Clotrimazole, an Antimycotic Drug, Inhibits the Sarcoplasmic Reticulum Calcium Pump and Contractile Function in Heart Muscle*

Clotrimazole (CLT), an antimycotic drug, has been shown to inhibit proliferation of normal and cancer cell lines and its systemic use as a new tool in the treatment of proliferative disorders is presently under scrutiny (Benzaquen, L. R., Brugnara, C., Byers, H. R., Gattoni-Celli, S., and Halperin, J. A. (1995) Nature Med. 1, 534–540). The action of CLT is thought to involve depletion of intracellular Ca2+stores but the underlying mechanism has not been defined. The present study utilized membrane vesicles of rabbit cardiac sarcoplasmic reticulum (SR) to determine the mechanism by which CLT depletes intracellular Ca2+ stores. The results revealed a strong, concentration-dependent inhibitory action of CLT on the ATP-energized Ca2+ uptake activity of SR (50% inhibition with ∼35 μm CLT). The inhibition was of rapid onset (manifested in <15 s), and was accompanied by a 7-fold decrease in the apparent affinity of the SR Ca2+-ATPase for Ca2+ and a minor decrement in the enzyme’s apparent affinity toward ATP. Exposure of SR to CLT in the absence or presence of Ca2+ resulted in irreversible inhibition of Ca2+ uptake demonstrating that the Ca2+-bound and Ca2+-free conformations of the Ca2+-ATPase are CLT-sensitive. Introduction of CLT to the reaction medium subsequent to induction of enzyme turnover with Ca2+ and ATP resulted in instantaneous cessation of Ca2+ transport indicating that an intermediate enzyme species generated during turnover undergoes rapid inactivation by CLT. The inhibition of Ca2+ uptake by CLT was accompanied by inhibition of Ca2+-stimulated ATP hydrolysis and Ca2+-induced phosphoenzyme intermediate formation from ATP in the ATPase catalytic cycle. Phosphorylation of the Ca2+-deprived enzyme with Pi in the reverse direction of catalytic cycle and Ca2+ release from Ca2+-preloaded SR vesicles were unaffected by CLT. It is concluded that CLT depletes intracellular Ca2+ stores by inhibiting Ca2+ sequestration by the Ca2+-ATPase. The mechanism of ATPase inhibition involves a drug-induced alteration in the Ca2+-binding site(s) resulting in paralysis of the enzyme’s catalytic and ion transport cycle. CLT (50 μm) caused marked depression of contractile function in isolated perfused, electrically paced rabbit heart preparations. The contractile function recovered gradually following withdrawal of CLT from the perfusate indicating the existence of mechanisms in the intact cell to inactivate, metabolize, or clear CLT from its target site.

Clotrimazole (CLT) 1 is an antimycotic imidazole derivative widely used for the treatment of yeast infections and its fungistatic action has been attributed to the inhibition of sterol 14␣-demethylase, a microsomal cytochrome P-450-dependent enzyme (1,2). Imidazole antimycotics have also been shown to be potent inhibitors of many mammalian cytochrome P-450mediated reactions (3)(4)(5). Recently, CLT has been shown to have a remarkable ability to inhibit the proliferation of normal and cancer cell lines in vitro and in vivo (6), and the potential of this drug as a new tool in the treatment of proliferative disorders is now being explored. Evidence from mechanistic studies indicates that the growth-blocking property of CLT may arise from its ability to interfere with cellular Ca 2ϩ homeostasis. Thus, it has been shown that CLT depletes intracellular Ca 2ϩ stores in 3T3 cells (6), inhibits voltage-and ligandstimulated Ca 2ϩ influx mechanisms in GH3 cells, chromaffin cells, and thymocytes (7,8), as well as Ca 2ϩ -activated K ϩ channels in erythrocytes and thymocytes (9,10). The effects of CLT on Ca 2ϩ influx and Ca 2ϩ -activated K ϩ channels may be secondary to the drug-induced depletion of intracellular Ca 2ϩ stores (6). It has not been clarified whether the CLT-induced depletion of intracellular Ca 2ϩ stores is due to activation of Ca 2ϩ release from intracellular stores or due to inhibition of reuptake of Ca 2ϩ back into intracellular stores.
In muscle cells, the intracellular membrane system of SR plays a central role in the storage and release of Ca 2ϩ during the contraction-relaxation cycle. Upon myocyte excitation, Ca 2ϩ is released from the SR through the ryanodine receptor-Ca 2ϩ release channel to initiate muscle contraction (11)(12)(13). Subsequent muscle relaxation occurs upon sequestration of Ca 2ϩ back into the SR lumen by an SR-associated Ca 2ϩ -pumping ATPase (14 -17). During the translocation of Ca 2ϩ across the SR membrane, Ca 2ϩ -ATPase serves as an energy transducer and a carrier for Ca 2ϩ (15)(16)(17). The mechanism of Ca 2ϩ transport by the SR Ca 2ϩ -ATPase is recognized to involve cyclic transitions between two major conformational states, E 1 and E 2 (15)(16)(17). These two states differ in that the affinity for Ca 2ϩ is high in the E 1 conformation and low in the E 2 conformation, and in that the Ca 2ϩ -binding sites are exposed to the cytoplasmic side of SR in E 1 but to the luminal side of SR in E 2 . The catalytic and ion transport cycle begins with the binding of 2 mol of Ca 2ϩ ions followed by 1 mol of Mg 2ϩ -ATP to the E 1 form of the ATPase. An aspartic acid residue (Asp 351 ) in the active site is then phosphorylated by the terminal phosphate of ATP forming an acylphosphate. Phosphorylation of the enzyme results in a conformational change in the E 1 PCa 2ϩ phosphoenzyme intermediate to the E 2 PCa 2ϩ form which has decreased Ca 2ϩ affinity. The Ca 2ϩ -binding sites are now everted so that they face the SR lumen to which Ca 2ϩ is subsequently released.
The Mg 2ϩ -catalyzed hydrolysis of E 2 P devoid of bound Ca 2ϩ ions results in the release of P i into the cytoplasm leaving E 2 which isomerizes to E 1 to complete the cycle. The Ca 2ϩ -ATPase reaction mechanism is thought to be similar for different isoforms of this enzyme expressed in muscle and non-muscle tissues (18).
Given its robust Ca 2ϩ cycling properties, the SR membrane provides an excellent model system to investigate the mechanisms by which CLT depletes intracellular Ca 2ϩ stores. Here we present the results from studies using rabbit cardiac SR demonstrating a strong inhibitory action of CLT on the Ca 2ϩ ion transporting as well as energy transducing functions of the Ca 2ϩ -ATPase. On the other hand, CLT was found to have no effect on Ca 2ϩ release from Ca 2ϩ -preloaded SR vesicles. In isolated perfused beating rabbit hearts, infusion of CLT resulted in marked depression of contractile function.

EXPERIMENTAL PROCEDURES
Materials-45 CaCl 2 and [ 32 P]Na 2 PO 4 were purchased from New England Nuclear (Montreal, PQ, Canada), and [␥-32 P]ATP from Amersham (Oakville, ON, Canada). CLT was from Sigma. All other chemicals were from Sigma or BDH Chemicals (Toronto, ON, Canada).
Preparation of SR Vesicles-SR membrane vesicles were prepared from heart ventricles of New Zealand White rabbits (body weight 2.5-3 kg) as described previously (19). Following isolation, the SR vesicles were suspended in 10 mM Tris maleate (pH 6.8) containing 100 mM KCl and stored at Ϫ80°C after quick-freezing in liquid N 2 . Protein concentration was determined by the method of Lowry et al. (20) using bovine serum albumin as standard.
Ca 2ϩ Transport and Ca 2ϩ -ATPase Assays-ATP-dependent, oxalatefacilitated Ca 2ϩ uptake by SR was determined using the Millipore filtration technique as described previously (21). The standard incubation medium for Ca 2ϩ uptake (total volume 250 l) contained 50 mM Tris maleate (pH 6.8), 5 mM MgCl 2 , 5 mM NaN 3 , 120 mM KCl, 0.1 mM EGTA, 5 mM potassium oxalate, 5 mM ATP, 0.1 mM 45 CaCl 2 (ϳ8000 cpm/nmol; free Ca 2ϩ , 10.6 M), and SR (7.5 g of protein). In experiments where Ca 2ϩ concentration dependence was studied, the EGTA concentration in the assay medium was held constant at 0.1 mM and the amount of total 45 CaCl 2 added was varied in the range 1 to 280 M to yield the desired free Ca 2ϩ . Modifications to the standard incubation medium are specified in figure legends. Unless indicated otherwise, all assays were carried out at 37°C; the Ca 2ϩ transport reaction was initiated by the addition of ATP after preincubation of the rest of the assay components for 3 min. The initial free Ca 2ϩ concentrations in the assay medium were determined using the computer program of Fabiato (22). The data on Ca 2ϩ concentration dependence on Ca 2ϩ uptake were analyzed by nonlinear regression curve fitting using SigmaPlot scientific graph program (Jandel Scientific) run on an IBM-PC computer. The data were fitted to the equation, where v is the measured Ca 2ϩ uptake activity at a given Ca 2ϩ concentration, V max is the maximum activity reached, K 0.5 is the Ca 2ϩ concentration giving half of V max , and n is the equivalent to the Hill coefficient. Ca 2ϩ -ATPase activity of SR membranes was quantified as described previously (23) using the assay conditions specified below. In one series of experiments, Ca 2ϩ -ATPase assays were performed using an incubation medium identical to that used for Ca 2ϩ uptake, except that [␥-32 P]ATP was used instead of nonradioactive ATP and nonradioactive CaCl 2 was used instead of 45 CaCl 2 . Parallel assays were also performed in the absence of Ca 2ϩ (i.e. in the presence of 0.2 mM EGTA with no CaCl 2 added to the assay medium) and the Ca 2ϩ -ATPase activity (designated "Ca 2ϩ -stimulated ATPase activity" under "Results") was defined as the difference in ATP hydrolysis (liberation of 32 P i ) measured in the absence and presence of Ca 2ϩ . In another series of experiments, Ca 2ϩ -ATPase assays were performed using an incubation medium similar to that described above but with the exception that potassium oxalate was omitted and the Ca 2ϩ ionophore A23187 (dissolved in ethanol, final concentration of ethanol in assay ϭ 1%) was added to give a final concentration of 3 M. These assays were performed in the absence and presence of TG. When present, the concentration of TG in the assay medium was 0.1 M. 2 In these experiments, the TG-inhibit-able ATP hydrolysis was defined as the Ca 2ϩ -ATPase activity (designated "TG-sensitive Ca 2ϩ -ATPase activity" under "Results"). In both series of experiments, the ATPase reaction was initiated by the addition of ATP after preincubation of the rest of the assay components for 3 min at 37°C, and was allowed to proceed for 3 min.
Unless indicated otherwise, the Ca 2ϩ uptake and ATPase assays in the presence of CLT were performed by adding the drug during preincubation. CLT and TG were dissolved in Me 2 SO and control tubes contained an equivalent volume of Me 2 SO vehicle; the final concentration of Me 2 SO did not exceed 5%.
Phosphorylation with ATP-Steady-state levels of Ca 2ϩ -induced phosphoenzyme were measured using [␥-32 P] ATP as described previously (24). The reaction mixture (total volume 200 l) contained 50 mM Tris maleate (pH 6.8), 1 mM MgCl 2 , 120 mM KCl, 25 M [␥-32 P]ATP, 0.1 mM EGTA, 0.1 mM CaCl 2 , and SR (50 g of protein). The Ca 2ϩ dependence of phosphoenzyme formation was monitored in parallel assays in the absence of Ca 2ϩ and in the presence of 1 mM EGTA. The reaction was initiated by the addition of ATP following preincubation of the rest of the assay mixture for 3 min at 23°C. The reaction was allowed to proceed for 15 s and was stopped by adding 1 ml of trichloroacetic acid containing 1 mM ATP and 2 mM KH 2 PO 4 . The phosphoenzyme was quantified by Millipore filtration and liquid scintillation counting (24).
Phosphorylation with P i -Phosphorylation of SR Ca 2ϩ -ATPase with P i was performed as described previously (25). The reaction mixture (total volume 200 l) contained 50 mM Tris maleate (pH 6.8), 100 mM KCl, 10 mM MgCl 2 , 1 mM EGTA, 10% Me 2 SO, 4 mM [ 32 P]Na 2 PO 4 and SR (50 g). The reaction, initiated by the addition of [ 32 P]phosphate following preincubation of the rest of the assay mixture at 23°C for 3 min, was allowed to proceed for 30 s and was quenched with 1 ml of 10% trichloroacetic acid containing 4 mM Na 2 HPO 4 . The acid-denatured protein was recovered by centrifugation, the pellets were washed with 0.2 mM Na 2 HPO 4 , digested in 500 l of 50 mM Tris-HCl (pH 7.5) containing 2% sodium dodecyl sulfate, and the 32 P radioactivity was determined by liquid scintillation counting.
Ca 2ϩ Release Assay-45 Ca 2ϩ release rates from SR vesicles passively loaded with 45 CaCl 2 were determined by Millipore filtration (25). Passive 45 Ca 2ϩ loading was performed by incubating SR vesicles (1 mg of protein/ml) at 23°C for 40 min in a medium containing 50 mM Tris maleate (pH 6.8), 120 mM KCl, 1 mM 45 CaCl 2 , and 1 mM potassium oxalate. To initiate Ca 2ϩ release, aliquots of the 45 Ca 2ϩ -loaded vesicles were diluted into a Ca 2ϩ release medium (50 mM Tris maleate, pH 6.8, containing 2 mM MgCl 2 and 1 mM EGTA) that was preincubated for 5 min at 37°C. Subsequently aliquots of the incubation mixture were filtered through Millipore filters at 30-s intervals for a period of 5 min. The filters were washed with 3 ml of ice-cold 10 mM Tris maleate buffer (pH 6.8) containing 120 mM KCl, 10 mM MgCl 2 , and 10 M ruthenium red, dried at 60°C, and 45 Ca 2ϩ radioactivity was determined by liquid scintillation counting.
Heart Perfusion and Measurement of Contractile Function-Rabbits were anesthetized with sodium pentobarbitol (35 mg/kg, intravenous), the hearts were excised and immediately cannulated for retrograde aortic perfusion of the coronary arteries with mammalian Ringer solution consisting of 154 mM NaCl, 5 mM KCl, 2.2 mM CaCl 2 , 6 mM NaHCO 3 , and 5.5 mM dextrose. The perfusion buffer was equilibrated with 95% O 2 ,5% CO 2 , which maintained a pH of 7.4; the perfusion temperature was set at 37 Ϯ 0.2°C. The hearts were perfused at a constant flow rate of 25 ml/min using a peristaltic pump. After an initial 15-20 min of perfusion, when the spontaneous beating had stabilized, the atria were removed and the hearts were paced electrically at a rate of 120 beats/min with a Grass SD9 stimulator via a platinum wire electrode inserted into the epicardium, at double threshold voltage and a duration of 5 ms. A latex balloon-tipped cannula filled with degassed H 2 O was inserted into the lumen of the left ventricle for obtaining systolic left ventricular pressure (LVP) development. The cannula was connected via a pressure transducer (COBE, Bramalea, Canada) to a BioPac System Digital Monitor (model MP100) and a personal computer which allowed on-line monitoring of LVP and off-line calculation of developed pressure, rate of pressure development (ϩdP/dt), and rate of relaxation (ϪdP/dt).
Data Presentation-Unless specified otherwise, the experimental values represent the average of at least three independent experiments using separate SR preparations performed in duplicate. The data are presented as mean Ϯ S.E.

Effect of CLT on the Time Course of ATP-dependent Ca 2ϩ
Uptake by SR-The ATP-dependent, oxalate-facilitated Ca 2ϩ uptake by SR vesicles is a useful, commonly used parameter to measure the Ca 2ϩ -pump (Ca 2ϩ -ATPase) function of SR in vitro (15). Fig. 1 shows the time course of ATP-dependent Ca 2ϩ uptake by cardiac SR vesicles measured in the absence of CLT and in the presence of three selected concentrations of CLT. The rates of Ca 2ϩ uptake by SR was strongly inhibited in the presence of CLT; the degree of inhibition increased with increasing concentration of CLT.
CLT and SR Protein Concentration Dependence on the Inhibitory Action of CLT on Ca 2ϩ Uptake-In the experiments shown in Fig. 2A, the ATP-dependent Ca 2ϩ uptake was determined in the absence of CLT and in the presence of varying concentrations of CLT (16 -120 M); the amount of SR in the assay medium was constant at 30 g of protein/ml (i.e. 7.5 g of protein/250 l of assay medium, see "Experimental Procedures"). It is seen that CLT inhibited Ca 2ϩ uptake strongly and in a concentration-dependent manner. Under these conditions, 50% inhibition of Ca 2ϩ uptake was observed at ϳ35 M CLT; nearly complete inhibition of Ca 2ϩ uptake occurred at CLT concentrations of 80 -120 M.
The results presented in Fig. 2B demonstrate the influence of SR protein concentration in the assay medium on the inhibitory action of CLT on Ca 2ϩ uptake by SR. In these experiments, the SR protein concentration in the assay medium was varied from 0.02 to 0.32 mg/ml and Ca 2ϩ uptake was measured in the absence of CLT and in the presence of two selected concentrations of CLT (32 and 64 M). It can be seen that at a given concentration of CLT, increasing the concentration of SR in the reaction mixture leads to progressively less inhibition of Ca 2ϩ uptake. This relationship is more pronounced at the low (32 M) CLT concentration. Thus, the CLT concentration dependence of Ca 2ϩ uptake inhibition is an apparent function of the concentration of SR protein in the reaction mixture. It should be noted, however, that half-maximal inhibition of Ca 2ϩ uptake required a large stoichiometric excess of CLT over Ca 2ϩ -ATPase in the assay medium. For example, since the Ca 2ϩ -ATPase constituted about 10% of the membrane protein in the cardiac SR preparations used, 3 it could be estimated that 32 and 64 M CLT produced 50% inhibition of Ca 2ϩ uptake in the presence of ϳ75 and 200 nM Ca 2ϩ -ATPase, respectively, in the assay medium (Fig. 2B). Therefore, the CLT concentration dependence of Ca 2ϩ uptake inhibition does not reflect a strict stoichiometric relationship of the inhibitor and Ca 2ϩ -ATPase (see "Discussion").
Effect of CLT on Ca 2ϩ -ATPase Activity-Since CLT inhibited ATP-dependent Ca 2ϩ uptake by SR, the effect of CLT on Ca 2ϩ -ATPase activity (ATP hydrolysis) of SR was investigated. Incubation of SR vesicles with varying concentrations of CLT (16 -120 M) under the assay conditions identical to that used for Ca 2ϩ uptake led to concentration-dependent inhibition of Ca 2ϩ -stimulated ATPase activity (Fig. 3A). This inhibition of ATPase activity occurred at the same concentration range of CLT required for inhibition of Ca 2ϩ uptake (cf. Fig. 2A). Therefore the observed reduction of Ca 2ϩ uptake is a consequence of a primary inhibition of ATPase activity by CLT. The Ca 2ϩstimulated ATPase activity of SR measured in the absence of CLT in these experiments (Fig. 3A), however, exceeded the Ca 2ϩ uptake activity determined under identical assay conditions ( Fig. 2A). This excess ATP hydrolysis is apparently due to uncoupling of ATP hydrolysis and Ca 2ϩ transport prevailing in the isolated SR vesicles and/or contribution of other ATPase(s) unrelated to the Ca 2ϩ -pumping ATPase. In order to examine the specific effect of CLT on the SR Ca 2ϩ -pumping ATPase, additional experiments were performed where TG (0.1 M)sensitive Ca 2ϩ -ATPase activity of SR was determined in the absence of CLT and in the presence of varying concentrations of CLT. Also, the assays were performed in the absence of oxalate The results showed concentration-dependent inhibition of TGsensitive ATPase activity by CLT (Fig. 3B). The concentration of CLT required for half-maximal inhibition of TG-sensitive Ca 2ϩ -ATPase activity (ϳ36 M, Fig. 3B) and Ca 2ϩ -stimulated ATPase activity (ϳ30 M, Fig. 3A) was found to be essentially similar. The TG-sensitive ATPase activity measured in the absence of CLT and in the presence of ionophore (Fig. 3B) was about 4-fold higher than the Ca 2ϩ -stimulated ATPase activity measured in the absence of ionophore and in the presence of oxalate (Fig. 3A). Such difference in enzyme activity is likely due to stimulation of the SR Ca 2ϩ -ATPase by the ionophore (26,27). 4 At a submaximally effective concentration of CLT (32 M), inhibition of TG-sensitive Ca 2ϩ -ATPase activity was observed at varying Ca 2ϩ concentrations (Fig. 3B, inset). The inhibitory effect of CLT on Ca 2ϩ -ATPase activity was associated with decrements in maximal velocity (V max (nmol of P i /mg of protein/3 min): control, 2491 Ϯ 62; ϩ32 M CLT, 1573 Ϯ 31) as well as the enzyme's apparent affinity for Ca 2ϩ (K 0.5 for Ca 2ϩ (M): control, 0.8 Ϯ 0.06; ϩ32 M CLT, 2.8 Ϯ 0.10).
Effect of CLT on Ca 2ϩ Uptake by SR at Varying Concentrations of Ca 2ϩ and ATP-The results presented in Fig. 4 show the effect of two selected concentrations of CLT (32 or 64 M) on Ca 2ϩ uptake by SR at a wide range of Ca 2ϩ concentrations (9 nM-71 M). CLT inhibited Ca 2ϩ uptake at all Ca 2ϩ concentra-tions tested. With a submaximally effective concentration of CLT (32 M), the inhibitory effect could not be overcome with increasing Ca 2ϩ concentration. The kinetic parameters derived from the data shown in Fig. 4 are summarized in Table I. It can be seen that the inhibitory action of CLT is associated with decrements in the apparent affinity of the Ca 2ϩ -ATPase for Ca 2ϩ , Hill coefficient (n H ) for Ca 2ϩ , as well as V max of Ca 2ϩ transport.
When Ca 2ϩ uptake assays were performed in the presence of various ATP concentrations, CLT inhibition was found to be independent of ATP concentration (Fig. 5). Approximately 60% decrease in the V max of Ca 2ϩ uptake was observed in the presence of 32 M CLT (V max (nmol of Ca 2ϩ /mg of protein/min): control, 120; ϩ32 M CLT, 50). This inhibitory action of CLT also appeared to involve a modest decrease in the apparent affinity of the Ca 2ϩ -ATPase for ATP (K 0.5 for ATP (mM): control, 0.11; ϩ32 M CLT, 0.20).
Blockade of Ca 2ϩ Uptake by Addition of CLT during Ca 2ϩ -ATPase Turnover Cycle-To investigate the effect of CLT on Ca 2ϩ -ATPase during its turnover, CLT was added to the Ca 2ϩ uptake assay medium 90 s after initiating Ca 2ϩ -ATPase turnover by the addition of ATP. The time course of Ca 2ϩ uptake was monitored prior to, and following the addition of CLT, for several minutes. It was found that addition of CLT (32 or 64 M) during the turnover cycle of Ca 2ϩ -ATPase resulted in apparently instantaneous inhibition of further Ca 2ϩ uptake by SR vesicles (Fig. 6). No subsequent increase in Ca 2ϩ content of SR vesicles was observed even after prolonged incubation.
Effect of CLT on Ca 2ϩ -dependent Phosphoenzyme Formation-One of the well characterized intermediate steps in Ca 2ϩ -ATPase reaction pathway is the formation of a phosphoenzyme intermediate (EP) upon the sequential binding of Ca 2ϩ followed by ATP to the ATPase on the cytoplasmic side of the SR (see Introduction). Since CLT inhibited the overall reaction of Ca 2ϩactivated ATP hydrolysis by the SR Ca 2ϩ -ATPase (Fig. 3), the effect of CLT on Ca 2ϩ -dependent enzyme phosphorylation with ATP was examined. The results showed that in SR vesicles incubated with Ca 2ϩ and ATP, EP formation was strongly inhibited by CLT (Fig. 7, panel A).
Effect of CLT on Ca 2ϩ -ATPase Phosphorylation with P i -A 4 Measurement of TG (0.1 M)-sensitive ATPase activity in the absence of ionophore and presence of oxalate in the assay medium (i.e. under the standard Ca 2ϩ uptake assay conditions employed in this study) gave a value of 720 Ϯ 75 nmol of P i /mg of protein/3 min which is not significantly different from the Ca 2ϩ -stimulated ATPase activity measured in the absence of CLT (cf. Fig. 3A). Therefore, the "Ca 2ϩstimulated" and "TG-sensitive" ATPase activities measured likely represent the enzymatic function of the same SR Ca 2ϩ -ATPase.  characteristic functional difference between Ca 2ϩ bound and Ca 2ϩ -free conformations of the Ca 2ϩ -ATPase is that the former undergoes phosphorylation with ATP but not P i whereas the latter undergoes phosphorylation with P i but not ATP (15). Since we observed an inhibitory action of CLT on Ca 2ϩ -dependent enzyme phosphorylation with ATP (Fig. 7, panel A) and Ca 2ϩ -stimulated ATP hydrolysis (Fig. 3), the effect of CLT on phosphorylation of the Ca 2ϩ -free ATPase with P i was also investigated. Interestingly, it was observed that CLT did not exert any inhibitory effect on enzyme phosphorylation with P i even at a high concentration of 100 M (Fig. 7, panel B).
Irreversible Nature of the Inhibitory Action of CLT on the SR Ca 2ϩ -ATPase-In order to assess whether the inhibitory effect of CLT on the SR Ca 2ϩ -ATPase was reversible, the following experiment was performed. SR vesicles were incubated with CLT (80 M) for 3 min at 37°C in the absence or presence of Ca 2ϩ . Subsequently, the SR vesicles were recovered by centrifugation, washed extensively with 10 mM Tris maleate buffer containing 100 mM KCl (pH 6.8), and the time course of Ca 2ϩ uptake was determined under standard assay conditions. SR vesicles subjected to the same experimental protocol but without CLT in the incubation medium served as control for this experiment. The results from this experiment showed that exposure of the SR to CLT (in the absence or presence of Ca 2ϩ ) resulted in an apparently irreversible inhibition of Ca 2ϩ -ATPase function (Fig. 8).
Effect of CLT on Ca 2ϩ Release-The possibility that CLT may also influence Ca 2ϩ release from the SR was investigated by determining the effect of CLT on unidirectional Ca 2ϩ release from passively Ca 2ϩ -loaded SR vesicles. As shown in Fig. 9, CLT (10 or 80 M) did not influence the rate of Ca 2ϩ release from the SR. Therefore the inhibitory effect of CLT on Ca 2ϩ uptake observed in this study can be attributed entirely to inhibition of the SR Ca 2ϩ -ATPase.
Effect of CLT on Cardiac Contractile Function-In view of the strong inhibitory action of CLT on Ca 2ϩ uptake by cardiac SR observed in vitro, it was of considerable interest to examine the effect of this drug on cardiac contractile function. In isolated, electrically paced rabbit heart preparations perfused at a constant perfusate flow rate, CLT (50 M) produced marked depression of contractile function as evidenced by decrements in developed LVP as well as maximum rates of pressure development and relaxation ( Fig. 10 and Table II). These effects were discernible within 1 min after initiating perfusion with CLT. Interestingly, reperfusion with normal buffer following CLT, resulted in gradual recovery of contractile function. DISCUSSION The results presented here demonstrate that micromolar concentrations of CLT strongly inhibits Ca 2ϩ transport in isolated cardiac SR vesicles. The inhibition of Ca 2ϩ transport is a consequence of primary inhibition of the SR Ca 2ϩ -ATPase by  5. Effect of CLT on ATP-dependent Ca 2؉ uptake by SR at varying ATP concentrations. SR vesicles were preincubated for 3 min at 37°C in the standard assay medium (see "Experimental Procedures") in the absence of CLT (q) or in the presence of 32 M CLT (f). The Ca 2ϩ uptake reaction was initiated by the addition of ATP and was allowed to proceed for 1 min. In panel A, Ca 2ϩ uptake activity is plotted against ATP concentration; panel B shows the double reciprocal plot of the data for the ATP concentration range 0.1-5 mM. The data represent mean Ϯ S.E. of three experiments using separate SR preparations. CLT. Furthermore, CLT was found not to influence Ca 2ϩ release from Ca 2ϩ -preloaded SR vesicles. Taken together, these findings suggest strongly that the ability of CLT to deplete intracellular Ca 2ϩ stores observed in other cell types (6) likely stems from a "thapsigargin-like" inhibitory action of this drug on the sarco(endo)plasmic reticulum Ca 2ϩ -ATPase.
Analysis of the characteristics of Ca 2ϩ -ATPase inhibition by CLT has provided insights into the mechanism of action of this drug. CLT inhibits the SR Ca 2ϩ -ATPase rapidly, and in an apparently irreversible fashion since even after extraction of the inhibitor from the incubation medium, Ca 2ϩ -ATPase did not regain its ion transporting activity (Fig. 8). The inhibitory action of CLT was accompanied by a striking decrease in the affinity of the Ca 2ϩ -ATPase for Ca 2ϩ (Table I) as well as diminished cooperativity of Ca 2ϩ binding to the enzyme (as judged from lower value for the Hill coefficient). These findings suggest that the inhibitory action of CLT is associated with a major alteration in the functional properties of the Ca 2ϩ -binding sites located in the transmembrane region of the ATPase (16,17). Since the inhibitory effect of CLT could not be overcome by increasing the concentration of free Ca 2ϩ , CLT inhibition is non-competitive with respect to Ca 2ϩ . Thus, the structural perturbation in the Ca 2ϩ -ATPase produced by CLT not only affects the enzyme's affinity for Ca 2ϩ but also causes a decrease in the maximal rate of Ca 2ϩ transport. The CLTinduced changes in kinetic characteristics, together with the irreversible nature of CLT inhibition demonstrates that CLT is not a Ca 2ϩ -chelating agent decreasing the concentration of free Ca 2ϩ available in the medium, but rather it appears to produce structural perturbations in the Ca 2ϩ -ATPase, which impact adversely on enzyme function. It must be noted that halfmaximal inhibition of Ca 2ϩ transport required a large stoichiometric excess of CLT over Ca 2ϩ -ATPase in the assay medium (Fig. 2B). On the other hand, introduction of CLT during Ca 2ϩ -ATPase turnover cycle resulted in instantaneous cessation of Ca 2ϩ transport (Fig. 6), and extensive washing of the SR membranes after exposure to CLT did not result in recovery of Ca 2ϩ -ATPase function (Fig. 8). From these observations it is not clear whether CLT-induced structural perturbations in the Ca 2ϩ -ATPase stem from direct interaction of the drug with the Ca 2ϩ -ATPase or indirectly due to partitioning of the drug in the membrane vesicles. It is possible that several molecules of CLT bind to hydrophobic "cavities" in the Ca 2ϩ -ATPase, and to accommodate the intruding CLT, the ATPase undergoes structural rearrangement that results in loss of its enzymatic and ion transport functions. Such a mechanism has been suggested for the inhibitory action of anesthetics on Ca 2ϩ -ATPases (28), and direct anesthetic binding to the SR Ca 2ϩ -ATPase has been demonstrated recently (29). It is noteworthy that CLT inhibited the energy transducing and ion transporting functions of the Ca 2ϩ -ATPase at the same concentration range. Thus, impaired coupling of catalytic and ion transport events does not contribute to the inhibitory action of CLT on the SR Ca 2ϩ pump. The inhibition of Ca 2ϩ transport by CLT was found to be independent of ATP concentration. Interestingly, while the Ca 2ϩ -dependent phosphoenzyme formation with ATP was strongly inhibited by CLT, the Ca 2ϩ -independent phosphoenzyme formation with P i in the reverse direction of the catalytic cycle was unaffected by CLT. These findings suggest that CLT does not induce major structural alteration at the catalytic site located in the extramembranous region of the Ca 2ϩ -ATPase (16,17). This situation is similar to that encountered upon derivatization of SR Ca 2ϩ -ATPase with the fluorescent carbodiimide NCD4 which also results in inhibition of Ca 2ϩ -dependent ATP utilization but not Ca 2ϩ -independent phosphoenzyme formation with P i (30). Evidence has been obtained suggesting that NCD4 induces a perturbation within or near the transmembrane region of the ATPase (at a relatively large distance from the catalytic site) that interferes with Ca 2ϩ binding (30). Also, it has been found by site-directed mutagenesis that conservative mutations of any of 6 amino acid residues in the transmembrane domain of the ATPase interfere with activa-  9. Effect of CLT on Ca 2؉ release from Ca 2؉ -loaded SR vesicles. SR vesicles passively loaded with 45 CaCl 2 were transferred to a Ca 2ϩ release medium to initiate Ca 2ϩ release. Ca 2ϩ release was monitored in the absence of CLT (E) and in the presence of 10 (q) or 80 (OE) M CLT in the Ca 2ϩ release assay medium (see "Experimental Procedures"). The figure shows the Ca 2ϩ content of SR vesicles (expressed as % of initial Ca 2ϩ load) at various time intervals following incubation in the Ca 2ϩ release medium. The initial Ca 2ϩ load of SR vesicles (prior to initiating Ca 2ϩ release) was 572 nmol of Ca 2ϩ /mg of protein. Results from a single experiment are shown here. Similar results were obtained in two additional experiments. tion of this enzyme by Ca 2ϩ (31). These observations are consistent with the view that CLT-induced structural perturbation involving the Ca 2ϩ -binding sites of the ATPase contributes to the inhibition of ATP hydrolysis and Ca 2ϩ transport.
Comparison of the observed characteristics of Ca 2ϩ -ATPase inhibition by CLT with those reported for other inhibitors of the SR Ca 2ϩ -ATPase such as TG and CPA, reveals certain similarities as well as striking differences. (i) Both TG and CPA react with and stabilize the Ca 2ϩ -deprived, E 2 form of the enzyme (32)(33)(34)(35)(36). Our finding that exposure of the Ca 2ϩ -ATPase to CLT in the absence or presence of Ca 2ϩ results in irreversible inhibition of the enzyme (Fig. 8) suggests that both the Ca 2ϩdeprived E 1 state and the Ca 2ϩ -liganded E 2 state of the ATPase are CLT-sensitive. (ii) The protective effect of Ca 2ϩ against TG and CPA inhibition of the Ca 2ϩ -ATPase is lost upon induction of enzyme turnover with ATP, and an intermediate species generated during turnover is highly susceptible to inhibition by these drugs (33,34,36). We found that introduction of CLT to the reaction medium during the turnover cycle of the Ca 2ϩ -ATPase results in apparently instantaneous cessation of Ca 2ϩ transport (Fig. 6). It is likely that an intermediate enzyme species generated during turnover undergoes rapid inactivation by CLT. In any case, the inhibitory action of CLT is manifested even when the catalytic and ion transport sites of the enzyme are occupied. (iii) Ca 2ϩ -dependent enzyme phosphorylation with ATP is inhibited by TG (33), CPA (36), and CLT (Fig. 7, panel A). On the other hand, phosphorylation of the Ca 2ϩ -deprived enzyme with P i is inhibited by TG (33) but not CLT (Fig. 7, panel B). This difference suggests that structural perturbations introduced by TG, but not CLT, affect the nucleotide-binding site in the catalytic domain of the ATPase. Consistent with this view, CLT was found to cause only a slight decrease in the enzyme's affinity toward ATP (Fig. 5) when compared with the much greater decrements in ATP binding affinity observed upon exposure of the ATPase to TG (37). (iv) The Ca 2ϩ -binding affinity of the ATPase is markedly decreased upon interaction of the enzyme with TG (32,33,38), CPA (34), and CLT (Table I). Thus, a structural perturbation affecting the properties of the Ca 2ϩ -binding sites appears to be a common feature underlying the mechanism of action of all three drugs. A recent study which examined the TG sensitivity of chimeric Ca 2ϩ -ATPase/Na ϩ ,K ϩ -ATPase molecules has suggested the transmembrane segments M3 and/or M4 as the potential TG target site in the Ca 2ϩ -ATPase (39). Location of the CLT-binding sites will shed more light on the role played by the region of the Ca 2ϩ -ATPase protein that is recognized by this drug.
CLT was found to cause marked depression of contractile function in the isolated perfused beating heart ( Fig. 10 and Table II). The inhibition of contractile function was not due to compromised coronary flow because the perfusate flow rate was held constant during perfusion. Furthermore, we observed that under these conditions, CLT (50 M) produced a modest decrease (ϳ20%) in coronary perfusion pressure, suggesting a vasodilator (not constrictor) effect (results not shown). Interestingly, the contractile function recovered gradually following withdrawal of CLT from the perfusion medium. This finding contrasts with the apparently irreversible effect of CLT on the SR Ca 2ϩ -ATPase observed in vitro and suggests the existence of mechanisms in the intact cell to inactivate, metabolize, or clear the drug from its target site. This observation and the effects and mechanisms of action of CLT on SR function described here might be of relevance in the design and evaluation of CLT and/or its derivatives for their therapeutic applications in vivo. The possibility that the CLT-induced impairment in cardiac contractile function is due to mechanisms other than the inhibitory action of CLT on the SR Ca 2ϩ pump observed in vitro cannot be discounted, however. The potential direct or indirect effects of CLT at the level of myofilaments and on ion transport systems other than the SR Ca 2ϩ -ATPase (e.g. plasma membrane Ca 2ϩ -ATPase, Na ϩ ,K ϩ -ATPase, Na ϩ /Ca 2ϩ -exchanger) remain to be investigated.  Table II. LVP, left ventricular pressure.

TABLE II
Effects of CLT on cardiac contractile function Contractile function was assessed in isolated perfused, electrically paced (120 beats/min) rabbit heart as described under "Experimental Procedures." Segments of 20 consecutive contractions such as those depicted in Fig. 10 were analyzed to obtain the average value shown for each parameter. Similar finding were obtained in two additional isolated heart preparations. LVP, left ventricular pressure (systolic); ϩdP/ dt, maximum rate of pressure development; ϪdP/dt, maximum rate of relaxation.