Departamento de Bioquímica y Biología Molecular A, Facultad de Veterinaria, Universidad de Murcia, Campus de Espinardo, 30071 Espinardo, Murcia, Spain
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ABSTRACT |
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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
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INTRODUCTION |
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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--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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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.
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MATERIALS AND METHODS |
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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(-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
[-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 [Data presentation. The experimental data points represent means ± SE of at least three independent determinations, each performed in duplicate.
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RESULTS |
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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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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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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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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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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 [-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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DISCUSSION |
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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+ 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 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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