 6 MgCl₂, 100 NaCl, 10 KCl, and 2.5 phosphoenolpyruvate plus 10 IU/ml pyruvate kinase. Phosphoenolpyruvate and pyruvate kinase were used as an ATP-regenerating system to maintain the concentration of ATP in the incubation medium. The reaction for measuring the total ATPase activity was started by adding 0.025 ml of 80 mM Tris·ATP, pH 7.4, and was terminated after 10 min with 0.5 ml of ice-cold 12% trichloroacetic acid. Mg²⁺-ATPase activity was estimated as the difference between the activities with and without Mg²⁺ (in the absence of Na⁺ and K⁺) in the medium. All measurements were carried out in duplicate. Na⁺-K⁺-ATPase activity was calculated as the difference between the total ATPase and Mg²⁺-ATPase activities.

Statistical analysis. Data are expressed as means ± SE. Statistical analysis was performed with MicroCal Origin version 6 (MicroCal Software, Northampton, MA). Differences between two groups were evaluated by Student's t-test. The data from more than two groups were evaluated by one-way ANOVA followed by the Newman-Keuls test. Values of P < 0.05 were considered statistically significant unless otherwise indicated in the text.

Drugs and chemicals. Ouabain, verapamil, diltiazem, vanadate, 5-(N-methyl-N-isobutyl) amiloride (MIA), Ni²⁺, lidocaine, tetrodotoxin (TTX), Gd³⁺, La³⁺, and SKF-96365 were purchased from Sigma-Aldrich, whereas KB-R7943 was purchased from Tocris Biosciences (Ellisville, MO). Fura 2-AM, fluo 3-AM, and SBFI-AM were purchased from Molecular Probes (Eugene, OR). Collagenase (type II, 265 U/mg) was purchased from Worthington Biochemical (Freehold, NJ). All other reagents were of analytical grade and purchased either from Sigma-Aldrich or Fisher Scientific (Fair Lawn, NJ).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Effect of ouabain on [Ca²⁺]_i in isolated cardiomyocytes. To examine the effect of ouabain on [Ca²⁺]_i, cardiomyocytes were exposed to different concentrations of ouabain (0.1–2 mM) inside the cuvette for 10 min before the addition of KCl (30 mM) and real-time tracings were plotted. It can be seen from the representative tracings in Fig. 1A that ouabain (1 mM) increased the basal [Ca²⁺]_i and augmented the KCl-mediated increase in [Ca²⁺]_i. As shown in Fig. 1, B–E, the increase in basal [Ca²⁺]_i and augmentation of KCl response were dependent on the concentration of ouabain over the range of 0.1–2 mM. In fact, it can be seen in Fig. 2 that a 10-min incubation period with ouabain (1 mM) produced maximal increases in both basal [Ca²⁺]_i and augmentation of KCl response, because no additional increase in [Ca²⁺]_i was observed after 20 min of treatment. Furthermore, treatment with ouabain for 1 and 5 min caused significantly less increase in [Ca²⁺]_i compared with 10 min (Fig. 2). Because submaximal increase in [Ca²⁺]_i was observed at 1 mM ouabain and the 10-min incubation period was sufficient to increase the [Ca²⁺]_i in the presence of ouabain, all further experiments were performed by using 1 mM ouabain for 10 min in the absence and presence of various pharmacological interventions.

View larger version (26K): [in this window] [in a new window]   Fig. 1. Modification of basal intracellular Ca2+ concentration ([Ca2+]i) and KCl-induced increase in [Ca2+]i in cardiomyocytes by different concentrations of ouabain. Cardiomyocytes were incubated with different concentrations of ouabain in cuvette for 10 min before the addition of 30 mM KCl. The ouabain-induced increase in basal [Ca2+]i was calculated by subtracting the value in the absence of ouabain from that in its presence. Similarly, ouabain-induced increase in KCl response was calculated by subtracting the respective value in the absence and presence of ouabain. A: representative tracing showing changes in [Ca2+]i due to ouabain before and after KCl. B: effect of different concentrations of ouabain on basal [Ca2+]i. C: ouabain-induced increase in basal [Ca2+]i. D: effect of different concentrations of ouabain on KCl-induced increase in [Ca2+]i. E: ouabain-induced augmentation of KCl-induced increase in [Ca2+]i. Values are means ± SE of 4 hearts. *P < 0.05 vs. value in the absence of ouabain.

View larger version (40K): [in this window] [in a new window]   Fig. 2. Modification of basal [Ca2+]i and KCl-induced increase in [Ca2+]i in cardiomyocytes by incubation with ouabain at different time periods. Cardiomyocytes were incubated with 1 mM ouabain in cuvette for 1–20 min before the addition of 30 mM KCl. The ouabain-induced increase in basal [Ca2+]i was calculated by subtracting the value in the absence of ouabain from that in its presence. Similarly, ouabain-induced increase in KCl response was calculated by subtracting the respective value in the absence and presence of ouabain. A and B: effect of ouabain on basal [Ca2+]i and ouabain-induced increase in basal [Ca2+]i, respectively, after 1, 5, 10, and 20 min incubation with ouabain. C and D: effect of ouabain on KCl-induced increase in [Ca2+]i and ouabain-induced augmentation of KCl-induced increase in [Ca2+]i, respectively, after 1, 5, 10, and 20 min incubation with ouabain. Values are means ± SE of 4 hearts. *P < 0.05 vs. value in the absence of ouabain.

View larger version (53K): [in this window] [in a new window]   Fig. 3. Effect of different concentrations of ouabain on basal and KCl-mediated increase in fluo 3 fluorescence in single cardiomyocytes measured by confocal microscopy. A: representative pictures showing the effect of different concentrations of ouabain (0–5 mM) on increase in basal fluo 3 fluorescence. B: representative pictures showing the effect of different concentrations of ouabain (0–5 mM) on KCl-induced increase in fluo 3 fluorescence. C: basal [Ca2+]i expressed by F/Fo ratio, where F is the intensity of fluorescence at any time point and Fo is the fluorescence intensity before the addition of ouabain. D: ouabain-induced augmentation of KCl-mediated increase in [Ca2+]i expressed by F/Fo ratio. The whole cell was selected as the region of interest for monitoring the changes in fluorescence. Values are means ± SE of 4 preparations. *P < 0.05 vs. value in the absence of ouabain.

View larger version (41K): [in this window] [in a new window]   Fig. 4. Effect of ouabain on Na+-K+-ATPase activity and SBFI fluorescence for intracellular Na+ concentration ([Na+]i) in cardiomyocytes. Cardiomyocytes were incubated with 0.5–2 mM ouabain in cuvette for 10 min before the addition of 30 mM KCl. Ouabain-induced augmentation of KCl response was calculated by subtracting the value for KCl-induced increase in [Ca2+]i in the absence of ouabain from that in its presence. A: effect of different concentrations of ouabain (0.5–2 mM) on Na+-K+-ATPase activity in isolated sarcolemmal (SL) vesicles. B: effect of ouabain (0.5–2 mM) on basal SBFI fluorescence for [Na+]i. C: effect of ouabain (0.5–2 mM) on KCl-induced increase in SBFI fluorescence for [Na+]i. D: effect of lidocaine (LID; 50 µM), 5-(N-methyl-N-isobutyl) amiloride (MIA; 5 µM), and their combination on ouabain (1 mM)-mediated augmentation of KCl response in terms of SBFI fluorescence for [Na+]i. Values are means ± SE of 4 hearts. *P < 0.05 vs. value in the absence of ouabain; #P < 0.05 vs. control.

View larger version (35K): [in this window] [in a new window]   Fig. 5. Effect of ouabain on basal and KCl-mediated increase in [Ca2+]i in cardiomyocytes treated with 0, 10, and 105 (control) mM Na+. Cardiomyocytes were incubated with 1 mM ouabain in cuvette for 10 min before the addition of 30 mM KCl. Ouabain-induced augmentation of KCl response was calculated by subtracting the value for KCl-induced increase in [Ca2+]i in the absence of ouabain from that in its presence. A: basal [Ca2+]i in cardiomyocytes. B: ouabain-induced increase in basal [Ca2+]i. C: KCl-induced increase in [Ca2+]i. D: ouabain-induced augmentation of KCl response. Values are means ± SE of 4 preparations. *P < 0.05 vs. control.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The experiments described in this study also reveal that inhibition of SL Na⁺-K⁺-ATPase as well as the increase in both [Na⁺]_i and [Ca²⁺]_i in cardiomyocytes due to ouabain were produced in a concentration-dependent manner. In fact, a highly significant linear relation was observed between ouabain-mediated increases in [Na⁺]_i and [Ca²⁺]_i in both quiescent and KCl-depolarized cardiomyocytes. Dependency of the ouabain-induced increase in basal [Ca²⁺]_i as well as augmentation of KCl-induced increase in [Ca²⁺]_i on Na⁺ was evident from our observations that these ouabain-induced changes in [Ca²⁺]_i were markedly attenuated when cardiomyocytes were treated in the absence of Na⁺ or with a medium containing very low concentrations (10 mM) of extracellular Na⁺. Furthermore, TTX and lidocaine, which are known to block SL Na⁺ channels and the entry of extracellular Na⁺ (24, 37, 46), prevented the ouabain-mediated increase in [Ca²⁺]_i in both quiescent and KCl-depolarized cardiomyocytes. It should be noted that lidocaine also reduced the level of [Na⁺]_i in cardiomyocytes.

In addition, treatment with MIA, which inhibits NHE (45), depressed the ouabain-induced alterations in both [Ca²⁺]_i and [Na⁺]_i; MIA has also been reported to decrease the ouabain-induced increase in [Ca²⁺]_i (26). It should be pointed out that, unlike TTX and lidocaine, MIA was found to increase the basal as well as the KCl-mediated increase in [Ca²⁺]_i. Because this action of MIA on [Ca²⁺]_i has been shown to be due to accumulation of H⁺ and subsequent release Ca²⁺ from SR in cardiomyocytes (34), such effects on release of Ca²⁺ from SR (20, 23) are considered to be responsible for the reversal of the ouabain-induced increase in [Ca²⁺]_i after treatment with MIA. Nonetheless, our findings regarding the reduction of the ouabain-induced increase in [Ca²⁺]_i by MIA, lidocaine, and TTX suggest the involvement of both SL NCX and SL Na⁺ channels in mobilization of Ca²⁺ in cardiomyocytes by ouabain.

Because both the ouabain-induced increase in [Ca²⁺]_i and augmentation of the KCl response were found to be dependent on the extracellular concentration of Ca²⁺, it is likely that the entry of Ca²⁺ into cardiomyocytes is of critical importance for the occurrence of ouabain-induced increase in [Ca²⁺]_i. The participation of SL NCX (reverse mode) in promoting the entry of Ca²⁺ by ouabain is evident from our observation that ouabain augmented the low-Na⁺-induced (70 and 35 mM Na⁺) increase in [Ca²⁺]_i, which has been reported to occur as a consequence of the activation of SL NCX (30). Furthermore, the involvement of SL NCX (reverse mode) in permitting the ouabain-mediated influx of Ca²⁺ from the extracellular compartment is apparent because Ni²⁺ and KB-R7943, blockers of NCX (36), attenuated the ouabain-induced changes in [Ca²⁺]_i. By employing different electrophysiological and biophysical methods, various investigators (18, 31, 39) have also shown that SL NCX is intimately involved in eliciting the increase in [Ca²⁺]_i due to Na⁺-K⁺-ATPase inhibition by cardiac glycosides . It is pointed out that decreasing the Na⁺ gradient across the SL membrane, as a consequence of either elevated level of [Na⁺]_i due to the action of ouabain or by decreasing the concentration of extracellular Na⁺ (70 and 35 mM), is also known to inhibit the efflux of Ca²⁺ through SL NCX (2, 6), and thus the contribution of the forward mode of NCX in inducing changes in basal [Ca²⁺]_i and KCl-induced increase in [Ca²⁺]_i in cardiomyocytes cannot be ruled out.

Because the ouabain-induced changes in [Ca²⁺]_i were depressed by 50% by inhibiting the SL NCX with a maximal concentration of KB-R7943, it appears that mechanisms other than the activation of SL NCX by an increased level of [Na⁺]_i may be participating in the ouabain-induced increase in [Ca²⁺]_i as well as augmentation of the KCl-mediated increase in [Ca²⁺]_i in cardiomyocytes. Because lidocaine and SKF-96365 were found to exert additive effects in reducing ouabain-induced increase in basal Ca²⁺ when used in combination with KB-R7943, it appears that SL Na⁺ channels and SL SOC may also be involved in producing changes in [Ca²⁺]_i due to ouabain. Because decreasing the concentration of extracellular Na⁺ from 105 mM to 35 mM augmented the ouabain-induced increase in [Ca²⁺]_i, and because Na⁺ and Ca²⁺ compete for their entry at slow Ca²⁺ channels in the SL membrane (10), it is possible that the augmentation of the ouabain response is due to reduced Na⁺ antagonism for the entry of Ca²⁺ through slow Ca²⁺ channels or some nonselective cation channels.

Depression of the ouabain-induced changes in [Ca²⁺]_i by Ca²⁺ antagonists verapamil and diltiazem, which are known to decrease the entry of Ca²⁺ into myocardium (30), suggests the participation of SL L-type Ca²⁺ channels in the ouabain action. In this regard, previous studies (13) have shown that cardiac glycosides produce a progressive depolarization and changes in membrane potential in atrioventricular node cells, which can be seen to permit Ca²⁺ entry through SL L-type Ca²⁺ channels. Ouabain-mediated depolarization of cardiomyocytes by inhibition of Na⁺-K⁺-ATPase leading to secondary stimulation of Ca²⁺ influx through voltage-operated L-type Ca²⁺ channels (21) may also be the mechanism for such an effect. Although Pittner et al. (29) have denied the role of L-type Ca²⁺ channels in the ouabain-mediated increase in [Ca²⁺]_i in endothelial cells, differences in Ca²⁺ handling in various cell types may be the reason for such a variable effect of ouabain.

Because the inhibition of SL Ca²⁺ pump ATPase by low concentrations of vanadate (34) did not affect the ouabain-induced changes in [Ca²⁺]_i, it appears that the SL Ca²⁺ pump may not participate in eliciting the ouabain-mediated alterations in [Ca²⁺]_i in cardiomyocytes. On the other hand, the involvement of SL SOC in the ouabain response was evident from the observation that SKF-96365, a known blocker of SOC (43), attenuated the ouabain-mediated increase in [Ca²⁺]_i. Note that specific blockers of SOC in vascular smooth muscle cells such as La³⁺ and Gd³⁺ (16, 43) did not alter the ouabain-induced increase in [Ca²⁺]_i. Although Hunton et al. (16) have reported that SOC are sensitive to SKF-96365, La³⁺_, and Gd³⁺, Uehara et al. (43) have shown that pharmacological characteristics of SOC of cardiomyocytes differ from other cell types, with a strong inhibition by SKF-96365, moderate inhibition by Gd³⁺_, and no inhibition by La³⁺. Because SKF-96365 is known to inhibit nonselective cation channels (49), their contribution to ouabain responsiveness cannot be ruled out on the basis of results obtained from the present study.

Cardiac glycosides have also been reported to promote the entry of Ca²⁺ by increasing the SL Ca²⁺ stores by a direct interaction with the SL membrane (10). Thus it is suggested that SL L-type Ca²⁺ channels and other nonselective cation channels or SL SOC are involved in elevating [Ca²⁺]_i in cardiomyocytes by ouabain.

In view of the well-known beneficial and toxic effects within a narrow range of doses of cardiac glycosides on the heart, it is difficult to clearly indicate the significance of the observed increase in [Ca²⁺]_i in cardiomyocytes. The initial and moderate increase in [Ca²⁺]_i in cardiomyocytes due to ouabain can be seen to produce the positive inotropic effect, whereas excessive and sustained increase in [Ca²⁺]_i would produce intracellular Ca²⁺ overload and may account for the toxic effect of cardiac glycosides. In this regard, it should be pointed out that intracellular Ca²⁺ overload has been reported to depress cardiac contractile force development in the failing heart (10). Nonetheless, from the results described in this study as well as from the foregoing discussion, it appears that different SL sites that control [Na⁺]_i as well as [Ca²⁺]_i may be participating in this process.

Although the action of ouabain on SL NCX is likely to be mediated through the increased level of [Na⁺]_i in cardiomyocytes, different SL channels may become open due to changes in membrane potential as a consequence of inhibition of SL Na⁺-K⁺-ATPase. Furthermore, SL NHE and Na⁺ channels may also be involved in raising the [Ca²⁺]_i during the action of ouabain by regulating the entry of extracellular Na⁺. Because the reversal of ouabain action by MIA did not occur in the KCl-induced increase in [Ca²⁺]_i, and because lidocaine as well as SKF-96365 did not produce an additive affect on the KCl-induced increase in [Ca²⁺]_i, it is likely that mechanisms for the regulation of [Ca²⁺]_i in the quiescent and depolarized cardiomyocytes are different from each other. In this regard, note that KCl has been shown to promote Ca²⁺ influx through L-type Ca²⁺ channels, to activate Ca²⁺-induced Ca²⁺ release from SR, and to stimulate SL NCX (reverse mode) for producing the increase in [Ca²⁺]_i in cardiomyocytes (30). Thus it is likely that the ouabain-induced increase in [Ca²⁺]_i in depolarized cardiomyocytes may involve Ca²⁺-induced Ca²⁺ release from SR in addition to the effects on SL L-type Ca²⁺ channels, as well as on SL NCX due to elevated levels of [Na⁺]_i.

On the other hand, ouabain-induced increase in basal [Ca²⁺]_i in quiescent cardiomyocytes may be due to the effect of an increase in [Na⁺]_i on SL NCX, L-type Ca²⁺ channels, and SL SOC. It should be mentioned that an elevated level of [Na⁺]_i due to the inhibition of SL Na⁺-K⁺-ATPase has been reported to promote the entry of Ca²⁺ through L-type Ca²⁺ channels (38) and to cause the release of Ca²⁺ from mitochondria (10). SR stores have also been indicated to raise the [Ca²⁺]_i in cardiomyocytes due to the action of cardiac glycosides (20, 23). In fact, in view of the critical role of SR Ca²⁺-release channels and Ca²⁺-pump mechanisms in regulating the [Ca²⁺]_i (4), extensive studies regarding the role of SR in the action of ouabain in cardiomyocytes remain to be carried out for fully understanding the mechanisms of increase in [Ca²⁺]_i on the inhibition of SL Na⁺-K⁺-ATPase.

We emphasize that some caution should be exercised while interpreting the results described in this study in terms of the selectivity of different pharmacological agents for specific sites. In this context, agents like vanadate at low concentrations have been shown to inhibit Na⁺-K⁺-ATPase in the outer medulla of dog kidneys (9), but no such effect on SL Na⁺-K⁺-ATPase activity was observed in rat hearts at the concentrations employed in the present study (40). Decreased access of vanadate (V+5 valency state) into intact cardiomyocytes as observed in dog kidneys (8) may be the reason for no effect on Na⁺-K⁺-ATPase activity at low concentrations. Since KB-R7943 at concentrations >10 µM can affect the forward and reverse mode of NCX (17), it is difficult to differentiate its effect at higher concentrations (25 and 50 µM). On the other hand, KB-R7943 at concentrations 10 µM has been shown to be a specific inhibitor of the reverse mode of NCX (11). Although some investigators have shown the effect of 10 µM KB-R7943 on L-type Ca²⁺ channels (5), the results vary depending on the type of preparation employed (5, 17). Furthermore, MIA (1–10 µM) has been considered to inhibit NHE specifically as demonstrated previously (34).

Because SKF can inhibit the nonselective cation channels and SOC (43, 49), further studies are needed to understand the direct involvement of SOC in the ouabain response. Nonetheless, on the basis of data presented in this study and in light of the available literature, we believe that the increase in [Ca²⁺]_i in cardiomyocytes due to inhibition of Na⁺-K⁺-ATPase by ouabain not only involves NCX but also other sites such as NHE, L-type Ca²⁺ channels, SOC, and Na⁺ channels, which directly or indirectly stimulate during the action of ouabain. A scheme depicting different sites subsequent to the action of ouabain on Na⁺-K⁺-ATPase leading to increase in [Ca²⁺]_i is shown in Fig. 6.

View larger version (15K): [in this window] [in a new window]   Fig. 6. Schematic representation of events showing increase in [Ca2+]i due to the inhibition of SL Na+-K+-ATPase by ouabain. Inhibition of SL Na+-K+-ATPase leads to an increase in [Na+]i, which stimulates Na+/Ca2+ exchanger (NCX) and causes an increase in [Ca2+]i. The involvement of the Na+/H+ exchanger (NHE) and SL Na+ channels is indicated in increasing [Na+]i and thus ultimately in elevating the levels of [Ca2+]i. In addition, the stimulation of L-type Ca2+ channels and store-operated Ca2+ channels (SOC) caused an indirect increase in [Ca2+]i. Furthermore, the electrogenic nature of Na+-K+-ATPase provides depolarization signals (DS) leading to an increase in Na+ channels, NHE, SOC, and L-type Ca2+ channel activity.

	ACKNOWLEDGMENTS

 
This study was supported by a grant from the Canadian Institute of Health Research. H. K. Saini was a predoctoral fellow of the Heart & Stroke Foundation of Canada.

	FOOTNOTES

 

Address for reprint requests and other correspondence: Dr. N. S. Dhalla, Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Tache Ave., Winnipeg, MB, Canada R2H 2A6 (e-mail: nsdhalla{at}sbrc.ca)

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. Section 1734 solely to indicate this fact.

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