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Am J Physiol Heart Circ Physiol 290: H373-H380, 2006. First published September 9, 2005; doi:10.1152/ajpheart.00613.2005
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Involvement of Na+/Ca2+ exchanger in catecholamine-induced increase in intracellular calcium in cardiomyocytes

Harjot K. Saini, Onkar N. Tripathi, Shetuan Zhang, Vijayan Elimban, and Naranjan S. Dhalla

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

Submitted 8 June 2005 ; accepted in final form 5 September 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Although sarcolemmal (SL) Na+/Ca2+ exchanger is known to regulate the intracellular Ca2+ concentration ([Ca2+]i), its involvement in catecholamine-induced increase in [Ca2+]i is not fully understood. To gain some information in this regard, isolated rat cardiomyocytes were treated with different agents, which are known to modify Ca2+ movements, in the absence or presence of a {beta}-adrenoceptor agonist, isoproterenol, and [Ca2+]i in cardiomyocytes was determined spectrofluorometrically with fura-2 AM. Treatment with isoproterenol did not alter [Ca2+]i in quiescent cardiomyocytes, whereas the ATP (purinergic receptor agonist)-induced increase in [Ca2+]i was significantly potentiated by isoproterenol. Unlike ryanodine and cyclopiazonic acid, which affect the sarcoplasmic reticulum function, SL L-type Ca2+ channel blockers verapamil and diltiazem, as well as a SL Ca2+-pump inhibitor, vanadate, caused a significant depression in the isoproterenol-induced increase in [Ca2+]i. The SL Na+/Ca2+ exchange blockers amiloride, Ni2+, and KB-R7943 also attenuated the isoproterenol-mediated increase in [Ca2+]i. Combination of KB-R7943 and verapamil showed additive inhibitory effects on the isoproterenol-induced increase in [Ca2+]i. The isoproterenol-induced increase in [Ca2+]i in KCl-depolarized cardiomyocytes was augmented by low Na+; this augmentation was significantly depressed by treatment with KB-R7943. The positive inotropic action of isoproterenol in isolated hearts was also reduced by KB-R7943. These data suggest that in addition to SL L-type Ca2+ channels, SL Na+/Ca2+ exchanger seems to play an important role in catecholamine-induced increase in [Ca2+]i in cardiomyocytes.

sodium/calcium exchanger; L-type calcium channels; calcium-pump adenosine triphosphatase; calcium-release channels; cardiomyocyte calcium handling

IT IS NOW WELL KNOWN that, unlike quiescent cardiomyocytes, the stimulation of {beta}-adrenoceptors ({beta}-ARs) by catecholamines causes a marked increase in the intracellular Ca2+ concentration ([Ca2+]i) in electrically stimulated (35) or KCl-depolarized cardiomyocytes (26). On the other hand, the stimulation of purinergic receptors by extracellular ATP augments [Ca2+]i in quiescent cardiomyocytes (8, 27). Previous studies by De Young and Scarpa (9, 10) have shown that ATP-induced increase in [Ca2+]i is potentiated by norepinephrine. This increase of the ATP response by norepinephrine treatment has been demonstrated to be due to Ca2+ entry through the sarcolemmal (SL) L-type Ca2+ channels. In this regard, it is pointed out that norepinephrine and other catecholamines, including isoproterenol, have been shown to phosphorylate the L-type Ca2+ channels and enhance the inward Ca2+ current as a consequence of increased production of cAMP and the activation of protein kinase A (PKA) (36, 37). The increased Ca2+ entry through the SL membrane further triggers the release of Ca2+ from the sarcoplasmic reticulum (SR) stores and thus results in the catecholamine-mediated increase in [Ca2+]i in electrically stimulated cardiomyocytes (16). In contrast, the norepinephrine-mediated potentiation of the ATP response in quiescent cardiomyocytes has been reported to be independent of Ca2+ release from the SR Ca2+ stores (9). It has been shown that the stimulation of {beta}-ARs by isoproterenol causes an increase in the SL Na+/Ca2+ exchange activity in guinea pig ventricular cardiomyocytes (22) and rabbit Purkinje fibers (13). On the other hand, in frog atrial fibers and ventricular cardiomyocytes, an increase (23) or decrease (12) in Na+/Ca2+ exchange activity was observed on stimulation by isoproterenol, respectively. Nonetheless, the participation of Na+/Ca2+ exchange in the catecholamine-mediated increase in ATP response in rat ventricular cardiomyocytes is not known. Accordingly, the present study was undertaken to test the hypothesis that SL Na+/Ca2+ exchanger is involved in the isoproterenol-mediated stimulation of the ATP-induced intracellular Ca2+ mobilization. For this purpose, fura-2 AM-loaded isolated rat cardiomyocytes were treated with isoproterenol, a {beta}-AR agonist (26), in the absence or presence of different antagonists of SL Na+/Ca2+ exchanger before the addition of ATP. In addition, the effects of other agents that are known to modify Ca2+ transport in both SL and SR (24, 33) were tested to gain some information regarding the participation of different sites in the catecholamine-induced increase in [Ca2+]i in cardiomyocytes. Because low Na+ has been shown to stimulate the SL Na+/Ca2+ exchange activity in isolated cardiomyocytes (24), some experiments were also performed in the presence of low Na+ to further confirm the contribution of Na+/Ca2+ exchanger in the catecholamine-mediated increase in [Ca2+]i. The effect of KB-R7943, an inhibitor of SL Na+/Ca2+ exchanger (28), on the positive inotropic action of isoproterenol was investigated by employing isolated rat heart preparations.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolation of cardiomyocytes. Ventricular myocytes were isolated according to the method as described previously (33, 34). For this purpose, 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, and perfused for 5 min with Ca2+-free buffer (pH 7.4) 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. 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 30-min recirculation period, the hearts were removed from the cannula. The ventricles were cut into small pieces and subjected to another 15-min digestion in a fresh collagenase solution in the presence of 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 filtered through a 200-µm nylon mesh. The myocytes were resuspended for 5 min in buffers containing gradually increasing extracellular Ca2+ concentration (250, 500, 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, Ontario, Canada) exclusion method. The unstained, stained, and total number of cells were counted by Neubauer chamber. The final cell suspension had 80–85% viable cardiomyocytes, which were used for the measurement of fluorescence. All protocols were approved by the University of Manitoba Animal Care Committee in accordance with the standards of the Canadian Council on Animal Care.

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 cuvette was adjusted to 0.3 million cells/ml for all the experimental groups. The alteration in fluorescence intensity was 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 levels were calculated as described previously (24, 34). Treatment with isoproterenol (100 µM) was performed by incubating the fura-2 AM-loaded cells in a buffer containing isoproterenol for 5 min at room temperature before the measurement of fluorescence. Treatments with different pharmacological agents for the modulation of [Ca2+]i were performed by incubating the fura-2 AM-loaded cells in the buffer containing the desired concentration of pharmacological agent for 10 min before the measurement of fluorescence; the cells were treated with ryanodine and cyclopiazonic acid (CPA) for 20 min before the determination of [Ca2+]i. The concentrations of different pharmacological interventions for the present investigation were selected on the basis of our previous studies (24, 33, 34). For examining the effect of low Na+ on catecholamine-mediated potentiation of [Ca2+]i, cardiomyocytes were treated with Krebs-Henseleit (K-H) buffer (pH 7.4) containing 35 mM extracellular Na+ for 10 min at room temperature as described previously, and the osmolarity of the solution was maintained by adding choline chloride (24). It is pointed out that no change in cell viability was observed under different incubation conditions. The increase in [Ca2+]i at peak [Ca2+]i was calculated as the net increase above the basal value in each experiment. The difference between the responses in the presence and absence of isoproterenol treatment was taken as the isoproterenol-induced increase in [Ca2+]i.

Isolated rat heart preparations. Isolated rat hearts were perfused with an oxygenated K-H solution at a flow of 10 ml/min (26, 27). After stabilization for 20 min, a bolus injection of isoproterenol (1 µM) was given to the hearts in the absence or presence of different concentrations of KB-R7943, an inhibitor of the Na+/Ca2+ exchanger (28), and the left ventricular developed pressure (LVDP), as well as rates of pressure development and decay (±dP/dt), was measured by using a Biopac Data Acquisition System (Biopac Systems, Goleta, CA) as described previously (26, 27). The concentrations of isoproterenol and KB-R7943 used in this study were selected on the basis of previous observations (19, 32).

Statistical analysis. All results were expressed as means ± SE. Statistical analysis was performed by using Microcal Origin version 6 (Microcal Software, Northampton, MA). The 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 showing P < 0.05 were considered statistically significant.

Drugs and chemicals. Verapamil, diltiazem, amiloride, nickel chloride, ouabain, CPA, ryanodine, sodium vanadate, isoproterenol, ATP, propranolol, EGTA, and BSA were purchased from Sigma Chemicals (St. Louis, MO). KB-R7943, fura-2 AM, and collagenase (type II, 295 U/mg) were purchased from Tocris Biosciences (Ellisville, MO), Molecular Probes (Eugene, OR), and Worthington Biochemical (Freehold, NJ), respectively. All other reagents were of analytical grade and purchased either from Sigma Chemicals or Fisher Scientific (Fair Lawn, NJ).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of {beta}-AR stimulation on ATP-mediated intracellular Ca2+ mobilization. To examine the effect of {beta}-AR activation on ATP-induced Ca2+ mobilization, isolated cardiomyocytes were treated with isoproterenol, a {beta}-AR agonist (26), before the addition of ATP (50 µM). Representative tracings showing the effects of ATP and isoproterenol on [Ca2+]i are shown in Fig. 1. ATP caused a significant (43 ± 4.2%) increase in [Ca2+]i, which is in agreement with results from previous studies (26). This increase in [Ca2+]i by ATP was further augmented by 55 ± 5.7% on isoproterenol treatment. It is pointed out that isoproterenol-induced potentiation of ATP response, as shown in Fig. 1, was completely blocked by a {beta}-AR antagonist, propranolol (50 µM). It can also be seen from Fig. 1 that isoproterenol and propranolol treatments had no effect on basal [Ca2+]i. Preliminary experiments in the cardiomyocytes treated with different concentrations of isoproterenol (10, 25, 50, and 100 µM) revealed a concentration-dependent effect on the potentiation of ATP response; however, the maximal effect of isoproterenol treatment was seen at 100 µM under in vitro experimental conditions employed in this study. These observations are in agreement with those reported in our previous studies (26, 27).



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  		Fig. 1. Isoproterenol (Iso)-induced alteration in intracellular Ca2+ concentration ([Ca2+]i) in isolated cardiomyocytes. A: representative tracings showing the effect of ATP and Iso on [Ca2+]i in isolated cardiomyocytes; <- indicates the time when the preparation was exposed to 50 µM ATP. B: effect of propranolol (50 µM) on Iso- and ATP-induced increase in [Ca2+]i in isolated cardiomyocytes. Treatment with 100 µM Iso was carried out for 5 min before intracellular Ca2+ measurements. Each point represents mean ± SE of 4 experiments in each group. *P < 0.05 vs. control group; {dagger}P < 0.05 vs. ATP.

 
Effects of L-type Ca2+ channels and SL Ca2+-pump ATPase inhibitors on catecholamine-induced increase in [Ca2+]i. To investigate the involvement of L-type Ca2+ channels in catecholamine-mediated increase in [Ca2+]i, isolated cardiomyocytes were treated with verapamil and diltiazem, well-known L-type Ca2+ channel blockers (24). Both verapamil (1 and 10 µM) and diltiazem (1 and 10 µM) caused a significant attenuation of the ATP- or the isoproterenol-induced increase in [Ca2+]i (Table 1). On the other hand, basal [Ca2+]i remained unaltered in the presence of these agents (Table 1).


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  		Table 1. Effect of L-type Ca2+ channel blockers on Iso-mediated potentiation of ATP-induced increase in [Ca2+]i

 
The participation of SL Ca2+-pump ATPase in catecholamine-mediated increase in [Ca2+]i was determined by treating the isolated cardiomyocytes with low concentrations of vanadate (1 and 2 µM), which is known to inhibit SL Ca2+-pump ATPase (29). Vanadate caused a significant depression in isoproterenol-induced increase in [Ca2+]i, whereas the ATP-mediated increase in [Ca2+]i was augmented (Fig. 2). The results in Fig. 2 indicate that basal [Ca2+]i was not affected by vanadate treatment.



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  		Fig. 2. Effect of different concentrations of vanadate on Iso-induced increase in [Ca2+]i in isolated cardiomyocytes. A: effect of different concentrations of vanadate on basal [Ca2+]i. B: effect of different concentrations of vanadate on ATP-induced increase in [Ca2+]i. C: effect of different concentrations of vanadate on Iso-induced increase in [Ca2+]i. Iso-induced increase in [Ca2+]i is a difference between ATP-induced increase in the presence and absence of Iso. Each point represents mean ± SE of 4 experiments in each group. {dagger}P < 0.05 vs. ATP; *P < 0.05 vs. in absence of vanadate.

 
Role of intracellular Ca2+ stores in catecholamine-induced increase in [Ca2+]i. To test the contribution of intracellular Ca2+ stores in isoproterenol-induced increase in [Ca2+]i, isolated cardiomyocytes were treated with ryanodine, an agent that prevents the release of Ca2+ from SR by opening the Ca2+ release channel to subconductance level or by locking the channel (4, 25), and CPA, a well-known inhibitor of SR Ca2+-pump ATPase (6). Both ryanodine (2 and 4 µM) and CPA (20 and 40 µM) were observed to decrease the ATP-induced increase in [Ca2+]i, whereas the isoproterenol mediated increase in [Ca2+]i remained unaltered by these agents (Table 2). Basal [Ca2+]i was not changed by treatment with these agents (Table 2). It is pointed out that the ATP-induced increase in control cardiomyocytes in this group was 21 ± 3.4% higher than that of other experimental groups (Table 2). Incubation of cells for 20 min under control conditions required for experiments involving 20-min treatment with ryanodine or CPA instead of 10 min appears to be the reason for an increase in the sensitivity of control cardiomyocytes for ATP.


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  		Table 2. Effect of sarcoplasmic reticulum Ca2+ store depletion on Iso-mediated potentiation of ATP-induced increase in [Ca2+]i

 
Effects of Na+/Ca2+ exchange inhibitors on catecholamine-mediated increase in [Ca2+]i. To examine the role of the Na+/Ca2+ exchanger in isoproterenol-mediated increase in[Ca2+]i, cardiomyocytes were treated with amiloride and Ni2+, inhibitors of Na+/Ca2+ exchanger (2, 21). Results in Figs. 3 and 4 indicate that both ATP- and isoproterenol-mediated increase in [Ca2+]i were significantly attenuated by these treatments. Basal [Ca2+]i remained unaltered in cardiomyocytes treated with amiloride, whereas a significant decrease in basal [Ca2+]i was observed in Ni2+-treated cells (Figs. 3 and 4). Such a decrease in basal [Ca2+]i by Ni2+ seems to be due to some nonspecific effect of this agent because no such depression was seen by other inhibitors of Na+/Ca2+ exchanger. No attempt was made to understand the reason for such an effect of Ni2+ on basal [Ca2+]i. Because both amiloride and Ni2+ are nonspecific inhibitors of Na+/Ca2+ exchanger (5, 14), the cells were also treated with KB-R7943, a moderately specific inhibitor of Ca2+ entry mode of Na+/Ca2+ exchanger (28) to confirm the involvement of Na+/Ca2+ exchanger in the isoproterenol-mediated increase in [Ca2+]i. KB-R7943 (25 and 50 µM) caused a significant depression in both ATP- and isoproterenol-mediated increase in [Ca2+]i without having any effect on basal [Ca2+]i (Fig. 5). To further reveal the participation of Na+/Ca2+ exchanger in isoproterenol-mediated increase in [Ca2+]i, cardiomyocytes were treated with KB-R7943 (50 µM) in the presence of different concentrations of verapamil (0.25, 0.5, 1 µM). Unlike ATP-induced increase in [Ca2+]i, an additive inhibitory effect on isoproterenol-mediated increase in [Ca2+]i was observed after treatment with a combination of KB-R7943 and verapamil (Table 3).



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  		Fig. 3. Effect of different concentrations of amiloride on Iso-induced increase in [Ca2+]i in isolated cardiomyocytes. A: effect of different concentrations of amiloride on basal [Ca2+]i. B: effect of different concentrations of amiloride on ATP-induced increase in [Ca2+]i. C: effect of different concentrations of amiloride on Iso-induced increase in [Ca2+]i. Iso-induced increase in [Ca2+]i is a difference between ATP-induced increase in the presence and absence of Iso. Each point represents mean ± SE of 4 experiments in each group. {dagger}P < 0.05 vs. ATP; *P < 0.05 vs. in absence of amiloride.

 


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  		Fig. 4. Effect of different concentrations of Ni2+ on Iso-induced increase in [Ca2+]i in isolated cardiomyocytes. A: effect of different concentrations of Ni2+ on basal [Ca2+]i. B: effect of different concentrations of Ni2+ on ATP-induced increase in [Ca2+]i. C: effect of different concentrations of Ni2+ on Iso-induced increase in [Ca2+]i. Iso-induced increase in [Ca2+]i is a difference between ATP-induced increase in the presence and absence of Iso. Each point represents mean ± SE of 4 experiments in each group. {dagger}P < 0.05 vs. ATP; *P < 0.05 vs. in absence of Ni2+. {ddagger}P < 0.05 vs. respective basal in the absence of Ni2+.

 


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  		Fig. 5. Effect of different concentrations of KB-R7943 (KBR) on Iso-induced increase in [Ca2+]i in isolated cardiomyocytes. A: effect of different concentrations of KBR on basal [Ca2+]i. B: effect of different concentrations of KBR on ATP-induced increase in [Ca2+]i. C: effect of different concentrations of KBR on Iso-induced increase in [Ca2+]i. Iso-induced increase in [Ca2+]i is a difference between ATP-induced increase in presence and absence of Iso. Each point represents mean ± SE of 4 experiments in each group. {dagger}P < 0.05 vs. ATP; *P < 0.05 vs. in absence of KBR.

 

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  		Table 3. Effect of combination of verapamil and KB-R7943 on Iso-induced potentiation of ATP-induced increase in [Ca2+]i

 
Involvement of Na+/Ca2+ exchanger in catecholamine-induced increase in [Ca2+]i. In view of the fact that low Na+ has been shown to cause an increase in [Ca2+]i by activation of Na+/Ca2+ exchanger (1, 3), the cells were exposed to 30 mM KCl, a known depolarizing agent, in the presence of low Na+ (35 mM). It can be seen from Fig. 6 that the KCl-mediated increase was further augmented in the presence of low Na+; this finding is in agreement with our previous observations (24). In addition, the isoproterenol-induced increase in [Ca2+]i in KCl-depolarized cardiomyocytes was potentiated by low Na+ (Fig. 6); this increase in [Ca2+]i by isoproterenol in KCl-depolarized cardiomyocytes was significantly depressed by different concentrations of KB-R7943 without any alteration in basal [Ca2+]i (Table 4).



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  		Fig. 6. Effect of low Na+ on Iso-induced alteration in [Ca2+]i in isolated cardiomyocytes. A: representative tracings showing the effect of low Na+ on KCl-induced increase in [Ca2+]i. B: effect of different concentrations of Na+ [90 mM (control) and 35 mM (low Na+)] on KCl-induced increase in [Ca2+]i. C: effect of low Na+ on Iso-induced increase in [Ca2+]i in presence of KCl. Iso-induced increase in [Ca2+]i is a difference between KCl-induced increase in presence and absence of Iso. Each point represents mean ± SE of 4 experiments in each group. *P < 0.05 vs. respective control value.

 

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  		Table 4. Effect of different concentrations of KB-R7943 on Iso-induced increase in [Ca2+]i by low Na+ in KCl-depolarized cardiomyocytes

 
Modification of positive inotropic effect of isoproterenol by inhibition of Na+/Ca2+ exchanger. Treatment of the isolated heart with different concentrations of KB-R7943 was found to depress the LVDP (Table 5). The data in Table 5 show that the positive inotropic effect of isoproterenol measured in terms of an increase in LVDP was significantly depressed by treatment with different concentrations of KB-R7943. Likewise, the basal [Ca2+]i as well as the increase in ±dP/dt by isoproterenol were also depressed by KB-R7943 (data not shown). It is also pointed out that the positive inotropic effect of isoproterenol was completely blocked by 10 µM propranolol (data not shown).


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  		Table 5. Effect of different concentrations of KB-R7943 on LVDP in isolated rat heart with or without stimulation by 1 µM Iso

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we have shown that ATP causes an increase in [Ca2+]i in isolated cardiomyocytes, an effect that was potentiated by treatment with isoproterenol. This finding is in agreement with previous observations (27, 37). The isoproterenol-mediated increase in [Ca2+]i, unlike that due to ATP, was markedly inhibited by propranolol, indicating the involvement of {beta}-ARs in the isoproterenol-mediated increase in [Ca2+]i. Similarly, Zheng et al. (36) have shown that the isoproterenol-induced increase in the ratio of changes in fluorescence level ({Delta}FATP/{Delta}FKCl) was significantly attenuated by propranolol after the addition of ATP and KCl. The effect of {beta}-AR blockade on isoproterenol-induced Ca2+-mobilization may be of some specific nature because basal [Ca2+]i remained unaltered in the presence of propranolol. The involvement of extracellular Ca2+ entry by L-type Ca2+ channels in the isoproterenol-mediated increase in [Ca2+]i was evident from the observation that low concentrations (1 µM) of verapamil and diltiazem, which are sufficient to block the SL L-type Ca2+ channels (24), produced a marked reduction in the isoproterenol-induced increase in [Ca2+]i. The ATP-induced increase in [Ca2+]i was also attenuated by treatment with these antagonists. Thus an increase in the amplitude of inward Ca2+ current through the SL L-type Ca2+ channels (36) and {beta}-AR-cAMP-PKA pathway-mediated phosphorylation of L-type Ca2+ channels (20) may be the mechanism of isoproterenol- and ATP-mediated increase in Ca2+-influx. Although {beta}-AR stimulation-mediated Ca2+ entry by L-type Ca2+ channels has been linked with Ca2+ release from the SR Ca2+ stores (30), the isoproterenol-mediated potentiation of the ATP-induced increase in [Ca2+]i seems to be independent of Ca2+ release from the intracellular stores because ryanodine, an inhibitor of the SR Ca2+ release channel (4, 25), did not affect the isoproterenol-mediated increase in [Ca2+]i. Similarly, De Young and Scarpa (9) have shown that the potentiation of ATP response by norepinephrine, a nonspecific {beta}-AR agonist, remained unaltered by depletion of the intracellular Ca2+ stores by ryanodine. The involvement of SR Ca2+ stores in the isoproterenol-mediated increase in [Ca2+]i was further ruled out by the observation that CPA, a well-known inhibitor of the SR Ca2+-pump ATPase (6), did not alter the isoproterenol responsiveness in isolated cardiomyocytes. On the other hand, the ATP-mediated increase in [Ca2+]i was depressed in the presence of both ryanodine and CPA, demonstrating that SR Ca2+ stores play an important role in the ATP-mediated increase in [Ca2+]i.

The contribution of SL Ca2+-pump ATPase, which is known to cause efflux of intracellular Ca2+ (7), in the ATP-mediated increase in [Ca2+]i was apparent from the observation that low concentrations of vanadate (1 and 2 µM), a SL Ca2+-pump ATPase blocker (29), caused a significant elevation of the [Ca2+]i. In contrast, the isoproterenol-mediated potentiation of the ATP-induced increase in [Ca2+]i was attenuated by vanadate treatment. Because the level of [Ca2+]i is determined by a balance between Ca2+ influx and Ca2+ efflux, and catecholamines are known to augment both these processes (7, 20), the observed decrease in the potentiation of ATP-induced increase in [Ca2+]i by isoproterenol in the presence of vanadate may be due to greater stimulation of Ca2+ efflux than Ca2+ influx. Such an effect of isoproterenol in the vanadate-treated preparations may occur as a consequence of release of the inhibitory action of vanadate on the SL Ca2+-pump ATPase by isoproterenol. This and other possibilities such as the status of Ca2+-pump mechanisms in the SR membrane in the presence of vanadate remain to be examined for a meaningful conclusion.

Because high concentrations of verapamil and diltiazem (10 µM) have been shown to depress the SL Na+/Ca2+ exchange activity (31), it is possible that inhibition of the isoproterenol- or ATP-mediated increase in [Ca2+]i by these agents may be due to a nonspecific inhibition of Na+/Ca2+ exchanger in addition to the SL L-type Ca2+ channels. The involvement of Na+/Ca2+ exchanger in catecholamine-mediated increase in [Ca2+]i was evident on the use of amiloride and Ni2+, inhibitors of Na+/Ca2+ exchanger, which caused a marked attenuation of the isoproterenol-mediated increase in [Ca2+]i; similar depression in ATP response was also observed by these blockers. It is pointed out that amiloride has also been reported to cause inhibition of Na+/H+ exchanger (5); therefore, the involvement of Na+/H+ exchanger in the isoproterenol- or ATP- mediated increase in [Ca2+]i cannot be ruled out. The participation of Na+/Ca2+ exchanger in the catecholamine-mediated increase in [Ca2+]i was further demonstrated by treatment of cardiomyocytes with KB-R7943, a specific inhibitor of Ca2+ influx mode of Na+/Ca2+ exchanger (28). Both isoproterenol- and ATP-mediated increases in [Ca2+]i were depressed by KB-R7943 treatment. From these observations it seems likely that extracellular Ca2+ entry via SL Na+/Ca2+ exchanger plays a crucial role in the ATP- or isoproterenol-mediated increase in [Ca2+]i. This observation was further confirmed by the finding that the combination of KB-R7943 and verapamil showed an additive effect on the isoproterenol-mediated increase in [Ca2+]i. This experiment indicates that both SL L-type Ca2+ channels and SL Na+/Ca2+ exchanger may be involved in eliciting the increase in [Ca2+]i due to isoproterenol. Although under physiological conditions Na+/Ca2+ exchanger is an important mechanism of Ca2+ efflux (17), an increase in intracellular Na+ or a change in transsarcolemmal potential causes the reversal of the exchanger to mediate Ca2+ entry (3). In this context, ATP has been shown to cause depolarization of the membrane (8, 36) and the induction of 22Na+ influx in the isolated cardiomyocytes (15). In addition, PKA-mediated phosphorylation of Na+/Ca2+ exchanger subsequent to increase in cAMP concentration by isoproterenol treatment (22) may be the mechanism of catecholamine-induced increase in [Ca2+]i.

Although low Na+ has been shown to cause an increase in Ca2+ entry in isolated cardiomyocytes by activation of SL Na+/Ca2+ exchanger (24), the contribution of Na+/Ca2+ exchanger in catecholamine-induced increase in [Ca2+]i was observed in the presence of low Na+. KCl, a known depolarizing agent, was found to cause a significant increase in [Ca2+]i in presence of low Na+; this is in agreement with our previous observations (24). In addition, isoproterenol-mediated potentiation of KCl response was further augmented by low Na+. It is pointed out that this increase in [Ca2+]i by isoproterenol treatment in KCl-depolarized cardiomyocytes was markedly attenuated by KB-R7943, indicating that Ca2+ influx through Na+/Ca2+ exchanger may be involved in the catecholamine-mediated increase in [Ca2+]i. Furthermore, the positive inotropic effect of isoproterenol in terms of increase in LVDP, which is known to be associated with an increase in [Ca2+]i (11), was significantly depressed in the presence of KB-R7943; this observation confirms the contribution of the reverse mode of Na+/Ca2+ exchanger in the isoproterenol-mediated increase in [Ca2+]i. The depressant effect of KB-R7943 on the basal cardiac function in the isolated rat heart is in agreement with a previous report (19) and is likely to be due to the inhibition of SL Na+/Ca2+ exchanger.

In conclusion, our observations suggest that SL Na+/Ca2+ exchanger plays an important role in catecholamine-mediated increase in [Ca2+]i besides the Ca2+ entry through L-type Ca2+ channels. However, it cannot be determined on the basis of data presented here that the increase in [Ca2+]i by catecholamines is induced by a decrease in Ca2+ efflux or an increase in Ca2+ influx by Na+/Ca2+ exchanger, as Na+/Ca2+ exchanger inhibitors like amiloride and Ni2+ are known to depress both the transport modes of the exchanger (14, 18). Additionally, isoproterenol has been shown to activate both the inward and outward current generated by Na+/Ca2+ exchanger (22). Nonetheless, the results of the present study in terms of inhibition of isoproterenol response by a selective inhibitor of Ca2+ entry mode of Na+/Ca2+ exchanger, KB-R7943, particularly when stimulated by low Na+, indicate an important role of the reverse mode of Na+/Ca2+ exchanger in the catecholamine-mediated increase in [Ca2+]i. Although the results in this study indicate that both SL L-type Ca2+ channels and SL Na+/Ca2+ exchanger may participate in eliciting the isoproterenol-induced increase in [Ca2+]i in isolated cardiomyocytes, the exact contribution of each site cannot be determined on the basis of pharmacological approaches employed here.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The work reported in the article was supported by a grant from the Heart and Stroke Foundation of Manitoba.


   ACKNOWLEDGMENTS
 
H. K. Saini is a predoctoral fellow of the Heart and Stroke Foundation of Canada, and S. Zhang is a scholar of the Heart and Stroke Foundation of Canada. O. N. Tripathi was a visiting scientist from Central Drug Research Institute, Lucknow, India.


   FOOTNOTES
 

Address for reprint requests and other correspondence: N. S. Dhalla, Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Tache Ave., Winnipeg, Manitoba, 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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Allen DG, Eisner DA, Lab MJ, and Orchard CH. The effects of low sodium solutions on intracellular calcium concentration and tension in ferret ventricular muscle. J Physiol 345: 391–407, 1983.[Abstract/Free Full Text]
  2. Antolini M, Trevisi L, Debetto P, and Luciani S. Effect of amiloride on sodium-calcium exchange activity in rat cardiac myocytes. Pharmacol Res 27: 227–231, 1993.[CrossRef][ISI][Medline]
  3. Bers DM. Mechanisms contributing to the cardiac inotropic effect of Na+ pump inhibition and reduction of extracellular Na+. J Gen Physiol 90: 479–504, 1987.[Abstract/Free Full Text]
  4. Bers DM. Ryanodine and the calcium content of cardiac SR assessed by caffeine and rapid cooling contractures. Am J Physiol Cell Physiol 253: C408–C415, 1987.[Abstract/Free Full Text]
  5. Cargnelli G, Bova S, Cannas S, Debetto P, and Luciani S. Effect of amiloride on inotropic and toxic actions of ouabain in guinea pig left atria. Eur J Pharmacol 292: 103–110, 1994.[ISI][Medline]
  6. Chiesi M, Wrzosek A, and Grueninger S. The role of the sarcoplasmic reticulum in various types of cardiomyocytes. Mol Cell Biochem 130: 159–171, 1994.[CrossRef][ISI][Medline]
  7. Choi HS and Eisner DA. The effects of inhibition of the sarcolemmal Ca2+-ATPase on systolic calcium fluxes and intracellular calcium concentration in rat ventricular myocytes. Pflügers Arch 437: 966–971, 1999.[CrossRef][ISI][Medline]
  8. Christie A, Sharma VK, and Sheu SS. Mechanism of extracellular ATP-induced increase of cytosolic Ca2+ concentration in isolated rat ventricular myocytes. J Physiol 445: 369–388, 1992.[Abstract/Free Full Text]
  9. De Young MB and Scarpa A. ATP receptor-induced Ca2+ transients in cardiac myocytes: sources of mobilized Ca2+. Am J Physiol Cell Physiol 257: C750–C758, 1989.[Abstract/Free Full Text]
  10. De Young MB and Scarpa A. Extracellular ATP induces Ca2+ transients in cardiac myocytes, which are potentiated by norepinephrine. FEBS Lett 223: 53–58, 1987.[CrossRef][ISI][Medline]
  11. Endoh M. The effects of various drugs on the myocardial inotropic response. Gen Pharmacol 26: 1–31, 1995.[ISI][Medline]
  12. Fan J, Shuba YM, and Morad M. Regulation of cardiac sodium-calcium exchanger by {beta}-adrenergic agonists. Proc Natl Acad Sci USA 93: 5527–5532, 1996.[Abstract/Free Full Text]
  13. Han X and Ferrier GR. Contribution of Na+-Ca2+ exchange to stimulation of transient inward current by isoproterenol in rabbit cardiac purkinje fibers. Circ Res 76: 664–674, 1995.[Abstract/Free Full Text]
  14. Hinde AK, Perchenet L, Hobai IA, Levi AJ, and Hancox JC. Inhibition of Na+/Ca2+ exchange by external Ni in guinea-pig ventricular myocytes at 37°C, dialysed internally with cAMP-free and cAMP-containing solutions. Cell Calcium 25: 321–331, 1999.[CrossRef][ISI][Medline]
  15. Hori T, Yoshizumi M, Kitagawa T, Houchi H, Tamaki T, and Katoh I. Extracellular adenosine 5'-triphosphate induces Ca2+ efflux from freshly isolated adult rat cardiomyocytes: possible involvement of Na+/Ca2+ exchange mechanism. Life Sci 61: 1679–1689, 1997.[CrossRef][ISI][Medline]
  16. Hussain M and Orchard CH. Sarcoplasmic reticulum Ca2+ content, L-type Ca2+ current and the Ca2+ transient in rat myocytes during {beta}-adrenergic stimulation. J Physiol 505: 385–402, 1997.[CrossRef][ISI][Medline]
  17. Kimura J, Noma A, and Irisawa H. Na+-Ca2+ exchange current in mammalian heart cells. Nature 319: 596–597, 1986.[CrossRef][Medline]
  18. Lu J, Liang Y, and Wang X. Amiloride and KB-R7943 in outward Na+/Ca2+ exchange current in guinea pig ventricular myocytes. J Cardiovasc Pharmacol 40: 106–111, 2002.[CrossRef][ISI][Medline]
  19. Magee WP, Deshmukh G, Deninno MP, Sutt JC, Chapman JG, and Tracey WR. Differing cardioprotective efficacy of the Na+/Ca2+ exchanger inhibitors SEA0400 and KB-R7943. Am J Physiol Heart Circ Physiol 284: H903–H910, 2003.[Abstract/Free Full Text]
  20. McDonald TF, Pelzer S, Trautwein W, and Pelzer DJ. Regulation and modulation of calcium channels in cardiac, skeletal, and smooth muscle cells. Physiol Rev 74: 365–507, 1994.[Free Full Text]
  21. O'Neill SC, Valdeolmillos M, and Eisner DA. The effects of nickel on contraction and membrane current in isolated rat myocytes. Q J Exp Physiol 73: 1017–1020, 1988.[Abstract/Free Full Text]
  22. Perchenet L, Hinde AK, Patel KC, Hancox JC, and Levi AJ. Stimulation of Na+/Ca2+ exchange by the {beta}-adrenergic/protein kinase A pathway in guinea-pig ventricular myocytes at 37°C. Pflügers Arch 439: 822–828, 2000.[CrossRef][ISI][Medline]
  23. Rakotonirina A and Soustre H. Effects of isoprenaline on tonic tension and Na+-Ca2+ exchange in frog atrial fibres. Gen Physiol Biophys 8: 313–326, 1989.[ISI][Medline]
  24. Rathi SS, Saini HK, Xu YJ, and Dhalla NS. Mechanisms of low Na+-induced increase in intracellular calcium in KCl-depolarized rat cardiomyocytes. Mol Cell Biochem 263: 151–162, 2004.[CrossRef][ISI][Medline]
  25. Rousseau E, Smith JS, and Meissner G. Ryanodine modifies conductance and gating behavior of single Ca2+ release channel. Am J Physiol Cell Physiol 253: C364–C368, 1987.[Abstract/Free Full Text]
  26. Saini HK and Dhalla NS. Defective calcium handling in cardiomyocytes isolated from hearts subjected to ischemia-reperfusion. Am J Physiol Heart Circ Physiol 288: H2260–H2270, 2005.[Abstract/Free Full Text]
  27. Saini HK, Elimban V, and Dhalla NS. Attenuation of extracellular ATP response in cardiomyocytes isolated from hearts subjected to ischemia-reperfusion. Am J Physiol Heart Circ Physiol 289: H614–H623, 2005.[Abstract/Free Full Text]
  28. Satoh H, Ginsburg KS, Qing K, Terada H, Hayashi H, and Bers DM. KB-R7943 block of Ca2+ influx via Na+/Ca2+ exchange does not alter twitches or glycoside inotropy but prevents Ca2+ overload in rat ventricular myocytes. Circulation 101: 1441–1446, 2000.[Abstract/Free Full Text]
  29. Shah KR, Matsubara T, Foerster DR, Xu YJ, and Dhalla NS. Mechanisms of inotropic responses of the isolated rat hearts to vanadate. Int J Cardiol 52: 101–113, 1995.[CrossRef][ISI][Medline]
  30. Song LS, Wang SQ, Xiao RP, Spurgeon H, Lakatta EG, and Cheng H. Beta-adrenergic stimulation synchronizes intracellular Ca2+ release during excitation-contraction coupling in cardiac myocytes. Circ Res 88: 794–801, 2001.[Abstract/Free Full Text]
  31. Takeo S, Elimban V, and Dhalla NS. Modification of cardiac sarcolemmal Na+-Ca2+ exchange by diltiazem and verapamil. Can J Cardiol 1: 131–138, 1985.[Medline]
  32. Wang X, Sentex E, Saini HK, Chapman D, and Dhalla NS. Upregulation of {beta}-adrenergic receptors in heart failure due to volume overload. Am J Physiol Heart Circ Physiol 289: H151–H159, 2005.[Abstract/Free Full Text]
  33. Xu YJ, Panagia V, Shao Q, Wang X, and Dhalla NS. Phosphatidic acid increases intracellular free Ca2+ and cardiac contractile force. Am J Physiol Heart Circ Physiol 271: H651–H659, 1996.[Abstract/Free Full Text]
  34. Xu YJ, Shao Q, and Dhalla NS. Fura-2 fluorescent technique for the assessment of Ca2+ homeostasis in cardiomyocytes. Mol Cell Biochem 172: 149–157, 1997.[CrossRef][ISI][Medline]
  35. Yu Z, Quamme GA, and McNeill JH. Depressed [Ca2+]i responses to isoproterenol and cAMP in isolated cardiomyocytes from experimental diabetic rats. Am J Physiol Heart Circ Physiol 266: H2334–H2342, 1994.[Abstract/Free Full Text]
  36. Zheng JS, Christie A, De Young MB, Levy MN, and Scarpa A. Synergism between cAMP and ATP in signal transduction in cardiac myocytes. Am J Physiol Cell Physiol 262: C128–C135, 1992.[Abstract/Free Full Text]
  37. Zheng JS, Christie A, Levy MN, and Scarpa A. Ca2+ mobilization by extracellular ATP in rat cardiac myocytes: regulation by protein kinase C and A. Am J Physiol Cell Physiol 263: C933–C940, 1992.[Abstract/Free Full Text]



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