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ANESTHETIC PHARMACOLOGY

Regulation of Intracellular Calcium by Bupivacaine Isomers in Cardiac Myocytes from Wistar Rats

Departamento de Farmacologia Básica e Clínica, ICB, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Serviço de Anestesiologia, Universidade Federal Fluminense, Rio de Janeiro, Brazil

Address correspondence and reprint requests to Gisele Zapata-Sudo, MD, PhD, Departamento de Farmacologia Basica e Clinica, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude, Instituto de Ciencias Biomedicas, Bloco J, Sala 14, Rio de Janeiro, Brazil, 21941-590. Address e-mail to gsudo{at}farmaco.ufrj.br.

	Abstract

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In this study we investigated the effects of a racemic mixture of bupivacaine (RS(±)bupivacaine) and its isomers (S(-)bupivacaine and R(+)bupivacaine) on the Ca²⁺ handling by ventricular myocytes from Wistar rats. Single ventricular myocytes were enzymatically isolated and loaded with the fluorescent Ca²⁺ indicator fura 2-am to estimate intracellular Ca²⁺ concentration during contraction and relaxation cycles. S(-)bupivacaine (10 µM) significantly increased peak amplitude and the rate of increase of Ca²⁺ transients in 155% ± 54% (P < 0.05) and 194% ± 94% (P < 0.01) of control. However, exposure to R(+)bupivacaine had no effect on either peak amplitude or rate of increase at any concentration tested. Saponin-skinned ventricular fibers were used to investigate the effect of bupivacaine on the intracellular Ca²⁺ regulation by sarcoplasmic reticulum (SR) and on the Ca²⁺ sensitivity of contractile system. S(-), R(+), and RS(±)bupivacaine induced Ca²⁺ release from SR (P < 0.01). In SR-disrupted skinned ventricular cells, bupivacaine and its isomers (5 mM) increased the sensitivity of contractile system to Ca²⁺. S(-), RS(±), and R(+)bupivacaine significantly increased pCa₅₀ from 5.8 ± 0.1, 5.8 ± 0.1, and 5.8 ± 0.1, to 6.1 ± 0.1 (P < 0.05), 6.0 ± 0.1 (P < 0.05), and 6.1 ± 0.1 (P < 0.05). Ca²⁺ release from SR through RyR₂ activation could explain the increase of Ca²⁺ transients in cardiac cells. Increased intracellular Ca²⁺ in cardiac myocytes display a stereoselectivity to S(-)bupivacaine.

	Introduction

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The use of the isolated S(-) isomer of bupivacaine instead of the racemic mixture of S(-) and R(+) isomers (RS(±)bupivacaine) decreased the risk of systemic toxicity during local anesthesia (1,2). The eletrophysiologic effects of RS(±)bupivacaine in cardiac cells is a major concern because its arrhythmogenic action is an important factor responsible for cardiac arrest with bupivacaine overdose. R(+)bupivacaine is more potent at inhibiting both Na⁺ (3,4) and K⁺ (5) channels than the S(-) isomer, although the inhibition of Ca²⁺ channels showed no difference (6). This can explain the more pronounced effects of the isomer R(+) and RS(±)bupivacaine in the heart compared with S(-)bupivacaine. Besides the electrophysiological actions, RS(±)bupivacaine induces cardiac depression (7,8), which can be attributed to its interference in the membrane channels and/or to an action in the cell. Intracellular effects of local anesthetics (LA) have not been extensively studied; the effects of the different isomers of bupivacaine have been studied even less. Shoshan-Barmatz and Zchut (9), using binding techniques, showed that some LA, such as lidocaine, QX314, and prilocaine, stimulate the binding of ryanodine to its receptor in skeletal muscle (increase the affinity of [3H] ryanodine binding), which results in opening of the ion channel to induce calcium release from the sarcoplasmic reticulum (SR). There are no conclusive studies on the effects of RS(±)bupivacaine on intracellular calcium regulation and its possible consequences to the heart. Takahashi (10) demonstrated that RS(±)bupivacaine (0.5 to 50.0 mM) increased the permeability of SR vesicles from rabbit masseter muscle to Ca²⁺, an effect partially attributed to the influence on ryanodine-sensitive calcium channel. Komai and Lokuta (11) showed that RS(±)bupivacaine has a biphasic effect on [3H]ryanodine binding to skeletal muscle microsomes isolated from swine, enhancing [3H]ryanodine binding at 5 mM and inhibiting it at 10 mM. The authors suggested that an increase in ryanodine receptor (RyR) channel activity could contribute to the myotoxicity of RS(±)bupivacaine. RS(±)bupivacaine inhibited [3H]ryanodine binding to cardiac microsomes, indicating that its effect depends on the RyR isoform. There is uncertainty, however, about the effects of bupivacaine stereoisomers on SR function and on the contractile system.

This study investigated the stereoselective effect of RS(±)bupivacaine on intracellular calcium handling. Calcium transients in fura-2 loaded ventricular myocytes and calcium release/uptake from SR in isolated cardiac skinned fibers from rats were used to compare the effects of bupivacaine and its isomers on the regulation of intracellular Ca²⁺. Also, we tested their actions on the Ca-sensitization of contractile proteins in SR-disrupted cardiac fibers.

	Methods

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The ethics and animal investigation committee at our institution (Universidade Federal do Rio de Janeiro) approved the protocols used in this study that investigated the effects of bupivacaine and its isomers on the Ca²⁺ handling in cardiac cells.

Isolation of single cardiac cells was as described previously (6). Briefly, hearts from male Wistar rats (200–250g) anesthetized with ether were rapidly excised and attached to a modified Langendorff system. Ventricular myocytes were enzymatically isolated through retrograde perfusion with an oxygenated nominally Ca²⁺-free Tyrode solution containing (in mM) NaCl, 142; MgCl₂, 1.0; KCl, 4; HEPES, 10; glucose, 10; and collagenase type II, 120 U · mL^–1, pH 7.4 at 34.0°C ± 0.1°C. After 10 min perfusion at 8 mL/min, the enzyme was washed out by 20 min perfusion of the same solution except for the addition of 0.2 mM CaCl₂. The left ventricle was separated and isolated myocytes were obtained by gentle agitation in the Tyrode solution containing 1.25 mM CaCl₂.

Isolated myocytes were placed in a horizontal chamber (1 mL) mounted on the stage of an inverted microscope (Nikon Diaphot 300; Nikon, Tokyo, Japan) and loaded with the fluorescent Ca²⁺ indicator fura 2-am (Molecular Probes, Eugene, OR) at a final concentration of 4 µM for 20 min at room temperature (22°C–25°C). The cells were then perfused with the Tyrode solution containing 1.25 mM CaCl₂ for 5 min at 0.5 mL/min to allow the dye to be completely washed out. A single myocyte was selected and field stimulated (Nihon Kohden, SEN 3201) at 0.5 Hz through a pair of platinum electrodes placed in the chamber. Ventricular myocytes were excited at 340 nm and 380 nm through a high-speed dual-wavelength scanning illuminator (xenon arc lamp, 75 W) at a speed of 650 ratios/s (Delta Scan; Photon Technology International, Lawrenceville, NJ). Emitted light was filtered at 510 nm and detected by a photomultiplier tube (Photon Technology International). The ratio of the fluorescence signals (f340/f380) used to estimate intracellular Ca²⁺ concentration ([Ca²⁺]_i) was processed using Felix software (Photon Technology International). Peak amplitude (PA), rate of increase, and rate to 50% decay (RD₅₀) of Ca²⁺ transients were measured before (control) and after addition of increasing concentrations of S(-), RS(±), and R(+)bupivacaine (1.0 and 10.0 µM). A different concentration of each LA (unknown to the experimenter) was tested on each preparation preceded and was followed by a control without drug.

Cardiac fascicles approximately 0.30 mm in diameter and 1-2 mm long were excised from the subendocardium of the left ventricular wall from Wistar rats at room temperature in an oxygenated (95% O₂ and 5% CO₂), nominally Ca²⁺-free buffered saline. The composition of the solution was (in mM) NaCl, 130; KCl, 5; MgCl₂, 1; NaH₂PO₄, 0.5; NaHCO₃, 24; glucose, 5.6, pH 7.0. The ends of the fascicles were attached to two hooks, one connected to a micromanipulator and the other to a force transducer (model FT-03; Grass, West Warwick, RI). After mounting in a horizontal tissue chamber (internal volume of 1 mL), the fascicle was stretched to 120% of the resting length using a binocular stereo microscope. To obtain skinned myocardial fibers, the bundle was immersed for 5 min in the presence of the detergent saponin (0.5% v/v, Merck Chemical CO, Darmstadt, Germany) dissolved in a relaxing solution (solution R) (12). Solution R was composed of (mM): K propionate, 185; Mg acetate, 2.5; imidazole propionate, 10; K₂Na₂ATP, 5 and K₂-ethylene glycol-bis(ß-amino-ethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 5 (pH 7.0). This treatment destroys the sarcolemma without affecting the integrity of contractile proteins and maintains the SR membrane intact (13). In the experiments during which the goal was to investigate the direct effect of bupivacaine and isomers on the Ca²⁺ sensitivity of contractile system, the SR membranes were also disrupted by a further 60-min exposure to solution R containing the non-ionic detergent octyl phenoxy polyethoxyethanol (Triton X-100, 1% v/v; Sigma Chemical CO, St. Louis, MO) (14). This treatment does not interfere with the maximal activated tension of the fibers (13). Throughout all experiments, the temperature was maintained at 22.0°C ± 0.5°C.

After the skinning procedure, the maximum contractile response of the fibers (Po) was determined by exposure to a washout solution (solution W) containing 15.85 µM Ca²⁺ (pCa 4.8). Solution W had the same composition as solution R except that it did not contain EGTA. Exposure to solution R to induce relaxation of fibers was performed immediately after the observation of maximal Ca²⁺-activated contracture; continuously monitored tension was recorded on a chart recorder (model 7400; Grass). The same protocol was repeated at the end of each experiment, and if Po declined by 20% or more the experiment was discarded. After the initial Po measurement, a SR loading cycle was performed as follows (Fig. 1): SR was depleted of Ca²⁺ using solution R containing 20 mM caffeine. Caffeine induces complete Ca²⁺ release from SR (15) and the presence of EGTA (5 mM) in solution R prevented Ca²⁺ re-uptake into SR, keeping SR completely empty. Fibers were then exposed to solution of pCa 6.8 (0.16 µM Ca²⁺) for 3 min to promote Ca²⁺ uptake into SR. Solutions used to load the SR were prepared from solution W, adding adequate concentrations of K₂EGTA and CaK₂EGTA, keeping constant the total EGTA concentration at 5 mM. Association constants used to calculate the different ratios of K₂EGTA/CaK₂EGTA to obtain the desired pCa were those reported by Orentlicher et al. (16). For other ligands, the constants were those used by Fabiato and Fabiato (17). Ca²⁺ loading into SR was then evaluated from the tension induced by exposure of the fibers to solution W containing 20 mM caffeine; then solution R was added to the bathing medium to induce relaxation (Fig. 1). Experiments in which the caffeine-induced contracture was less than 70% of Po were rejected. To investigate the effect of bupivacaine and its isomers on Ca²⁺-release from SR, each LA (0.1–10 mM) was added to solution W after the SR loading cycle. The tension induced by each LA was expressed as percentage of Po. The dose-response curves were compared among RS(±) bupivacaine and isomers. The effect of LA (5 mM) on the SR Ca²⁺ release was also investigated in the presence of procaine 40 mM, which is a Ca²⁺ release inhibitor.

View larger version (9K): [in this window] [in a new window]   Figure 1. Representative tracings showing maximal response of the fiber (pCa 4.8) determined by exposure to a washout solution (W) containing 15.85 µM Ca2+ (pCa 4.8) followed by exposure to a relaxing solution (R). Caffeine-induced contracture was then obtained after sarcoplasmic reticulum (SR) loading cycle, which consisted of 2 steps: SR depletion of Ca2+ with exposure to solution R containing caffeine (20 mM) followed by Ca2+ uptake into the SR consequent to exposure of pCa 6.8 for 3 min. Calibration bars: vertical, 15 mg; horizontal, 1 min.

To investigate the influence of bupivacaine and isomers on Ca²⁺ uptake into SR, the loading cycle was repeated in the presence of procaine (40 mM) with or without increasing concentrations of LA (0.1, 0.5, 1.0, and 5.0 mM). Procaine was present in the loading solution to prevent Ca²⁺ release from SR induced by LA during the loading period. Ca²⁺ content was then evaluated by exposure to caffeine.

Effects of RS(±)bupivacaine and isomers on Ca-sensitization of the contractile proteins were studied in SR-disrupted preparations. The efficiency of the treatment with Triton X-100 was confirmed for each experiment by the inhibition of more than 90% of caffeine-induced tension after the SR loading cycle. The pCa versus isometric tension curves were performed in the absence (control) and presence of 5.0 mM of R(+), S(-), and RS(±)bupivacaine. The pCa value that induced 50% of maximal tension (pCa₅₀) was calculated for each experiment in the absence or presence of LA using the Hill equation.

The PA, rate of increase, and RD variables determined in the fura-2 fluorescence measurements were expressed as percentages of control values. Kruskal-Wallis and Dunn's tests were used for analysis of critical difference between variables. Tension amplitudes measured in skinned fibers were expressed as percentage of Po. Comparisons among S(-), R(+), and RS(±)bupivacaine effects in the Ca²⁺ release from SR were evaluated for statistical significant difference using one-way analysis of variance and Bonferroni's test for critical differences. Kruskal-Wallis test followed by Student-Newman-Keuls test were used for comparisons between pCa₅₀ values. The Ca²⁺ concentration-response curve was fitted to the equation y = y_max · Ca²⁺ⁿ (Ca²⁺ⁿ + k_0.5), where y is the percentage of isometric tension, n the Hill coefficient, and k_0.5 the Ca²⁺ concentration producing 50% of maximum tension. Data are expressed as mean ± sd. Differences were considered significant if P < 0.05.

	Results

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Figure 2A shows representative tracings of Ca²⁺ transients obtained on enzymatically isolated ventricular myocytes loaded with fura-2 am and stimulated at 0.5 Hz. The transients were recorded in the absence (control) and presence of S(-)bupivacaine, 1 µM. The ratio of the fluorescence signals emitted by dual excitation of fura-2 (f340/f380) was increased in the presence of S(-)bupivacaine (1 µM) (Fig. 2A). Compared with control, both S(-) and RS(±)bupivacaine increased PA of Ca²⁺ transients. In contrast, R(+)bupivacaine did not significantly alter the PA of Ca²⁺ transients in the isolated ventricular myocyte (Fig. 2B, upper panel). S(-)bupivacaine induced an increase in PA of 146% ± 39% and 155% ± 54% at 1 and 10 µM, respectively. The same results were observed with RS(±)bupivacaine, which increased 148% ± 37% and 165% ± 57% of control PA. The diastolic Ca²⁺ transient was similar for all myocytes used, 0.43 ± 0.03 (n = 15), 0.44 ± 0.03 (n = 15), and 0.42 ± 0.03 (n = 15) for S(-), RS(±), and R(+)bupivacaine, respectively. Rate of increase to the peak and RD₅₀ were also analyzed. Changes in these variables induced by LA were expressed as percentage of control and are summarized in Figure 2B. S(-) and RS(±)bupivacaine (1 and 10 µM) significantly increased the rate of increase (P < 0.05); however, exposure to R(+)bupivacaine had no effect on rate of increase at any concentration tested (Fig. 2B, left lower panel). S(-)bupivacaine increased rate of increase in 187% ± 81% (1 µM) and 194% ± 94% (10 µM) of control. Also, RS(±)bupivacaine induced an increase of 167% ± 83% and 164% ± 99% at 1 and 10 µM, respectively. These effects were totally reversed after washout of the LA. R(+)bupivacaine did not significantly alter rate of increase of Ca²⁺ transients when compared with control: 100% ± 33% and 115% ± 93% of control for 1 and 10 µM. RD₅₀ was not significantly different among LA (Fig. 2B, right lower panel).

View larger version (30K): [in this window] [in a new window]   Figure 2. A, Representative tracing of Ca2+ transients of ventricular myocytes electrically stimulated at 0.5 Hz in the absence (control) and presence of S(-) bupivacaine (1 µM). Cells were enzymatically isolated from rat heart and loaded with fura-2 am. Data are expressed as ratio of the fluorescence signals emitted by dual excitation of the dye (f340/f380). B, Effects of RS(±)bupivacaine and isomers (1 and 10 µM) in the peak amplitude (PA), rate of increase, and rate of 50% decay RD50 of Ca2+ transients. Data are expressed as mean ± sd (n = 15). *P < 0.05 compared with control.

The effects of bupivacaine and its isomers on Ca²⁺ release from SR were investigated in saponin-skinned ventricular fibers after the SR loading procedure. At 1.0 µM, the contractions induced by RS(±), R(+), and S(-) bupivacaine were 26% ± 7%, 19% ± 6%, and 33% ± 11% of maximum contractile response of the fiber. The S(-)bupivacaine-induced tension was greater than the contractile response observed in the presence of 1.0 µM of R(+)bupivacaine. Figure 3A shows representative recording of the contracture induced by S(-) bupivacaine. Initially, the Po was observed during exposure to solution W with pCa 4.8 followed by complete relaxation when in the presence of solution R. Then, increasing concentrations of S(-)bupivacaine (1, 5 and 10 mM) induced contractures after SR preload with Ca²⁺ (pCa 6.8). The concentration-response curves for the LA-induced contracture are shown in Figure 3B. S(-), R(+), and RS(±)bupivacaine induced tension in a dose-dependent manner. At 1 and 10 mM, S(-)bupivacaine induced contracture of 31% ± 6% and 57% ± 9% of Po, respectively. Similar results were observed with RS(±)bupivacaine. R(+)bupivacaine induced smaller contracture than S(-) and RS(±)bupivacaine. Procaine (40 mM), which blocks the Ca²⁺ release from SR (23–25), inhibited the increase in tension induced by S(-)bupivacaine (Fig. 4). These results suggest that RS(±)bupivacaine and isomers can activate Ca²⁺ release mechanism from preloaded SR and also that this action is regulate by a stereoselective process.

View larger version (16K): [in this window] [in a new window]   Figure 3. Effects of RS(±)bupivacaine and isomers on rat ventricular chemically-skinned fibers after sarcoplasmic reticulum (SR) loading. A, The maximum contractile response of the fibers (nominated Po) was determined by exposure to a washout solution containing 15.85 µM Ca2+ (pCa 4.8) followed by exposure to solution R to induce relaxation. Isometric tension tracings were then observed in the presence of increasing concentrations of S(-) bupivacaine (1, 5 and 10 mM) after preload of sarcoplasmic reticulum (SR) with pCa 6.8 for 3 min. During the plateau of the S(-)bupivacaine-induced contracture, the fibers were also exposed to solution R. Calibration bars: vertical, 15 mg; horizontal, 40 s. B, Dose-contractile response curves for RS(±)bupivacaine and isomers. Data are expressed as mean ± sd (n = 8). * P < 0.05 compared with S(-)bupivacaine.

View larger version (10K): [in this window] [in a new window]   Figure 4. S(-)bupivacaine-induced contracture after sarcoplasmic reticulum (SR) loading in chemically skinned fibers in the absence and presence of procaine (40 mM). Initially, maximal response was determined by exposing fibers to solution containing 15.85 µM Ca2+ (pCa 4.8). Release of Ca2+ stored in the SR by S(-)bupivacaine (5 mM) was observed after preload of SR with solution of pCa 6.8. Relaxation was induced by exposure to solution R (arrow). The same Ca2+-loading cycle was repeated followed by exposure to S(-)bupivacaine and procaine (40 mM). Calibration bars: vertical, 15 mg; horizontal, 40 s.

The rate of adenosine triphosphate-dependent Ca²⁺ uptake by the SR was investigated in the absence and presence of RS(±)bupivacaine and isomers (0.1 – 5 mM). The amount of Ca²⁺ into SR was estimated by the caffeine-induced tension after SR loading with solution W of pCa 6.8 during 1 min. Ca²⁺ loading cycle was then repeated in the absence or presence of increasing concentrations of RS(±)bupivacaine and isomers. There was no significant change in the caffeine-induced tension when S(-), R(+), or RS(±)bupivacaine was present during the SR loading cycle (Fig. 5).

View larger version (49K): [in this window] [in a new window]   Figure 5. Effects of RS(±)bupivacaine and isomers on the sarcoplasmic reticulum (SR) Ca2+-uptake in chemically skinned fibers. The amount of Ca2+ into the SR was evaluated by the caffeine (20 mM)-induced contracture after SR loading with pCa 6.8 for 1 min in the absence (control) and presence of each local anesthetic. Data are expressed as mean ± sd (n = 6).

Chemically skinned fibers treated with Triton X-100 (SR-disrupted cells) were used to investigate whether RS(±)bupivacaine and its isomers could alter Ca²⁺ sensitivity of the contractile system. Figure 6 shows the pCa-tension curves obtained in the absence (control) and in the presence of 5 mM of S(-), RS(±), and R(+)bupivacaine. There was no significant difference between the pCa₅₀ values obtained in the control groups. At 5 mM, S(-), RS(±), and R(+) bupivacaine shifted the pCa-tension curves to the left resulting in an increase in pCa₅₀. S(-), RS(±), and R(+)bupivacaine significantly increased pCa₅₀ from 5.8 ± 0.1, 5.8 ± 0.1, 5.8 ± 0.1 to 6.1 ± 0.1, 6.0 ± 0.1, and 6.1 ± 0.1 (Fig. 6). No significant difference on pCa₅₀ was observed at a smaller concentration than 1 µM of any LA used. Data suggest that only at large concentration of either RS(±)bupivacaine or isomers increase on sensitivity of contractile proteins to Ca²⁺ can be observed in skinned cardiac cells.

View larger version (28K): [in this window] [in a new window]   Figure 6. Isometric tension of chemically skinned fibers treated with triton X-100 (sarcoplasmic reticulum [SR]-disrupted cells) exposed to increasing Ca2+ concentrations (pCa 6.8–4.8) in the absence (control) and presence of RS(±)bupivacaine and isomers. The pCa value that induced 50% of maximal tension (pCa50) was increased in the presence of all tested local anesthetics. Data were fitted to a Hill plot where the slopes (n) were not significantly altered by RS(±)bupivacaine and its isomers. Data are expressed as mean ± sd (n = 6). *P < 0.05 compared with control values (absence of local anesthetics) at the same concentration of Ca2+.

	Discussion

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The mechanisms responsible for the cardiac depression induced by a large blood concentration of racemic bupivacaine are unclear. In one study (18), the S(-)isomer of bupivacaine was less toxic than the R(+)isomer in producing depression of myocardial contractility, but no clear explanation was offered. Thus, the present study investigated the influence of bupivacaine and its isomers to the Ca²⁺ regulation of cardiac cells.

Ca⁺² transients of electrically stimulated isolated ventricular myocytes were recorded before and after treatment with S(-), R(+), and RS(±)bupivacaine. S(-) and RS(±)bupivacaine, but not R(+)bupivacaine, increased PA and rate of increase measured in Ca²⁺ transients, but these effects were independent of drug concentration. These findings suggest that S(-)isomer, but not R(+)bupivacaine, could increase the intracellular Ca²⁺ concentration during each electrically stimulated contraction. We suggest a stereoselective ability of bupivacaine to increase Ca²⁺ transients in isolated ventricular myocytes. RS(±)bupivacaine was as effective as S(-)bupivacaine, which suggested that 50% of S(-) isomer molecules was enough to promote an increase in intracellular calcium concentration. The main question is which source of Ca²⁺ is mobilized into myoplasm, as RS(±)bupivacaine and enantiomers block L-type Ca²⁺ channels (6). Thus, an increase of intracellular Ca²⁺ mobilization could be a consequence of increased Ca²⁺ uptake into SR and/or release of Ca²⁺ from SR through the RyR calcium release channels (RyR₂). Mio et al. (19) described that bupivacaine (3 µM) reduced Ca²⁺ transients in thapsigargin-treated isolated myocytes. As thapsigargin abolishes SR function, the effect of bupivacaine was explained by inhibition of Ca²⁺ influx into ventricular myocytes. To test the hypothesis that bupivacaine isomers induced Ca²⁺ release from SR, we investigated the regulation of intracellular Ca²⁺ in rat ventricular saponin-skinned cells. Initially, we showed that S(-), RS(±), and R(+)bupivacaine induced contracture of cardiac skinned fibers when added after SR Ca²⁺ loading, which could be a consequence of Ca²⁺ release from SR through the RyR₂. This conclusion is based on the fact that procaine, a RyR inhibitor, inhibited the bupivacaine-induced contracture. Similar results were observed in skeletal muscle (20), in which bupivacaine (1–15 mM) promoted release of Ca²⁺ from SR in skeletal skinned fibers from BALB/c mice. Bupivacaine also increases ryanodine binding in swine skeletal and cardiac microsomes, which suggests that bupivacaine can activate RyR (11).

Another important aspect of our experiments was that the tension observed after SR Ca⁺² loading was higher in the presence of S(-) than of R(+) bupivacaine, which is in accordance with the stereoselective increase of Ca²⁺ transients observed in the presence of S(-)bupivacaine. Despite the Ca²⁺ release from SR in skinned cardiac fibers induced by R(+)bupivacaine, this isomer did not increase Ca²⁺ transients at a smaller concentration. That could be the reason for the high toxicity of R(+)bupivacaine.

We also investigated the effect of RS(±)bupivacaine and isomers in the Ca²⁺ uptake function of SR in chemically skinned fibers. SR loading was performed in the absence (control) and in the presence of S(-), R(+), or RS(±) bupivacaine (0.1–5.0 mM). SR Ca²⁺ content was evaluated by the caffeine-induced tension after different loading conditions, which was not altered by any LA tested. RS(±)bupivacaine and isomers did not interfere with the removal of Ca²⁺ from cytoplasm into SR. This was previously suspected because the RD₅₀ measured in the Ca²⁺ transients experiments was not altered by LA. Based on these results, we could exclude that the increase in Ca²⁺ mobilization observed in Ca²⁺ transient experiments could be a consequence of increased Ca²⁺ loaded into SR.

Isometric tension of SR-disrupted cells exposed to increasing Ca²⁺ concentrations was not altered by RS(±)bupivacaine and isomers at 1 µM (data not shown). However, Ca²⁺-induced contractures were greater when in the presence of 5 mM of RS(±), S(-), and R(+)bupivacaine, suggesting a Ca²⁺-sensitizing effect of all LA tested. A previous study (20) demonstrated that bupivacaine increased sensitivity of the contractile system to Ca²⁺ in skeletal fibers at the same range of concentrations used in this work. In contrast, Mio et al. (19) described that bupivacaine (1–100 µM) reduced sensitivity of contractile proteins to Ca²⁺ in skinned cardiac cells.

In conclusion, the present work demonstrates that S(-)isomer, but not R(+)bupivacaine, significantly increases Ca²⁺ transients in fura-2 loaded ventricular myocytes. The main mechanism proposed is an increase of Ca²⁺ release from SR through RyR₂ activation. Our data indicate that the increase of intracellular Ca²⁺ in cardiac myocytes displays a stereoselectivity for S(-)bupivacaine being the isomer more potent than R(+)bupivacaine to release Ca²⁺ from SR. This could explain the decreased toxicity of S(-)bupivacaine to induce cardiac depression. Only at millimolar concentrations of RS(±)bupivacaine or its isomers can an increase on the sensitivity of contractile system to Ca²⁺ be observed.

	Footnotes

 
Supported, in part, by Cristalia Produtos Quimicos e Farmaceuticos Ltda (Cristália), Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação Universitária Jose Bonifácio (FUJB), Programa Pronex, Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ).

Accepted for publication October 13, 2005.

	References

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