Damiani, Carlos Estevan Nolf PhD*†; Moreira, Cleci Menezes MSc*; Zhang, Heng Tao PhD‡; Creazzo, Tony Louis PhD‡; Vassallo, Dalton Valentim MD, PhD*§
It is now recognized that the sarcolemmal Ca2+ channel plays a key role in the development of hypertension, cardiac hypertrophy, calcium overload, and arrhythmias. For this reason the use of Ca2+ channel blockers represents one of the main pharmacological interventions for the management of hypertension. Eugenol, a natural pungent substance, is the main constituent of clove oil and among its properties appears to act as a calcium antagonist in both cardiac and smooth muscles.1-3
Eugenol affects contractions in skeletal, cardiac, and smooth muscles. Previous studies4 showed that the effects of eugenol at low concentrations (0.1 to 2.5 mM) blocked the contracture induced by 80 mM K+ in skeletal muscles. The authors suggested the possibility that some of the blockade of the potassium-induced contracture might be due to an inhibition of Ca2+-induced Ca2+ release from the sarcoplasmic reticulum (SR). Recent reports suggest that eugenol has a vasodilator activity by inhibiting potassium ([K+]0)-induced contraction of rabbit thoracic aorta.1 These results also suggested that the inhibition of the [K+]0-induced contraction by eugenol was not associated with [Ca2+]i. Others have shown that eugenol was able to inhibit both [K+]0- and phenylephrine (10−7 M)-induced contractions of rat thoracic aorta.3 With respect to the actions of eugenol on the heart, it has been shown that intravenous administration in dogs caused a strong but transient reduction in arterial blood pressure and myocardial contractile force, without changing the heart rate.5 Eugenol also reduces force development and acts as a Ca2+ channel blocker in the heart.2
However, the underlying mechanisms for the cardiac actions of eugenol are not completely elucidated yet, and since toxic actions were reported6,7 putative effects on the contractile machinery could also occur. Hence, the present study was designed to reevaluate whether the mechanisms that produce the negative inotropic actions of eugenol are only dependent on a Ca2+ channel blocker action or are also dependent on actions on the contractile proteins.
MATERIALS AND METHODS
Male Wistar rats weighing 250 to 300 g were used in this study. Care and use of laboratory animals were in accordance to NIH guidelines. The rats received 500 units of heparin i.p. and after 10 minutes they were anesthetized with 45 mg/kg of sodium pentobarbital i.p.. The hearts were removed rapidly after thoracotomy and perfused with Krebs solution through the aortic stump to permit a proper selection and dissection of the left ventricle papillary muscles. The papillary muscles were immersed in a 50-mL water-jacketed bath maintained at 29 ± 1°C and gassed with (95% O2 and 5% CO2) Krebs bicarbonate buffer solution, at 29 ± 1°C. Muscles stretched to Lmax (muscle length at which active tension is maximal) were stimulated by isolated rectangular pulses (10 to 15 V, 12 milliseconds duration) through a pair of platinum electrodes placed along the entire extension of the muscle. The standard stimulation rate was 0.5 Hz (steady-state). Recording started after 60 minutes to permit the beating preparation to adapt to the new environmental conditions.
The bathing solution was a modified Krebs solution with the following composition (in mM):120 NaCl, 5.4 KCl, 1.2 MgCl2 6H2O, 1.25 CaCl2 2H2O, 2.0 NaH2PO4H2O, 1.2 Na2SO4, 27 NaHCO3, 11 Glucose. The following variables were analyzed: peak isometric force (F), time to peak tension (TTP), relaxation time (RT), relative potentiation (RP) obtained after pauses of 15, 30, and 60 seconds, before and after eugenol treatment under steady-state stimulation.
A stock solution (10−1 M) was prepared with eugenol solubilized in a mixture of Tween 80 (5%) and distilled fresh water. In this case, the volume of the solubilizing and solubilized agents was never greater than 1% of Krebs solution volume. Preliminary experiments with Tween 80 showed no effects on isometric contractions developed by papillary muscles (data not shown). Muscles were stretched to Lmax and kept under steady-state conditions. Developed force (F) was measured with an isometric force transducer (Nihon-Kohden, TB 612T, Tokyo) and recorded on a chart recorder (Nihon-Kohden, RM- 6200, Tokyo), and normalized to the muscle cross-sectional area (g/mm2). Considering the papillary muscle as a cylinder and tissue density as 1, the cross-sectional area was calculated by dividing the muscle length at Lmax by its weight. To avoid the possibility of a hypoxic core we performed experiments at low temperature (29 ± 1°C) as previously described.8 Pause intervals of various durations (15, 30, and 60 seconds) were used and the results are presented as relative potentiation (the amplitude of post-rest contractions divided by steady-state contractions).
The following protocols were used:
(1) The effects of eugenol (0.01, 0.03, 0.05, 0.1, 0.3, and 0.5 mM) on cardiac contractions were tested in 16 preparations (cross-sectional area = 1.16 ± 0.09 mm2) using the protocol previously described.
(2) Under steady-state conditions force was measured at different Ca2+ concentrations (0.62, 1.25, 2.5, and 3.75 mM) in the absence (cross-sectional area = 1.04 ± 0.10 mm2; n = 7) and in the presence of eugenol (0.1 mM) (cross-sectional area = 1.14 ± 0.14 mm2; n = 6). Still under steady-state conditions, the effect of eugenol (0.1 mM) was investigated in another group of papillary muscles (cross-sectional area = 1.12 ± 0.02 mm2, n = 8), under the positive inotropic effect produced by (−) isoproterenol (5 ng/ml).
(3) The influence of eugenol (0.01, 0.05, 0.1, and 0.5 mM) on the contractile machinery was studied in another group of papillary muscles (cross-sectional area = 1.18 ± 0.07 mm2, n = 11) during tetanic stimulations. Tetanic tension was obtained after treatment with 5 mM caffeine (for 20 minutes, at a rate of 10 Hz, for 15 seconds duration) as previously described.9 Between each tetanic stimulus the muscles remained under steady-state stimulation for at least 10 minutes. To insure that the effects of eugenol were not dependent on time, a time control was performed in another group of papillary muscles (cross-sectional area = 1.04 ± 0.03 mm2, n = 18) under the same previous conditions. To observe the recovery of muscle contraction, at the end of these experiments the preparations were washed twice with Krebs solution and kept at steady-state conditions. After 60 minutes, the tension was measured.
(4) To evaluate if eugenol is capable to affect myosin ATPase activity, the effects of this compound were assayed according to previous reports.10,11 Myosin was prepared from minced and homogenized left ventricles (n = 7), extracted briefly with KCl− phosphate buffer (0.3 M KCl, 0.2 M phosphate buffer, pH 6.5).12 After precipitation of myosin and muscle residues by 15-fold dilution with water, the muscle residue was separated by filtration using cheesecloth. This procedure filters fragments of cells including membranes. The supernatant containing the myosin was centrifuged at 33,000 g for 30 minutes. After decantation of the supernatant the precipitate was redissolved in 0.6 M KCl to dilute myosin under high ionic strength and 1 mL of water was added for each gram of tissue to produce a new precipitation. The material was again centrifuged at 33,000 g for 30 minutes and the muscle residue was separated by filtration. The material was redissolved again in 14 mL of water per gram of tissue, centrifuged, and filtered as before. The precipitate was dissolved in 50 mM HEPES, pH 7, and 0.6 M KCl plus 50%, vol/vol, glycerol and placed at −20°C. To use the stocked myosin it was diluted in water (1:12) and centrifuged at 3000 rpm for 15 minutes. The precipitate was resuspended in 50 mM HEPES, pH 7, and 0.6 M KCl, and centrifuged at 3000 rpm again. The supernatant was used.
Myosin ATPase activity was assayed by measuring Pi liberation from 1 mM ATP in the presence of 50 mM HEPES, pH 7, 0.6 M KCl, 5 mM CaCl2, or 10 mM EGTA in the absence (n = 7) and presence of eugenol (0.1 and 0.5 mM) (n = 6) in a final volume of 200 μL. Under this high ionic strength and no Mg2+ in the medium, only myosin activity was measured and there is no significant Ca2+-ATPase activity from sarcoplasmic reticulum membranes, which require high Mg2+ and low Ca2+ concentrations. The nucleotide was added to the reaction mixture and preincubated for 5 minutes at 30°C. The reaction was initiated by adding the enzyme fraction (3 to 5 μg protein) to the reaction mixture. Incubation time and protein concentration were chosen to ensure the linearity of the reaction. The reaction was stopped by the addition of 200 μL of 10% trichloroacetic acid. Controls with addition of the enzyme preparation after addition of trichloroacetic acid were used to correct for nonenzymatic hydrolysis of the substrate. All samples were in duplicates. The enzyme activity was calculated as the difference between the activities observed in the presence of Ca2+ and in the presence of 10 mM EGTA. Inorganic phosphate was determined by the method of Chan et al.13 The specific activity was reported as nmol Pi released per min per mg of protein unless otherwise stated. Protein was measured by the Coomassie blue method according to Bradford14 using bovine serum albumin as standard.
(5) To investigate the effects of eugenol on the calcium channels of the heart muscle, we performed voltage clamp experiments.
Single ventricular myocytes were enzymatically isolated from male Sprague-Dawley rats (250-300 g) as described by Mitra and Morad15 with some modifications. The animal was anesthetized with sodium pentobarbital (45 mg/kg, i.p.) and anticoagulated with heparin (500 IU, i.p.). Then the heart was rapidly removed and perfused with oxygenated Tyrode's solution (36 ± 0.5°C) for 5 minutes at a constant flux of 10 mL/min through an aortic cannula in a Langendorff apparatus. The Tyrode's solution contained (in mM): NaCl 150; KCl 5.4; MgCl2 6H2O 1.2; CaCl2 1.8; HEPES 5 and glucose 5.5 (pH 7.4 with NaOH). It was followed by 5 minutes perfusion with low Na+, Ca2+-free solution, which contained (in mM): NaCl 100; KCl 10; K2HPO4 1.2; MgSO4 4; HEPES 10; Taurine 50 and glucose 20 (pH 6.9 with KOH). Seven minutes of perfusion with enzyme solution, which contained 0.75 mg/ml collagenase type II, 0.1% albumin, 2 mg pronase, and 100 μM CaCl2 in 40 mL low Na+ solution initiated enzymatic digestion. Ventricular myocytes were released by mincing the ventricles into small fragments in a low Na+, solution containing 200 μM CaCl2 followed by gentle stirring in flask until the supernatant became cloudy with cells. A 5 mL aliquot of the supernatant was filtered and diluted with 45 mL of KB solution, which contained (in mM): KOH 70; KCl 40; MgCl2 3; EGTA 0.5; Glutamic acid 50; Creatine 5; Pyruvic acid 5; Succinic acid 5; Taurine 20; HEPES 10; and Glucose 10 (pH 7.3 adjusted with KOH) filtered with a sterile filter. The preparation was kept refrigerated and can be used for 3 days without any significant change in the observed parameters during this period.16,17
Aliquots of cells were allowed for 20 minutes to settle in a shallow bath mounted on the stage of an inverted microscope (Olympus-IMT-2). The cells were then perfused at room temperature (22-25°C) at a flow rate of 2 to 3 mL/min for 5 minutes before current measurements were attempted. The extracellular solution contained (in mM): NaCl 110; KCl 5.4; MgCl2 6H2O 1.2; CaCl2 0.5; CsCl 30; HEPES 10; and Glucose 5 at a pH 7.4 adjusted with NaOH. To inactivate the fast sodium current, 30 μM tetrodotoxin (TTX) was added to the extracellular solution before the experiments.
Borosilicate pipettes made from 7052 Garner glass (Garner Glass Co., Claremont, CA) were pulled using a horizontal Flaming-Brown micropipette puller (Model P-97, Sutter Instrument Co.). The pipette tips were fire-polished (model MF-83; Narishige Scientific Instrument Laboratories, Tokyo, Japan) to produce 1 to 4 MΩ tip resistances when filled with the pipette solution. Pipettes were back-filled with internal solution and used immediately. The pipette solution contained (in mM): NaCl 5; K aspartate 140; MgCl2 6H2O 2; EGTA 5; CaCl2 0,062; HEPES 10; Na2 ATP 5; Na2 GTP 0.3 (pH 7.2 adjusted with NaOH). Single myocytes were voltage clamped with an Axopatch 200B Amplifier (Axon Instruments Foster City, CA) using the whole-cell configuration of the patch clamp technique as reported before.18,19 Offset potential were compensated by zeroing the patch clamp amplifier before seal formation with the electrode in the bath. Voltage clamp protocols were elicited and digitized data were acquired by an Axon Instruments Digidata 1322A. After gigaseal formation, negative pressure was applied to rupture the patch membrane and established the whole-cell configuration. For patch clamp studies, the following protocols were performed:
Time Course of the Response to Eugenol
The following protocol was performed to show the effect of eugenol at different concentrations (0.1 and 0.5 mM) on calcium current (ICa) recorded in a rat myocyte. In all conditions, the cell was depolarized every 5 seconds from −80 mV to −30 mV for 30 milliseconds followed by depolarization from −80 mV to 0 mV for 200 milliseconds and repeated for up to 20 minutes using the same cell. To study the possible effect of eugenol on ICa, extracellular solution was regularly changed by extracellular solution plus eugenol (0.1 and/or 0.5 mM). The Na+ current was blocked by depolarization every 5 seconds to −30 mV and by the addition of 30 μM tetrodotoxin (TTX) to the extracellular solution.20
Effects of Eugenol on Calcium Inward Current (ICa)
To measure effects on the L-type calcium inward current (ICa,L) the mean current-voltage (I-V) relationships of cells from control and eugenol-treated were performed as previously described.19,21 The holding potential of the cell was set to −80 mV followed by a 400-second depolarization step, varying in steps of 10 mV, from −80 mV to +50 mV in the absence and presence of eugenol (0.1 and 0.5 mM). The ICa was filtered at 1 kHz through a Bessel filter and digitalized at 2 kHz. The calcium inward current was evaluated as the difference between the peak inward current and the current at the end of the 400-millisecond clamp step. In the extracellular solution, 30 mM CsCl was used to block K+ currents. T-type Ca2+ current blockade was not attempted as this current is not present in adult rat ventricular myocytes.22,23
Pentobarbital sodium 30 mg (Cristalia - Produtos Químicos Farmacêuticos Ltda., São Paulo, SP, Brazil); Heparin 5000 U.I. (Roche Q.F.S.A., Rio de Janeiro, RJ, Brazil; Sigma); Eugenol (Sigma; Fluka); Pronase (Fluka); Collagenase type 2 (Worthington). Albumin bovine; Tetrodotoxin; Na2 ADP; Na2 ATP; (−) Isoproterenol hydrochloride and Caffeine anhydrous were purchased from Sigma Chemical Co. (St. Louis, MO). All other reagents used were of analytical grade from Sigma; E. Merk (Darmstadt, Germany) or Reagen (Rio de Janeiro, RJ, Brazil).
The results are presented as mean ± SEM with n indicating the number of observations. Values were analyzed using ANOVA (one- and two-way). When ANOVA revealed a significant difference, the Tukey test was applied (* as P < 0.05 and # as P < 0.01 were taken as significant). The analysis of the data and the plotting of figures were carried out using softwares GraphPad Prism™ (version 2.0, GraphPad Software, San Diego, CA) and GB-STAT (version 4.0, Dynamic Microsystem Inc., Silver Spring, MD).
Figure 1 shows the effects of eugenol at different concentrations, on isometric force developed by papillary muscles. The results demonstrated a concentration-dependent inhibition of force with nearly a 50% reduction in 0.1 mM eugenol compared with the control. In addition, there was a concentration-dependent reduction of time parameters including time to peak tension (TTP) and relaxation time (RT) of the isometric contraction (Fig. 2). This reduction was more evident on TTP than on RT.
Post-rest potentiation was used to determine if eugenol affects the function of the SR. Post-rest contractions (PRCs) obtained after 15-, 30-, and 60-second pauses were recorded and analyzed as relative potentiation. The increase of eugenol concentration produced smaller steady-state contractions but a progressive increase in relative potentiation (Fig. 3).
The control of contractility in the heart can be exerted through changes in the amount of extracellular calcium concentration [Ca2+]0.24 Therefore, the dependence of force development upon changes in [Ca2+]0 (0.62, 1.25, 2.5, and 3.75 mM) in the absence and presence of eugenol was investigated. Figure 4 shows that in control conditions [Ca2+]0 increased the force in a concentration-dependent way. However, in the presence of eugenol the force was reduced at each Ca2+ concentration.
The influence of eugenol on a positive inotropic intervention using isoproterenol (ISO) was investigated. Figure 5 shows that at low Ca2+ concentration the negative inotropic effect elicited by eugenol was reversed by ISO (5 ng/ml).
We also investigated if the depressant effect of eugenol was mediated via solely Ca2+ influx or whether it could directly affect contractile proteins. To examine this possibility we tested the effects of eugenol on tetanic contractions. Tetanic contractions developed a fast upstroke (tetanic peak force-TPF) followed by a slow decay and a plateau (tetanic plateau force-TPF). Figure 6 shows that eugenol, in a concentration-dependent way, depressed the tetanic contractions developed by papillary muscles. This effect was greater in the tetanic peak force (TPF) than in the tetanic plateau force (TPL). However, tetanic tension could decay under control conditions as a function of time similar to the observed eugenol effect. Figure 7 shows that no differences of tetanic peak force or plateau force were observed during time control experiments of tetanic contractions.
Since tension development depends on myosin ATPase activity11 eugenol could reduce the activity of this enzyme. Our results suggested that eugenol (0.1 and 0.5 mM) did not affect the myosin Ca2+-ATPase activity (Fig. 8).
Several authors have reported toxic actions of eugenol, and as it produced a depressant effect on force, it was necessary to investigate if it was reversible or not. Therefore, at the end of the experiment with tetanic contractions, papillary muscles were washed twice and force measured again after 60 minutes (control: 1.10 ± 0.06 g/mm2; n = 8) (tetanus-final: 0.18 ± 0.07 g/mm2; n = 9) (recuperation: 0.84 ± 0.07 g/mm2; n = 8). Note that at the concentrations of eugenol used the force reversibility was close to 70% of the control.
The inhibitory effects of eugenol on the calcium inward current (ICa) were investigated in voltage-clamp experiments on isolated single rat ventricular myocytes. Figure 9 shows the inhibitory effect of eugenol on the current-voltage relationship. Eugenol 0.1 mM decreased the current amplitude in the entire potential range tested without shifting the current-voltage relationship. At 0.5 mM, eugenol almost completely blocked the calcium inward current. Figure 10 summarizes the effects of eugenol on the calcium inward current (A) and shows that eugenol 0.5 mM was able to inhibit ICa by 97%.
Results presented here show that eugenol is a negative inotropic agent that does not depress the myosin ATPase and the SR activities. Results also suggest that the negative inotropic effect is only dependent on a calcium channel blockade since eugenol reduces ICa,L, and reduces tetanic tension and the positive inotropic effects produced by Ca2+ and ISO.
Calcium required for contraction comes from two sources: (1) the extracellular Ca2+ influx through voltage-gated slow Ca2+ channels and (2) the Ca2+ pool in the SR lumen. Ca2+ entry from the extracellular pool triggers the release of Ca2+ from the SR. It is estimated that about 30% of the Ca2+ required to elevate the [Ca]i to the level required for maximum force development is derived from Ca2+ influx across the sarcolemma and the remaining 70% is Ca2+ derived from SR.25
It was reported that eugenol depresses isometric force development in papillary muscles2 and intravenous injections of eugenol (0.05-1.5 mL) caused transient decrease in myocardial contractile force.5 These results are in agreement with the concentration-dependent depression of the isometric force produced by eugenol in papillary muscles described in this report. Eugenol also affected the temporal parameters of the muscle contractions. As has been described for other Ca2+ channel blockers,26 both TTP and RT were reduced by eugenol.
The force developed during the contraction of the cardiac muscle is altered in response to changes in rate and rhythm. In cardiac muscle, the contractions occurring after short pauses are potentiated. In several mammalian species these post-rest contractions (PRCs) usually become reduced in force as the rest periods increase. The rat cardiac muscle is an exception since post-rest contractions increase its force as the rest period increases.27 PRCs depend on pause durations and on the amount of calcium stored at intracellular sites and the relative participation of the SR is more important for PRCs than for steady-state contractions (SSCs). Therefore, if PRCs are more dependent on stored intracellular Ca2+ than SSCs, maneuvers, which modify the transmembrane Ca2+ influx, should affect SSCs more than PRCs. In our experiments, although the steady-state contractions were progressively depressed, the PRCs were potentiated in all pause durations tested. The result suggests that the effects of eugenol were much more dependent on membrane Ca2+ influx through voltage-sensitive channels than on Ca2+ released from SR. Similar results were reported in the presence of verapamil and manganese (both are Ca2+ channel blockers), which reduce the rate of transmembrane Ca2+ influx during activation than by Ca2+ release from SR.27 Therefore, if Ca2+ entry is partially blocked, the relative participation of the tension development of the first contraction after a pause increases because of Ca2+ storage in SR, when compared with the steady-state contraction.27
It is well known that force is dependent on extracellular calcium influx.28 So, when calcium was added to the solution the force was increased in a concentration-dependent way. However, in the presence of eugenol the force was reduced. Therefore, the results suggest that eugenol was reducing the calcium influx through the sarcolemma.
The β-adrenoceptors when stimulated by catecholamines activate a sarcolemmal adenylate cyclase through a Gs protein promoting an increase in cyclic AMP concentration. Cyclic AMP increases the transmembrane Ca2+ influx29 and produces a faster Ca2+ uptake by the SR. Hence, a stronger myocardial contraction occurs and temporal parameters of contractions are reduced (TTP and RT).8 Considering that the rat myocardium saturates its positive inotropic response at extracellular Ca2+ concentrations lower than that for other species8 protocols were performed in the presence of low extracellular Ca2+ concentrations (0.62 mM). We, therefore, investigated if the negative inotropic response induced by eugenol was reversed by ISO. The depressant effect of eugenol on force, in preparations at low Ca2+ concentration (0.62 mM) and treated with ISO, was observed but the positive inotropic effect of ISO was still present. This result suggested that the negative inotropic effect induced by eugenol was counteracted by the treatment with ISO.
Tetanus was used to evaluate eugenol's effects on contractile proteins. Tetanic stimulation obtained after inhibition of SR activity with caffeine or ryanodine has been used to produce maximal activation on the contractile machinery in the intact myocardium.9,24,30,31 Caffeine acts by emptying the SR of its calcium content and the sustained exposure to caffeine prevents SR Ca2+ reuptake.30 Relaxation in the rat myocardium is a process mainly dependent on SR Ca2+ reuptake,32 and since rat myocardium has action potentials with short duration and short refractory period these features could facilitate the development of tetanic contractions in this species.30,33,34 Then, the rat myocardium tetanic tension is only dependent on the sarcolemmal Ca2+ influx and on the activity of the contractile proteins.35
Our results showed that the peak force and the plateau force developed by papillary muscles treated with eugenol and under tetanic stimulations were depressed in a concentration-dependent way. To be sure that the elicited force tension decay was not related to a cellular fatigue, time control experiments were performed. The results showed no difference in tension decay along the time course of the experiments. Thus, the action of eugenol rather than time, reduced the tension developed by papillary muscles during tetanic stimulations. These results suggest that eugenol was reducing the sarcolemmal Ca2+ influx or depressing myosin ATPase activity.
To investigate if tetanic tension reduction was also dependent on the effects of the contractile proteins, eugenol actions on myosin ATPase activity were studied. Eugenol, at different concentrations (0.1 and 0.5 mM), did not affect the myosin Ca2+-ATPase activity, reinforcing the suggestion of its Ca2+ channel blocker action.
The calcium channel blocking effect of eugenol was demonstrated in voltage clamp experiments by measuring the ICa in isolated rat cardiac myocytes. Eugenol reduced the ICa without changing the current-voltage relationship. So, ICa inhibition might be regarded as the main mechanism for the negative inotropic effect of this clove oil component. These results, in accordance to those observed in eugenol's effects on the mechanical activity of rat papillary muscles are also in accordance to those observed for eugenol (180 μmol−1) in guinea-pig ventricular myocytes.2
However, other authors reported possible cytotoxic effects of eugenol,6,7 which could produce irreversible effects. To investigate if eugenol's effects, at the concentrations used, were irreversible or not, at the end of the experiments with tetanic stimulation muscles were washed twice with Krebs solution. Sixty minutes later it was possible to observe that muscle contractions were about 70% of control force, suggesting that the concentrations used did not produce permanent damage of myocytes.
In conclusion, our results suggest that: (1) eugenol depresses myocardial contractility in a concentration-dependent way and also reduces time parameters of the isometric contractions; (2) the negative inotropic effect of eugenol was reversed by ISO and Ca2+; (3) the depressant effect of eugenol is not accompanied by a reduction of the relative potentiation suggesting no interference with SR activity; (4) the depressant action of eugenol on the contraction, with no interference to the SR function reinforces the suggested reduction of the Ca2+ influx; (5) the depressant effect of eugenol on tetanic contractions suggests a putative action on the contractile proteins, but it was not observed on the myosin ATPase activity; (6) eugenol depresses the calcium inward current suggesting that this compound acts as a calcium channel blocker. These results suggest that the negative inotropic action eugenol seems to result from a single action as a calcium channel blocker. It does not interfere with the sarcoplasmic reticulum activity and has no effects on contractile proteins.
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