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(Hypertension. 2003;41:1386.)
© 2003 American Heart Association, Inc.

Scientific Contributions

Ca²⁺ Mobilization Induced by Ouabain in Thymocytes Involves Intracellular and Extracellular Ca²⁺ Pools

Juliana Echevarria-Lima; Elizabeth Giestal de Araújo; Leopoldo de Meis; Vivian M. Rumjanek

From Laboratório de Imunologia Tumoral, Departamento de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Brazil (J.E.-L., V.M.R.); Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil (J.E.-L.); Laboratório de Bioenergética, Departamento de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Brazil (L.d.M.); and Laboratório de Cultura de Tecidos Hertha Meyer, Departamento de Neurobiologia, Universidade Federal Fluminense, Brazil (E.G.d.A.).

Correspondence to Vivian M. Rumjanek, Laboratório de Imunologia Tumoral, Departamento de Bioquímica Médica, ICB, Centro de Ciências da Saúde -Universidade Federal do Rio de Janeiro, Cidade Universitária. Rio de Janeiro, Brazil. E-mail vivian{at}bioqmed.ufrj.br

	Abstract

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Immune dysfunction has been reported in hypertensive rats, and circulating levels of ouabain are increased in some experimental models of hypertension. Ouabain is an inhibitor of the Na⁺/K⁺-ATPase capable of diverse effects on cells of the immune system, but its mode of action on these cells is still unknown. The levels of cytoplasmic calcium ions play an important role in cell signaling, and ouabain may induce an increase in intracellular calcium indirectly through the Na⁺/Ca²⁺ exchanger. The current work examined the possibility that this drug could be exerting its effects on thymocytes through calcium mobilization and an increase in the cytosolic calcium concentration. Intracellular calcium was evaluated by using Balb-c mouse thymocytes loaded with FURA-2. Both intracellular and extracellular calcium pools were mobilized by ouabain (3 to 1000 nmol). The influx of extracellular calcium depended on the Na⁺/Ca²⁺ exchanger and on voltage-dependent calcium channels, as it was inhibited by amiloride and benzamil, consistent with the inhibition of the Na⁺/K⁺ pump. In addition, the increase of calcium from intracellular stores was extremely rapid. Furthermore, an increase in cytosolic calcium levels was obtained with the combination of ouabain and thapsigargin, which was greater than that seen with either drug alone. Our data suggest that low concentrations of ouabain may be acting on thymocytes through a mechanism different from the traditional inhibition of the Na⁺/K⁺-ATPase, as the cytosolic calcium rise was partly dependent on the release from intracellular stores.

Key Words: ouabain • calcium • ions • hypertension, experimental • lymphocytes

	Introduction

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Cardiotonic steroids have been used clinically to treat congestive heart failure. Ouabain is a member of this class of drugs that is widely used in clinical practice. In 1960, Skou¹ showed that ouabain is an inhibitor of the Na⁺/K⁺-ATPase. The Na⁺/K⁺-ATPase consists of -, ß-, and -subunits. The -subunit contains the binding sites for Na⁺ and K⁺, ATP, and ouabain.² Four -isoforms (1, 2, 3, and 4) with different sensitivities to ouabain have been identified.³ In 1990, Noel et al⁴ showed that 1 (ouabain-resistant), 2 (moderately ouabain-sensitive), and 3 (ouabain-sensitive) isoforms of Na⁺/K⁺-ATPase -subunit are present in rodent tissues, although the 1-isoform is by far the most prevalent.

The existence of an endogenous substance with cardiotonic properties similar to digitalis has been suggested for some time.⁵ In 1991, Hamlyn and coworkers⁶ isolated from blood and adrenal glands a glycoside that was indistinguishable from ouabain isolated from plants. Later, this endogenous ouabain was detected not only in the adrenals⁷ but also in urine, hypothalamus, and pituitary.^8–11 It has been recently postulated that endogenous ouabain is involved in the pathogenesis of hypertension,^12–14 although this has been questioned by other authors.^15,16

The circulating levels of ouabain are increased in experimental models and in human hypertension.¹² However, the ouabain levels in hypertensinogenic rats are only 1 nmol/L.¹³ Changes in ouabain plasma concentrations affect the contractility, growth rate, and differentiation of a great number of cells in a tissue-specific way.¹⁷ Immune dysfunction has also been reported in spontaneously hypertensive rats. These animals have reduced numbers of mature and immature thymocytes and decreased proliferative responses, and this immune dysfunction advances with age.^18,19 Although a correlation exists between immunologic depression and hypertension, the mechanisms involved in this effect need to be elucidated.

In 1968, Quasel and Kaplan²⁰ showed that the proliferation of lymphocytes was inhibited when these cells were stimulated with phytohemagglutinin in the presence of ouabain. They suggested that Na⁺/K⁺-ATPase enhanced monovalent cation transport in stimulated lymphocytes. After those initial studies, different workers, including our group, investigated the effect of ouabain on cells of the immune system, showing inhibition of human and rodent lymphocyte proliferation induced by different mitogens.^21–24 Moreover, it was also demonstrated by us²⁵ that ouabain blocked the progression of the cell cycle from G1 to S in lymphocytes stimulated with mitogens. This is consistent with the fact that other events that result from the proliferative stimulus on lymphocytes and allow cycle progression, such as the production of interleukin 2 (IL-2) and its receptor CD25, are also inhibited by ouabain.^22,26 Ouabain was also capable of inducing apoptosis of peripheral blood lymphocytes and Jurkat cells,^27,28 although there is conflicting evidence from other cell types with regard to apoptosis induction.²⁵ These results suggest that endogenous ouabain may play a physiological role in the immune response if a high enough local concentration is reached.

Although there is evidence that ouabain increases cytosolic Ca²⁺ in muscle cells,^12,29 platelets,³⁰ and mature lymphocytes,³¹ there are no reports to our knowledge of the effect produced by ouabain on intracellular Ca²⁺ levels in thymocytes. However, thymocytes might have nearly 4 times more Na⁺/K⁺-ATPase activity than mature lymphocytes³² as well as capacity of regulating Na⁺ contents.³³

It has been suggested that the increase in cytosolic Ca²⁺ produced by ouabain is secondary to the reduction of the Na⁺/Ca²⁺ exchange-coupled Ca²⁺ efflux caused by lowering of the Na⁺ gradient when the accumulation of intracellular Na⁺ occurs as a result of the inhibition of Na⁺/K⁺-ATPase.³⁰ Human peripheral blood lymphocytes have been reported to have Na⁺/Ca²⁺ exchanger.^34,35 Under physiological conditions, the role of the Na⁺/Ca²⁺ exchanger is to pump Ca²⁺ out of the cell; however, under conditions in which cytosolic Na⁺ is increased, the exchanger can carry out the net influx of Ca²⁺.

Although a correlation exists between immunologic depression and hypertension, the mechanism involved in this effect needs to be elucidated. As the intracellular Ca²⁺ concentration is a determinant in cell signaling and apoptosis induction, the aim of this work was to investigate whether in mouse thymocytes ouabain is involved in calcium mobilization.

	Methods

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Materials
The culture medium RPMI 1640 (Sigma) was used to suspend the isolated thymocytes. Ouabain (Sigma) was dissolved in RPMI medium just before use. In some experiments, the calcium ionophore A23187 (Sigma) was used at a concentration of 2.5 µmol/L and EGTA at 10 mmol/L. Thapsigargin (Sigma) was dissolved as a stock solution in dimethylsulfoxide (DMSO; Sigma) at a concentration of 100 µg/mL. The ionic channel inhibitors amiloride (RBI) and benzamil (RBI) were dissolved in DMSO in stock solutions of 5 mmol/L and 2.5 mmol/L, respectively.

Animals and Preparation of the Thymocyte Suspension
Inbred male Balb-c mice (3 to 4 weeks old) were used in all experiments. Animals were housed in a temperature-controlled room and received water and food ad libitum. During all the experiments performed, animals were treated in accordance with published COBEA regulations for animal laboratory use. Animals were killed, and their thymi were removed. Thymocytes were isolated, centrifuged, and resuspended in RPMI 1640 medium supplemented with 5x10^-5 M ß-mercaptoethanol (Sigma), penicillin 60 mg/L (Sigma), streptomycin 100 mg/L (Sigma), and 10% fetal calf serum (Gibco); the pH was adjusted to 7.4. Cell viability was measured by Trypan blue dye exclusion.

Calcium Concentration Measurements
Cytoplasmic calcium concentrations were determined by using a modification of the method of Scharff et al³⁶ and Grynkiewicz et al.³⁷Thymocytes were incubated for 40 minutes at room temperature with 1.5 µmol/L of the calcium-sensitive fluorescent probe FURA2-AM (acetoxymethyl ester form of FURA-2) (Molecular Probes) in supplemented RPMI medium. Cells were washed twice and resuspended in PBS with 1 mmol/L CaCl₂ (pH 7.4). In all experiments, the cell number was adjusted at 1x10⁶ cells/mL and used in the specific assays. In some experiments, cells were resuspended in Ca²⁺-free PBS with 10 mmol/L EGTA. After 100 seconds, cells were stimulated with the different reagents. Calcium measurement was performed in a F4500 fluorometer (Hitachi). Excitation was set at 340 nm and emission at 505 nm, with entry and exit slits of 5 nm. Results are plotted as fluorescence intensity versus time (600 seconds).

The free Ca²⁺ concentration in the cell was titrated with the use of calcium calibration buffer KIT#2 (Molecular Probes). The fluorescence obtained in the presence of different Ca²⁺ and EGTA standard solutions yielding different free Ca²⁺ concentrations was measured by using an amount of fluorophore on the same order as that detected with the cell suspension. The standard mixtures of Ca²⁺ and EGTA were obtained from calcium calibration buffer kit #2.

Plasma-Membrane Potential Measurement
Thymocytes were incubated in the presence or absence of ouabain (100 nmol/L, 100 µmol/L, and 10 mmol/L) in RPMI medium for 6 hours at 37°C with 5% of CO₂. After this, the changes in plasma membrane potential were measured by flow cytometry, using an anionic lipophilic potential-sensitive dye, bis-oxonol(bis-[1,3-dibutylbarbituric acid] trimethineoxonol, DiBAC₄³ (Molecular Probes). According to Brauner et al,³⁸ the dye has a fluorescence response of 1%/mV. Plasma membrane potentials were measured by the method described by Mann et al.³⁹ Thymocytes were incubated for 30 minutes with or without 150 nmol/L DiBAC₄³ at 37°C with 5% CO₂ and were immediately examined by flow cytometry with a FACS (Becton Dickinson and Co), with excitation performed with a 488-nm argon laser and fluorescent emission detected at 530 nm in the FL-1 channel. Ten thousand cells were examined under each condition, and all flow cytometry analyses were accomplished with the WINMDI software (http://facs. scripps.edu/software.htlm). An increase in DiBAC₄³ fluorescence at 530 nm indicates cellular depolarization.

Statistical Analysis
Values given are mean±SD of the mean. Statistical significance was calculated by 1-way ANOVA followed by Bonferroni t-test, and probability values <0.05 were considered significant.

	Results

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Ouabain-Induced Intracellular Ca²⁺ Mobilization
Thymocytes exposed to ouabain (3, 10, 30, 100, and 1000 nmol/L) exhibited a rapid and concentration-dependent rise in cytosolic Ca²⁺ levels ([Ca²⁺]_i) (Figure 1A). In resting thymocytes in the absence of ouabain, [Ca ²⁺]_i was 20 nmol/L (Table 1). The maximum elevation of intracellular Ca²⁺ concentration was obtained with 100 nmol/L ouabain when [Ca ²⁺]_i of 78 nmol/L was measured (Table 1). A concentration x10 greater (1000 nmol/L) did not induce any further increase.

View larger version (29K): [in this window] [in a new window]   Figure 1. Ca2+ mobilization induced by ouabain (OUA). Isolated thymocytes from 3- to 4-week-old mice were exposed to different doses of OUA (, 3 nmol/L; , 10 nmol/L; •, 30 nmol/L; , 100 nmol/L; , 1000 nmol/L). Arrow shows the moment OUA was added. A, Cells exposed to OUA (3 to 1000 nmol/L). B, Cells exposed to OUA (100 nmol/L) in the presence () or absence () of extracellular Ca2+. On the right-hand side are Ca2+ fluorescence traces of the graph on the left. This figure is representative of 5 different experiments. Excitation and emission were 340 nm and 505 nm, respectively.

View this table: [in this window] [in a new window]   TABLE 1. Effect of Ouabain on Cytosolic Ca2+ Levels

Our results are consistent with those of Zhu et al⁴⁰ and Aizman et al,⁴¹ who showed that nanomolar ouabain concentrations were capable of increasing [Ca²⁺]_i in vascular smooth muscle cells and renal epithelial cells, respectively. To evaluate external Ca²⁺ participation in the intracellular Ca²⁺ increase observed in thymocytes, these cells were exposed to ouabain (100 nmol/L) in the absence of external Ca²⁺ (Ca²⁺-free PBS with 10 mmol/L EGTA). The effect induced by ouabain was 40% smaller but was still present (Figure 1B).

The results above suggest that the ouabain effect in cell Ca²⁺ depends on both intracellular and extracellular pools of this ion. Thus, the next experiments were performed to evaluate the contribution of each of these pools to the ouabain-induced rise in [Ca²⁺]_i. To evaluate the dependence on extracellular Ca²⁺, 2 inhibitors of the Na⁺/Ca²⁺ antiporter system, amiloride and benzamil, were used, and to evaluate the contribution of intracellular Ca²⁺, thapsigargin, an inhibitor of the endoplasmic reticulum Ca ²⁺-ATPase, was used.

Influence of Voltage-Dependent Ca²⁺ Channels, Na⁺ Channels, and Na⁺/Ca²⁺ Exchange Mechanism on Ouabain-Induced Ca²⁺ Mobilization
To establish the extracellular Ca²⁺ participation in ouabain-induced Ca²⁺ mobilization, thymocytes were stimulated with ouabain (100 nmol/L) in the presence of amiloride (50 µmol/L) (inhibitor of voltage-dependent Ca²⁺ channels and of Na⁺/Ca²⁺ antiporter system) or benzamil (25 µmol/L) (inhibitor of Na⁺ channels and of Na⁺/Ca²⁺ antiporter system).⁴² In the presence of these inhibitors the increase in [Ca²⁺]_i produced by ouabain was significantly diminished, to a level similar to that seen in the absence of external Ca²⁺ (Table 2). These findings suggest that there is involvement of the Na⁺/Ca²⁺ antiporter system and/or calcium channels in the Ca²⁺ mobilization induced by ouabain in mouse thymocytes. The effect of ouabain on voltage-dependent Ca²⁺ channels was suggested by Haddy and Overbeck,⁴³ and the role of the Na⁺/Ca²⁺ exchanger in the ouabain-induced [Ca²⁺]_i increase has already been postulated in muscle cells.^12,29,44 Thus, our results corroborate those in the literature and suggest that external Ca²⁺ is involved in part of the effect produced by ouabain. However, they also suggest that the intracellular stores are important contributors.

View this table: [in this window] [in a new window]   TABLE 2. Effect of Inhibitors of Ca2+ Channels, Na+ Channels, and Na+/ Ca2+ Exchanger on Cytosolic Ca2+ Levels Produced by Ouabain

Ca²⁺ Mobilization Induced by Thapsigargin
Inhibition of the endoplasmic reticulum Ca²⁺-ATPase by the use of thapsigargin leads to Ca²⁺ depletion of this organelle.⁴⁵ Treatment of isolated mouse thymocytes with 5, 10, 50, and 100 nmol/L of thapsigargin, a specific inhibitor of endoplasmic reticulum Ca²⁺-ATPase, caused a sustained increase in intracellular Ca²⁺ in the cytosol (Figure 2A). The Ca²⁺ mobilization induced by thapsigargin was concentration-dependent, but no difference was observed between 50 and 100 nmol/L, which produced an increase of 150 nmol/L in Ca²⁺ cytosolic levels. The stimulation of thymocytes with thapsigargin (50 nmol/L) in saline (PBS) without Ca²⁺ and with 10 mmol/L EGTA resulted in a lower peak, with an increase of 64 nmol/L (Figure 2B). This is in agreement with the observation that sustained Ca²⁺ levels require the entry of external Ca²⁺ stimulated by depletion of endoplasmic reticulum stores.⁴⁶

View larger version (31K): [in this window] [in a new window]   Figure 2. Ca2+ mobilization induced by thapsigargin (TG). Isolated thymocytes from 3- to 4-week-old mice were exposed to different doses of TG (, 5 nmol/L; •, 10 nmol/L; , 50 nmol/L; , 100 nmol/L). Arrow shows the moment TG was added. A, Cells exposed to TG (5 to 100 nmol/L). B, Cells exposed to TG (50 nmol/L) in the presence () or absence () of extracellular Ca2+. On the right-hand side are Ca2+ fluorescence traces of the graph on the left. This figure is representative of 5 different experiments. Excitation and emission were 340 nm and 505 nm, respectively.

Effect of Thapsigargin on Ouabain-Induced Increase in Intracellular Ca²⁺
To investigate the possibility that the simultaneous stimulation of thymocytes with ouabain and thapsigargin produced modifications in intracellular Ca²⁺ mobilization, in the next experiments, cells were stimulated with thapsigargin (50 nmol/L) and ouabain (3 to 100 nmol/L). Cells stimulated with thapsigargin plus ouabain (3, 10, and 30 nmol/L) exhibited [Ca²⁺]_i similar to those treated with thapsigargin alone (Table 3). However, with 100 nmol/L and 1000 nmol/L ouabain in the presence of thapsigargin, the increase in [Ca²⁺]_i was greater than that seen with thapsigargin alone (Table 3). Two hypotheses may explain the results with the higher doses of ouabain: The involvement of intracellular Ca²⁺ stores different from endoplasmic reticulum, or ouabain, induces the opening of Ca²⁺ channels and activation of the Na⁺/Ca²⁺ exchanger, leading to Ca²⁺ entry and a further increase in [Ca²⁺]_i.

View this table: [in this window] [in a new window]   TABLE 3. Effect of Combination of Ouabain and Thapsigargin on Cytosolic Ca2+ Levels

Effect of the Combination of Ouabain and Thapsigargin in the Absence of Extracellular Ca²⁺
To analyze whether the high levels of [Ca²⁺]_i obtained with the combination of ouabain and thapsigargin were a result of extracellular Ca²⁺ influx, thymocytes were exposed to thapsigargin (50 nmol/L), followed 10 minutes later by the addition of ouabain (100 nmol/L) (Figure 3A). When both procedures were performed in the absence of external Ca²⁺ (Figure 3B), the intracellular Ca²⁺ levels attained after ouabain stimulation of cells pretreated with thapsigargin were not different from those seen in cells not treated with ouabain and treated only with thapsigargin. This result suggests that the further increase in [Ca²⁺]_i that occurs with the combination depends on extracellular Ca²⁺ influx and not on other intracellular pools.

View larger version (27K): [in this window] [in a new window]   Figure 3. Cytosolic Ca2+ concentration induced by ouabain (OUA) and the combination OUA with thapsigargin (TG) in the presence and absence of external Ca2+. Isolated thymocytes from 3- to 4-week-old mice were used. Cells were exposed to 50 nmol/L TG and 100 nmol/L OUA in the presence (A) or absence (B) of Ca2+. Values of untreated thymocytes were 20.6±3.3 [Ca2+]i. *P<0.05 vs OUA, **P<0.05 vs TG.

Effect of Ouabain on Plasma-Membrane Potential
Inhibition of Na⁺/K⁺-ATPase may lead to depolarization of the plasma membrane. To evaluate the plasma membrane potential of thymocytes in the presence of ouabain, these cells were incubated for 6 hours with or without ouabain (100 nmol/L, 100 µmol/L, and 10 mmol/L). At 10 mmol/L, ouabain caused depolarization (Figure 4), probably by inhibiting the Na⁺/K⁺-ATPase, similar to what has been described by Mann et al.³⁹ However, lower concentrations of ouabain did not alter the membrane potential despite the fact that 100 nmol/L was already capable of inducing a [Ca²⁺]_i increase in these cells, suggesting a novel mode of action for ouabain.

View larger version (31K): [in this window] [in a new window]   Figure 4. Flow cytometry profile of the effect of ouabain (OUA) on plasma membrane potential. All thymocytes, with the exception of the saline control (CTR-PBS), were preincubated with DiBAC43 and exposed or not exposed to ouabain (100 nmol/L, 100 µmol/L, and 10 mmol/L) for 6 hours. A, Control cells in the absence of ouabain and exposed to 50 mmol/L KCl as positive control. B, Cells exposed to 100 nmol/L OUA. C, Cells exposed to 100 µmol/L OUA. D, Cells exposed to 10 mmol/L OUA.

	Discussion

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Our results demonstrate for the first time that ouabain induces an increase in cytosolic Ca²⁺ in thymocytes. Furthermore, we were able to show that this cytosolic Ca²⁺ rise was produced by low (nanomolar) concentrations of ouabain and depended on intracellular and extracellular stores. These results are consistent with other reports. Zhu et al⁴⁰ showed that nanomolar concentrations of ouabain are capable of increasing [Ca²⁺]_i in vascular smooth muscle cells, Weiss et al⁴⁷ showed that nanomolar concentrations of ouabain significantly increase the responsiveness of rat arteries to vasoconstrictors. Satoh and Nakazato⁴⁸ and Monteith and Blaustein⁴⁹ showed that low doses of ouabain alter neuronal Ca²⁺ metabolism.

In a number of cell types, Ca²⁺ mobilization induced by ouabain needs extracellular Ca²⁺.¹² Our results with amiloride and benzamil suggest that part of the effect produced by ouabain on thymocytes requires participation of the Na⁺/Ca²⁺ antiporter system and of voltage-dependent Ca²⁺ channels and may also involve Na⁺ channels. The effect of ouabain on voltage-dependent Ca²⁺ channels was suggested by Haddy and Overbeck in 1976⁴³ and was later demonstrated in other models.^29,40,41,44

Wacholtz and collaborators^34,35 proposed that the Na⁺/Ca²⁺ exchanger regulates changes in [Ca²⁺]_i in activated T-lymphocytes, and it has been described that in peripheral blood lymphocytes, ouabain is capable of altering intracellular Na⁺ concentration, thereby affecting the Na⁺/Ca²⁺ exchanger activity and leading to Ca²⁺ accumulation in the cytosol.^12,29 It has also been shown that in peripheral blood mononuclear cells, amiloride and amlodipine can suppress the effect exerted by ouabain on cytokine production,^50,51 suggesting the participation of extracellular Ca²⁺ in this ouabain effect. Thus, our results are consistent with the literature and suggest that external Ca²⁺ is involved in part of the effect produced by ouabain on thymocytes. However, the extracellular Ca²⁺ influx accounted for less than half of the total increase in [Ca²⁺]_i. Therefore, our results indicate that Ca²⁺ mobilization induced by ouabain depends on intracellular Ca²⁺ stores as well.

Inside the cells, Ca²⁺ can be stored in cellular organelles such as endoplasmic reticulum and mitochondria, and the removal of cytosolic Ca²⁺ is carried out by pumps and exchange systems.⁵² The function of the sarco/endoplasmic reticulum Ca²⁺-ATPase pump is to sequester the Ca²⁺ introduced into the cytoplasm.⁵³ Thapsigargin was used in the current study because it inhibits Ca²⁺ uptake by endoplasmic reticulum, depletes this intracellular store of Ca²⁺, and activates extracellular Ca²⁺ entry.^45,46 The combination of ouabain and thapsigargin promoted a greater intracellular Ca²⁺ mobilization compared with either drug alone. However, when thymocytes were pretreated with thapsigargin without extracellular Ca²⁺ and subsequently exposed to ouabain, still in the absence of external Ca²⁺, they did not show a further increase in [Ca²⁺]_i, maintaining the levels obtained with thapsigargin alone. These results confirm the involvement of extracellular pools by ouabain and suggest that endoplasmic reticulum is also involved in ouabain-induced [Ca²⁺]_i mobilization. It has been suggested that ouabain can activate receptors for ryanodine and IP3 in the endoplasmic reticulum and that this organelle therefore could be the source of internal Ca²⁺ mobilized by ouabain.^41,54 Thus, it seems that in thymocytes, ouabain releases Ca²⁺ from the same intracellular store as thapsigargin (endoplasmic reticulum) and in addition opens other Ca²⁺ channels (voltage-dependent) and induces activation of the Na⁺/Ca²⁺ exchanger.

We were unable to alter thymocyte plasma membrane potential with low concentrations of ouabain, although 10 mmol/L did have an effect, as also reported by Mann and coworkers.³⁹ These results support the idea that low concentrations of ouabain affect Ca²⁺ distribution in thymocytes without totally inhibiting Na⁺/K⁺-ATPase activity. This is consistent with the suggestion that the interaction of ouabain with Na⁺/K⁺-ATPase can elicit a signaling cascade of events that is independent of changes in intracellular Na⁺ and K⁺ concentrations and may lead to a rise in intracellular Ca²⁺.⁵⁵

Our results show that in thymocytes, the increase in [Ca²⁺]_i induced by 100 nmol/L ouabain was of the order of 78 nmol/L and that this increase depended on both intracellular and extracellular Ca²⁺ mobilization. Our data suggest that ouabain may also be acting on murine thymocytes through a mechanism different from the traditional inhibition of the Na⁺/K⁺-ATPase, as the cytosolic [Ca²⁺]_i rise was partly dependent on the release from intracellular stores and was not related to membrane depolarization.

Perspectives
Immune dysfunction has been reported in patients with essential hypertension⁵⁶ and in spontaneously hypertensive rats,^{18,19,57–59} which have reduced numbers of thymocytes.^18,19 Low levels of ouabain have recently been implicated in hypertension, and the present work shows that low concentrations of ouabain markedly affect cytosolic Ca²⁺ in mouse thymocytes. This increase in Ca²⁺ levels is not accompanied by a measurable depolarization of the plasma membrane and depends on both intracellular and extracellular calcium pools. Similarly, levels of ouabain that only partially inhibit the Na⁺/K⁺-ATPase are capable of producing Ca²⁺ oscillations in another system and acting as a signal transducer.⁴¹ Our results suggest a possible mechanism of action of ouabain in thymocytes. Further studies are necessary to relate the effects we observed in [Ca²⁺]_i with those described on thymocytes of hypertensive rats.

	Acknowledgments

 
This work was supported by FAPERJ (Fundação de Amparo a Pesquisa do Rio de Janeiro), PRONEX, and CNPq (Brazilian National Research Council). Juliana Echevarria-Lima was a recipient of a PhD Fellowship from CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior). We would like to acknowledge Clarice Kirszberg for her help with the manuscript.

Received November 14, 2002; first decision November 27, 2002; accepted April 8, 2003.

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