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Meeting Abstracts  |   July 1995
Halothane and Isoflurane Alter the Calcium sup 2+ Binding Properties of Calmodulin
Author Notes
  • (Levin) Intern.
  • (Blanck) Vice-Chair of Research; Professor of Anesthesiology; Professor of Pharmacology; and Professor of Physiology and Biophysics.
  • Received from the Department of Anesthesiology, Cornell University Medical College, New York, New York. Submitted for publication July 15, 1994. Accepted for publication March 13, 1995. Presented in part at the meeting of the Biophysical Society, New Orleans, Louisiana, March 7, 1994.
  • Address reprint requests to Dr. Blanck: Department of Anesthesiology, The Hospital for Special Surgery, 535 East 70th Street, New York, New York 10021.
Article Information
Meeting Abstracts   |   July 1995
Halothane and Isoflurane Alter the Calcium sup 2+ Binding Properties of Calmodulin
Anesthesiology 7 1995, Vol.83, 120-126.. doi:
Anesthesiology 7 1995, Vol.83, 120-126.. doi:
Methods: The fluorescence emission of calmodulin was obtained over a range of Calcium2+ concentrations (10 sup -7 - 10 sup -4 M) in the presence and absence of halothane and isoflurane. The intrinsic tyrosine fluorescence of calmodulin was measured at an excitation wavelength of 280 nm and an emission wavelength of 320 nm. Fluorescence measurements were carried out in 50 mM hydroxyethylpiperazineethane sulfonic acid, 100 mM KCl, and 2 mM ethyleneglycol-bis-(beta-aminoethyl ether) tetraacetic acid at pH 7.0 and 37 degrees Celsius. Experiments were performed in polytetrafluorethylene-sealed cuvettes so that the volatile anesthetic concentrations remained constant. The titration data were analyzed in two ways. The data were fit to the Hill equation by using nonlinear regression analysis to derive the Hill coefficient and the dissociation constant. The data were also analyzed by two-way analysis of variance with multiple comparisons to determine statistically significant effects. Volatile anesthetic concentrations were measured by gas chromatography.
Results: The presence of volatile anesthetics altered the Calcium2+ -binding affinity of calmodulin in a dose-dependent fashion. At 0.57% (0.25 mM) halothane and 1.7% (0.66 mM) isoflurane, the affinity of calmodulin for Calcium2+ relative to control was decreased. However, at higher concentrations of both anesthetics, the affinity for Calcium2+ was increased. When the volatile anesthetics were allowed to evaporate from the experimental solutions, the observed rightward shift of the calmodulin-Calcium2+ binding curve for Calcium2+ at low concentrations of the anesthetics returned to the control position. The leftward shift seen at high concentrations of the anesthetics was irreversible after evaporation of 8.7% (3.3 mM) isoflurane and 5.7% (2.5 mM) halothane.
Conclusions: These data demonstrate a complex interaction of two hydrophobic volatile anesthetics with calmodulin. A biphasic effect was observed both for halothane and for isoflurane. Calmodulin, an EF-hand Calcium2+ -binding protein, undergoes a conformational shift when binding Calcium2+, exposing several hydrophobic residues. These residues may be sites at which the anesthetics act.
Key words: Anesthetics, volatile: halothane; isoflurane. Calcium-binding proteins: calmodulin. Ions: calcium. Measurement techniques: fluorescence emission.
THE mechanisms through which the volatile anesthetics alter perceptions of pain, levels of consciousness, myocardial contractility, vascular resistance, and alveolar ventilation remain unknown. Several clues, however, suggest that these anesthetics may accomplish their varied and complex tasks by affecting Calcium2+ homeostasis. [1] .
Evidence indicates that halothane depresses myocardial contractility by altering voltage-dependent Calcium2+ channels and by changing Calcium2+ release from the sarcoplasmic reticulum. [2-5] Further studies suggest that volatile anesthetics may affect signal transduction as well, by interfering with the Calcium2+ -binding protein calmodulin. [6,7] Stepwise and sequential binding of Calcium sup 2+ [8,9] to calmodulin selectively activates a host of enzymes, including phosphodiesterase, myosin light-chain kinase, calmodulin-dependent kinase, calcineurin, erythrocyte Calcium2+ -adenosine triphosphatase, brain adenylyl cyclase, phosphorylase kinase, and nitric oxide synthase. [10] .
Halothane may inhibit some of these enzymes by specific interactions with calmodulin. Nosaka and Wong found that in the presence of calmodulin, volatile anesthetics weakly reduced the activity of myosin light-chain kinase. [6] Moreover, Rudnick et al. suggested that halothane may potentiate antitumor activity of gamma interferon by binding to calmodulin and altering its interaction with Calcium2+ -dependent kinases. [7] .
Although the volatile anesthetics have been implicated in derangements of calmodulin-Calcium2+ signaling pathways, there are no data that show direct effects of these anesthetics on the Calcium2+ -binding properties of calmodulin. In this study we monitored the intrinsic tyrosine fluorescence of calmodulin to measure the effects of halothane and isoflurane on Calcium2+ binding.
Calmodulin, a 16,700-Da monomer, is a well-characterized member of the EF-hand family of Calcium2+ -binding proteins. [8] It contains two tyrosine molecules, located within two of the four Calcium sup 2+ -binding loops in the protein. [8] When calmodulin binds Calcium sup 2+, a conformational change occurs and enhancement of tyrosine fluorescence results. [11-13] .
Analysis of fluorescence emission over a range of Calcium2+ concentrations allowed us to estimate dissociation constants (Kd) and Hill coefficients (n) for calmodulin's Calcium2+ -binding sites in the presence and absence of volatile anesthetics and to compare them with known Calcium2+ -binding data. We took advantage of the volatility of these anesthetics to determine whether their effects on calmodulin were reversible or irreversible.
Materials and Methods
Bovine brain calmodulin was obtained from Sigma Chemical (St. Louis, MO) and Calbiochem (La Jolla, CA) and purity was confirmed by gel electrophoresis. The fluorescence emission of calmodulin at 320 nm was measured over a range of Calcium2+ concentrations (10 sup -7 - 10 sup -4 M) in the presence and absence of halothane and isoflurane. Solutions of fixed Calcium2+ concentrations were formulated according to calculations based on the program of Fabiato and Fabiato [14] as modified by Berman.* Halothane and isoflurane concentrations from 0.5-8.7% (0.25-5 mM) were generated by adding aliquots of anesthetic-saturated buffer to the experimental solutions. Anesthetic concentrations were verified by gas chromatography.
The intrinsic tyrosine fluorescence of calmodulin was measured in a spectrofluorometer (SLM-Aminco 8000, Rochester, NY) at an excitation wavelength of 280 nm and an emission wavelength of 320 nm. The fluorescence of calmodulin is known to increase on incubation with Calcium sup 2+. In control titration experiments, small aliquots of CaCl2were added to a fluorescence cuvette containing 1 micro Meter calmodulin, 50 mM hydroxyethylpiperazineethane sulfonic acid, 100 mM KCl, and 2 mM ethyleneglycol-bis-(beta-aminoethyl ether) tetraacetic acid, at 37 degrees Celsius and pH 7.0. Over the range of the titration, pH varied less than 0.25 units. This variation has been shown not to significantly alter the affinity of calmodulin for Calcium2+. [8] Titrations in the presence of anesthetic were carried out with known concentrations of halothane or isoflurane added from saturated solutions to cuvettes containing 2 ml buffer; the cuvettes were sealed with polytetrafluorethylene to maintain constant volatile anesthetic concentrations. Aliquots of CaCl2were added with a Hamilton syringe through the seal. In the reversibility experiments, the control and anesthetic additions were performed as described above, except that samples were kept in the dark after 15 min of exposure, and the cuvettes were unsealed and stirred for 1 h in a Nitrogen2atmosphere before CaCl2aliquots were added. This procedure allowed time for the anesthetics to evaporate from the calmodulin-containing solutions. The absence of anesthetic was verified by gas chromatography. The cuvettes were then resealed, and Calcium2+ titrations were carried out. Final concentrations of anesthetic were confirmed by gas chromatography.
The fluorescence data were normalized as follows. The fluorescence of calmodulin before CaCl2additions (F0) was subtracted from intrinsic tyrosine fluorescence of calmodulin (F), and the difference F - F0was divided by the maximum fluorescence (Fmax) achieved after saturation with Calcium2+. The result, Fn, equal to (F - F0)/Fmax, was plotted versus Calcium2+ concentration. All titration data were fit by nonlinear regression to the Hill equation, Fn= [Ca2+]n/([Ca2+]n+ Kdsup n). Parameters derived from nonlinear regression analyses were used with the Enzfitter program (R. J. Leatherbarrow, Elsevier Biosoft, Cambridge, UK) to generate the curves shown in Figure 1, Figure 2, Figure 3and Figure 4. The statistical significance for the differences between anesthetic treatment and control were determined by two-way analysis of variance with multiple comparisons by using the Student-Neuman-Keuls method. [15] P values less than 0.05 were considered statistically significant.
Figure 1. The effect of halothane on the fluorescence response of calmodulin from Sigma Chemical (CaMs) to Calcium2+. The intrinsic tyrosine fluorescence of CaMswas measured (lambdaexcitation= 280 nm and lambdaemission= 320 nm) in the presence of 0 (squares), 0.57% (circles), and 5.7% (triangles) halothane. Normalized fluorescence is plotted as a function of free Calcium2+ concentration (molarity). Each data point is the mean plus/minus SD of three independent determinations.
Figure 1. The effect of halothane on the fluorescence response of calmodulin from Sigma Chemical (CaMs) to Calcium2+. The intrinsic tyrosine fluorescence of CaMswas measured (lambdaexcitation= 280 nm and lambdaemission= 320 nm) in the presence of 0 (squares), 0.57% (circles), and 5.7% (triangles) halothane. Normalized fluorescence is plotted as a function of free Calcium2+ concentration (molarity). Each data point is the mean plus/minus SD of three independent determinations.
Figure 1. The effect of halothane on the fluorescence response of calmodulin from Sigma Chemical (CaMs) to Calcium2+. The intrinsic tyrosine fluorescence of CaMswas measured (lambdaexcitation= 280 nm and lambdaemission= 320 nm) in the presence of 0 (squares), 0.57% (circles), and 5.7% (triangles) halothane. Normalized fluorescence is plotted as a function of free Calcium2+ concentration (molarity). Each data point is the mean plus/minus SD of three independent determinations.
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Figure 2. The effect of isoflurane on the intrinsic tyrosine fluorescence response of calmodulin from Sigma Chemical (CaMs) to Calcium2+ in the presence of 0 (squares), 1.7% (circles), and 8.7% (triangles) isoflurane. Each data point is the mean plus/minus SD of three independent determinations.
Figure 2. The effect of isoflurane on the intrinsic tyrosine fluorescence response of calmodulin from Sigma Chemical (CaMs) to Calcium2+ in the presence of 0 (squares), 1.7% (circles), and 8.7% (triangles) isoflurane. Each data point is the mean plus/minus SD of three independent determinations.
Figure 2. The effect of isoflurane on the intrinsic tyrosine fluorescence response of calmodulin from Sigma Chemical (CaMs) to Calcium2+ in the presence of 0 (squares), 1.7% (circles), and 8.7% (triangles) isoflurane. Each data point is the mean plus/minus SD of three independent determinations.
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Figure 3. The reversibility of halothane's effects on the Calcium2+ -binding affinity of calmodulin from Sigma Chemical (CaMs). CaMswas exposed to 0.57% and 5.7% halothane, which then was allowed to evaporate. Curves for titrations with CaMsexposed to 0.57% (circles) and 5.7% (triangles) halothane that had been allowed to evaporate are compared with control data (squares). The 0.57% halothane reversed curve is coincident with the control curve. Similar results were found with isoflurane.
Figure 3. The reversibility of halothane's effects on the Calcium2+ -binding affinity of calmodulin from Sigma Chemical (CaMs). CaMswas exposed to 0.57% and 5.7% halothane, which then was allowed to evaporate. Curves for titrations with CaMsexposed to 0.57% (circles) and 5.7% (triangles) halothane that had been allowed to evaporate are compared with control data (squares). The 0.57% halothane reversed curve is coincident with the control curve. Similar results were found with isoflurane.
Figure 3. The reversibility of halothane's effects on the Calcium2+ -binding affinity of calmodulin from Sigma Chemical (CaMs). CaMswas exposed to 0.57% and 5.7% halothane, which then was allowed to evaporate. Curves for titrations with CaMsexposed to 0.57% (circles) and 5.7% (triangles) halothane that had been allowed to evaporate are compared with control data (squares). The 0.57% halothane reversed curve is coincident with the control curve. Similar results were found with isoflurane.
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Figure 4. (A) The Calcium2+ titration of 1 micro Meter calmodulin from Calbiochem (CaMc) with 0 (squares), 0.57% (circles), and 5.7% (open triangles) halothane. (B) The irreversibility of the changes in CaM sub c after exposure and evaporation of 5.7% halothane. Squares = control; open triangles = 5.7% halothane; filled triangles = titration after evaporation of 5.7% halothane.
Figure 4. (A) The Calcium2+ titration of 1 micro Meter calmodulin from Calbiochem (CaMc) with 0 (squares), 0.57% (circles), and 5.7% (open triangles) halothane. (B) The irreversibility of the changes in CaM sub c after exposure and evaporation of 5.7% halothane. Squares = control; open triangles = 5.7% halothane; filled triangles = titration after evaporation of 5.7% halothane.
Figure 4. (A) The Calcium2+ titration of 1 micro Meter calmodulin from Calbiochem (CaMc) with 0 (squares), 0.57% (circles), and 5.7% (open triangles) halothane. (B) The irreversibility of the changes in CaM sub c after exposure and evaporation of 5.7% halothane. Squares = control; open triangles = 5.7% halothane; filled triangles = titration after evaporation of 5.7% halothane.
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Results
Calcium2+ binding was measured by the change in intrinsic tyrosine fluorescence of calmodulin. The change in fluorescence was plotted as a function of Calcium2+ and fit by nonlinear regression analysis to the Hill equation to yield a value for Kdand a value for n. Calmodulin from two sources was used in the experiments: calmodulin purchased from Sigma Chemical (CaMs) and calmodulin purchased from Calbiochem (CaMc). These two preparations were characterized by different Kdand n values (Table 1), a difference that we attribute to greater Calcium2+ binding to CaMsthan to CaMcbefore the Calcium2+ titration and fluorescence measurement. Despite this quantitative difference, the qualitative changes in Calcium2+ binding observed on exposure to halothane and isoflurane were essentially identical. The fluorescence spectra of Calcium2+ -bound calmodulin at low and high anesthetic concentrations were identical to the spectra of control samples.
Table 1. The Effect of Halothane and Isoflurane on Calcium Binding to Calmodulin
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Table 1. The Effect of Halothane and Isoflurane on Calcium Binding to Calmodulin
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Volatile anesthetics altered the Calcium2+ -binding affinity of CaMsin a dose-dependent, biphasic fashion. Titrations were carried out at halothane concentrations of 0.57% (0.25 mM), 1.14% (0.5 mM), 2.27% (1.0 mM), and 5.68% (2.5 mM) and at isoflurane concentrations of 1.74% (0.66 mM) and 8.68% (3.3 mM). At low concentrations of halothane and isoflurane, the affinity of calmodulin for Calcium2+ was decreased. At 1.74% isoflurane, for example, Kdwas increased to 6.99 x 10 sup -7 from 5.89 x 10 sup -7 M, indicating lower affinity. However, at higher concentrations of both anesthetics, the affinity for Calcium2+ was increased. In the presence of 8.68% isoflurane, the Kdfor Calcium2+ decreased from 5.89 x 10 sup -7 to 5.09 x 10 sup -7 M (Figure 1and Figure 2and Table 1).
When Calcium2+ titrations of CaMswere carried out in the presence of 0.57% halothane and 1.74% isoflurane, the Calcium2+ -calmodulin binding curve shifted to the right. These changes were statistically significantly different from control at the P < 0.05 level, as determined by two-way analysis of variance. After exposure of calmodulin to anesthetic for 15 min followed by evaporation for 1 h to remove the anesthetic, the rightward shift was completely reversed, and Calcium2+ binding reverted to control values. At higher concentrations of isoflurane (8.7%) and halothane (5.7%), the leftward shift of the Calcium2+ -calmodulin binding curve induced by the anesthetics also was statistically significantly different from control. The leftward shift persisted even after the concentration of anesthetic in solution was measured to be zero, both for high halothane and for high isoflurane concentrations (Figure 3).
Titrations also were carried out with CaMc. The control titration resulted in a Kdof 1.76 plus/minus 0.66 x 10 sup -6 M and n = 1.81 plus/minus 0.095. The same effect was observed for CaMcas for CaMswhen exposed to halothane and isoflurane. Figure 4(A) demonstrates the shift to the right at 0.57% (0.25 mM) halothane and the marked shift to the left at 5.7% (2.5 mM) halothane. Furthermore, with evaporation of halothane (5.7%, 2.5 mM), there was no reversibility (Figure 4(B)). The response to isoflurane of CaMcwas qualitatively similar to that observed with CaMs(data not shown).
The effect of low and high concentrations of halothane on CaM sub c were compared by two-way analysis of variance. The data sets obtained from Calcium2+ titrations during exposure to 0.57% and 5.68% halothane were statistically significantly different from the control data set (P < 0.05).
Discussion
We have shown, by using intrinsic tyrosine fluorescence emission data, that the volatile anesthetics halothane and isoflurane alter calmodulin's Calcium2+ -binding properties. At low concentrations of anesthetic these effects appear reversible, whereas at higher concentrations they are irreversible for both isoflurane and halothane.
Our observations are based on the phenomenon that calmodulin in the presence of Calcium2+ undergoes a conformational shift with a corresponding enhancement of fluorescence. [9] Several sources of evidence support this finding. Spectroscopy studies have shown that Calcium2+ induces structural changes in the third and fourth Calcium sup 2+ -binding domains of calmodulin, where the two tyrosine residues lie. [16] Proteolysis studies have corroborated these findings by noting that calmodulin's third domain undergoes folding in the presence of Calcium2+, rendering the protein resistant to trypsin digestion. [17] Moreover, pKaanalysis has shown that the environment of tyrosine 138, which resides in calmodulin's fourth domain, is altered in the presence of Calcium2+. [18,19] .
The conformational change that occurs in the presence of Calcium2+, in addition to increasing fluorescence emission, exposes hydrophobic regions in calmodulin. [20] These hydrophobic surfaces have been implicated as necessary for calmodulin's stimulatory effects on other enzymes. [10] The same hydrophobic residues are also thought to be the sites where a host of lipophilic molecules, including the phenothiazines, interact with calmodulin to block its enzyme-activating steps. [20-25] Perhaps by binding to these hydrophobic sites the lipophilic volatile anesthetics alter calmodulin's Calcium2+ -binding properties.
We have shown two distinct effects of the volatile anesthetics on Calcium2+ binding. First, at low concentrations of anesthetic, calmodulin bound Calcium2+ with decreased affinity in a reversible fashion. In the presence of halothane and isoflurane, calmodulin may adopt a conformation that reduces its ability to bind Calcium2+. A reduction in the amount of Calcium2+ -bound calmodulin implies that the amount of calmodulin in the Calcium2+ -modified conformation available to activate other enzymes is decreased. Thus, the ability of the volatile anesthetics to promote amnesia, analgesia, respiratory depression, and muscle relaxation in a reversible fashion may result in part from temporary inhibition of the calmodulin-activated enzymes that normally regulate neural, respiratory, and skeletal muscle systems. [1] .
In contrast, the second observed effect of the volatile anesthetics on calmodulin occurred at higher concentrations of halothane and isoflurane, much greater than concentrations used clinically. We observed a dose-dependent increase in affinity for Calcium2+. It is conceivable that at concentrations greater than some critical level, the hydrophobic anesthetics alter calmodulin's folding so that Calcium2+ binding is favored. At high concentrations of both anesthetics, the change in Calcium2+ binding was not reversed, even after the anesthetic concentrations were measured to be zero by gas chromatography. These studies were performed in the dark to prevent the possibility that photoactivation of the anesthetics would lead to a covalent interaction.
One factor in our experimental system that may account for an irreversible alteration in calmodulin structure and Calcium2+ binding is pH change. We observed a decrease in pH in our experimental solutions from 7.00 to 6.75 over the course of Calcium2+ additions in all titration experiments. Three observations, however, make it doubtful that pH shifts explain our results. First, data from Haiech et al. suggest that the pH decrease we encountered is not sufficient to alter Calcium2+ binding. Only at pH less than 6.0 is the affinity of calmodulin for Calcium2+ significantly altered. [8] Second, as noted by Haiech et al., a pH decrease confers a concomitant decrease in Calcium2+ affinity. [8] At high concentrations of anesthetics, we saw an increase in Calcium2+ affinity despite a slight pH decrement. Third, although we did observe a decrease in Calcium2+ -binding affinity at low concentrations of halothane and isoflurane, this decrease cannot be ascribed to a pH change, because although the pH change was irreversible, the decrease in Calcium2+ affinity that we observed reversed as the anesthetic evaporated.
Titrations in the presence of anesthetics were achieved by the liquid addition of buffer saturated with halothane or isoflurane. An artifact that may lead to the appearance of "irreversibility" may result from contamination of anesthetic-saturated buffers with Calcium2+. We therefore estimated the amounts of contaminating Calcium2+ in the saturated buffers by using the Calcium2+ -fluorescent dyes Calcium sup 2+ green and fura 2. We found slight amounts of contaminating Calcium sup 2+ in the anesthetic-saturated buffers, but they were much less than the amounts that could explain the marked shift to the left seen at high halothane and isoflurane concentrations.
Our finding that the volatile anesthetics can alter the conformation of a specific protein are corroborated by related results from a study by Franks and Lieb. They demonstrated that halothane and other general anesthetics can inhibit soluble firefly luciferase by binding at a hydrophobic pocket in the luciferase molecule. [26] Taken together, these experiments lend further credence to the theory that general anesthetics can act by specific interactions with target protein molecules and that nonspecific associations with the lipid bilayer component of cell membranes may not be required.
Although we now report significant effects of the volatile anesthetics on calmodulin's ability to bind Calcium2+, Blanck et al. were unable to show similar effects of halothane on troponin C. [27] Troponin C and calmodulin, both members of the EF-hand family of Calcium sup 2+ -binding proteins, share as much as 76% homology. In light of the considerable similarity between these two proteins, it is reasonable to expect that the volatile anesthetics may affect both similarly. However, given the dose-dependent, biphasic nature of the effects of the volatile anesthetics on calmodulin, we suggest that a significant alteration in Calcium2+ binding was not observed in the case of troponin C because of the concentration of halothane used in that experiment. In the troponin experiment, Calcium2+ binding was measured in the presence of 1.0 and 0.9 mM halothane. These concentrations are between the lower and higher concentrations of anesthetic that alternately yield a decrease or an increase in Calcium2+ binding in the case of calmodulin and may be pertinent to the lack of an observed alteration on Calcium2+ binding to troponin C.
In summary, by measuring the Calcium2+ -dependent fluorescence emission of calmodulin, we found that low concentrations of halothane and isoflurane reversibly decreased calmodulin's Calcium2+ affinity. At higher anesthetic concentration, however, Calcium2+ binding was increased in an irreversible fashion both by halothane and by isoflurane.
The authors thank Arthur Silvers, Ph.D., for suggestions for the statistical analysis of the Calcium2+ -binding curves; Velicia White for expert technical assistance; and Damian Blanck for the calculations. The authors also thank Olaf S. Andersen, M.D., for helpful discussions regarding protein conformation.
* Personal communication.
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Figure 1. The effect of halothane on the fluorescence response of calmodulin from Sigma Chemical (CaMs) to Calcium2+. The intrinsic tyrosine fluorescence of CaMswas measured (lambdaexcitation= 280 nm and lambdaemission= 320 nm) in the presence of 0 (squares), 0.57% (circles), and 5.7% (triangles) halothane. Normalized fluorescence is plotted as a function of free Calcium2+ concentration (molarity). Each data point is the mean plus/minus SD of three independent determinations.
Figure 1. The effect of halothane on the fluorescence response of calmodulin from Sigma Chemical (CaMs) to Calcium2+. The intrinsic tyrosine fluorescence of CaMswas measured (lambdaexcitation= 280 nm and lambdaemission= 320 nm) in the presence of 0 (squares), 0.57% (circles), and 5.7% (triangles) halothane. Normalized fluorescence is plotted as a function of free Calcium2+ concentration (molarity). Each data point is the mean plus/minus SD of three independent determinations.
Figure 1. The effect of halothane on the fluorescence response of calmodulin from Sigma Chemical (CaMs) to Calcium2+. The intrinsic tyrosine fluorescence of CaMswas measured (lambdaexcitation= 280 nm and lambdaemission= 320 nm) in the presence of 0 (squares), 0.57% (circles), and 5.7% (triangles) halothane. Normalized fluorescence is plotted as a function of free Calcium2+ concentration (molarity). Each data point is the mean plus/minus SD of three independent determinations.
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Figure 2. The effect of isoflurane on the intrinsic tyrosine fluorescence response of calmodulin from Sigma Chemical (CaMs) to Calcium2+ in the presence of 0 (squares), 1.7% (circles), and 8.7% (triangles) isoflurane. Each data point is the mean plus/minus SD of three independent determinations.
Figure 2. The effect of isoflurane on the intrinsic tyrosine fluorescence response of calmodulin from Sigma Chemical (CaMs) to Calcium2+ in the presence of 0 (squares), 1.7% (circles), and 8.7% (triangles) isoflurane. Each data point is the mean plus/minus SD of three independent determinations.
Figure 2. The effect of isoflurane on the intrinsic tyrosine fluorescence response of calmodulin from Sigma Chemical (CaMs) to Calcium2+ in the presence of 0 (squares), 1.7% (circles), and 8.7% (triangles) isoflurane. Each data point is the mean plus/minus SD of three independent determinations.
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Figure 3. The reversibility of halothane's effects on the Calcium2+ -binding affinity of calmodulin from Sigma Chemical (CaMs). CaMswas exposed to 0.57% and 5.7% halothane, which then was allowed to evaporate. Curves for titrations with CaMsexposed to 0.57% (circles) and 5.7% (triangles) halothane that had been allowed to evaporate are compared with control data (squares). The 0.57% halothane reversed curve is coincident with the control curve. Similar results were found with isoflurane.
Figure 3. The reversibility of halothane's effects on the Calcium2+ -binding affinity of calmodulin from Sigma Chemical (CaMs). CaMswas exposed to 0.57% and 5.7% halothane, which then was allowed to evaporate. Curves for titrations with CaMsexposed to 0.57% (circles) and 5.7% (triangles) halothane that had been allowed to evaporate are compared with control data (squares). The 0.57% halothane reversed curve is coincident with the control curve. Similar results were found with isoflurane.
Figure 3. The reversibility of halothane's effects on the Calcium2+ -binding affinity of calmodulin from Sigma Chemical (CaMs). CaMswas exposed to 0.57% and 5.7% halothane, which then was allowed to evaporate. Curves for titrations with CaMsexposed to 0.57% (circles) and 5.7% (triangles) halothane that had been allowed to evaporate are compared with control data (squares). The 0.57% halothane reversed curve is coincident with the control curve. Similar results were found with isoflurane.
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Figure 4. (A) The Calcium2+ titration of 1 micro Meter calmodulin from Calbiochem (CaMc) with 0 (squares), 0.57% (circles), and 5.7% (open triangles) halothane. (B) The irreversibility of the changes in CaM sub c after exposure and evaporation of 5.7% halothane. Squares = control; open triangles = 5.7% halothane; filled triangles = titration after evaporation of 5.7% halothane.
Figure 4. (A) The Calcium2+ titration of 1 micro Meter calmodulin from Calbiochem (CaMc) with 0 (squares), 0.57% (circles), and 5.7% (open triangles) halothane. (B) The irreversibility of the changes in CaM sub c after exposure and evaporation of 5.7% halothane. Squares = control; open triangles = 5.7% halothane; filled triangles = titration after evaporation of 5.7% halothane.
Figure 4. (A) The Calcium2+ titration of 1 micro Meter calmodulin from Calbiochem (CaMc) with 0 (squares), 0.57% (circles), and 5.7% (open triangles) halothane. (B) The irreversibility of the changes in CaM sub c after exposure and evaporation of 5.7% halothane. Squares = control; open triangles = 5.7% halothane; filled triangles = titration after evaporation of 5.7% halothane.
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Table 1. The Effect of Halothane and Isoflurane on Calcium Binding to Calmodulin
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Table 1. The Effect of Halothane and Isoflurane on Calcium Binding to Calmodulin
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