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Meeting Abstracts  |   May 1995
Inhibition of Sodium/Calcium Exchange and Calcium Channels of Heart Cells by Volatile Anesthetics 
Author Notes
  • (Haworth) Senior Scientist.
  • (Goknur) Research Associate.
  • Received from the Department of Anesthesiology, University of Wisconsin Medical School, Madison, Wisconsin. Submitted for publication September 6, 1994. Accepted for publication February 1, 1995. Supported by United States Public Health Service grant HL33652 and a Grant-in-aid from the University of Wisconsin Foundation. Presented in part at the annual meetings of the American Society of Anesthesiologists, San Francisco, California, October 26–30, 1991, and Washington, DC, October 9–13, 1993.
  • Address reprint requests to Dr. Haworth: Department of Anesthesiology, University of Wisconsin Clinical Science Center, 600 Highland Avenue, Madison, Wisconsin 53792.
Article Information
Meeting Abstracts   |   May 1995
Inhibition of Sodium/Calcium Exchange and Calcium Channels of Heart Cells by Volatile Anesthetics 
Anesthesiology 5 1995, Vol.82, 1255-1265. doi:
Anesthesiology 5 1995, Vol.82, 1255-1265. doi:
Key words: Anesthetics, volatile: enflurane; halothane; isoflurane. Ions: calcium; sodium. Ions, calcium: channels.
VOLATILE anesthetics exert profound effects on the heart that are thought to be caused largely by their effect on Calcium2+ movements during the cardiac cycle. [1 ] Of interest, the various anesthetics affect cardiac function in different ways and to different degrees, suggesting a spectrum of actions at the subcellular level. Because Calcium2+ flux into and out of the heart cell is mediated primarily by Calcium2+ channels and by the Sodium sup +/Calcium2+ exchanger, we sought to measure and compare the effect of halothane, isoflurane, and enflurane on these two systems.
We were particularly interested in measuring the effects of the anesthetics on the Sodium sup +/Calcium2+ exchanger, because in previous work [2 ] we had found that the inhibition of the exchanger by octanol was strongly potentiated by extracellular Sodium sup +. In these studies Sodium sup +/Calcium2+ exchange activity was measured from the rate of45Calcium2+ uptake by Sodium sup +-loaded adult rat heart cells, in the presence of ouabain to inhibit the Sodium sup + pump. This assay was judged to be specific for Sodium sup +/Calcium2+ exchange on the basis of its Sodium sup + dependence and the insensitivity of the Calcium2+ uptake to the Calcium2+ channel blocker verapamil under these conditions of Sodium sup + loading. [2 ].
To measure the inhibition of Calcium2+ channel activity by anesthetics, we have used a unique assay developed in this laboratory: Calcium2+ channel activity is measured from the rate of uptake of trace concentrations of54Manganese2+ induced by electric stimulation of the cells in suspension. [3 ] The principal of this assay is that trace concentrations of Manganese2+ can pass through Calcium2+ channels without significantly inhibiting them, and Manganese2+ is not carried by the Sodium sup +/Calcium2+ exchanger. The rate of Manganese4+ efflux from cells is negligible. This results in a linear rate of54Manganese4+ uptake induced by electric stimulation. Because the uptake was found to be stimulated by isoproterenol and inhibited by verapamil we were able to conclude that this uptake is a specific measure of Calcium2+ channel activity. [3 ] An advantage of this assay over electrophysiologic methods is that the measurement is done under more physiologic conditions, without disturbing the intracellular composition or clamping the membrane potential.
Materials and Methods
Cell Isolation
This protocol was approved by our institutional Animal Care Committee. Female retired breeder rats were anesthetized by intraperitoneal injection with 30 mg sodium thiamylal. The chest was opened and the heart removed. Heart cells were isolated from the excised hearts according to our original method, [4 ] as modified. [5 ] According to the modification (condition 5 in Table 2of that report [5 ]), the perfusion buffers contained 25 mM N-2- hydroxyethylpiperazine-N'-ethanesulfonic acid (HEPES), adjusted to pH 7.4 with NaOH, in place of bicarbonate, plus basal Eagle's medium amino acids. Calcium2+(1 mM) was restored to the recirculating perfusate 15 min after enzyme addition. This method gave a high yield of cells with a high percentage (74.3 plus/minus 6.0%) of rod-shaped cells, when the cells were resuspended in our standard HEPES buffer medium, which contains 1 mM Calcium2+. [5 ] The remaining cells, 25.7%, were round. Only 2.5% of the round cells in this preparation exclude trypan blue. [6 ] Thus the contribution of round cells to Sodium sup +- sensitive45Calcium2+ uptake and verapamil-sensitive54Manganese4+ uptake, both of which require an intact sarcolemma, is expected to be small.
HEPES Buffer Medium
The buffer was composed of (in millimolar units) NaCl 118, KCl 4.8, HEPES 25, KH2PO41.2, MgSO41.2, CaCl21.0, pyruvic acid 5, and glucose 1 l and (in micromolar units) insulin 1, adjusted to pH 7.4 with NaOH.
Anesthetic Solutions
For each experiment a stock solution of anesthetic- equilibrated HEPES buffer was prepared by adding liquid anesthetic to a glass bottle containing glass beads and filled with a known volume of buffer. The stoppered bottle was shaken vigorously until the anesthetic was visibly dissolved. The values used for minimum alveolar concentration (MAC) and other relevant conversion factors are given in Table 1.
Table 1. Rat MAC Values Used in the Present Study
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Table 1. Rat MAC Values Used in the Present Study
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Verification of Anesthetic Concentration
Anesthetic concentrations were also monitored by gas chromatography of head-space extracts [7 ] of anesthetic-buffer mixtures incubated in the chamber under the conditions described below. Such measurements showed that loss of anesthetic from the chamber over the duration of Calcium2+ uptake or54Manganese4+ uptake was negligible.
Measurement of Sodium sup +/Calcium sup 2+ Exchange Activity by sup 45 Calcium sup 2+ Uptake
Sodium sup + Loading. Cells were resuspended (4–5 mg protein/ml) in HEPES buffer medium without Magnesium2+ or Calcium sup 2+, and containing 0.1 mM EDTA. Ouabain (1 mM) was added, and cells were incubated 10 min. Suspensions were maintained aerobic by equilibration with air in a shaking incubator at 37 degrees Celsius. We have previously found these conditions to cause complete equilibration of monovalent ions across the sarcolemma via Calcium2+ channels, [8 ] resulting in complete Sodium sup + loading. After 10 min Magnesium2+(1.3 mM) was restored, extra ouabain (2.5 mM final) and ruthenium red (12.5 micro Meter) were added, and cells were stored on ice until used. The extra ouabain and the ruthenium red concentrations were such as to give final concentrations of 1 mM and 5 micro Meter, respectively, under conditions of45Calcium2+ uptake (see below). Ruthenium red was included to prevent any contribution to measured uptake of Calcium2+ by mitochondria of damaged cells. Length of storage on ice (< 1 h) did not affect measured rates of45Calcium2+ uptake. An aliquot of cells was additionally exposed to rotenone (4 micro Meter) plus carbonylcyanide p- trifluoromethoxyphenylhydrazone (2 micro Meter) for 8 min before storage on ice, to deplete them of adenosine triphosphate (ATP). [9 ] ATP depletion blocks45Calcium2+ uptake by Sodium sup +/Calcium sup 2+ exchange and hence allowed us to use this condition to define zero uptake. [9 ].
sup 45 Calcium2+ Uptake. Time-course Experiments.45Calcium2+ uptake was measured on cell suspensions in a 5-ml glass syringe placed on a rocking mixer and kept at 37 degrees Celsius in an incubator. This arrangement prevented loss of the volatile anesthetic from the cell suspension during45Calcium2+ uptake. Sodium sup +-loaded cells (2 ml) were drawn into the syringe with an air space and incubated at 37 degrees for 2 min. The air space was then excluded, and buffer (3 ml) with or without halothane was drawn into the syringe. After 2 min incubation with mixing,45Calcium2+ uptake was initiated by adding Calcium2+,45Calcium2+, and3H sub 2 O to give a free Calcium2+ concentration of 1 mM, and final isotope concentrations of 0.3 and 1 micro Ci/ml respectively. At intervals 0.5-ml aliquots of cell suspension were dispensed from the syringe into plastic tubes where they were immediately centrifuged through a bromododecane layer into perchloric acid, as previously described. [10 ] The3H2O was included to allow automatic compensation for dispensing errors. [10 ] Because no air space was created when the aliquots were dispensed, anesthetic concentration remained constant during the time course of45Calcium2+ uptake.
Dose-Response Curves. Dose response curves were generated by measuring45Calcium2+ uptake for a fixed time (2 min) by cell suspensions incubated with various concentrations of anesthetic in water-jacketed chamber at 37 degrees. The chamber, volume 1.5 ml, was stirred continuously and was free of significant air space when closed with a glass stopper. This chamber [11 ] was the same one we use for electric field stimulation of cells in suspension. Sodium sup +-loaded cells (0.6 ml) were put into the chamber, followed by (0.9 - x) ml buffer and x ml anesthetic-equilibrated buffer, and the chamber was closed with the stopper. The volume x was varied in steps of 0.1 ml to give different final concentrations of anesthetic.45Calcium2+ uptake was initiated by introducing 4 micro liter45Calcium2+ through the bleed hole in the stopper with a Hamilton syringe. The addition contained Calcium2+,45Calcium2+, and3H sub 2 O to give a free Calcium2+ concentration of 1 mM, and final isotope concentrations of 0.3 and 1 micro Ci/ml, respectively. After 2 min the stopper was removed, and 0.5-ml aliquot was taken for immediate centrifugation. This chamber allowed better control of suspension temperature than the syringe system used in time-course experiments, but only a single sample could be taken for a well-defined concentration of anesthetic, because taking a sample created an air space. The rate of ATP-depletion-sensitive45Calcium2+ uptake in the presence of anesthetic was expressed as a percentage of the rate of ATP-depletion- sensitive45Calcium2+ uptake in the absence of anesthetic. Anesthetics had no effect on baseline45Calcium2+ uptake in ATP-depleted cells (Figure 1).
Figure 1. Inhibition by halothane (open triangles and solid triangles) of Calcium2+ uptake by Sodium sup +-loaded cells, measured with sup 45 Calcium2+. Values shown are the means and standard deviations of three experiments. ATP adenosine triphosphate; MAC - minimum alveolar concentration.
Figure 1. Inhibition by halothane (open triangles and solid triangles) of Calcium2+ uptake by Sodium sup +-loaded cells, measured with sup 45 Calcium2+. Values shown are the means and standard deviations of three experiments. ATP adenosine triphosphate; MAC - minimum alveolar concentration.
Figure 1. Inhibition by halothane (open triangles and solid triangles) of Calcium2+ uptake by Sodium sup +-loaded cells, measured with sup 45 Calcium2+. Values shown are the means and standard deviations of three experiments. ATP adenosine triphosphate; MAC - minimum alveolar concentration.
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Measurement of Calcium Channel Activity by sup 54 Manganese sup 2+ Uptake
Cells after isolation were suspended (4–5 mg protein/ml) in our standard HEPES buffer medium and maintained aerobically at 37 degrees in a shaking incubator. Aliquots were exposed to isoproterenol (1 micro Meter) for 3 min and transferred to the stimulation chamber. [11 ] This was the same chamber as was used for the measurement of45Calcium2+ uptake by Sodium sup +/Calcium2+ exchange (see above). Ruthenium red (5 micro Meter) was added, and verapamil if needed (10 micro Meter), followed by (0.9 - x) ml buffer. Ruthenium red was included to prevent any contribution to measured uptake of54Manganese2+ by mitochondria of damaged cells. The chamber was then closed with the stopper and x ml anesthetic-equilibrated buffer was added through the bleed hole. The volume x was varied in steps of 0.1 ml to give different final concentrations of anesthetic.54Manganese sup 2+(5 micro Meter final concentration, 0.3 micro Ci/ml) was introduced through the bleed hole in the stopper with a Hamilton syringe, and after 30-s electric field stimulation of the suspension at 5 Hz was initiated. After 2 min the stopper was removed, and a 1 ml aliquot was taken and added to 80 micro liter 40 mM ethyleneglycolbis-(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid. The mixture was incubated for 2 min on ice, and two 0.5 ml aliquots of the mixture were centrifuged as for the measurement of45Calcium2+ uptake. The rate of verapamil-sensitive54Manganese2+ uptake in the presence of anesthetic was expressed as a percentage of the rate of verapamil-sensitive54Manganese2+ uptake in the absence of anesthetic. The magnitude of verapamil-sensitive54Manganese2+ uptake was 18.4 plus/minus 2.2. pmol54Manganese2+/mg protein, and the magnitude of verapamil-insensitive54Manganese1+ uptake was 20.0 plus/minus 1.2 pmol54Manganese2+/mg protein. Anesthetics had no effect on the latter, the baseline54Manganese sup 2+ uptake in the presence of verapamil (data not shown).
Data Analysis
Values shown are the mean and standard deviation of experimental points about that mean, using experimental points from at least three experiments. Data from each experiment were converted to percentage inhibition before data from different experiments were pooled.
Data were fit to the Hill equation [12 ]:Equation 1where v = the initial rate of45Calcium2+ uptake, expressed as a percentage of the rate measured without anesthetic; n = the slope; and IC50= the concentration of anesthetic producing 50% inhibition. Because the Hill plot distorts errors, the fit was weighted by weight (w), where:Equation 2where sdh = the standard deviation of v/(100 - v), which is given by Equation 3where sdv = the standard deviation of the measurement v.
Test for Linearity of Data. The test statistic for lack of fit of data to a straight line is given by the ratio of the mean square error for lack of fit to the mean square error for pure error. [13 ].
The test for different slopes of linear fits in Figure 1was by analysis of covariance. [14 ].
Results
When 1 mM free Calcium2+ containing45Calcium2+ was added to Sodium sup +-loaded cells, the rate of45Calcium sup 2+ uptake was rapid for 2 min, and was near zero in cells after ATP depletion (Figure 1), as was observed previously. [9 ] We concluded in that study that the intercept of45Calcium2+ uptake measured on ATP-depleted cells corresponds to45Calcium2+ that is bound to the extracellular surface, and thus can be considered as the baseline for intracellular uptake of Calcium2+. When halothane (0.88 mM, 3 MAC) was present,45Calcium2+ uptake by cells with ATP was reduced by half, whereas there was no effect on the cells without ATP (Figure 1). Because we were interested in measuring the initial rate of45Calcium2+ uptake and the effect of anesthetics on the initial rate, we tested the data in Figure 1for linearity (see Methods). First we fit the data for ATP-depleted cells to a straight line, to give a y-axis intercept that we could use as a point of origin for the other data. We then tested whether the other data deviated significantly from a fit to a straight line that went through this point of origin. We found that the control curve was not significantly different from linear for as much as 2 min (F = 0.653; F = 7.71 for P < 0.05), and the curve for uptake with 3 MAC halothane was not significantly different from linear for as much as 5 min (F = 0.567; F = 4.07 for P < 0.05). The slopes of the best fit lines were, however, significantly different (P < 0.05) between conditions, except for the two ATP depletion conditions.
A difficulty with the experimental design of Figure 1was that temperature control was uncertain, because the syringe had to be removed from the incubator for samples to be taken. Also, we wished to study the effect of anesthetics at several different concentrations. Therefore, we used water-jacketed closed chambers we had previously developed for subjecting cells in suspension to electric field stimulation, [11 ] to measure the effect of anesthetic on45Calcium2+ uptake by Sodium sup +-loaded cells. Preliminary experiments using a similar chamber containing on Oxygen2electrode showed that the cell suspension remained aerobic for the duration of the sup 45 Calcium2+ uptake measurement, which we chose as 2 min on the basis of the result in Figure 1. Also, we determined by gas chromatography that anesthetic concentrations in the chamber remained constant for the duration of the45Calcium2+ uptake measurement.
With these chambers we examined the concentration dependence of the inhibition of Sodium sup +/Calcium2+ exchange-mediated45Calcium2+ uptake by the anesthetics halothane, isoflurane, and enflurane. We found (Figure 2) that although all these anesthetics inhibited45Calcium2+ uptake completely, they varied considerably in their potency, when concentrations were expressed in units of MAC (Figure 2(A)). In contrast, when expressed in millimolar units, a similar concentration dependence of45Calcium2+ uptake inhibition was seen for all three anesthetics (Figure 2(B)). A further feature of the dose response curves was their bell shape, suggesting a multisite action of the anesthetics. This was more quantitatively evident when the data were plotted as a Hill plot (Figure 3). The slope in the region > 50% inhibited had a best fit value of -3.46 plus/minus 0.04, indicating at least four sites of action of the anesthetic. [12 ] For comparison, the data we obtained previously for inhibition of the Sodium1/Calcium2+ exchanger by octanol under these conditions are also shown. The slope for octanol in the region > 50% inhibition was - 1.78 plus/minus 0.06, suggesting at least two sites of inhibition. Studies on the reversibility of this inhibition were not undertaken.
Figure 2. Concentration dependence of inhibition by anesthetics of45Calcium2+ uptake by a Sodium sup +-loaded cells. Concentrations are expressed (A) as rat minimum alveolar concentration (MAC) and (B) in millimolar units. Conversion factors are given in Table 1.
Figure 2. Concentration dependence of inhibition by anesthetics of45Calcium2+ uptake by a Sodium sup +-loaded cells. Concentrations are expressed (A) as rat minimum alveolar concentration (MAC) and (B) in millimolar units. Conversion factors are given in Table 1.
Figure 2. Concentration dependence of inhibition by anesthetics of45Calcium2+ uptake by a Sodium sup +-loaded cells. Concentrations are expressed (A) as rat minimum alveolar concentration (MAC) and (B) in millimolar units. Conversion factors are given in Table 1.
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Figure 3. Hill plot of inhibition by halothane and octanol of45Calcium2+ uptake by Sodium sup +-loaded cells. Lines are best linear fits to data for < 50% inhibition and > 50% inhibition. The octanol data have been reported previously. [2 ].
Figure 3. Hill plot of inhibition by halothane and octanol of45Calcium2+ uptake by Sodium sup +-loaded cells. Lines are best linear fits to data for < 50% inhibition and > 50% inhibition. The octanol data have been reported previously. [2].
Figure 3. Hill plot of inhibition by halothane and octanol of45Calcium2+ uptake by Sodium sup +-loaded cells. Lines are best linear fits to data for < 50% inhibition and > 50% inhibition. The octanol data have been reported previously. [2 ].
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When the effect of the anesthetics on Calcium2+ channel activity was assessed with the54Manganese4+ uptake assay, an inhibitory action was observed but with characteristics different from those of the inhibition of Sodium sup +/Calcium2+ exchange (Figure 4). When anesthetic concentrations were expressed as MAC, halothane, isoflurane, and enflurane were approximately equipotent (Figure 4(A)). Expressed in millimolar units, enflurane was considerably weaker than the other two (Figure 4(B)). Also, the shape of the curves by inspection was less bell-shaped. We have previously found that the uptake of54Manganese4+ induced by electric stimulation was linear to 5 min. [3 ] The uptake time of 2 min used here is therefore within the linear range, as for45Calcium2+ uptake by Sodium sup +/Calcium2+ exchange, and the concentration dependence of anesthetic effects on these two systems may be compared with validity.
Figure 4. Concentration dependence of inhibition by anesthetics of stimulation-induced Manganese4+ uptake by normal cells, measured with54Manganese4+ uptake was measured after 2 min of electric stimulation. Concentrations are expressed (A) as rat minimum alveolar concentration (MAC) and (B) in millimolar units. Conversion factors are given in Table 1.
Figure 4. Concentration dependence of inhibition by anesthetics of stimulation-induced Manganese4+ uptake by normal cells, measured with54Manganese4+ uptake was measured after 2 min of electric stimulation. Concentrations are expressed (A) as rat minimum alveolar concentration (MAC) and (B) in millimolar units. Conversion factors are given in Table 1.
Figure 4. Concentration dependence of inhibition by anesthetics of stimulation-induced Manganese4+ uptake by normal cells, measured with54Manganese4+ uptake was measured after 2 min of electric stimulation. Concentrations are expressed (A) as rat minimum alveolar concentration (MAC) and (B) in millimolar units. Conversion factors are given in Table 1.
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To compare the action of the anesthetics at the exchanger and at Calcium24channels, we first derived best fit IC50values for each curve from a linear fit of the Hill plot data for > 50% inhibition, and then plotted the combined data for all anesthetics on a Hill plot, with concentrations for each anesthetic normalized by expression as a factor of its own IC50. Figure 5shows these plots, for inhibition at the exchanger (Figure 5(A)) and at Calcium sup 2+ channels (Figure 5(B)). The visual impression of a steeper concentration dependence for inhibition of the exchanger (Figure 2) than for inhibition of Calcium2+ channels (Figure 4) is born out quantitatively in this plot (Figure 5) as a steeper slope at > 50% inhibition, being -3.87 plus/minus 0.50 for Sodium sup +/Calcium2+ exchange and -1.73 plus/minus 0.19 for Calcium2+ channels, for data pooled for halothane, isoflurane, and enflurane (Table 2). The IC50values found are given in Table 3.
Figure 5. Hill plot of inhibition by anesthetics of (A)45Calcium sup 2+ uptake by Sodium sup +-loaded cells and (B) stimulation-induced sup 54 Manganese4+ uptake by normal cells. Concentrations of anesthetics are expressed relative to their concentrations producing 50% inhibition (IC50)(Table 3). Lines are best linear fits to data for > 50% inhibition; the slopes of these lines are given in Table 2. v - the initial rate of uptake, expressed as a percentage of the rate measured without anesthetic.
Figure 5. Hill plot of inhibition by anesthetics of (A)45Calcium sup 2+ uptake by Sodium sup +-loaded cells and (B) stimulation-induced sup 54 Manganese4+ uptake by normal cells. Concentrations of anesthetics are expressed relative to their concentrations producing 50% inhibition (IC50)(Table 3). Lines are best linear fits to data for > 50% inhibition; the slopes of these lines are given in Table 2. v - the initial rate of uptake, expressed as a percentage of the rate measured without anesthetic.
Figure 5. Hill plot of inhibition by anesthetics of (A)45Calcium sup 2+ uptake by Sodium sup +-loaded cells and (B) stimulation-induced sup 54 Manganese4+ uptake by normal cells. Concentrations of anesthetics are expressed relative to their concentrations producing 50% inhibition (IC50)(Table 3). Lines are best linear fits to data for > 50% inhibition; the slopes of these lines are given in Table 2. v - the initial rate of uptake, expressed as a percentage of the rate measured without anesthetic.
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Table 3. Concentration of Anesthetic Required for 50% Inhibition (IC sub 50)
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Table 3. Concentration of Anesthetic Required for 50% Inhibition (IC sub 50)
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Table 2. Comparison of Hill Plot Slopes for Inhibition by Anesthetics of the Sodium/Calcium Exchanger and Calcium Channels
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Table 2. Comparison of Hill Plot Slopes for Inhibition by Anesthetics of the Sodium/Calcium Exchanger and Calcium Channels
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Finally, because the data for inhibition of the exchanger were obtained on separate days and with different heart cell preparations from that obtained for inhibition of the Calcium2+ channel, we sought to confirm our essential findings by measuring both kinds of inhibition in the same preparation, for all anesthetics, on the same day. To do this we chose a single level of anesthetic, 1.5 MAC. We found (Figure 6) a similar level of inhibition of Calcium2+ channels with each anesthetic, and a greater degree of inhibition of Sodium sup +/Calcium2+ exchange by enflurane than by isoflurane, consistent with the results of Figure 2and Figure 4.
Figure 6. Comparison of the inhibition by 1.5 minimum alveolar concentrations of anesthetics of45Calcium2+ uptake by Sodium sup +-loaded cells and5+ Manganese2+ uptake by stimulated normal cells, measured in the same preparations. **P 0.016, a significantly greater degree of inhibition of sup +5 Calcium2+ uptake by enflurane than by isoflurane, by a pooled t test.
Figure 6. Comparison of the inhibition by 1.5 minimum alveolar concentrations of anesthetics of45Calcium2+ uptake by Sodium sup +-loaded cells and5+ Manganese2+ uptake by stimulated normal cells, measured in the same preparations. **P 0.016, a significantly greater degree of inhibition of sup +5 Calcium2+ uptake by enflurane than by isoflurane, by a pooled t test.
Figure 6. Comparison of the inhibition by 1.5 minimum alveolar concentrations of anesthetics of45Calcium2+ uptake by Sodium sup +-loaded cells and5+ Manganese2+ uptake by stimulated normal cells, measured in the same preparations. **P 0.016, a significantly greater degree of inhibition of sup +5 Calcium2+ uptake by enflurane than by isoflurane, by a pooled t test.
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Discussion
The assays for Calcium2+ channel activity and Sodium sup +/Calcium2+ exchange activity used here do not appear to overlap in what they measure, because Manganese2+ cannot enter the cell through the Sodium sup +/Calcium2+ exchanger, and Calcium2+ influx by Sodium sup +-loaded cells under the conditions used here was not inhibited by verapamil. We should, however, consider the possibility that although the5+ Manganese2+ uptake is a measure of Calcium sup 2+ channel activity, the inhibition of5+ Manganese2+ uptake seen with anesthetics may be indirect. This could occur if the anesthetics were inhibiting Sodium sup + channels, and Sodium sup + channel activity was required to trigger Calcium2+ channel activity under our conditions of electric field stimulation. We have indeed obtained some evidence in previous experiments that inhibition of Sodium sup + channels with tetrodotoxin causes a reduction in Calcium2+ channel activation in this system. [15 ] We also found that this Sodium sup + channel dependence could be overcome by beta-adrenergic stimulation of the cells with isoproterenol. [15 ] For the current experiments we therefore used cells treated with isoproterenol, and in control experiments (not shown) found that5+ Manganese2+ uptake under these conditions was insensitive to tetrodotoxin. We therefore conclude that the inhibitory action of the anesthetics on5+ Manganese2+ uptake observed here truly reflects their inhibition of Calcium2+ channel activity.
A 66% inhibition of Sodium sup +/Calcium2+ exchange activity by 3% halothane has recently been reported in neonatal rabbit ventricular myocytes, [16 ] measured electrophysiologically at 23 degrees Celsius under conditions of physiologic concentrations of extracellular Sodium sup +. This level of inhibition is comparable, and actually somewhat greater, than the inhibition we report here (Table 3).
An inhibitory action of halothane on Calcium2+ currents has been observed in rat, [17 ] guinea pig, [18 ] and dog [19 ] heart cells. These studies have shown degrees of inhibition of Calcium sup 2+ currents ranging from about 57% reduction with 0.88 mM halothane in dog cells stimulated at 0.1–0.2 Hz at room temperature [19 ] to only 20% reduction in guinea pig cells stimulated at 0.3 Hz at 37 degrees Celsius, for the same liquid phase concentration. [18 ] This range may be caused by differences in species and conditions of measurement. Also, anesthetics were found to inhibit Calcium2+ channels much more strongly at a stimulation frequency of 3 Hz than at 0.3 Hz. [16 ] The degree of inhibition of Calcium2+ channels found in rat heart cells using a switched voltage clamp at room temperature was 35%, with 1% gas phase halothane (0.63 mM at 20 degrees Celsius). [17 ] That is similar to the degree of inhibition observed here, where 0.46 mM halothane (1.46 MAC at 37 degrees Celsius) caused about 30% inhibition of verapamil-sensitive5+ Manganese4+ uptake. It is possible that the similarity of our result masks a cancellation of a tendency for the anesthetic to be more potent at the higher rate of stimulation and to be less potent at the higher temperature. The degree of inhibition found here with the5+ Manganese4+ method is more than that found with guinea pig cells at 37 degrees Celsius and less than that found with dog cells at room temperature. [19 ].
Bosnjak et al. also found that equianesthetic concentrations of halothane, isoflurane, and enflurane each produced a similar degree of inhibition of Calcium2+ current in dog heart cells. [19 ] Our results with rat heart cells are in agreement with this observation (Figure 4). The significance of this may be that cardiac Calcium2+ channels possess similar structural properties to the channels responsible for anesthesia. This similarity of anesthetic action is in striking contrast to the very different potency of the anesthetics as inhibitors Sodium sup +/Calcium2+ exchange (Figure 2). A further difference in action is indicated by the steeper Hill plots for inhibition of the exchanger than for inhibition of Calcium2+ channels (Figure 5). The steeper Hill plot for inhibition of the exchanger (Figure 5(A)) suggests more sites of action for the volatile anesthetic on the exchanger than at the Calcium2+ channel. [12 ] A potentially important consequence of this difference is that their relative degree of inhibition will vary with anesthetic concentration. At higher concentrations of anesthetic, inhibition of the exchanger will become increasingly important.
What light do our observations shed on the mechanisms of the observed effect of these anesthetics on the heart?
First, we consider how the action of anesthetics on Calcium sup 2+ channels and on Sodium sup +/Calcium2+ exchange may contribute to their inotropic effect. Inotropy in the heart is controlled to a large extent by the regulation of the access of Calcium sup 2+ to the myofilaments. The inotropic effect of anesthetics is thought to be mediated primarily through their effect on Calcium2+ homeostasis, because their effect on the myofilaments in skinned muscle preparations [20,21 ] and intact preparations [22,23 ] is relatively small. Calcium2+ for activation of the myofilaments comes from two sources: Calcium2+ influx from outside the cell and Calcium2+ released from the sarcoplasmic reticulum (SR), an intracellular store. [24,25 ] Recent experiments with thapsigargin, a specific inhibitor of SR Calcium2+ uptake, show that the contribution of the two sources of Calcium2+ to myofilament activation can be equally significant, and also that the contribution of each source varies between species. [26,27 ] Calcium2+ influx affects contractile strength not only by direct activation of the myofilaments but also by triggering SR Calcium2+ release, [28 ] and further by replenishing SR Calcium sup 2+ stores. Calcium2+ channels and the Sodium sup +/Calcium2+ exchanger are the only two known pathways for Calcium2+ influx in the heart. Because the exchanger is electrogenic, exchanging three Sodium sup + ions for one Calcium2+ ion, Calcium2+ influx is favored by membrane depolarization. [29 ] However, although the ability of Calcium2+ channels to mediate Calcium2+-induced Calcium2+ release during excitation has long been recognized, the role of the exchanger in this process is only just emerging. [30–33 ] With regard to SR filling, evidence has been gained for a role of Calcium2+ influx both by Calcium2+ channels [34–36 ] and by the Sodium sup +/Calcium2+ exchanger [37,38 ] in replenishing SR Calcium2+ stores. Thus Calcium2+ channels and the Sodium sup +/Calcium2+ exchanger both can potentially play a role in all three inotropic actions of Calcium2+ influx: direct activation of the myofilaments, triggering SR Calcium2+ release, and SR filling.
In addition to the role of the exchanger in Calcium2+ influx, its role in Calcium2+ efflux must also be considered, because the effect of inhibition of the exchanger on inotropy will be a consequence of the net change in flux as a result of inhibition of both influx and efflux. A major role for the exchanger in Calcium2+ efflux is generally accepted. The only other known efflux mechanism is the sarcolemmal Calcium2+ pump, and its contribution appears to be small. [39 ] Because at steady state the Calcium2+ entering through Calcium2+ channels and the Sodium sup +/Calcium2+ exchanger must equal the Calcium2+ leaving through the Calcium2+ pump and the Sodium sup +/Calcium2+ exchanger, the amount of Calcium2+ leaving through the exchanger would appear to be greater than the amount entering through the exchanger if, as would commonly be assumed, the amount entering through Calcium2+ channels was greater than the amount leaving through the Calcium2+ pump. An inhibition of the exchanger would thus be expected to have a positive inotropic effect, although it is possible that total Calcium2+ efflux by Sodium sup +/Calcium2+ exchange may be affected less by a partial inhibition of the exchanger than total Calcium2+ influx by Sodium sup +/Calcium2+ exchange.
In light of the above considerations, we can evaluate how the inhibition of the Calcium2+ channels and Sodium sup +/Calcium2+ exchange by anesthetics would be expected to affect inotropy. Because Calcium2+ channels have no role in Calcium2+ efflux, the inhibition of Calcium2+ channels by anesthetics would be expected to have a negative inotropic effect. The effect of inhibition of the exchanger, on the other hand, is uncertain, depending on the relative magnitude of the role of the exchanger in Calcium2+ influx and efflux. A role in both directions may mean that the inotropic effect of an inhibition would tend to cancel out.
The inotropic effect of halothane, isoflurane, and enflurane on rat papillary muscle has been investigated by Lynch and Frazer. [40 ] Halothane and enflurane decreased the force of contraction by 28% and 24% respectively at 2 MAC and 3 Hz, whereas the negative inotropic effect of isoflurane was only 7%. A similarly small negative inotropic effect of isoflurane relative to that of halothane at high (but physiologic) frequencies of stimulation has also been noted with guinea pig [41 ] and rabbit. [42 ] In contrast, the reduction of Calcium sup 2+ channel activity observed here (at 4 Hz) was similar for all three anesthetics, being 30, 39 and 36%, for halothane, enflurane, and isoflurane respectively. This suggests to us that at these high frequencies of stimulation a partial inhibition of the Calcium2+ channel may have little effect on inotropy, and thus the negative inotropic effect of halothane and enflurane under these conditions would have little to do with inhibition of the Calcium2+ channel. Moreover, because we saw a similar degree of inhibition of the Sodium sup +/Calcium2+ exchanger by isoflurane as for Calcium2+ channels, the lack of inotropic effect of isoflurane would suggest that Calcium2+ influx by this route also is not limiting contractility at high stimulation frequencies. The negative inotropic effect of halothane and enflurane under these conditions may rather be related to their effect on the SR. Consistent with this, studies of potentiated state contractions, the strength of which is thought to reflect SR function, show a strong depressant effect of halothane, some effect of enflurane, and no effect of isoflurane. [43,44 ] At low frequencies of stimulation, the anesthetics inhibited contraction more strongly. Halothane, isoflurane, and enflurane at 2 MAC were reported to inhibit rested state contractions by 53%, 26%, and 41%, respectively. [45 ] The degree of inhibition of force by isoflurane is particularly stronger under these conditions than at high frequency, and is much more like the degree of inhibition of the Calcium2+ channel. The inhibition by halothane and enflurane is still stronger than that by isoflurane, reflecting their inhibition at the SR as well as the Calcium2+ channel. Terrar and Victory [18 ] also concluded from their studies on guinea pig myocytes that the negative inotropic action of halothane was in part caused by inhibition of Calcium2+ channels and in part caused by a reduction in SR Calcium2+ stores.
Thus, in summary, the inotropic action of halothane, isoflurane, and enflurane on the rat heart can be understood in terms of their inhibition of Calcium2+ channels and SR at low frequencies of stimulation and their inhibition of SR at high frequencies of stimulation. It is not clear if any of the observed inotropic effects can be attributed to an action on the Sodium sup +/Calcium2+ exchanger.
The action of anesthetics on Sodium sup +/Calcium2+ exchange may, however, contribute to their effect on relaxation. Relaxation in the heart is accomplished by Calcium2+ removal from the myofilaments, primarily by the SR, in species that have a well- developed SR but also by Calcium2+ extrusion from the cell by Sodium sup +/Calcium2+ exchange, as described above. Therefore, anesthetics may slow relaxation, especially in species with less well- developed SR. This possibility is consistent with the observations of Lynch and Frazer, [40 ] who found that 2 MAC enflurane consistently delayed relaxation in the frog, whereas isoflurane and halothane did not, and that enflurane had no effect on relaxation in the rat.
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Figure 1. Inhibition by halothane (open triangles and solid triangles) of Calcium2+ uptake by Sodium sup +-loaded cells, measured with sup 45 Calcium2+. Values shown are the means and standard deviations of three experiments. ATP adenosine triphosphate; MAC - minimum alveolar concentration.
Figure 1. Inhibition by halothane (open triangles and solid triangles) of Calcium2+ uptake by Sodium sup +-loaded cells, measured with sup 45 Calcium2+. Values shown are the means and standard deviations of three experiments. ATP adenosine triphosphate; MAC - minimum alveolar concentration.
Figure 1. Inhibition by halothane (open triangles and solid triangles) of Calcium2+ uptake by Sodium sup +-loaded cells, measured with sup 45 Calcium2+. Values shown are the means and standard deviations of three experiments. ATP adenosine triphosphate; MAC - minimum alveolar concentration.
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Figure 2. Concentration dependence of inhibition by anesthetics of45Calcium2+ uptake by a Sodium sup +-loaded cells. Concentrations are expressed (A) as rat minimum alveolar concentration (MAC) and (B) in millimolar units. Conversion factors are given in Table 1.
Figure 2. Concentration dependence of inhibition by anesthetics of45Calcium2+ uptake by a Sodium sup +-loaded cells. Concentrations are expressed (A) as rat minimum alveolar concentration (MAC) and (B) in millimolar units. Conversion factors are given in Table 1.
Figure 2. Concentration dependence of inhibition by anesthetics of45Calcium2+ uptake by a Sodium sup +-loaded cells. Concentrations are expressed (A) as rat minimum alveolar concentration (MAC) and (B) in millimolar units. Conversion factors are given in Table 1.
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Figure 3. Hill plot of inhibition by halothane and octanol of45Calcium2+ uptake by Sodium sup +-loaded cells. Lines are best linear fits to data for < 50% inhibition and > 50% inhibition. The octanol data have been reported previously. [2 ].
Figure 3. Hill plot of inhibition by halothane and octanol of45Calcium2+ uptake by Sodium sup +-loaded cells. Lines are best linear fits to data for < 50% inhibition and > 50% inhibition. The octanol data have been reported previously. [2].
Figure 3. Hill plot of inhibition by halothane and octanol of45Calcium2+ uptake by Sodium sup +-loaded cells. Lines are best linear fits to data for < 50% inhibition and > 50% inhibition. The octanol data have been reported previously. [2 ].
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Figure 4. Concentration dependence of inhibition by anesthetics of stimulation-induced Manganese4+ uptake by normal cells, measured with54Manganese4+ uptake was measured after 2 min of electric stimulation. Concentrations are expressed (A) as rat minimum alveolar concentration (MAC) and (B) in millimolar units. Conversion factors are given in Table 1.
Figure 4. Concentration dependence of inhibition by anesthetics of stimulation-induced Manganese4+ uptake by normal cells, measured with54Manganese4+ uptake was measured after 2 min of electric stimulation. Concentrations are expressed (A) as rat minimum alveolar concentration (MAC) and (B) in millimolar units. Conversion factors are given in Table 1.
Figure 4. Concentration dependence of inhibition by anesthetics of stimulation-induced Manganese4+ uptake by normal cells, measured with54Manganese4+ uptake was measured after 2 min of electric stimulation. Concentrations are expressed (A) as rat minimum alveolar concentration (MAC) and (B) in millimolar units. Conversion factors are given in Table 1.
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Figure 5. Hill plot of inhibition by anesthetics of (A)45Calcium sup 2+ uptake by Sodium sup +-loaded cells and (B) stimulation-induced sup 54 Manganese4+ uptake by normal cells. Concentrations of anesthetics are expressed relative to their concentrations producing 50% inhibition (IC50)(Table 3). Lines are best linear fits to data for > 50% inhibition; the slopes of these lines are given in Table 2. v - the initial rate of uptake, expressed as a percentage of the rate measured without anesthetic.
Figure 5. Hill plot of inhibition by anesthetics of (A)45Calcium sup 2+ uptake by Sodium sup +-loaded cells and (B) stimulation-induced sup 54 Manganese4+ uptake by normal cells. Concentrations of anesthetics are expressed relative to their concentrations producing 50% inhibition (IC50)(Table 3). Lines are best linear fits to data for > 50% inhibition; the slopes of these lines are given in Table 2. v - the initial rate of uptake, expressed as a percentage of the rate measured without anesthetic.
Figure 5. Hill plot of inhibition by anesthetics of (A)45Calcium sup 2+ uptake by Sodium sup +-loaded cells and (B) stimulation-induced sup 54 Manganese4+ uptake by normal cells. Concentrations of anesthetics are expressed relative to their concentrations producing 50% inhibition (IC50)(Table 3). Lines are best linear fits to data for > 50% inhibition; the slopes of these lines are given in Table 2. v - the initial rate of uptake, expressed as a percentage of the rate measured without anesthetic.
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Figure 6. Comparison of the inhibition by 1.5 minimum alveolar concentrations of anesthetics of45Calcium2+ uptake by Sodium sup +-loaded cells and5+ Manganese2+ uptake by stimulated normal cells, measured in the same preparations. **P 0.016, a significantly greater degree of inhibition of sup +5 Calcium2+ uptake by enflurane than by isoflurane, by a pooled t test.
Figure 6. Comparison of the inhibition by 1.5 minimum alveolar concentrations of anesthetics of45Calcium2+ uptake by Sodium sup +-loaded cells and5+ Manganese2+ uptake by stimulated normal cells, measured in the same preparations. **P 0.016, a significantly greater degree of inhibition of sup +5 Calcium2+ uptake by enflurane than by isoflurane, by a pooled t test.
Figure 6. Comparison of the inhibition by 1.5 minimum alveolar concentrations of anesthetics of45Calcium2+ uptake by Sodium sup +-loaded cells and5+ Manganese2+ uptake by stimulated normal cells, measured in the same preparations. **P 0.016, a significantly greater degree of inhibition of sup +5 Calcium2+ uptake by enflurane than by isoflurane, by a pooled t test.
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Table 1. Rat MAC Values Used in the Present Study
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Table 1. Rat MAC Values Used in the Present Study
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Table 3. Concentration of Anesthetic Required for 50% Inhibition (IC sub 50)
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Table 3. Concentration of Anesthetic Required for 50% Inhibition (IC sub 50)
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Table 2. Comparison of Hill Plot Slopes for Inhibition by Anesthetics of the Sodium/Calcium Exchanger and Calcium Channels
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Table 2. Comparison of Hill Plot Slopes for Inhibition by Anesthetics of the Sodium/Calcium Exchanger and Calcium Channels
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