Myocardial contractility is regulated by intracellular concentration of free Ca2+ ([Ca2'],) and myofilament Ca2+ sensitivity. The objective of this study was to elucidate the direct effects of thiopental on cardiac excitation-contraction coupling using individual, field-stimulated ventricular myocytes.
Freshly isolated rat ventricular myocytes were loaded with the Ca2+ indicator, fura-2, and placed on the stage of an inverted fluorescence microscope in a temperature-regulated bath. [Ca2+], (340/380 ratio) and myocyte shortening (video-edge detection) were monitored simultaneously in individual cells field-stimulated at 0.3 Hz. Amplitude and timing of myocyte shortening and [Ca2+l, were compared before and after addition of thiopental. Intracellular pH was measured with the pH indicator, BCECF (500/440 ratio). Real-time uptake of Ca2+ into isolated sarcoplasmic reticulum vesicles was measured using fura-2 free acid in the extravesicular compartment. One hundred thirty-two cells were studied.
Field stimulation increased [Ca2+]i from 85 + 10 nM to 355 + 22 nM (mean + SEM). Myocytes shortened by 10% of resting cell length (127 + 5 tlm). Times to peak [Ca2+], and shortening were 139 + 6 and 173 + 7 msec, respectively. Times to 50% recovery for [Ca2+], and shortening were 296 + 6 and 290 + 6 ms, respectively. Addition of thiopental (30-1,000 /lM) resulted in dose-dependent decreases in peak [Ca2+]i and myocyte shortening. Thiopental altered time to peak and time to 50% recovery for [Ca2+], and myocyte shortening and inhibited the rate of uptake of Ca2+ into isolated sarcoplasmic reticulum vesicles. Thiopental did not, however, alter the amount of Ca2+ released in response to caffeine in sarcoplasmic reticulum vesicles or intact cells. Thiopental (100 uM) increased intracellular pH and caused an upward shift in the dose-response curve to extracellular Ca2+ for shortening, with no concomitant effect on peak [Ca2+],. These effects were abolished by ethylisopropyl amiloride, an inhibitor of Na+-H+ exchange.
Thiopental has a direct negative inotropic effect on cardiac excitation-contraction coupling at the cellular level, which is mediated by a decrease in [Ca2+],. Thiopental also increases myofilament Ca2+ sensitivity via alkalinization of the cell, which may partially offset its negative inotropic effect.
USE of thiopental for induction of anesthesia is sometimes associated with hypotension and cardiac depression. [1,2]Because of concomitant changes in preload, afterload, baroreflex activity, and central nervous system activity after induction, the direct effects of thiopental on cardiac contractility are difficult to ascertain in vivo. In vitro studies provide a more direct approach for examining the specific effects of thiopental on myocardial contractility. Thiopental causes myocardial depression in isolated perfused hearts and isometrically contracting papillary muscles. [5–11]Inhibition of transsarcolemmal Ca2+influx [6,8,11–14]and uptake [11,15]of Ca2+by, or release [9,11]of Ca2+from, the sarcoplasmic reticulum (SR) have been suggested as potential cellular mechanisms of action of thiopental. Other reports have indicated, however, that thiopental inhibits K+currents, [12,16]increases duration of action potential, [11,16]and enhances myofilament Ca2+sensitivity but does not alter uptake of Ca2+by isolated SR vesicles. Therefore, thiopental could exert direct effects on the cardiomyocyte through multiple sites of action. The functional consequences and mechanisms of action of thiopental at the cellular level have not been established, however.
This study evaluated the direct effects of thiopental on excitation - contraction coupling in individual freshly isolated rat ventricular myocytes. This experimental model allowed us to simultaneously measure changes in the amplitude and timing of intracellular Ca2+transients and myocyte shortening. We tested the hypothesis that thiopental would reduce myocyte shortening via a decrease in concentration of intracellular free Ca2+([Ca2+]i). We also investigated the effects of thiopental on the handling of Ca2+by the SR, myofilament Ca (2+) sensitivity, and intracellular pH (pHi).
Methods and Materials
Preparation of Ventricular Myocytes
Isolated adult ventricular myocytes from rat hearts were obtained as previously described. The hearts were excised, cannulated via the aorta, attached to a modified Langendorff perfusion apparatus, and perfused with oxygenated (95% O2/5% CO2) Krebs-Henseleit buffer (37 [degree sign]C) containing the following (in mM): 118 NaCl, 4.8 KCl, 1.2 MgCl2, 1.2 KH2PO4, 1.2 CaCl2, 37.5 NaHCO3, and 16.5 dextrose, pH 7.35. After a 5-min equilibration period, the perfusion buffer was changed to a Ca2+-freeKrebs-Henseleit buffer (120 ml) containing 30 mg collagenase type II (lot #M6C152, 347 U/ml; Worthington Biochemical Corp., Freehold, NJ). After digestion will collagenase (20 min), the ventricles were minced and shaken in Krebs-Henseleit buffer, and the resulting cellular digest was washed, filtered, and resuspended in phosphate-free HEPES-buffered saline (HBS) containing the following (in mM): 118 NaCl, 4.8 KCl, 1.2 MgCl (2), 1.25 CaCl2,; 11.0 dextrose, 25.0 HEPES, and 5.0 pyruvate, pH .35. The solution was vigorously bubbled immediately before use with 100% O2. Typically, 6–8 x 106cells per rat heart were obtained using this procedure. Viability, as assessed by the percent of cells retaining a rod-like shape with no blebs or granulations, was routinely 80–90%. Myocytes were suspended in HBS (1 x 106cells/ml) and stored in an O2hood until used.
Measurements of Contractility and [Ca2+]i
Simultaneous measurement of contraction and [Ca2+]iwas performed as previously described. Ventricular myocytes (0.5 x 106cells/ml) were incubated in HBS containing 2 [micro sign]M fura-2/acetoxy methylester (AM) at 37 [degree sign]C for 20 min. Fura-2-loaded ventricular myocytes were placed in a temperature regulated (28 [degree sign]C) chamber (Bioptechs, Inc., Butler, PA) mounted on the stage of an Olympus IX-70 inverted fluorescence microscope (Olympus America, Lake Success, NY). This temperature was used because it minimizes loss of dye from the cells and is commonly used by other investigators. [20,21]In addition, it reduces the likelihood of spontaneous contractions of Ca2+overload, which can result in contracture and cessation of contractility before completion of the protocol. The volume of the chamber was 1.5 ml. The cells were superfused continuously with HBS at a flow rate of 2 ml/min and field stimulated via bipolar platinum electrodes at a frequency of 0.3 Hz and for 5 ms using a Grass SD9 stimulator (Grass Instruments, West Warwick, RI). Myocytes exhibiting a rod-shaped appearance with clear striations, no membrane blebs, and a negative staircase of twitch performance on stimulation from rest were chosen for study.
Fluorescence measurements were performed on single ventricular myocytes using a dual-wavelength spectrofluorometer (Deltascan RFK6002; Photon Technology International, South Brunswick, NJ) at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. The cells also were illuminated with red light at a wavelength higher than 600 nm for simultaneous video-edge detection. An additional postspecimen dichroic mirror deflects light at wavelengths >600 nm into a CCD video camera (Phillips VC 62505T; Marshall Electronics, Culver City, CA) for measurement of myocyte shortening and relengthening. The fluorescence sampling frequency was 100 Hz, and data were collected using software from Photon Technology International (Felix). [Ca2+]iwas estimated by comparing the cellular fluorescence ratio with fluorescence ratios acquired using fura-2 (free acid) in buffers containing known concentrations of Ca2+.
Simultaneous measurement of cell shortening was monitored using a video-edge detector (Crescent Electronics, Sandy, UT) with a 16-ms temporal resolution. The video-edge detector was calibrated using a stage micrometer so that cell lengths during shortening and relengthening could be monitored. Lab View (National Instruments, Austin, TX) was used for acquisition of data regarding cell shortening using a sampling rate of 100 Hz.
Analysis of Intracellular Ca2+Transients and Contractile Data
Fluorescence data for [Ca2+]iwere imported into Labview, in which [Ca2+]iand myocyte contractile responses were analyzed synchronously and simultaneously. The following parameters were calculated for each individual contraction: diastolic [Ca2+]iand cell length; systolic [Ca2+]iand cell length; change in [Ca2+]iand twitch amplitude; time to peak (Tp) for [Ca2+]iand peak shortening; and time to 50%(Tr) diastolic [Ca2+]iand 50% relengthening. Parameters from 15 contractions ([Ca2+]iand shortening) were averaged to obtain mean values at baseline and in response to the various interventions. Averaging the parameters over time minimizes beat-to-beat variation.
Myocyte length in response to field stimulation was measured (in micrometers) and is expressed as the change from resting cell length (twitch amplitude). Changes in twitch amplitude in response to the interventions are expressed as a percent of baseline shortening. Changes in timing were measured in milliseconds and were normalized to changes in amplitude. Changes in [Ca2+]iwere measured as the change in the 340/380 ratio from baseline. Changes in the 340/380 ratio in from baseline. Changes in the 340/380 ratio in response to the interventions are expressed as a percent of the control response in the absence of any intervention.
Measurement of Intracellular pH
Intracellular pH was measured in myocytes using the acetoxy methylester form of 2', 7'-bis-(2-carboxy-ethyl)-5, 6-carboxyfluorescein (BCECF/AM; Texas Fluorescence Labs, Inc., Austin, TX). Loading of BCECF/AM into ventricular myocytes was identical to the procedure described for fura-2; however, fluorescence measurements were performed using excitation wavelengths of 440 and 500 nm and an emission wavelength of 530 nm. The fluorescence sampling frequency was 10 Hz, and data were collected as described for [Ca2+]i. The ratio of 500 to 440 nm fluorescence values was used to estimate pHi. At the end of the experiment, the excitation ratio from each cell was calibrated in situ by exposing cells to solutions of different pH. Each solution contained (in mM unless otherwise stated) K+140, MgCl21.0, HEPES 4.0, EGTA 2.0, 2,3-butanedione monoxime 30, BAPTA-AM (Molecular Probes, Inc., Eugene, OR) 50 [micro sign]M, and nigericin 14 [micro sign]M and was titrated to different pH values (6.7, 6.8, 7.0, 7.2, 7.4, and 7.8) using 1.0 N KOH. A linear relationship existed between pH and fluorescence ratios over the pH range 6.5–7.8.
Purification of Sarcoplasmic Reticulum Vesicles
Freshly isolated adult rat hearts were homogenized in 5 volumes of 3-[N-morpholino]propanesulfonic acid (MOPS) buffer (10 mM, pH 7.4, 4 [degree sign]C) containing sucrose (290 mM), NaN3(3 mM), dithiothreitol (1 mM), pepstatin A (1 [micro sign]M), leupeptin (1 [micro sign]M) and phenylmethylsulfonyl fluoride (0.8 mM), pH 7.4, using a homogenizer (Brinkmann Polytron, Westbury, NY). The homogenate was centrifuged at 7,500 g (20 min). The supernatant was saved and centrifuged again at 40,000 g (60 min). The resultant pellet was suspended in three volumes of MOPS (10 mM, Ph 6.8, 4 [degree sign]C) containing KCl (600 mM), NaN3(3 mM) dithiothreitol (1 mM), and protease inhibitors. The material was centrifuged at 140,000 g (40 min) and the final pellet resuspended in a Ca2+-freesucrose buffer and stored at -80 [degree sign]C until used.
Measurement of Uptake and Content of Ca2+by the Sarcoplasmic Reticulum in Sarcoplasmic Reticulum Vesicles
Double-distilled tap water was deionized using a Milli-Q reagent water system (Millipore Corp., Bedford, MA) and further purified by dual ion exchange chromatography and a Ca2+Sponge-S (Molecular Probes) to remove residual Ca2+. A buffering system representing intracellular conditions and capable of regenerating adenosine triphosphate was used for suspending the vesicles and contained the following (in mM): HEPES 20, KCl 100, NaCl 5, MgCl25, and creatine phosphate 5 (pH 7.2, 37 [degree sign]C) and creatine phosphokinase (0.4 U/ml). Oxalate (10 mM) was added to act as a Ca (2+) precipitating anion inside the vesicles to minimize leakage of Ca2+and maintain the Ca2+gradient across the vesicular membrane. The solutions were prepared using an iterative solution mixing program (Solwin v2.0, Philadelphia, PA). Binding constants for the ionic compounds were corrected for temperature and ionic strength. CaCl2was added back to the buffer to yield a concentration of free Ca2+of 1 [micro sign]M (pCa 6).
Measurements of uptake and release of Ca2+were examined in real time using suspensions of SR vesicles and 2 [micro sign]M fura-2 free acid (Texas Fluorescence Labs) in these extravesicular compartment. Fluorescence experiments were performed using dual wavelength fluorometry in a temperature-regulated sample compartment (37 [degree sign]C). Microcuvettes (250 [micro sign]l) were washed in EGTA (2 mM) solution to remove all Ca2+and then thoroughly rinsed with Ca2+-freebuffer and allowed to dry. For studies on uptake of Ca2+, adenosine triphosphate (1 mM) was added to the vesicular suspension to trigger the uptake of Ca2+into the vesicles, which was measured as a decrease in the fluorescence signal (340/380 ratio) from the extravesicular compartment. Caffeine (20 mM) was used to release Ca2+from the vesicles to examine vesicular content of Ca2+. Fluorescence data were collected using Felix at a sampling frequency of 20 Hz. The rate of uptake of Ca2+was measured as the decrease in the fluorescence signal over 60 s in the presence or absence of thiopental. Addition of thiopental (30–1,000 [micro sign]M) did not alter the pH of the suspension buffer.
Protocol 1: Dose-dependent Effects of Thiopental on [Ca2+](i) and Myocyte Shortening. Changes in myocyte shortening and [Ca2+](i) during exposure to thiopental were determined. Baseline measurements were collected from individual myocytes for 1.5 min in the absence of any intervention. Myocytes were exposed to four concentrations of thiopental (30, 100, 300, and 1,000 [micro sign]M) by exchanging the buffer in the dish with new buffer containing thiopental at the desired concentration. Data were acquired for 1.5 min after a 5-min equilibration period in the presence of the anesthetic agent.
Protocol 2: Effect of Thiopental on Ca2+Stores in Sarcoplasmic Reticulum. To determine whether thiopental alters release of Ca (2+) from intracellular Ca2+stores, we measured caffeine-induced release of Ca2+in the presence or absence of the anesthetic agent. Baseline values for [Ca2+]iwere measured in individual, field-stimulated myocytes for 1.5 min. Thiopental (100 and 1,000 [micro sign]M) was then added to the superfusion buffer and allowed to equilibrate for 5 min. Field stimulation of the myocyte was discontinued, and caffeine (20 mM) was applied to the cell 15 s later. Peak [Ca2+]iinduced by caffeine was compared with peak [Ca2+]ibefore addition of thiopental and is reported as a percent of the control amplitude.
Protocol 3: Effect of Thiopental on Myofilament Ca2+Sensitivity. To determine whether thiopental alters myofilament Ca2+sensitivity, we examined the dose-response curve to the extracellular concentration of Ca2+([Ca2+]o) in the presence of absence of thiopental. Baseline parameters were collected from individual myocytes for 1.5 min. Dose-response curves for [Ca2+]owere performed by exchanging the buffer in the dish with a new buffer containing the desired [Ca2+]o. Data were acquired for 1.5 min after establishment of a new steady state. Dose-response curves for [Ca2+]owere then performed in the presence of thiopental (100 [micro sign]M). This concentration was chosen because we wanted to assess myofilament Ca2+sensitivity in the absence of any significant cardiac depression. Cells were allowed to stabilize for 5 min after addition of each intervention. The data for cell shortening were plotted against [Ca2+]oand peak [Ca2+]i. The relative contribution of the Na+-H+exchanger in mediating changes in myofilament Ca2+sensitivity was assessed using the protocol, except the cells were pretreated (5 min) with 1 [micro sign]M 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) before addition of thiopental.
Protocol 4: Effect of Thiopental on Intracellular pH. To determine the effects of thiopental on pHi, separate experiments were performed in myocytes loaded with BCECF/AM. Myocytes were first perfused with the same HBS used for baseline superfusion to estimate the effects of Cl-/HCO3(-) exchange and Na+/HCO3-exchange on pHi. The superfusate was then switched to HBS containing 30 or 100 [micro sign]M thiopental for 15 min. To further assess the contribution of Na+-H+exchange to thiopental-induced alkalinization, additional BCECF/AM-loaded myocytes were perfused with EIPA (1 [micro sign]M). As described earlier, all experiments were performed using HBS with an extracellular pH of 7.4. The pH (i) was measured at baseline and after 5 min of exposure to 1 [micro sign]M EIPA, followed by 15 min of exposure to 0, 30, or 100 [micro sign]M thiopental in the presence of EIPA.
We have verified that thiopental has no effect on the fura-2 or BCECF signal at the concentrations tested. This was confirmed in separate experiments using fura-2 or BCECF free acid in HBS and examining whether thiopental altered the 340/380 or 500/440 ratios. In addition, thiopental had no effect on the pH of the bath solution within the range 30–1,000 [micro sign]M. EIPA alone did not cause any changes in baseline pHi; however, EIPA interfered with fura-2 fluorescence measurements, so it was not possible to assess its effect on peak [Ca2+]iin response to increasing [Ca (2+)]o.
Statistical Analysis and Data Presentation
Each experimental protocol was performed on multiple myocytes from the same heart and repeated in at least four hearts. Results obtained from myocytes in each heart were averaged so that all hearts were weighted equally. The dose-dependent effects of thiopental on myocyte shortening and [Ca2+]iwere assessed using one-way analysis of variance with repeated measures and the Bonferroni/Dunn post hoc test or Student's t test when appropriate. Comparisons between groups were made by two-way analysis of variance. Results are expressed as means +/- SEM. Differences were considered statistically significant at P < 0.05.
Sodium thiopental, caffeine, EIPA, and nigericin were purchased from Sigma Chemical Co. (St. Louis, MO). Thiopental was dissolved fresh each day before use.
Baseline Parameters for Myocyte Shortening and [Ca2+]i
One hundred thirty-two cells were used for the study. Baseline [Ca (2+)]iwas 85 +/- 10 nM and the diastolic cell length was 127 +/- 5 [micro sign]m. Peak [Ca2+]iwas 355 +/- 22 nM. Twitch amplitude was 10%(12.8 +/- 0.8 [micro sign]m) of the baseline diastolic resting cell length. Time to peak (Tp) for [Ca2+]iand shortening were 139 +/- 6 and 173 +/- 7 ms, respectively. Time to 50% recovery (Tr) for [Ca2+]iand shortening were 296 +/- 6 and 290 +/- 6 ms, respectively.
Effect of Thiopental on Myocyte Shortening and [Ca2+]i
(Figure 1A) demonstrates that addition of thiopental to a single, field-stimulated ventricular myocyte resulted in dose-dependent inhibition of myocyte shortening and a concomitant decrease in peak [Ca2+]i. The myocardial depressant effects of thiopental were reversed completely after washout. Individual contractions and [Ca2+]itransients are illustrated in Figure 1B. Thiopental had no effect on resting [Ca2+]ior cell length. The summarized data are shown in Figure 2. Thiopental caused dose-dependent decreases in myocyte shortening and peak [Ca2+]i. At the highest concentration tested, thiopental (1,000 [micro sign]M) decreased myocyte shortening and peak [Ca2+]ito 37 +/- 3% and 58 +/- 7% of control, respectively.
Thiopental prolonged Tp for [Ca2+]iat all concentrations tested (Figure 3). The lowest dose of thiopental (30 [micro sign]M) decreased Tp for shortening, whereas 1,000 [micro sign]M thiopental increased Tp for shortening. Thiopental increased Tr for [Ca2+]iand shortening at the highest concentration tested (1,000 [micro sign]M).
Effect of Thiopental on Uptake and Content of Ca2+in Isolated Sarcoplasmic Reticulum Vesicles
We assessed the extent to which thiopental altered the initial rate of uptake and content of Ca2+into isolated SR vesicles. The rate of uptake of Ca2+by the vesicles was measured in real time as a decrease in the 340/380 ratio from the extravesicular compartment. Caffeine (20 mM) was used to release Ca2+from the vesicles. Figure 4A demonstrated that addition of thiopental caused dose-dependent inhibition in the rate of uptake of Ca2+into the SR vesicles. The total amount of Ca (2+) released from the vesicles in response to caffeine, however, was unaltered (98 +/- 3% of control) by thiopental (Figure 4A). The summarized data for the effects of thiopental on the rate of uptake of [Ca2+] into SR vesicles are shown in Figure 4B. Thiopental (30 [micro sign]M) decreased the rate of uptake of Ca2+into the vesicles by 28 +/- 4%. Higher concentrations of thiopental (300 [micro sign]M) decreased the rate of uptake into the SR vesicles by 60 +/- 6%.
Effect of Thiopental on Caffeine-induced Release of Ca2+from the SR in Myocytes
We also assessed the extent to which thiopental altered the amount of Ca2+released from the SR in response to caffeine (20 mM) in intact myocytes. In control myocytes, rapid exposure to caffeine caused a transient increase in [Ca2+]i, which was 99 +/- 5% of peak [Ca2+]iinduced by field stimulation. Thiopental (100 and 1,000 [micro sign]M) did not alter the amplitude of the cafeine-releasable pool of Ca2+compared with that observed with the control response to the caffeine (Figure 5).
Effect of Thiopental on the Dose-Response Curve to [Ca2+](o)
The effect of [Ca2+]oon myocyte shortening and peak [Ca (2+)]iwas examined in the presence and absence of thiopental. Increasing [Ca2+]ofrom 1 to 4 mM (control, without thiopental) resulted in a dose-dependent increase in shortening and a concomitant increase in peak [Ca2+]i(Figure 6). Thiopental (100 [micro sign]M) caused an upward shift in the dose-response curve to the [Ca2+](o) for shortening, with no concomitant effect on peak [Ca2+]i(Figure 6). In addition, thiopental caused a significant leftward shift in the relationship between peak [Ca2+]iand cell shortening (Figure 7).
Effect of Thiopental on Intracellular pH
The effects of thiopental on pHi, in cardiac myocytes are shown in Figure 8. Baseline pHiwas 7.08 +/- 0.01. Thiopental (30 and 100 [micro sign]M) caused a significant increase in pHiafter only 1 min, and this increase lasted for at least 15 min. To assess the potential contribution of Na+-H+exchange on this thiopental-induced alkalinization, additional cells were exposed to 1 [micro sign]M EIPA for 5 min (to inhibit Na+-H+exchange) and then exposed to 100 [micro sign]M thiopental for 15 min during continued exposure to EIPA. In the presence of EIPA, 100 [micro sign]M thiopental failed to cause intracellular alkalinization in myocytes (Figure 8).
Effect of EIPA on the Thiopental-induced Upward Shift in the Dose-Response Curve to [Ca2+]o
We assessed the contribution of the Na+-H+exchanger in mediating the thiopental-induced upward shift in the dose-response curve to [Ca2+]ofor myocyte shortening. The results are summarized in Figure 9. In the presence of EIPA (1 [micro sign]M), thiopental (100 [micro sign]M) failed to cause an upward shift in the dose-response curve to [Ca2+]o.
This is the first study to simultaneously assess the effects of thiopental on cardiomyocyte contractility and [Ca2+]iat the cellular level and to examine the effects of thiopental on pHi. Thiopental decreased shortening and peak [Ca2+]ibut increased pH (i) in a dose-dependent fashion; however, the concentrations of thiopental required to achieve cardiac depression in this model are higher than those encountered clinically.
Binding of Thiopental to Plasma Proteins
All anesthetic agents bind to plasma proteins, reducing the concentration of anesthetic agent available to bind to tissues. The peak concentration in plasma of thiopental during induction of general anesthesia has been estimated at 50–300 [micro sign]M. [23,24]Assuming the high degree of protein binding (83–86%), the peak concentration of free thiopental would not normally exceed 50 [micro sign]M; however, the microkinetic behavior within the vascular space has not been defined. In addition, small changes in the amount or binding capacity of proteins could result in significant increases in the free plasma concentration of anesthetic agents. Not only is there uncertainty in calculating the in vivo concentration of thiopental during normal circumstances but the concentration in free plasma would certainly be higher when concentration of protein in serum is reduced (e.g., hemodilution, liver disease, hypoproteinemia).
Effect of Thiopental on Contractility and [Ca2+]i
Myocardial depression by thiopental has been reported previously in humans, [1,2]animals, and isolated cardiac tissue. [4,6–11,16,26,27]Although the use of isolated cardiac tissue is useful for examining the direct effects of thiopental on contractility, the concentrations required to achieve significant contractile depression are variable depending on the species, cardiac preparation, and perfusion medium. In buffer-perfused cardiac preparations from several different species, the concentrations of thiopental required to cause a 50% depression in contractility are variable, ranging from 12–400 [micro sign]M. [4–6,8,11,13,26,28,29]In blood-perfused preparations in which protein binding reduces the free concentration available to interact with the tissue, concentrations of thiopental ranging from 70–568 [micro sign]M were required to cause a 50% depression in contractility. [1,10,30,31]The concentration of free thiopental in these studies, however, was likely in the range of 20–90 [micro sign]M. These differential results are likely attributable to intraspecies variation; experimental preparation; or concomitant changes in neural, humoral, or locally derived factors.
In our study, the negative inotropic effect of thiopental was reversible on washout, indicating that the effect was not toxic. Inhibition of transsarcolemmal entry of Ca2+is thought to be one possible explanation. Thiopental attenuated peak [Ca2+]iin ferret papillary muscle and inhibited the L-type Ca2+current in rat and guinea pig ventricular myocytes. In this study, the inhibitory effects of thiopental were correlated with a reduction in peak [Ca2+](i), which is likely mediated, at least in part, via inhibition of sarcolemmal L-type Ca2+currents.
Effect of Thiopental on Time to Peak and Time to 50% Recovery for Myocyte Shortening and [Ca2+]i
Changes in the timing parameters would suggest alterations in ion channel activity, SR cycling of Ca2+, or myofilament Ca2+sensitivity. Several reports indicate that thiopental alters the dynamics of Ca2+in the SR. [8,11]Our results are consistent with a decrease in the rate of release of Ca2+from the SR [9,11]or inhibition of the uptake of Ca2+by the SR. [11,15,32]Alternatively, inhibition of K (+) currents by thiopental [12,16]could prolong Tp for [Ca2+]i. The decrease in Tp for shortening observed with thiopental (30 [micro sign]M), in the presence of an increase in Tp for [Ca2+]i, suggests that thiopental may increase myofilament Ca2+sensitivity. This may be attributable to the intracellular alkalinization and enhanced affinity of troponin C for Ca2+. Prolongation of Tp and Tr for both [Ca2+]iand shortening were observed at the highest concentration tested, along with a decrease in peak [Ca2+]i, which is consistent with direct inhibition of uptake of Ca2+by the SR. Alternative explanations include an indirect effect on uptake of Ca2+by the SR because of a decrease in peak [Ca2+]i. An increase in the affinity of troponin C for Ca2+could also prolong Tr. Therefore, thiopental likely has multiple sites of action at the cellular level.
Effect of Thiopental on Uptake and Content of Ca2+in Isolated Sarcoplasmic Reticulum Vesicles
Thiopental caused a dose-dependent inhibition or no inhibition on the uptake of Ca2+into SR vesicles from dog and rabbit SR, respectively. In our preparation, thiopental decreased the rate of uptake of Ca2+into the SR vesicles in a dose-dependent manner. This could, in part, explain the increase in Tp and Tr and the depression in peak [Ca2+]iobserved in this study. A decrease in the rate of uptake could ultimately result in a decrease in the content of Ca2+in the SR depending on the amount of time allowed for uptake. We observed no significant difference in total vesicular content of Ca2+, despite differences in the initial rate of uptake. These results are consistent with changes in the dynamics of Ca2+in the SR in response to thiopental with no net change in total content of Ca2+in the SR. [8,9,15]Species differences or differences in methodologic approaches may account for the differences between studies. 
Effect of Thiopental on Caffeine-induced Release of Ca2+from the Sarcoplasmic Reticulum in Myocytes
Rapid application of caffeine (20 mM) to quiescent myocytes resulted in a [Ca2+]isignal similar in amplitude to that observed with field stimulation. Thiopental, however, did not alter the amplitude of the caffeine-induced [Ca2+]itransient even at the highest concentration tested. These results are consistent with our data obtained in the isolated SR vesicles indicating no change in content of Ca2+in the SR after treatment with thiopental. It has been proposed that thiopental may inhibit release of Ca2+by the SR without decreasing the content of Ca (2+) in the SR. Our results indicate that thiopental does not exert a negative inotropic effect by altering the content of Ca2+in the SR.
Effect of Thiopental on the Dose-Response Curve to [Ca2+](o)
Because alterations in myofilament Ca2+sensitivity can alter contractility, we examined whether thiopental altered dose - response curves to [Ca2+]o. This protocol allowed for paired comparisons of [Ca2+]iand contractile amplitude in the same cell in the presence or absence of thiopental, over a range of more than one [Ca2+]o. Thiopental caused an upward shift in the dose-response curve to [Ca2+](o) for shortening, with no concomitant effect on [Ca2+]i. Because an increase in [Ca2+]oresults in an increase in [Ca2+]i, these data suggest that the negative inotropic effect of thiopental is diminished when [Ca2+]iis increased. Therefore, thiopental may increase the maximal response of the myofilament to Ca2+as [Ca2+]iincreases. Thiopental also caused a leftward shift in the cell shortening versus peak [Ca2+]irelationship, indicating an increase in the affinity of the myofilament for Ca2+. Enhanced myofilament Ca2+sensitivity by thiopental has been suggested previously. The Ca2+-sensitizingeffect of thiopental observed in our study may partially offset the negative inotropic effect of a reduction in [Ca2+]i.
Effect of Thiopental on Intracellular pH
One possible mechanism for an increase in myofilament Ca2+sensitivity is intracellular alkalinization. [34–36]Our results demonstrate that in HBS (absence of CO2and HCO3), thiopental increases steady-state pHi. The increase in steady-state pHioccurred during the first minute after application of the anesthetic agent and was completely blocked by EIPA, an inhibitor of Na+-H+exchange. The time course for the change in pHicorrelated with the time course for the changes in the inotropic state of the cell. Taken together, our data suggest that the thiopental-induced intracellular alkalinization is mediated by stimulation of Na+-H+exchange. Possible involvement of thiopental-induced changes in Na+-HCO3-symport or Cl--HCO3-exchange is unlikely, because Na+-HCO3-symport is inactive in ventricular myocytes bathed in HCO3--free solution. [37,38]Moreover, sodium-dependent Cl--HCO3-exchange has not been found in adult mammalian ventricular myocytes. The magnitude of the change in pHidemonstrated with 100 [micro sign]M thiopental (Delta pHi, 0.05) is similar to that reported for 50 [micro sign]M phenylephrine (Delta pHi, 0.06)and 100 pM endothelin (Delta pHi, 0.08)in cardiac myocytes. The increase in pHialso may be partially responsible for the decrease in peak [Ca2+]i, because alkalosis has been shown to diminish the amplitude of the [Ca2+](i) transient. 
Effect of EIPA on the Thiopental-induced Upward Shift in the Dose - Response Curve to [Ca2+]o
If intracellular alkalinization mediates the thiopental-induced upward shift in the dose - response curve to [Ca2+]ofor shortening, then this effect should be reversed by inhibiting the Na+-H (+) exchanger with EIPA. Our results indicate that inhibition of Na+-H+exchange activity with EIPA prevents the thiopental-induced increase in myofilament Ca2+sensitivity. These data suggest that the negative inotropic effect of thiopental is diminished when [Ca2+]ois increased, and this effect is abolished by EIPA. This result is similar to other reports in which inhibition of Na+-H+exchange with amiloride attenuates endothelin-and phenylephrine-enhanced myofilament Ca2+sensitivity in rat ventricular myocytes.
There are several factors that could explain why high doses of thiopental were required to achieve a significant decrease in myocyte shortening. First, our results clearly demonstrate that thiopental has multiple actions on regulation of [Ca2+]i. It is conceivable that the effects of thiopental on uptake of Ca2+by the SR and Tp for [Ca2+]icould counteract the direct cardiodepressant action resulting from reduced Ca2+entry. Moreover, the negative inotropic effect of thiopental is likely masked by the concomitant increase in myofilament Ca2+sensitivity. Second, it is possible that the temperature used in this study (28 [degree sign]C) is “cardioprotective” and could contribute to the requirement for higher concentrations of thiopental compared with physiologic temperatures (37 [degree sign]C). In addition, enzymatic activity (e.g., SR Ca2+adenosine triphosphatase) is likely to be reduced to a greater extent than ion transport processes. This could mask thiopental-induced alterations in Tr for [Ca2+]iin the intact cardiomyocyte with low doses of thiopental, while still exhibiting a prolongation of Tp for [Ca2+]i. The isolated SR vesicle experiments performed at physiologic temperature (37 [degree sign]C) support this idea. A third factor could involve differences in regulation of [Ca2+]iregarding the role of the Na+-Ca2+exchanger and the SR Ca2+pump in rat cardiomyocytes compared with other species (guinea pig, rabbit, cat, ferret)in which cardiac depression is achieved with lower doses of thiopental. Therefore, it may be difficult to compare our results with data obtained in these species. Further, certain effects of thiopental on excitation - contraction coupling may thus be exaggerated, whereas others may be underestimated. Finally, the use of unloaded myocyte shortening as a measure of the inotropic state of the heart may be a limitation because the force developed during contraction is unknown. Unloaded myocytes may be less likely to show contractile depression in response to anesthetic agents. Despite these limitations, there is remarkable consistency between the qualitative results obtained from unloaded cells and multicellular preparations, although quantitative differences (e.g., shifts in dose dependence) may exist.
The inhibitory effect of thiopental on myocyte shortening involves a decrease in the availability of [Ca2+]i, which may be partially counteracted by a concomitant increase in myofilament Ca2+sensitivity. This latter effect is likely attributable to intracellular alkalinization via activation of Na+-H+exchange. Thiopental altered Tp and Tr for [Ca2+]iand shortening. These changes in timing are likely attributable to alterations in myofilament Ca2+sensitivity or uptake of Ca2+by the SR.
The authors thank Beth Summers and Cindy Shumaker for excellent technical support and Ronnie Sanders for outstanding work in preparing the manuscript.