Introduction

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Physiological regulation of the Ca²⁺ pump of the cardiac sarcoplasmic reticulum (SR) occurs through phosphorylation/dephosphorylation of phospholamban (PLN), which is also present on the SR membrane. PLN is one of several proteins that are phosphorylated as a result of a -adrenergically induced increase in cyclic AMP-dependent protein kinase (PKA) activity upon sympathetic stimulation of the myocardium (Wolska et al., 1996). The PLN-mediated increase in the rate of removal of cytoplasmic Ca²⁺ by the SR Ca²⁺ pump during muscle relaxation and, hence, greater retention of intracellular Ca²⁺ within the SR lumen available for subsequent release are major components of the inotropic and relaxation-promoting (lusitropic) effects of -adrenergic agonists.

The kinetic mechanism of regulation by PLN of the Ca²⁺ pump, when studied in vitro, involves increases in V_max(Ca) and the apparent affinity of the pump for Ca²⁺ (Antipenko et al., 1997b; Kargacin et al., 1998). It is unclear, however, how these kinetic effects, particularly the former, relate to the catalytic cycle. During this cycle (Inesi, 1985), an equilibrium that is thought to exist between two major conformational states of the enzyme, E₂ and E₁, is shifted in favor of E₁ in the presence of Ca²⁺, which allows binding of 2 Ca²⁺ (2Ca·E₁). E₁ also binds ATP with high affinity (E₁·ATP), and in the presence of Ca²⁺ , an acylphosphoprotein intermediate is formed (2Ca·E₁·P). Translocation of 2 Ca²⁺ across the membrane occurs during the conversion of 2Ca·E₁·P to 2Ca·E₂·P, whereupon 2 Ca²⁺ are released into the SR lumen as a result of a low affinity of E₂ for Ca²⁺. Decomposition of E₂P allows release of P_i on the cytoplasmic side of the membrane. ATP may also bind to the pump at reduced affinity and accelerate certain steps in the catalytic cycle (Dupont et al., 1985; Gould et al., 1986; Mignaco et al., 1996). Such ATP is called regulatory nucleotide in contrast to catalytic nucleotide, which binds with high affinity (i.e., at submicromolar ATP concentrations). An increase in the apparent affinity of the pump for Ca²⁺ as a result of PLN phosphorylation or treatment of SR membranes with anti-PLN monoclonal antibodies to remove the inhibition by PLN is attributable to an increase in the rate of a conformational change in the enzyme associated with binding of the first of bound 2 Ca²⁺ (Cantilina et al., 1993). Another explanation, however, is required for the effect of PLN on V_max(Ca).

Information regarding the mechanism by which the Ca²⁺ pump may be regulated by PLN or other means may be obtained from the study of certain plant-derived compounds that are stimulatory in cardiac microsomes under certain conditions (Kobayashi et al., 1987, 1988; Patil et al., 1996; Berrebi-Bertrand et al., 1997). Thus, in a recent study with crude cardiac microsomes (Berrebi-Bertrand et al., 1997), ellagic acid, a polyphenol, increased V_max(Ca) of microsomal Ca²⁺ uptake and produced a slight apparent decrease in K_m(Ca) that failed to attain statistical significance (p >.05). In the same study, an analog of gingerol, namely 1-(3,4-dimethoxyphenyl)-3-dodecanone, also increased V_max(Ca) but markedly increased K_m(Ca). Both compounds decreased the Hill coefficient. These reported kinetic effects on the Ca²⁺ pump resemble effects observed in our previous study with jasmone in assays of Ca²⁺ uptake and Ca²⁺-ATPase activity (Antipenko et al., 1997a). However, in the study by Berrebi-Bertrand et al. (1997), although ellagic acid significantly increased V_max(Ca) of Ca²⁺ uptake, it failed to produce the expected parallel increase in Ca²⁺-ATPase activity. Also, the marked decrease in K_m(Ca) of Ca²⁺ uptake obtained with 1-(3,4-dimethoxyphenyl)-3-dodecanone was much reduced and statistically insignificant in assays of Ca²⁺-ATPase activity in the same study. Hence, the actions of the various compounds tested are unclear. Part of the uncertainty regarding their actions on the SR Ca²⁺ pump stems from the use of heterogeneous membrane preparations, which have also resulted in discordant results regarding the kinetic effects of PLN phosphorylation on the cardiac SR Ca²⁺ pump (Antipenko et al., 1999).

In the present study, we investigated the effects of gingerol and ellagic acid on the Ca²⁺ pump using purified (light) cardiac SR vesicles. The effect of gingerol on the kinetic properties of the pump is of particular interest because gingerol produces lusitropic and inotropic effects in isolated cardiac muscle similar to those of the -adrenergic agonist isoproterenol (Shoji et al., 1982; Kobayashi et al., 1988). The contribution of the SR Ca²⁺ pump to the physiological actions of -adrenergic agents is generally attributed to the ability of PLN phosphorylation to decrease the K_m(Ca) of Ca²⁺ uptake or Ca²⁺-ATPase activity (MacLennan and Toyofuku, 1996; Kadambi and Kranias, 1997). Contrary to the expectation on this basis, we observed no significant effect of gingerol on K_m(Ca) of Ca²⁺ uptake. We report, however, a significant increase in V_max(Ca) and no change in the Hill coefficient. We compare the kinetic effects of these compounds in PKA-phosphorylated and unphosphorylated microsomes and relate our findings to the effects of PLN on the pump.

	Experimental Procedures

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The investigation conforms to the "Guide for the Care and Use of Laboratory Animals" (National Academy Press, Washington, D.C., 1996).

Materials. Light SR vesicles were obtained by fractionation of crude microsomes prepared from canine left ventricle on a sucrose density gradient (Antipenko et al., 1997a). Fast skeletal muscle light SR vesicles were similarly prepared from the adductor longus muscle of New Zealand White rabbits. Microsomal protein was estimated by the biuret procedure with BSA as the standard. The gingerol used in the present study was 6-gingerol or 1-(4'-hydroxy-3'-methoxyphenyl)-5-hydroxydecan-3-one (Wako BioProducts, Richmond, VA). Okadaic acid (sodium salt) was obtained from Calbiochem (La Jolla, CA). Ellagic acid (see Berrebi-Bertrand et al., 1997, for structural formula) was purchased from Sigma Chemical Co. (St. Louis, MO). All other reagents were obtained as described previously (Antipenko et al., 1997a,b).

Ca²⁺ Uptake Measurements. Microsomal Ca²⁺ uptake was assayed using ⁴⁵Ca and a filtration procedure. Briefly, the standard reaction mixture consisted of 40 mM histidine-HCl, pH 6.8, at 37°C, 0.12 M KCl, 5 mM NaN₃, 4 mM phospho(enol)pyruvate, 0.22 mg/ml pyruvate kinase, 2 mM MgCl₂, 5 mM oxalate-Tris, 1 µM okadaic acid, 1 mM ATP, and a CaCl₂-EGTA buffer to yield the Ca²⁺ concentrations shown in the text as determined using the computer program MaxChelator and the file constants BERS.CCM (Bers et al., 1994). The specific radioactivity was approximately 1.3 × 10⁵ Bq/µmol at 9 µM Ca²⁺ and was progressively increased to a maximum of 1.3 × 10⁶ Bq/µmol at 0.02 µM, the lowest Ca²⁺ concentration tested. The reaction mixture also contained either 160 U/ml PKA catalytic subunit or an equivalent amount of heat-denatured PKA catalytic subunit (Antipenko et al., 1997b). Microsomes (2.5-5.0 µg/ml microsomal protein) were added to the temperature-equilibrated reaction mixture to allow the phosphorylation or control reaction to proceed, and 2 min later, gingerol or ellagic acid was added to a final concentration of 50 µM unless otherwise specified in the text. The identical time course was used for control incubates containing dimethyl sulfoxide, the solvent for the two compounds. The final concentration of dimethyl sulfoxide was 2%, which had no detectable effect in any of the assays used in this study. After a further 2-min incubation, Ca²⁺ was added to start the Ca²⁺ uptake reaction. Aliquots were removed for filtration between 1 and 4 min after the start of the reaction. Ca²⁺ uptake rates, obtained as a function of Ca²⁺ concentration, were fitted to the Hill equation, V = V_max/[1 + (K_{m (L)}/[L])^N], by a nonlinear least-squares procedure using the SigmaPlot software package (Jandel Corp., San Rafael, CA), where L represents Ca²⁺ and N is the Hill coefficient. The presence of gingerol or ellagic acid had no detectable effect on the ATP-regenerating system or the enzyme-linked ATPase detection system used in the standard assay mixtures for measurement of Ca²⁺ uptake and Ca²⁺-ATPase activity. In some experiments, the ATP concentration was varied from 0.3 µM to 2 mM. MgATP concentrations, corresponding to MgATP², were calculated as described previously (Antipenko et al., 1997a).

Ca²⁺-ATPase Assay. The reaction mixture for the assay of ATPase activity was the same as in the Ca²⁺ uptake assay except for the addition of the enzymes necessary for following oxidation of NADH and the use of nonradiolabeled CaCl₂ (Antipenko et al., 1997a). Microsomes were added to the temperature-equilibrated reaction mixture, followed by the addition of 50 µM gingerol or vehicle 1 min later and a CaCl₂-EGTA buffer 2 min later. The time program on a Shimadzu UV160U recording spectrophotometer was started on the addition of the Ca²⁺. Reactions were linear with respect to protein concentration and to time for at least 5 min, the duration the reaction was followed. Ca²⁺-ATPase activity was taken as the difference in ATPase activity measured at 9 µM Ca²⁺ and at 2 mM EGTA.

E₂P Formation from P_i and E₂P Decomposition. Steady-state E₂P formation from P_i and E₂P decomposition were measured as described previously (Antipenko et al., 1997a) with the following procedural modifications. Microsomes (0.25 mg/ml) were added to the temperature-equilibrated reaction mixture, which included 1 µM okadaic acid, followed by the addition of gingerol or vehicle 1 min later. After a 5-min incubation, ³²P_i (2 mM) was added, and the reaction was terminated 15 s later by the addition of acid. E₂P decomposition was measured using a Bio-Logic QFM-5 system using drive sequences written specifically for these experiments (Antipenko et al., 1997a). The chase solution contained 50 µM gingerol or vehicle alone, so when one volume of the phosphorylation mixture including the ³²P-labeled microsomes (0.25 mg/ml) was combined with 16 volumes of chase solution, the final concentration of gingerol or vehicle was 47 µM or 2%, respectively.

Statistical Evaluation. Unless otherwise indicated, data are expressed as mean ± S.E.M. in three or four independent experiments with different microsome preparations. As a result of the well known microsomal variability, control and test experiments were generally carried out on the same day using the same microsome preparation. The statistical significance of differences between values was tested by Student's t test for paired variates, and p <.05 was taken as significant.

	Results

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A stimulation of Ca²⁺ uptake observed on incubation of light cardiac SR vesicles in the presence of gingerol attained a maximum of about 35% between 40 and 70 µM concentrations of the compound and diminished with further increases in concentration; the half-maximally effective concentration was approximately 9 µM (Fig. 1A). The stimulatory effect of gingerol was rapid, occurring within 1 min after addition to the reaction mixture. The magnitude of the stimulatory effect remained unchanged with incubation for up to 20 min, the longest period tested (Fig. 1B). Ellagic acid produced a generally similar pattern of concentration- (Fig. 1A) and time-dependent (data not shown) effects. A maximally stimulatory concentration of 50 µM of either compound was used in subsequent experiments.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Dependence of the stimulation of Ca2+ uptake on (A) the concentration of gingerol () or ellagic acid () and (B) the duration of incubation with gingerol. Microsomes were incubated for 2 min (A) in the presence of either the indicated concentrations of gingerol or ellagic acid or the vehicle alone or (B) in the presence of 50 µM gingerol or the vehicle alone for the indicated times before the addition of 9 µM Ca2+ to start the Ca2+ uptake reaction. Data points represent the mean ± S.E.M. of four independent experiments (A, ) or single experiments (A, ; B, ).

Gingerol clearly increased Ca²⁺ uptake at Ca²⁺ concentrations of 0.5 µM and higher when tested in unphosphorylated (control) microsomes, whereas at lower concentrations, an increase, if any, was not readily apparent (Fig. 2A). An almost identical pattern of effects was observed with ellagic acid (Fig. 2B). In microsomes phosphorylated with PKA, gingerol significantly increased Ca²⁺ uptake only at 9 µM Ca²⁺ (p <.05) (Fig. 2C). Differences between control and PKA-treated microsomes tested in the absence of gingerol were obscured by the fact that the experiments shown in Fig. 2, A and C, had been carried out in different microsome preparations and on different days. Thus, in these experiments, neither a 27% mean increase in V_max(Ca) (1.38 ± 0.10 versus 1.09 ± 0.10 µmol/mg·min) nor a 10% mean decrease in K_m(Ca) (0.48 ± 0.07 versus 0.43 ± 0.03 µM) attributable to treatment with PKA attained statistical significance in an unpaired Student's t test. To demonstrate the effect of PKA in experiments carried out concurrently with the same microsome preparation, we show data for paired control and PKA-treated microsomes taken from a previous publication (Fig. 2D). The optimized kinetic parameters obtained from nonlinear least-squares fits to the Hill equation (see Experimental Procedures) of the data presented in Fig. 2, A-C, are shown in Table 1. Significant increases in V_max(Ca) of Ca²⁺ uptake attributable to treatment of microsomes with gingerol and ellagic acid were obtained when assays were carried out in paired experiments (as described in Experimental Procedures). Neither gingerol nor ellagic acid produced a significant change in the K_m(Ca) in unphosphorylated microsomes, whereas in phosphorylated microsomes, gingerol increased K_m(Ca) by 28% and V_max(Ca) by 12% (Table 1). In the paired experiments shown in Fig. 2D, we had previously obtained a significant 44% increase in V_max(Ca) with PKA and a 14% decrease in K_m(Ca). Under none of the conditions tested was there a significant change in the Hill coefficient (Table 1).

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Comparative effects of gingerol, ellagic acid, and PKA on the Ca2+ concentration dependence of microsomal Ca2+ uptake assayed at 1 mM MgATP. Symbols represent the mean ± S.E.M. of paired experiments. For clarity in this and subsequent figures, only positive or negative error bars are shown. The lines are the results of fits of the data to the Hill equation (see Experimental Procedures). Mean ± S.E.M. of the optimized kinetic parameters obtained in separate fits of the data from each paired experiment are shown in Table 1. A, , no gingerol; , 50 µM gingerol; microsomes incubated in the presence of heat-denatured PKA (n = 4). B, same as A except that the symbols represent the presence and absence of 50 µM ellagic acid (n = 3). C, , no gingerol; , 50 µM gingerol; microsomes incubated in the presence of PKA (n = 4). D, , heat-denatured PKA; , PKA; no gingerol or ellagic acid (n = 3). The data shown in D were replotted from Fig. 3 in Antipenko et al. (1997b) with permission from the American Chemical Society. The absolute values for 100% Ca2+ uptake by control microsomes are given in Table 1. Also see Table 1 for statistical analyses.

View this table: [in this window] [in a new window]   TABLE 1 Kinetic parameters for Ca2+ uptake by control and phosphorylated cardiac microsomes: Effects of 50 µM gingerol and ellagic acid Values are mean ± S.E.M. of kinetic parameters obtained in the three or four experiments represented in each panel of Fig. 2. The last pair of values (control and phosphorylated microsomes, no addition) was reproduced from Table 1 in Antipenko et al. (1997b) with permission from the American Chemical Society.

To determine whether the effect of gingerol was the result of stimulation of the Ca²⁺ pump or of an inhibition of a parallel efflux pathway, Ca²⁺-ATPase activity was assayed in the presence and absence of 50 µM gingerol at saturating Ca²⁺ under conditions similar to those used in the Ca²⁺ uptake assay (Table 2). Gingerol increased Ca²⁺-ATPase activity to a comparable extent as Ca²⁺ uptake in both phosphorylated and control microsomes. An unchanged stoichiometric ratio approaching the theoretical value of 2 mol Ca²⁺ transported/mol ATP hydrolyzed indicates that gingerol produces no uncoupling of the pump from ATP hydrolysis. Moreover, the increase in Ca²⁺-ATPase activity attributable to gingerol was considerably reduced in the phosphorylated microsomes, which is in agreement with the Ca²⁺ uptake measurements. The finding of a significant, albeit reduced, increase in Ca²⁺-ATPase activity in phosphorylated microsomes compared with control microsomes eliminates any possibility that the increase in Ca²⁺ uptake seen in Fig. 2C at 9 µM Ca²⁺ was attributable to random experimental variation.

The foregoing experiments were carried out at 1 mM MgATP. Additional experiments were performed to determine the effect of gingerol on Ca²⁺ uptake at 0 to 10 µM MgATP to detect changes in Ca²⁺ uptake activity associated with regulatory nucleotide binding (Fig. 3). Ca²⁺ uptake accelerated markedly between 1.5 and 3 µM MgATP and to a lesser extent as the nucleotide concentration was increased further in both control and PKA-phosphorylated microsomes. This pattern of Ca²⁺ pump activation can be explained by nucleotide binding to the catalytic site on the Ca²⁺ pump protein between 0 and 1.5 µM MgATP, followed by regulatory nucleotide binding (Wakabayashi and Shigekawa, 1990; Lu et al., 1993) above 1.5 µM MgATP. In unphosphorylated (control) microsomes, gingerol had no detectable effect on Ca²⁺ uptake assayed at 0 to 1.5 µM MgATP, above which gingerol produced a marked inhibition that was statistically significant at 3 and 5 µM MgATP but was overcome as the MgATP concentration reached 10 µM. In phosphorylated microsomes, there was an apparent inhibition of Ca²⁺ uptake already below 1.5 µM MgATP, which became statistically significant at 1.5 µM. This inhibition was greatest between 3 and 5 µM, and, as in the unphosphorylated microsomes, the inhibition was overcome as the nucleotide concentration was increased to 10 µM.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Effect of 50 µM gingerol on microsomal Ca2+ uptake assayed at µM MgATP. Purified cardiac microsomes (8 µg/ml) were incubated in the presence of PKA (, ) or heat-denatured PKA (, ), followed by the addition of gingerol (, ) or the vehicle alone (, ) before measurement of Ca2+ uptake at 9 µM Ca2+ and the indicated MgATP concentrations. Data points represent mean ± S.E.M. of three independent experiments. *statistically significant difference at the p < .05 level when tested by Student's t test for paired variates.

Assays of Ca²⁺ uptake were also carried out at MgATP concentrations between 0.05 to 2.0 mM to detect any additional nucleotide-dependent changes in response to gingerol. The magnitude of the stimulatory effect of gingerol, although different in control (about 30-40%) and phosphorylated (about 12%) microsomes, remained relatively constant over the nucleotide concentration range tested (Fig. 4), and at 1 mM nucleotide, it was consistent with the stimulation seen in Figs. 1 and 2 and in Table 1. When fast skeletal muscle microsomes, which contain no PLN (Jorgensen and Jones, 1986), were tested, gingerol was found to stimulate Ca²⁺ uptake to the approximately the same extent as was observed in phosphorylated cardiac microsomes (i.e., about 12%) (Fig. 4).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Relative stimulation of Ca2+ uptake by gingerol in fast skeletal muscle light SR and cardiac light SR vesicles assayed at different MgATP concentrations. The experiments were carried out as described in the legend to Fig. 3. Shown is the percent increase in Ca2+ uptake attributable to the presence of 50 µM gingerol in the reaction mixture when assayed in rabbit fast skeletal muscle light SR vesicles () or phosphorylated () and unphosphorylated () canine cardiac light SR vesicles over the indicated range of MgATP concentrations. Data points are the mean ± S.E.M. of three independent experiments. A 100% Ca2+ uptake represents 2.10 ± 0.12 (), 1.62 ± 0.09 (), and 1.22 ± 0.17 () µmol Ca2+/mg·min. All differences in rates obtained in the presence and absence of gingerol in the three types of microsomes were significant (p < .05) when tested by Student's t test for paired variates in three independent experiments.

To determine whether an increase in E₂P decomposition during the catalytic cycle could contribute to the gingerol-induced increase in Ca²⁺ uptake observed at high Ca²⁺ concentration and millimolar ATP concentration, both the rate of E₂P decomposition and the steady-state E₂P formation from P_i were determined. Steady-state E₂P formation was significantly decreased by gingerol when measured at 15°C, but as the temperature was increased, the inhibition was greatly diminished (Table 3). In control microsomes, steady-state E₂P formation remained constant over the temperature range examined. The lower level of E₂P measured under steady-state conditions at 15°C and 50 µM gingerol is consistent with the significant acceleration in the rate of E₂P decomposition measured by the rapid-mixing and quench method (Fig. 5). The rapidity of the reaction relative to the time resolution of the available rapid kinetics instrumentation precluded similar measurements at 25° and 37°C.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Effect of gingerol on E2P decomposition. Unphosphorylated microsomes were incubated in the presence of 32Pi to form E2P and then mixed at 15°C in a QFM-5 rapid mixing system with 16 volumes of a chase solution containing either 50 µM gingerol () or the vehicle alone (). At the indicated times, the reaction was quenched with an equal volume of 10.3% trichloroacetic acid, as described elsewhere (Antipenko et al., 1997a). To obtain the 5-s time points, the reactants were mixed manually. To obtain the zero time points, the quench solution was added manually to the 32P-labeled microsomes. Each data point represents the average of two determinations. The lines are calculated from single exponential fits with the rate constants shown in the inset.

	Discussion

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We have shown that gingerol, like PLN phosphorylation, increases V_max(Ca) of Ca²⁺ uptake yet unlike phosphorylation, which decreases K_m(Ca), gingerol has no significant effect on K_m(Ca) and the Hill coefficient in unphosphorylated light cardiac SR vesicles (Fig. 3 and Table 1). An increase in V_max(Ca) of Ca²⁺ uptake by the SR may thus be an important component of the kinetic mechanism by which gingerol exerts the inotropic and lusitropic effects previously demonstrated in isolated myocardial preparations (Shoji et al., 1982; Kobayashi et al., 1988). The effects of gingerol and ellagic acid on the kinetic parameters obtained in the present study differ significantly from those reported by Berrebi-Bertrand et al. (1997) (see introduction). Possible reasons for discrepancies in such kinetic data, including the use of crude microsome preparations, were discussed in a previous report (Antipenko et al., 1997b).

PKA-phosphorylated cardiac microsomes resemble fast skeletal muscle microsomes in two respects relevant to the present discussion: 1) the magnitude of the gingerol-induced increase in Ca²⁺ uptake assayed at saturating Ca²⁺ and mM ATP was similar (i.e., about 12%) in phosphorylated cardiac and fast skeletal muscle microsomes (Fig. 4), and 2) gingerol increased K_m(Ca) of Ca²⁺ uptake, as shown in Table 1 for phosphorylated cardiac microsomes and reported by Kobayashi et al. (1987) for skeletal muscle microsomes. It is unclear whether a much-reduced gingerol-induced increase in Ca²⁺ uptake and Ca²⁺-ATPase activity in phosphorylated cardiac microsomes, compared with unphosphorylated microsomes, at saturating Ca²⁺ is related to incomplete PLN phosphorylation by PKA, a lack of PLN phosphorylation by calmodulin-dependent protein kinase, or additive effects of saturating levels of PKA phosphorylation and a direct action of gingerol on the Ca²⁺-ATPase, as discussed further below. The aforementioned similarities between phosphorylated cardiac and skeletal muscle microsomes might also be an indication of an as-yet-unidentified means of regulation involving homologous regions in the membrane domains of PLN and sarcoplipin (Odermatt et al., 1998), a protein that is found in fast skeletal muscle SR but lacks the cytoplasmic domain of PLN.

In view of the significant gingerol-induced increase in K_m(Ca) in the phosphorylated microsomes (Table 1), the E₂ + 2Ca²⁺  2Ca·E₁ transition in the catalytic cycle of the Ca²⁺-ATPase is a likely target of the inhibitory action of gingerol. An inhibition at this step would produce a decrease in the apparent affinity of the pump for Ca²⁺ , as was shown for PLN (Cantilina et al., 1993), which inhibits a substep of this transition. This conclusion is further supported by the finding that gingerol significantly inhibits Ca²⁺ uptake in the low micromolar MgATP concentration range (3-5 µM or less) (Fig. 3), at which the E₂ + 2Ca²⁺  2Ca·E₁ transition is likely to be accelerated as a result of regulatory nucleotide binding to the E₂ conformation (Wakabayashi and Shigekawa, 1990; Lu et al., 1993). The latter inhibition is completely overcome as the nucleotide concentration is increased to 10 µM at saturating Ca^2+. Thus, gingerol appears to inhibit either nucleotide binding or subsequent nucleotide-dependent activation of the pump in a competitive manner in contrast to PLN, whose inhibition of the pump is undiminished at ATP concentrations up to 2 mM (Lu et al., 1993).

A significant inhibition by gingerol was evident already at lower micromolar nucleotide concentrations in the phosphorylated microsomes (2 µM or less) than in the control microsomes (3 and 5 µM) (Fig. 3), suggesting that unphosphorylated PLN may partially protect against the inhibition and that removal of the inhibition by PLN, whether by proteolytic cleavage from the membrane or phosphorylation, renders the pump more vulnerable to inhibition. Consistent with this hypothesis are the previously reported greater inhibition of Ca²⁺ uptake by jasmone in trypsin-treated microsomes compared with control microsomes (Antipenko et al., 1997a) and the greater sensitivity of PKA-phosphorylated microsomes to the inhibitory effects of thapsigargin (Kijima et al., 1991). Moreover, submicellar concentrations of octaethylene glycol dodecyl monoether (C₁₂E₈), which affects major nucleotide-accelerated steps in the catalytic cycle and, in particular, inhibits E₂P decomposition (Champeil et al., 1986), almost completely eliminated the increase in V_max(Ca) of Ca²⁺ uptake associated with the removal of the inhibitory influence of PLN by trypsin treatment but caused no significant change in V_max(Ca) of control microsomes at millimolar ATP concentrations (Lu and Kirchberger, 1994). The inhibition of Ca²⁺ uptake by C₁₂E₈ seen in the presence of saturating Ca²⁺ in trypsin-treated microsomes initially became apparent as the nucleotide concentration was increased above 3 µM to 1 mM and hence was affecting a nucleotide-accelerated step in the catalytic cycle. At <3 µM MgATP (and saturating Ca²⁺), C₁₂E₈ increased V_max(ATP) of Ca²⁺ uptake associated with nucleotide binding to the catalytic site. This action is attributable either to dissociation of inhibitory PLN from the pump or to the known ability of C₁₂E₈ to accelerate the E₂ + 2 Ca²⁺  2Ca·E₁ transition and other major steps in the catalytic cycle.

The gingerol- or ellagic acid-induced V_max(Ca) at saturating Ca²⁺ may be interpreted as a direct action of the compounds on the pump related to their successful competition against the inhibition by PLN of two or more ATP-accelerated steps in the catalytic cycle. At saturating Ca²⁺ and millimolar ATP concentrations, although not measured in the present study, the nucleotide-accelerated transport step (i.e., the E₁P·2Ca  E₂P + 2Ca) may contribute most to rate limitation (Champeil and Guillain, 1986). Evidence reported by Hughes et al. (1996) suggests that this step is regulated by PLN. An acceleration of both the transport step and E₂P decomposition by jasmone may play a role in the previously demonstrated jasmone-induced increase in V_max(Ca) of Ca²⁺ uptake in cardiac microsomes at 25°C (Antipenko et al., 1997a). Like PLN phosphorylation (Antipenko et al., 1997b), gingerol also increases E₂P decomposition (Fig. 5). The more pronounced ATP-surmountable inhibition of Ca²⁺ uptake by gingerol at low micromolar ATP concentrations in phosphorylated microsomes (Fig. 3), as well as its increase in K_m(Ca) of Ca²⁺ uptake at mM ATP (Table 1) in such microsomes, suggests a direct action of gingerol on the pump at a nucleotide-related site.

Thus, gingerol, ellagic acid, and other structurally diverse compounds such as jasmone (Antipenko et al., 1997b) and C₁₂E₈ (Lu and Kirchberger, 1994) produce both stimulatory and inhibitory effects on the cardiac SR Ca²⁺-ATPase, all of which involve the interaction of nucleotide with the pump. The increase in V_max(Ca) of Ca²⁺ uptake produced by gingerol in otherwise untreated microsomes is similar to that produced by PLN phosphorylation when assayed at millimolar ATP at which the E₂ + 2Ca²⁺  2Ca·E₁ transition is already accelerated by regulatory nucleotide (Table 1). It is noteworthy that in control microsomes the K_m(Ca) of Ca²⁺ uptake does not differ significantly in the presence and absence of gingerol (Table 1) despite the suggested inhibitory effect of gingerol on the E₂ + 2Ca²⁺  2Ca·E₁ transition at either subsaturating Ca²⁺ or subsaturating MgATP. Such an inhibitory effect would be masked if gingerol removes the inhibitory effect of PLN with respect to K_m(Ca) resulting in no net observable change in this parameter.

The interactions among PLN, the Ca²⁺ pump protein, and a variety of structurally diverse hydrophobic compounds obviously are complex. The kinetic effect of the latter compounds on the Ca²⁺ pump may depend on their specific effects on a particular step in the catalytic cycle and whether this step is rate limiting or becomes rate limiting under a particular set of conditions. PLN and the inhibitors thapsigargin and cyclopiazonic acid (Plenge-Tellechea et al., 1998) all appear to bind to the E₂ conformation of the Ca²⁺ pump protein and hence will affect E₂ + 2Ca²⁺  2Ca·E₁ transition. Moreover, an effect of cyclopiazonic acid on Ca²⁺ pump turnover in fast skeletal muscle SR has been reported to be a function of the compound's stoichiometric relationship to the pump and the contribution to the observed kinetic parameters of two different reaction cycles, which are characterized by different kinetic parameters: one in the presence and the other in the absence of cyclopiazonic acid. Similarly, in cardiac SR, the observed V_max(Ca) may depend on the relative amounts of inhibitory monomeric PLN available for interaction with Ca²⁺ pump units on the membrane. Although studies using in vitro expression systems to study PLN-Ca²⁺ pump interaction rely on changes in K_m(Ca) as evidence of PLN-Ca²⁺ pump interaction, such studies have demonstrated marked inhibitory activity of monomeric PLN on the pump (Kimura et al., 1997).

Thus, an increasing amount of evidence, as described above, suggests a functional relationship between nucleotide binding and the stimulatory or inhibitory actions of various hydrophobic or amphoteric compounds, including PLN, on the cardiac SR Ca²⁺ pump. Definition of their precise relationship may be related to the long-standing question of whether each SR Ca²⁺-ATPase molecule contains one or two nucleotide binding sites (e.g., Dupont et al., 1985) that account for the effects of catalytic and regulatory nucleotide. In fast skeletal muscle SR, evidence exists for the presence of two simultaneous binding sites for nucleotide analogs that are capable of increasing the rate of Ca²⁺ pump turnover (Mignaco et al., 1996).