Inhibition of sarcoplasmic reticulum Ca2+-ATPase by miconazole

Antonio Lax, Fernando Soler, and Francisco Fernandez-Belda

Departamento de Bioquímica y Biología Molecular A, Facultad de Veterinaria, Universidad de Murcia, Campus de Espinardo, 30071 Espinardo, Murcia, Spain


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The inhibition of sarcoplasmic reticulum Ca2+-ATPase activity by miconazole was dependent on the concentration of ATP and membrane protein. Half-maximal inhibition was observed at 12 µM miconazole when the ATP concentration was 50 µM and the membrane protein was 0.05 mg/ml. When ATP was 1 mM, a low micromolar concentration of miconazole activated the enzyme, whereas higher concentrations inhibited it. A qualitatively similar response was observed when Ca2+ transport was measured. Likewise, the half-maximal inhibition value was higher when the membrane concentration was raised. Phosphorylation studies carried out after sample preequilibration in different experimental settings shed light on key partial reactions such as Ca2+ binding and ATP phosphorylation. The miconazole effect on Ca2+-ATPase activity can be attributed to stabilization of the Ca2+-free enzyme conformation giving rise to a decrease in the rate of the Ca2+ binding transition. The phosphoryl transfer reaction was not affected by miconazole.

calcium adenosine 5'-triphosphatase; sarcoplasmic reticulum membrane; imidazole antimycotics


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MICONAZOLE (Mic) and other antifungal agents bearing an imidazole ring are known to have fungistatic or fungicidal action depending on the concentration used. The pharmacological effect is attributed to inhibition of cytochrome P-450 14-alpha -demethylase, thus avoiding lanosterol demethylation. This is a necessary step in the formation of ergosterol, a critical component of the fungal membrane. An alternative mechanism may be inhibition of the respiratory chain electron transport (39).

N1-substituted imidazole drugs were initially described as potent inhibitors of different cytochrome P-450-dependent oxidative processes (3, 29, 34), even though other metabolic effects were later described. Of particular interest may be the effects on cellular Ca2+ homeostasis. Thus Mic and related compounds inhibit with high affinity the store-operated Ca2+ channel from rat thymocyte plasma membrane (1) and the Ca2+-dependent K+ channel from human erythrocyte (2). Therefore, the movement of Ca2+ and K+ across the plasma membrane is impaired. It is also known that clotrimazole depletes intracellular Ca2+ stores, an effect that has been associated with the arrest of cell proliferation in normal and cancer cell lines (4).

Other studies have shown that imidazole antimycotics release Ca2+ from thapsigargin-sensitive Ca2+ pools in rat thymic lymphocytes (24). They also inhibit Ca2+ uptake after thapsigargin-mediated depletion of intracellular Ca2+ stores (24). Additionally, econazole, Mic, and SKF-96365 inhibit the Ca2+-dependent ATPase activity in sarcoplasmic reticulum (SR) vesicles isolated from skeletal muscle (24). Inhibition of SR Ca2+-ATPase and cardiac muscle contraction by clotrimazole has also been reported (35).

With this in mind, we analyzed the inhibition mechanism of this key intracellular transport system, i.e., SR Ca2+-ATPase, by means of the imizadole-containing drug Mic.

Coupling between Ca2+ transport and ATP hydrolysis occurs through a cyclic sequence of phosphorylated and nonphosphorylated enzyme intermediates with or without bound Ca2+ (12, 15, 20). In a very basic reaction scheme (Fig. 1), the Ca2+-free nonphosphorylated enzyme E interacts with cytoplasmic (external) Ca2+ to form E · Ca2. The Ca2+-bound species interacts with ATP, leading to the steady-state accumulation of Ca2+-bound phosphoenzyme (EP · Ca2) plus ADP. Subsequently, a conformational transition involving reorientation of the Ca2+ sites ensures Ca2+ dissociation into the luminal (internal) space, while phosphoenzyme (EP) hydrolysis produces the release of inorganic phosphate and recovery of E with externally oriented Ca2+ binding sites.


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Fig. 1.   Minimal reaction cycle of the sarcoplasmic reticulum (SR) Ca2+-ATPase. 1, The Ca2+-free enzyme E binds Ca2+ at the cytoplasmic side, giving E · Ca2. 2, The enzyme with external Ca2+ bond is phosphorylated by ATP, giving phosphoenzyme (EP) · Ca2 plus ADP. 3, The sequential breakdown of the ternary complex produces Ca2+ dissociation inside the SR (Ca<UP><SUB>in</SUB><SUP>2+</SUP></UP>), as well as Pi release and recovery of the Ca2+-free enzyme with external orientation.

In the present study, we tested overall hydrolytic and transport activities under different experimental conditions. Furthermore, we focused on partial reactions related to ligand binding and conformational changes, such as Ca2+ binding and ATP binding/phosphorylation, that are critical to the ATP-dependent Ca2+ transport process.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SR preparation. Fast-twitch skeletal muscle was obtained from the hind leg of female New Zealand rabbits (body wt 2-2.5 kg). Microsomal vesicles were prepared according to Eletr and Inesi (10). Isolated samples were aliquoted and stored at -80°C until use.

Protein concentration. The SR membrane concentration refers to milligrams of total protein per milliliter and was measured by the Lowry et al. (19) procedure. Bovine serum albumin was used as a standard.

Ca2+ in media. The free Ca2+ concentration was adjusted by adding appropriate volumes of CaCl2 and/or ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) stock solutions, as described by Fabiato (11). The computer program used for calculation took into account the absolute stability constant for the Ca2+-EGTA complex (32), the EGTA protonation equilibria (6), the presence of Ca2+ ligands, and the pH of the medium. "Absence of Ca2+" or "Ca2+-free medium" means that there was a sufficiently low level of free Ca2+ to prevent EP formation from ATP or the expression of Ca2+-dependent ATPase activity.

Phospholipid vesicles. Liposomes were prepared by vortexing for 2 min at room temperature a mixture of 3 mg of egg yolk phosphatidylcholine (Avanti Polar Lipids) in 0.75 ml of medium containing 20 mM MOPS, pH 7.0, 80 mM KCl, and 5 mM MgCl2. The mixture was briefly sonicated to clarity and then centrifuged to remove unwanted material. Phospholipid was finally quantified by measuring the inorganic phosphate content (7).

Ca2+-ATPase activity. The initial rate of inorganic phosphate release was measured by the colorimetric method of Lin and Morales (17). The experiments were performed at 25°C. A typical reaction medium contained 20 mM MOPS, pH 7.0, 80 mM KCl, 5 mM MgCl2, 5 mM potassium oxalate, 2 mM EGTA, 1.92 mM CaCl2 (free Ca2+ was 10 µM), 0.05 mg SR protein/ml, 1 mM ATP, and a given Mic concentration when indicated. An ATP-regenerating system containing 2 mM phosphoenolpyruvate and 6 U/ml pyruvate kinase was included when the ATP concentration was lowered to 50 µM. The Ca2+ dependence was studied in a medium containing 1.5 µM A-23187 and 0.02 mg/ml membrane protein. EGTA concentration was 0.1 mM, and suitable CaCl2 concentrations were added to yield the desired free Ca2+. Experiments in the presence of phosphatidylcholine vesicles were carried out in the described 10 µM free Ca2+ medium containing 0.01 mg of SR protein/ml and a fixed Mic concentration of 30 µM. The precise composition of the reaction medium is described in the corresponding figure legends.

Ca2+ transport. The initial rate of Ca2+ transport was measured at 25°C with the aid of 45Ca2+ as a radioactive tracer (23). The composition of the reaction medium was that described for Ca2+-ATPase activity, although SR protein was 0.01 mg/ml and ~1,000 cpm 45Ca2+/nmol Ca2+ was included. The transport process was stopped by filtering 1 ml of sample aliquots (0.01 mg) through HAWP Millipore filters (Milford, MA) (0.45-µm pore diameter). The filters were rinsed with 10 ml of ice-cold medium containing 20 mM MOPS, pH 7.0, 80 mM KCl, 5 mM MgCl2, and 1 mM LaCl3 and then subjected to liquid scintillation counting. A blank assay, performed with a medium containing no ATP, was used to subtract nonspecific Ca2+ retained in the filter.

EP measurements. The accumulation of radioactive EP after addition of [gamma -32P]ATP was measured as described by de Meis (9). Samples were initially preincubated following different protocols.

Addition of Mic to E · Ca2. The initial Ca2+-containing medium, i.e., 20 mM MOPS, pH 7.0, 80 mM KCl, 20 mM MgCl2, 0.1 mM EGTA, either 0.103 mM CaCl2 (10 µM free Ca2+) or 1.1 mM Ca2+ (1 mM free Ca2+), and 0.05 mg SR/ml, was preincubated in an ice-water bath with a given Mic concentration and allowed to stand for a variable period of time. In some experiments, 20 mM MOPS, pH 7.0, was substituted with 20 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.0.

Addition of Ca2+ to E · Mic. SR vesicles (0.05 mg of SR/ml) equilibrated in a Ca2+-free medium containing 20 mM MOPS, pH 7.0, 80 mM KCl, 20 mM MgCl2, 0.1 mM EGTA, and a given Mic concentration were placed in an ice-water bath. After being mixed with 1.1 mM CaCl2, the samples were allowed to stand for a certain period of time. In some experiments, 20 mM MES, pH 6.0, was used instead of 20 mM MOPS, pH 7.0.

Phosphorylation of preincubated samples was initiated at the ice-water temperature by adding a given [gamma -32P]ATP concentration, usually 50 µM. The reaction was stopped 1 s later by adding 2 ml of ice-cold quenching solution containing 250 mM perchloric acid and 4 mM sodium phosphate. Samples were 1:1 diluted by the denaturing acid solution. Quenched samples were filtered through 0.45-µm nitrocellulose filters (HAWP; Millipore). Filters were rinsed with 35 ml of ice-cold medium containing 125 mM perchloric acid and 2 mM sodium phosphate and then solubilized and counted by the liquid scintillation technique. Suitable blank assays were made by adding the quenching solution before [gamma -32P]ATP.

Data presentation. The experimental data points represent means ± SE of at least three independent determinations, each performed in duplicate.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Ca2+-dependent ATP hydrolysis catalyzed by a preparation of skeletal SR vesicles is sensitive to the presence of Mic.

Dependence on ATP. The initial experiments were performed at neutral pH in the presence of 10 µM free Ca2+, 50 µM ATP, 5 mM Mg2+, and 5 mM oxalate. The membrane protein concentration was 0.05 mg/ml. Under these conditions, the hydrolysis rate was monotonically inhibited as the Mic concentration was raised. The K0.5 value for inhibition was 12 µM (Fig. 2A). The ATP concentration was kept constant during the measurements with an ATP-regenerating system. When the free Ca2+ concentration was maintained at 10 µM but the ATP concentration was increased to 1 mM, a more complex dependence became evident (Fig. 2B). In this case, the rate of hydrolysis was activated when the Mic concentration was <10 µM and inhibited when the drug concentration was higher. It is clear that the Mic effect is dependent on the phosphorylating substrate concentration.


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Fig. 2.   Effect of ATP on Ca2+-ATPase activity measured in the presence of miconazole (Mic). Hydrolytic activity was measured at 25°C in a medium containing 20 mM MOPS, pH 7.0, 80 mM KCl, 5 mM MgCl2, 5 mM potassium oxalate, 2 mM EGTA, 1.92 mM CaCl2, (10 µM free Ca2+), 0.05 mg SR/ml, and a given Mic concentration. A: reaction was started by the addition of 50 µM ATP; 2 mM phosphoenolpyruvate and 6 U/ml pyruvate kinase were also present. B: reaction was started by adding 1 mM ATP in the absence of the ATP-regenerating system.

The effect of Mic on Ca2+ transport was also measured under the conditions described for the hydrolysis experiments. The buffered medium was at a neutral pH, free Ca2+ was 10 µM, ATP was 1 mM, and Mg2+ was 5 mM. The membrane protein concentration was 0.01 mg/ml, and the Ca2+-chelating agent oxalate was also present. Here, again, the activation or inhibition by Mic was concentration dependent, i.e., a low Mic concentration produced activation, whereas higher concentrations exerted an inhibitory action (Fig. 3).


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Fig. 3.   ATP-dependent Ca2+ transport measured in the presence of Mic. SR vesicles (0.01 mg/ml) were equilibrated at 25°C in a medium containing 20 mM MOPS, pH 7.0, 80 mM KCl, 5 mM MgCl2, 5 mM potassium oxalate, 2 mM EGTA, 1.92 mM 45CaCl2 (~1,000 cpm/nmol), and a given Mic concentration. The transport process was initiated by the addition of 1 mM ATP and stopped by filtering 1-ml aliquots of reaction medium at different times. Filters were processed as described in MATERIALS AND METHODS.

Dependence on membrane protein. The influence of the membrane protein concentration on the effect of Mic was also explored. This was assayed by measuring initial rates of ATP hydrolysis at a neutral pH in a medium containing 10 µM free Ca2+, 1 mM ATP, 5 mM Mg2+, and 5 mM oxalate. When the ATP hydrolysis rate was studied as a function of Mic concentration using 0.01, 0.05, or 0.15 mg SR/ml, a family of curves was generated. The degree of inhibition induced by a given Mic concentration fell as the protein concentration was raised and vice versa (Fig. 4A). In other words, the K0.5 for inhibition increased progressively from 12 µM to 42 and 100 µM, respectively, as the membrane protein concentration was raised. We also selected conditions to observe hydrolytic activity inhibition in the standard 10 µM free Ca2+ medium using 0.01 mg protein/ml and 30 µM Mic. When the experiment was repeated in the presence of phosphatidylcholine vesicles, the enzyme activity was progressively protected as the phospholipid concentration was raised (Fig. 4B).


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Fig. 4.   Effect of membrane protein and exogenous lipid on Ca2+-ATPase activity measured in the presence of Mic. A: reaction was carried out at 25°C in a medium containing 20 mM MOPS, pH 7.0, 80 mM KCl, 5 mM MgCl2, 5 mM potassium oxalate, 2 mM EGTA, 1.92 mM CaCl2 (10 µM free Ca2+), and a given Mic concentration. The reaction was started by adding 1 mM ATP, and the SR protein concentration was 0.01 (), 0.05 (), or 0.15 mg/ml (open circle ). B: experiments were performed in the presence of 0.01 mg protein/ml and 30 µM Mic by using the described 10 µM free Ca2+ medium. A final concentration of phospholipid vesicles was also present when indicated.

Dependence on Ca2+. Initial rates of ATP hydrolysis were also measured in the presence of different free Ca2+ concentrations using SR vesicles leaky to Ca2+. A plot of Ca2+-ATPase activity vs. Ca2+ concentration, expressed as pCa, showed a bell-shaped dependence (Fig. 5). When the experiments were repeated in the presence of 30 µM Mic, half-maximal activation required a higher Ca2+ concentration and the maximal rate did not reach that observed in the absence of Mic.


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Fig. 5.   Effect of Mic on Ca2+-ATPase activity measured as a function of Ca2+ concentration. The reaction medium consisted of 20 mM MOPS, pH 7.0, 80 mM KCl, 5 mM MgCl2, 0.1 mM EGTA, 0.02 mg SR/ml, 1.5 µM A-23187, and a given CaCl2 concentration to yield a certain Ca2+ concentration (pCa). The reaction was initiated at 25°C by adding 1 mM ATP (). The reaction medium was supplemented with 30 µM Mic when indicated (open circle ). pCa is the negative logarithm of free Ca2+ expressed as molar concentration.

A more detailed characterization was undertaken by analyzing single steps of the enzyme reaction cycle (Fig. 1). Data were obtained by preincubating samples under different conditions followed by a short phosphorylation with 50 µM [gamma -32P]ATP to evaluate the accumulation of EP. The assays were carried out at the ice-water temperature, and the phosphorylation was stopped after 1 s. Preliminary experiments indicated that the Mic effect was the same when the phosphorylation time was prolonged (data not shown). The SR protein concentration in these assays was 0.05 mg/ml.

In one case, SR vesicles at a neutral pH and in the absence of free Ca2+ were equilibrated at the ice-water temperature for 5 min with 20 µM Mic. Then, 1 mM free Ca2+ was added and preincubation was maintained at the same temperature for different periods of time. The time-dependent effect was evaluated by measuring the accumulation of EP after Ca2+ addition. The enzyme capacity to be phosphorylated by ATP increased slowly in the minute time scale when 1 mM free Ca2+ was added to a medium containing SR vesicles in the presence of Mic (Fig. 6). The hyperbolic increase showed a value of ~2.5 nmol EP/mg protein 30 min after Ca2+ addition. It is also shown that EP reached maximal values of ~3.2 nmol/mg protein from the initial time point when preincubation was performed in the absence of Mic.


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Fig. 6.   Time-dependent EP accumulation after addition of Ca2+ to SR vesicles in the presence of Mic. SR vesicles (0.05 mg/ml) in a Ca2+-free medium containing 20 mM MOPS, pH 7.0, 80 mM KCl, 20 mM MgCl2, and 0.1 mM EGTA were mixed at the ice-water temperature for 5 min with 20 µM Mic. The addition of 1.1 mM Ca2+ (1 mM free Ca2+) marked the preincubation time 0. Phosphorylation of samples after different preincubation times was initiated by adding 50 µM [gamma -32P]ATP and stopped after 1 s by acid quenching. The experimental data show EP levels when the preincubation was performed in the absence () or presence (open circle ) of 20 µM Mic.

Alternatively, SR vesicles resuspended in a Ca2+-containing medium were set in an ice-water bath, and then 20 µM Mic was added. Incubation after Mic addition was prolonged for different periods of time and was finished by a 1-s phosphorylation step, with [gamma -32P]ATP added first and then acid solution. When the vesicles were initially resuspended in the presence of 10 µM free Ca2+, the enzyme very rapidly lost the capacity to be phosphorylated by ATP (Fig. 7A). Preincubation with 20 µM Mic for 10 s was sufficient to drastically decrease the phosphorylating capacity of the enzyme. Control experiments performed without Mic in the preincubation medium provided the maximal level of phosphorylation.


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Fig. 7.   Time-dependent EP accumulation after addition of Mic to SR vesicles in the presence of Ca2+. A: SR vesicles (0.05 mg/ml) in a 10-µM free Ca2+ medium containing 20 mM MOPS, pH 7.0, 80 mM KCl, 20 mM MgCl2, 0.1 mM EGTA, and 0.103 mM CaCl2 were preincubated at the ice-water temperature for a given period of time with 20 µM Mic (open circle ). B: SR vesicles (0.05 mg/ml) in a 1 mM free Ca2+ medium containing 20 mM MOPS, pH 7.0, 80 mM KCl, 20 mM MgCl2, 0.1 mM EGTA, and 1.1 mM CaCl2 were preincubated for a given period of time with 20 µM Mic (open circle ). EP was evaluated by 1-s phosphorylation at the ice-water temperature using 50 µM [gamma -32P]ATP. Control experiments without a Mic preincubation were performed and data are shown in A and B ().

Interestingly, when the vesicles were resuspended in a 1 mM free Ca2+ medium, the subsequent addition of 20 µM Mic produced only a small decrease in the accumulated EP. The EP level in the absence of Mic preincubation was 3.2 nmol/mg protein (Fig. 7B), and this value decreased to 2.6 nmol/mg protein after preincubation with 20 µM Mic.

Dependence on enzyme conformation and pH. The effect of Mic concentration on EP was measured at the ice-water temperature by performing both the preincubation of samples and phosphorylation by ATP under different conditions. When the experiments were performed at pH 7.0 and Mic was preincubated for 1 min with SR vesicles already in the presence of 1 mM free Ca2+, the effect of Mic concentration on EP was moderate (Fig. 8). As a reference, 57% of the maximal EP level was accumulated when 30 µM Mic was included in the preincubation medium. When the vesicles in the absence of Ca2+ were exposed to Mic and then preincubated for 1 min with 1 mM free Ca2+, the phosphorylating capacity of the enzyme was clearly lower. Thus 7 µM Mic produced a 50% inhibition of EP accumulation. Note that the same Mic concentration induced only a 12% inhibition when added to the vesicles in the presence of Ca2+. The inhibitory pattern at pH 6.0 was qualitatively similar, i.e., the inhibition was higher when Mic was added to the vesicles before Ca2+, although some differences were observed (Fig. 8B). Namely, preincubation for 1 min of SR vesicles in the presence of 1 mM free Ca2+ with 4 µM Mic inhibited EP accumulation by 3%, but, when 4 µM Mic was initially mixed with the vesicles in the absence of Ca2+ and the subsequent preincubation with 1 mM free Ca2+ lasted 1 min, EP accumulation was inhibited by 50%.


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Fig. 8.   Mic concentration effect on EP when samples were preincubated in the presence of Ca2+. A: experiments were performed at pH 7.0. The initial medium containing 20 mM MOPS, pH 7.0, 80 mM KCl, 5 mM MgCl2, 0.1 mM EGTA, 1.1 mM CaCl2 (1 mM free Ca2+), and 0.05 mg/ml SR protein was preincubated at the ice-water temperature for 1 min with a given Mic concentration (open circle ). Vesicles in a Ca2+-free medium containing 20 mM MOPS, pH 7.0, 80 mM KCl, 5 mM MgCl2, 0.1 mM EGTA, 0.05 mg/ml SR protein, and a given Mic concentration were preincubated at the ice-water temperature for 1 min with 1.1 mM CaCl2 (1 mM free Ca2+) (). B: experiments were performed at pH 6.0. SR vesicles in a 1-mM free Ca2+ medium were preincubated at the ice-water temperature for 1 min with a given Mic concentration (open circle ). In the absence of Ca2+, SR vesicles were mixed with a given Mic concentration and then preincubated with 1 mM free Ca2+ (). In all cases, samples after preincubation were phosphorylated for 1 s with 50 µM [gamma -32P]ATP.

Dependence on phosphorylation. The phosphorylation partial reaction was studied by measuring EP as a function of ATP concentration. In these experiments, SR vesicles were equilibrated at the ice-water temperature in the presence of 1 mM free Ca2+ before a given Mic concentration was added. The preincubation was maintained at the ice-water temperature for 1 min, and the subsequent 1-s phosphorylation step was performed using different [gamma -32P]ATP concentrations. Control data obtained with no previous Mic preincubation step showed that EP increased linearly when the radioactive ATP increased from 1 to 100 µM (Fig. 9A). The presence of 10 µM Mic during preincubation did not alter the dependence of EP on ATP concentration. When the Mic concentration was raised to 20 µM, EP accumulation was lower, although the dependence on ATP concentration was still linear. When the EP level is plotted on a relative scale as a function of the ATP concentration on a logarithmic scale, it is clear that Mic did not affect either the maximal level of phosphorylation or the apparent affinity for ATP (Fig. 9B).


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Fig. 9.   Effect of Mic on ATP phosphorylation rate. A: SR vesicles (0.05 mg/ml) in a medium containing 20 mM MOPS, pH 7.0, 80 mM KCl, 20 mM MgCl2, 0.1 mM EGTA, and 1.1 mM CaCl2 (1 mM free Ca2+) were phosphorylated at the ice-water temperature for 1 s with a given concentration of [gamma -32P]ATP (). In some experiments, preincubation for 1 min before phosphorylation at the ice-water temperature of SR vesicles in the 1 mM free Ca2+ medium with 10 µM () or 20 µM Mic (open circle ) was performed. B: EP levels in a relative scale were plotted as a function of the ATP concentration. Symbols are as defined in A.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A common feature of many different compounds affecting the Ca2+-ATPase activity of isolated SR vesicles is their hydrophobic character (8, 22, 26, 27, 33, 36). Some of these compounds, including high-affinity inhibitors (30, 37), exhibit only inhibitory action, whereas others (5, 25, 38) are known to exert activating effect. Mic can be included in a group (22, 26, 18) that exerts activation when used at a low concentration and inhibition when used at higher concentrations (Figs. 2B and 4A). The dual effect of Mic on Ca2+-ATPase activity can be explained by binding/interaction to two different sites of the enzyme.

Mic can also activate or inhibit the rate of Ca2+ transport, depending on the concentration used, as observed during ATP hydrolysis (Fig. 3). This confirms that the Mic effect is on the Ca2+-ATPase protein. However, the dependence of transport and hydrolysis on Mic concentration is not exactly the same (cf. Figs. 3 and 4A).

The hydrophobic nature of drugs usually promotes their incorporation into the membrane, and when this happens, activation or inhibition is dependent on both drug and membrane concentration. The Mic effect also shares this feature because the degree of inhibition is dependent on membrane protein concentration (Fig. 4A). One milligram of SR protein contains ~4 nmol of active enzyme, as deduced from the maximal phosphorylation level. Therefore, a membrane protein concentration of 0.05 mg/ml is equivalent to 0.2 µM Ca2+-ATPase. If we consider that the K0.5 for inhibition is 12 µM (Fig. 2A), a Mic-to-ATPase ratio of 60 can be calculated. This means that 50% inhibition of the enzyme activity requires the participation of 60 drug molecules. Therefore, Mic is not a high-affinity inhibitor of the enzyme (30), and the protecting effect when the membrane concentration is raised (Fig. 4A) can be attributed to drug partitioning. Incorporation of Mic into the lipidic phase was confirmed when phosphatidylcholine vesicles were included in the reaction medium (Fig. 4B). Full protection against inhibition was observed using 10 times the phospholipid concentration present in the sample of SR membrane.

A brief ATP phosphorylation at low temperature is a valuable tool for distinguishing between E · Ca2 and E accumulation, because only the Ca2+-bound enzyme can be phosphorylated by ATP (see Fig. 1). Using this approach, the partial reaction E + 2Ca2+ right-arrow E · Ca2 (transition 1 in Fig. 1) was studied. The Ca2+-free to Ca2+-bound transition in the absence of Mic takes place in the millisecond time scale (16). However, the transition from the Ca2+-free to the Ca2+-bound state in the presence of Mic is slow and requires the addition of mM Ca2+ (Fig. 6). This suggests that the enzyme is retained in the Ca2+-free conformation when Mic is added to SR vesicles in the absence of Ca2+.

Furthermore, the Ca2+-bound to Ca2+-free transition can be rapidly elicited by Mic when the vesicles are initially in the presence of low µM Ca2+ (Fig. 7A). This confirms the stabilization of the Ca2+-free enzyme by Mic. Nonetheless, the Ca2+-bound conformation can be stabilized in the presence of Mic when SR vesicles are previously equilibrated in the presence of millimolar Ca2+ (Fig. 7B). The final equilibrium of Ca2+ and Mic binding to the enzyme is independent of the order of addition, although the slow Ca2+ binding transition in the presence of Mic is responsible for the distinct behavior of the Ca2+-bound and Ca2+-free enzymatic forms that we observe. Stabilization of the Ca2+-free enzyme is the main effect of clotrimazole (35) and other high-affinity inhibitors of the enzyme (14, 31, 37).

The Mic effect on Ca2+ binding was exploited by performing 1-min preincubations as described in Fig. 8. This demonstrated that the phosphorylating capacity of the enzyme was lower when Mic was added to the enzyme in the absence of Ca2+, i.e., when Mic was added before millimolar concentrations of Ca2+. In contrast, the EP level was higher when Mic was added to the enzyme in the presence of Ca2+, i.e., when millimolar concentrations of Ca2+ were added before Mic. This is consistent with a decrease in the rate of the Ca2+ binding transition induced by Mic.

The existence of a pH-dependent equilibrium between enzymatic forms, which affects Ca2+ binding, has been proposed (13, 28). Acidic pH favors the accumulation of E, whereas alkaline pH favors the accumulation of E · Ca2. According to this idea, when Mic is added to the enzyme in the absence of Ca2+, the decrease in EP would be higher at pH 6 than at pH 7 because of the greater stabilization of the Ca2+-free enzyme at acidic pH (Fig. 8). When Mic was added to the enzyme in the presence of mM Ca2+, the inhibition was lower at pH 6 than at pH 7. This can be attributed to a slower enzyme turnover at acidic pH. The decrease in EP is due to the accumulation of Ca2+-free species after ATP addition.

The partial reaction E · Ca2 + ATP right-arrow EP · Ca2 + ADP (transition 2 in Fig. 1) was studied by preincubating SR vesicles in the presence of mM Ca2+ to saturate the transport sites and then phosphorylating with different ATP concentrations. When the data were plotted on a relative scale, it was clear that Mic did not affect either the maximal rate of phosphorylation or enzyme affinity for ATP (Fig. 9B). This indicates that Mic does not affect the ATP phosphorylation reaction. However, we noted that the inhibition of Ca2+-ATPase activity by Mic was lower in the presence of 1 mM ATP than in the presence of 50 µM (Fig. 2). The protective role on the overall catalytic cycle induced by mM ATP can be explained by the activating effect exerted by the substrate, leading to the steady-state accumulation of Ca2+-bound against Ca2+-free enzymatic species.

A dose of 200 mg of ketoconazole, taken once daily, is used as treatment or prophylaxis against fungal infection (21). The oral dose translates into a peak plasma concentration of up to 7 µg/ml, i.e., 13.2 µM (21). Our in vitro inhibition data with Mic show K0.5 values in a similar concentration range. Therefore, the inhibition of SR Ca2+-ATPase by Mic and an alteration of the cytoplasmic free Ca2+ may be involved in the antifungal activity and/or the adverse effects associated with the use of this drug.


    ACKNOWLEDGEMENTS

This study was funded by grants PB97-1039 from the Spanish Ministerio de Ciencia y Tecnologia and PI-22/00756/FS/01 from Fundacion Seneca de la Region de Murcia, Spain.


    FOOTNOTES

Address for reprint requests and other correspondence: F. Fernandez-Belda, Depto. de Bioquímica y Biología Molecular A, Facultad de Veterinaria, Univ. de Murcia, Campus de Espinardo, 30071 Espinardo, Murcia, Spain (E-mail: fbelda{at}um.es).

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.

First published February 20, 2002;10.1152/ajpcell.00580.2001

Received 5 December 2001; accepted in final form 12 February 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 283(1):C85-C92
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