Anesth Analg 2004;98:1013-1016
© 2004 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000104481.20813.35
ANESTHETIC PHARMACOLOGY
The Effects of Alfentanil on Cytosolic Ca2+ and Contraction in Rat Ventricular Myocytes
Mark D. Graham, BSc*,
Philip M. Hopkins, MD
, and
Simon M. Harrison, PhD*
*School of Biomedical Sciences and
Academic Unit of Anaesthesia, University of Leeds, Leeds, United Kingdom
Address correspondence and reprint requests to Simon M. Harrison, PhD, School of Biomedical Sciences, University of Leeds, Leeds, LS2 9JT, UK. Address e-mail to S.M.Harrison{at}Leeds.ac.uk
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Abstract
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Previous investigations of the effects of potent opioid analgesics on the heart have concentrated on effects on contraction magnitude and time course, but little is known about their effects on cytosolic Ca2+ regulation in cardiac tissue. In this study, we sought to assess the effects of alfentanil on contractility and the cytosolic Ca2+ transient in ventricular myocytes isolated from the rat ventricle by enzymatic dispersion. Cells were loaded with fura-2 and electrically stimulated at 1 Hz, and Ca2+ transients and contractions were recorded optically at 30°C. Alfentanil 10-8 and 10-7 M had no effect on the magnitude or time course of contraction or the cytosolic Ca2+ transient. In contrast, 10-6 M alfentanil induced a significant (P < 0.001) positive inotropic effect, increasing the mean (±SEM) unloaded shortening from 7.3 ± 1.3 µm to 8.7 ± 1.4 µm (an increase of 20%), with no change in the cytosolic Ca2+ transient. Myofilament Ca2+ sensitivity was significantly (P = 0.027) increased by 10-6 M alfentanil but unaffected at 10-7 M alfentanil. These data show that 10-6 M alfentanil, a concentration close to the maximum clinical free plasma concentration, induced a positive inotropic effect due to sensitization of the myofilaments to Ca2+ rather than to modified cytosolic Ca2+ regulation.
IMPLICATIONS: Alfentanil, at concentrations achieved in clinical practice, increased contraction in ventricular cells by a mechanism involving an increase in the sensitivity of the contractile apparatus to Ca2+.
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Introduction
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The synthetic opioid alfentanil, used for analgesia or anesthetic induction and supplementation, has a rapid onset and short duration but also induces a variety of hemodynamic effects. For example, it decreases mean arterial blood pressure and heart rate (1). Several studies have been performed to identify any direct effects of alfentanil on the heart; alfentanil reduced the spontaneous beating rate of right atrial preparations, consistent with observed bradycardia, in vivo and enhanced contractility in atrial tissue, but not in ventricular preparations, where resting, but not active, force was increased (2). In contrast, no effect of alfentanil on contractility was observed in isolated whole hearts (3), but a positive inotropic effect was seen in human atrial tissue (4). There are other inconsistencies with the observed lusitropic effects of alfentanil; it has been reported to prolong (2) or have no effect (4) on the time to peak contraction and relaxation.
No reports describing the effects of alfentanil on cytosolic Ca2+ have been published; therefore, the aim of these studies was to investigate the effects of alfentanil on cytosolic Ca2+ regulation to determine whether any changes in inotropic and lusitropic variables induced by alfentanil were secondary to altered Ca2+ regulation.
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Methods
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The technique used to prepare rat ventricular myocytes has been described previously in full (5). Briefly, rats (200–250 g) were killed by a blow to the head followed by cervical dislocation (Schedule 1 techniques sanctioned by the United Kingdom government Home Office), the heart was excised rapidly, and the aorta was cannulated. The heart was perfused retrogradely via the coronary arteries with "isolation solution" (see below for composition), which was supplemented with 750 µM CaCl2 and equilibrated with 100% oxygen to flush the coronaries of blood. Once the heart was beating regularly, the perfusate was changed to isolation solution supplemented with 100 µM Na2EGTA for 4 min. The heart was then perfused for 9 min with the isolation solution supplemented with 1 mg/mL collagenase (Type 1; Worthington Biochemical Corp., Lakewood, NJ) and 0.1 mg/mL protease (Type XIV; Sigma, Poole, UK), after which the ventricles were cut from the heart, finely chopped, and shaken in the collected enzyme solution (to which 1% bovine serum albumin was added) for 5-min intervals. Dissociated cells were harvested by filtration at the end of each 5-min digestion, and the remaining tissue was returned for further enzyme treatment. The dissociated cells were centrifuged at 30g for 40 s, resuspended in the 750 µM CaCl2 solution, and stored at room temperature until they were needed.
The isolation solution was composed of the following (mM): NaCl 130, KCl 5.4, MgCl2 1.4, NaH2PO4 0.4, HEPES 5, glucose 10, taurine 20, and creatine 10, pH 7.1 (NaOH), at 37°C. After dissociation, cells were stored in and subsequently perfused with a physiological salt solution of the following composition (mM): NaCl 140, KCl 5.4, MgCl2 1.2, NaH2PO4 0.4, HEPES 5, glucose 10, and CaCl2 1, pH 7.4 (NaOH), at 30°C. Alfentanil (RapifenTM; Janssen-Cilag, High Wycombe, UK) was diluted to the desired concentrations with the physiological salt solution. Unless stated otherwise, all solution constituents were from Sigma.
Freshly dissociated cells were transferred to a tissue chamber (volume, 0.1 mL) attached to the stage of an inverted microscope (Diaphot; Nikon, Tokyo, Japan). The cells were allowed to settle for several minutes onto the glass bottom of the chamber before being superfused at 
3 mL/min with the physiological salt solution. Solutions were delivered to the experimental chamber by magnetic drive gear metering pumps (Micropump, Concord, CA), and solution level and temperature were maintained by feedback circuits (6). All experiments were performed at 30°C.
Cells were stimulated electrically at a frequency of 1 Hz (stimulus duration, 2 ms) via 2 platinum electrodes situated in the sides of the chamber. Cell length was continuously digitized at 200 Hz and analyzed with Ionoptix software (Ionoptix).
Cells were loaded with fura-2 by gentle agitation of a 2-mL aliquot of cell suspension with 6.25 µL of 1 mM fura-2 AM in dimethyl sulfoxide for 12 min. After centrifugation as before, the supernatant was removed by suction, and the pellet of cells was resuspended in 750 µM Ca2+ solution. The fura-2–loaded cells were left for at least 30 min before use to allow deesterification of the dye to take place. These cells were then transferred to the tissue chamber and stimulated as above. To record Ca2+ transients, the fura-2–loaded cells were excited alternately with light at 2 wavelengths (340 and 380 nm), and fluorescence was detected at 510 nm by using a monochromator-based system (Cairn, Kent, UK). The ratio of fluorescence at 510 nm in response to excitation at 340 and 380 nm was used as a measure of the intracellular Ca2+ concentration. Ca2+ transients were digitized at 1 kHz and analyzed with Ionoptix software.
Data are presented as mean ± SEM, and statistical comparisons were performed with paired Student’s t-tests. If the data failed a normality test, they are presented as median (interquartile range) and were compared with paired Wilcoxon’s signed rank tests by using SigmaStat (Jandel Scientific, Erkrath, Germany). All figures were prepared by using SigmaPlot (Jandel Scientific).
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Results
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At 10-7 M (and 10-8 M; not shown), alfentanil had no effect on the extent of shortening, whereas at 10-6 M, alfentanil induced a positive inotropic effect that reached a new steady state within 30 s and was rapidly reversed on washout (Fig. 1). In 9 cells exposed to 10-6 M alfentanil, contraction increased significantly from 7.3 ± 1.3 µm under control conditions to 8.7 ± 1.4 µm with alfentanil (P < 0.001), an increase of 20%. No significant differences in either the time to peak (P = 1.0) or the time to half relaxation (P = 0.652) of the contraction were observed during exposure to 10-6 M alfentanil.
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Figure 1. Slow time base records of cell length before, during, and after a 1-min exposure to 10-7 M (A) and 10-6 M (B) alfentanil. Data were from different myocytes, and each myocyte was exposed to only one concentration of alfentanil.
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Figure 2 illustrates fast time base records of contraction and Ca2+ transients recorded simultaneously from a representative myocyte during exposure to 10-6 M alfentanil. Contraction was increased as described in Figure 1, but this was not associated with an increase in the cytosolic Ca2+ transient. In nine cells, the Ca2+ transient magnitude was unchanged (0.075 ± 0.013 fura-2 fluorescence units) under control and with 10-6 M alfentanil (P = 0.83).
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Figure 2. Superimposed fast time base records of cell length (A) and the cytosolic Ca2+ transient (B) from the same cell under control conditions and after equilibration with 10-6 M alfentanil.
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Data in Figure 2 suggest that myofilament Ca2+ sensitivity is enhanced by alfentanil. To investigate this, cell shortening was plotted against the cytosolic fura-2 ratio to gain an index of myofilament Ca2+ sensitivity (7,8). Figure 3A illustrates the relationship between shortening and the fura-2 ratio for the entire contraction-relaxation phases from a representative cell, and Figure 3B shows a linear regression of the final phase of relaxation, the slope of which is determined by myofilament Ca2+ sensitivity. These data show that in the presence of alfentanil, the slope of the regression is steeper; this indicates enhanced sensitivity of the myofilaments to Ca2+. In nine cells, the slope of this relationship was 8.0 µm/fluorescence unit (5.6–16.8 µm/fluorescence unit) under control conditions and was significantly increased by 10-6 M alfentanil (P = 0.027; Wilcoxon’s test) to 8.7 µm/fluorescence unit (5.5–26.5 µm/fluorescence unit).
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Figure 3. A, Plot of cell length against fura-2 fluorescence ratio under control conditions (•) and after equilibration with 10-6 M alfentanil ( . B, Linear regression of the final phases of relaxation. See text for further details.
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Discussion
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This is the first report to investigate the effect of alfentanil on both contraction and the Ca2+ transient in ventricular muscle. These data illustrate that 10-8 and 10-7 M alfentanil had no significant direct effects on contractility or the magnitude of the cytosolic Ca2+ transient. At 10-6 M, however, alfentanil led to a positive inotropic effect, which appears to result from enhanced myofilament Ca2+ sensitivity rather than from an increase in the magnitude of the cytosolic Ca2+ transient. In contrast to some previous reports (see the introduction), we observed no significant lusitropic effects of alfentanil on either the contraction or the Ca2+ transient at any of the concentrations tested.
Single ventricular myocytes, rather than multicellular preparations (e.g., papillary muscles or ventricular trabeculae), were used in this study to allow an investigation of the direct mechanism of action of alfentanil at the cellular level uncomplicated by potential effects of this drug on endothelial systems. Single myocytes have advantages over multicellular preparations: e.g., oxygen delivery to the cell is improved, and equilibration with anesthetic is rapid throughout the entire preparation, minimizing problems associated with anoxia and diffusion delays that could occur in multicellular preparations. Furthermore, single cells appear to respond to inotropic interventions in qualitatively the same way as multicellular preparations (9,10). These experiments were performed at 30°C on unloaded rat ventricular myocytes stimulated at 1 Hz. This temperature was chosen to maximize retention of fura-2 during experimental protocols to ensure a good signal to noise ratio, which is important for analysis of myofilament Ca2+ sensitivity. However, it should be noted that the balance between Ca2+ entry and efflux from the cell would differ slightly from that at 37°C.
Concerning the clinical relevance of this study, obviously caution has to be exercised when these data are extrapolated to human ventricular tissue. However, a previous study on the effects of alfentanil on human atrial tissue (4) also reported a positive inotropic effect. As such, it is plausible that the increase in myofilament Ca2+ sensitivity seen in these experiments may underlie the positive inotropic effect observed in human atrial muscle, although this would need to be confirmed. However, it is interesting that in the clinical situation, alfentanil is a cardiovascular depressant (1). This could be due to a greater inhibition of heart rate than increased ventricular contractility but in vivo may also be due to the effects of alfentanil on the vasculature. However, our data (and those of others) suggest that at concentrations close to the maximum clinical concentrations, alfentanil directly increases ventricular (and atrial) contractility as a consequence of increased myofilament Ca2+ sensitivity.
An important consideration in the interpretation of these data is the clinically relevant free concentrations of alfentanil. It has been reported that 91% of alfentanil is protein bound (11), and, in the same study, the maximum total plasma concentration was approximately to 600 ng/mL (equivalent to a free concentration of 130 nM). In other studies, larger concentrations have been used, resulting in larger free plasma concentrations (12), which may transiently exceed 2 µM (13). These data suggest that, at large clinical concentrations, alfentanil enhances ventricular contractility, which would help to offset its bradycardic effects on cardiac output.
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Acknowledgments
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This work was supported by a project grant from The British Heart Foundation (London, UK) and by the Medical Research Council (United Kingdom).
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References
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Accepted for publication October 14, 2003.
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Abstract
Introduction
