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Meeting Abstracts  |   May 1995
Effects of Propofol on Calcium sup 2+ Regulation by Malignant Hyperthermia-susceptible Muscle Membranes 
Bradley R. Fruen, PhD; James R. Mickelson, PhD; Timothy J. Roghair, BS; Lynn A. Litterer, BS; Charles F. Louis, PhD
Author Notes
  • (Freun) Research Associate, Department of Veterinary PathoBiology.
  • (Mickelson) Assistant Professor, Department of Veterinary PathoBiology.
  • (Roghair) Junior Scientist, Department of Veterinary PathoBiology.
  • (Litterer) Scientist, Department of Veterinary PathoBiology.
  • (Louis) Professor, Department of Veterinary PathoBiology and the Department of Biochemistry.
  • Received from the Department of Veterinary PathoBiology and the Department of Biochemistry, University of Minnesota, St. Paul, Minnesota. Submitted for publication August 17, 1994. Accepted for publication February 6, 1995. Supported by National Institutes of Health grant GM-31382.
  • Address reprint requests to Dr. Fruen: Department of Veterinary PathoBiology. University of Minnesota. 1988 Fitch Avenue. Room 295, St. Paul, Minnesota 55108.
Article Information
Meeting Abstracts   |   May 1995
Effects of Propofol on Calcium sup 2+ Regulation by Malignant Hyperthermia-susceptible Muscle Membranes 
Anesthesiology 5 1995, Vol.82, 1274-1282. doi:
Anesthesiology 5 1995, Vol.82, 1274-1282. doi:
Key words: Anesthetics, in travenous: propofol. Hyperthermia: malignant. Muscle, skeletal: calcium channels; sarcoplasmic reticulum. Receptors: ryanodine.
Calcium2+ CONCENTRATIONS in skeletal muscle are regulated by channels and pumps located in the transverse tubule and sarcoplasmic reticulum (SR) membranes. During muscle contraction, dihydropyridine receptor (DHPR) Calcium2+ channels in the transverse tubule undergo a conformational change in response to surface membrane depolarization that, in turn, effects the opening of ryanodine receptor (RYR) Calcium2+ channels in the SR. [1,2 ] Calcium2+ released to the myoplasm through RYR channels is subsequently resequestered to the SR by the action of the Calcium2+-adenosine triphosphatase (Calcium2+-ATPase) pump, allowing the relaxation of muscle.
There is now general agreement that skeletal muscle Calcium sup 2+ regulation is altered in the pharmacogenetic disease malignant hyperthermia (MH). [3 ] On exposure to inhalation anesthetics, MH- susceptible patients exhibit increased myoplasmic Calcium2+, muscle contracture, and accelerated metabolism. [3 ] Linkage of MH to mutations in the gene encoding the skeletal muscle RYR in a porcine model of MH [4 ] and in certain human families [5,6 ] has provided further evidence that defects in skeletal muscle Calcium2+ regulation are responsible for MH susceptibility. Nevertheless, the mechanisms by which certain anesthetics act to trigger an MH episode in susceptible patients are less clear.
Previous biochemical studies indicate that the interaction of anesthetics with the SR RYR may play an important role in triggering MH. [7,9 ] For example, we have shown that clinical concentrations of halothane, isoflurane, and enflurane increased the rate of RYR-mediated Calcium2+ efflux from porcine SR. [8 ] This effect of inhalation anesthetics of RYR activity was more pronounced on MH-susceptible than on normal muscle membrane preparations, consistent with an inherent defect in the RYR channel protein in porcine MH. [8 ] In contrast, the effects of inhalation anesthetics of DHPR and Calcium2+-ATPase function cannot explain the ability of these agents to increase myoplasmic Calcium2+ and trigger MH. Thus, dihydropyridine binding to MH-susceptible and normal muscle membranes was inhibited by clinical concentrations of inhalation anesthetics, [10 ] whereas Calcium2+ uptake by MH-susceptible and normal preparations was stimulated by clinical concentrations of these agents and inhibited by greater concentrations. [8 ] Existing evidence therefore points to the activation of RYR Calcium2+ channels being a primary mechanism by which inhalation anesthetics act to trigger an MH episode.
The intravenous anesthetic propofol appears to be a safe, nontriggering alternative to the inhalation anesthetics in the management of the MH-susceptible patient. Clinical reports indicate that MH-susceptible patients undergoing propofol anesthesia showed no signs of an MH response. [11–13 ] Similarly, propofol did not induce contractures during in vitro testing of MH-susceptible muscle [13,14 ] or trigger an MH episode in susceptible pigs. [15–17 ] To understand better how the effects of propofol on Calcium2+ regulation by MH- susceptible muscle may differ from the effects of MH-triggering inhalation anesthetics, we have investigated the effects of propofol on MH-susceptible and normal RYR, DHPR, and Calcium2+-ATPase function. Our results reveal differences between the effect of propofol and the previously described effects of inhalation anesthetics on RYR function that support the hypothesis that the activation of RYR Calcium sup 2+ channels by inhalation anesthetics is a primary mechanism for triggering MH in pigs and some susceptible patients.
Materials and Methods
Animals and Drugs
Pigs (30–45 kg) were obtained from the University of Minnesota Experimental Farm where they are part of a swine herd maintained for studies of the inheritance of MH susceptibility. The two breeds of pig used in this study were a Pietrain herd that is homozygous for the MH-susceptibility mutation on the basis of its ryr-l genotype (cytosine at base pair 1843 determined by the polymerase chain reaction diagnostic procedure of Fujii et al. [4 ]), and a Yorkshire herd that is homozygous normal (thymidine at base pair 1843). Animals were killed by intravenous administration of Sodium thiamylal (15 mg/ml; Parke- Davis, Morris Plains, NJ) followed by euthanasia solution (Schering- Plough, Kenilworth, NJ), in adherence with a protocol approved by the Institutional Animal Care Committee of the University of Minnesota. Pure propofol was generously provided by Zeneca Pharmaceuticals (Wilmington, DE). Propofol was stored in the dark, at 22 degrees Celsius, under Nitrogen2, in Teflon-sealed Reacti-vials (Pierce Chemical, Rockford, IL). Propofol stock solutions were prepared fresh daily in dimethyl sulfoxide. Concentrations of the dimethyl sulfoxide solvent were held constant through all treatment at less or equal to 2%. These concentrations of dimethyl sulfoxide had no significant effect on Calcium2+ flux or radioligand binding. [15 ] Calcium2+ and [sup 3 Hydrogen]ryanodine were purchased from du Pont-New England Nuclear (Boston, MA). [sup 3 Hydrogen]PN200–110 was purchased from Amersham (Arlington Heights, IL).
Membrane Preparation
SR vesicle preparations that are enriched in the junctional membrane proteins of the SR and the transverse tubule [18 ] were prepared from the longissimus dorsi muscle. In brief, the 2,600 g supernatant of a muscle homogenate was centrifuged at 10,000 g for 30 min. The resulting pellet was resuspended in 0.6 M KCI, 10% sucrose, 10 mM Tris-2-(N-morpholino)-ethanesulfonic acid (pH 6.8), incubated on ice for 1 h, and centrifuged at 100,000 g. The pellets were resuspended in 10% sucrose, 0.4 M KCI, and 10 mM piperazine-N,N'-bis (2-ethanesulfonic acid)(PIPES)(pH 6.8) and placed on discontinuous sucrose density gradients containing 0.4 M KCI and 10 mM PIPES, pH 6.8, in all layers. Membranes that banded at the 36–45% interface after 5 h centrifugation at 85,000 g were collected, frozen in liquid Nitrogen2, and stored at - 70 degrees Celsius. All isolation buffers contained 0.1 mM phenylmethylsulfonyl fluoride, 1 micro gram/ml leupeptin, and 1 micro gram/ml aprotinin to inhibit proteolysis.
sup 45 Calcium sup 2+ Efflux
SR vesicles (10–15 mg protein/ml) were passively loaded with sup 15 Calcium2+ to a concentration of 20–40 nmol/mg by incubation for 2 h at 22 degrees Celsius in 0.1 M KCI, 10 mM PIPES (adjusted to pH 6.5 with KOH) and 2 mM CaCl2(containing 0.1 mCi/ml [45]CaCl sub 2).45Calcium2+-loaded vesicles (2 micro liter) were placed on the side of a polystyrene tube containing 200 micro liter of Calcium2+ release medium (100 mM KCI, 10 mM PIPES, pH 6.5, and a CaCl2-ethyleneglycol-bis-(beta-aminoethyl ether) N,N,N',N'- tetraacetic acid (EGTA) buffer set to give 6 micro Meter ionized Calcium sup 2+). Non-specific efflux was measured in a release medium containing 10 mM EGTA, 10 mM MgCl2, and 10 micro Meter ruthenium red. Calcium sup 2+ efflux was initiated with rapid mixing and stopped after 1 s (timed with a metronome) by the addition of 20 micro liter of a Calcium sup 2+ release-inhibiting medium (10 mM MgCl2, 10 mM EGTA, 100 mM KCI, 10 micro Meter ruthenium red, and 10 mM PIPES, pH 6.5), followed by rapid filtration onto 0.45-micro meter filters (Millipore, Bedford, MA) and washing with ice-cold release-inhibiting medium. Experiments were performed at pH 6.5 to slow the rate of Calcium2+-induced Calcium sup 2+ efflux, allowing for accurate measurements of first-order rate constants [19 ] in the presence of micromolar Calcium2+.
Radioligand Binding
[sup 3 Hydrogen]Ryanodine binding was measured at equilibrium essentially as described previously [20 ] in media containing 0.2 mg SR protein/ml, 0.1 M KCl, 10 mM PIPES (adjusted to pH 7.0 with Tris), 0.5 mM phenylmethylsulfonyl fluoride, 100 nM [sup 3 Hydrogen]ryanodine, and a CaCl2-EGTA buffer set to give 6 micro Meter ionized Calcium sup 2+. After 16-h incubations at 22 degrees Celsius, membranes were filtered (GF/B filters, Whatman, Hillsboro, OR) and washed with 8 ml ice-cold 0.1 M KCI, 10 mM PIPES buffer (pH 7.0). Nonspecific binding was measured in the presence of 20 micro Meter nonradioactive ryanodine and was unaffected by propofol (1–300 micro Meter).
[sup 3 Hydrogen]PN200–110 binding was measured at equilibrium in media containing 10 micro gram SR membrane protein/ml, 50 mM Tris-Cl buffer (pH 7.4), and 0.2 or 2.5 nM [sup 3 Hydrogen]PN200–110, as described. [10 ] After 45-min incubations at 22 degrees Celsius, vesicles were filtered onto filters (GF/B, Whatman) that had been pretreated for 20 min with 0.5% polyethyleneimine. Filters were washed with 20 ml ice-cold 200 mM choline chloride, 20 mM Tris-Cl buffer (pH 7.4). Nonspecific binding was measured in the presence of 1 micro Meter nifedipine.
Calcium sup 2+-Adenosine Triphosphatase
Membranes (25 micro gram protein/ml) were incubated at 22 degrees Celsius in media containing 100 mM KCI, 10 mM PIPES (pH 7.0), 10 micro Meter of the Calcium2+ ionophore A-23187, and 5 mM Magnesium adenosine triphosphate. Calcium2+-ATPase activity was defined as the difference between phosphate liberation in the presence of 100 micro Meter CaCl2and that in the presence of 1 mM EGTA.45Calcium sup 2+ EGTA, uptake was measured in media containing 20 micro gram SR membrane protein/ml, 100 mM KCI, 10 mM PIPES (pH 7.0), 50 mM phosphate, 5 mM Magnesium adenosine triphosphate, 100 micro Meter45CaCl, and 100 micro Meter ruthenium red (to inhibit Calcium2+ efflux through RYR channels [8 ]). After 2-min incubations at 22 degrees Celsius, samples were filtered through 0.45-micro Meter membranes (HA, Millipore) and washed with ice-cold KCI/PIPES buffer (pH 7.0). Data were corrected for radio-activity accumulated in the absence of Magnesium adenosine triphosphate.
Analysis of Data
All enzyme activity and ligand binding measurements on each membrane preparation were performed in duplicate and duplicate observations were averaged. Reported values are means obtained from three or four MH-susceptible or three or four normal pigs. Means over all propofol concentrations were analyzed using a one-way analysis of variance. When appropriate, Fisher's least-significant difference test was used to compare effects of individual propofol concentrations with controls (no propofol) to determine the threshold concentration at which propofol produced a significant effect (P < 0.05). We have previously demonstrated that the MH-susceptible and normal membrane preparations used in this study do not significantly differ in regard to PN200–110 binding and Calcium2+-ATPAse activity. [8,10,18 ] Therefore, analyses of the effect of propofol on [sup 3 Hydrogen]PN200–110 binding and Calcium2+-ATPase function are presented as percentages of control activities in the absence of propofol, to emphasize the similarity of effects on MH-susceptible and normal muscle membranes.
Results
Effect of Propofol on Ryanodine Receptor Activity
The effect of propofol on Calcium2+ efflux from45Calcium2+-filled SR vesicles was determined in conditions at which RYR-mediated efflux was maximally activated by micromolar Calcium2+ and in conditions at which RYR-mediated efflux was inhibited by EGTA, Magnesium2+, and ruthenium red. In this way, effects of propofol on Calcium2+ efflux could be attributed to effects on RYR channel activity or to effects on other, undefined factors contributing to membrane vesicle Calcium2+ permeability (termed nonspecific efflux).
Rate constants of nonspecific Calcium2+ efflux in the presence of RYR inhibitors were similar for MH-susceptible (Figure 1(A)) and normal SR vesicles (Figure 1(B)). In contrast, rate constants of Calcium2+ efflux in the presence of 6 micro Meter Calcium2+ were significantly greater for MH-susceptible (Figure 1(A)) than for normal vesicles (Figure 1(B)), consistent with the specific defect in RYR-mediated Calcium2+ efflux in porcine MH, [20,21 ] Propofol concentrations to 200 micro Meter had no significant effect on Calcium2+ efflux rate constants from either MH- susceptible or normal vesicles. The greatest propofol concentration examined (500 micro Meter) significantly increased the rate constants of nonspecific Calcium2+ efflux (1.1 S sup -1 increase for MH- susceptible vesicles, 0.9 s sup -1 increase for normal vesicles). Rate constants of Calcium2+ efflux in the presence of 6 micro Meter Calcium2+ were increased by the same magnitude (1.1 s sup -1 increase for MH-susceptible vesicles, 0.8 s sup -1 increase for normal vesicles), so that this increase in Calcium2+ efflux rate could be attributed entirely to the effect of high propofol concentrations on nonspecific Calcium2+ efflux. Thus, correcting the data for the effect of propofol on nonspecific Calcium2+ efflux (Figure 1, insets) showed that RYR-mediated Calcium2+ efflux was not significantly affected by any concentration of propofol.
Figure 1. Effect of propofol on the rate constant of Calcium2+ efflux from sarcoplasmic reticulum vesicles. Malignant hyperthermia (MH)-susceptible (A) and normal (B) vesicles were loaded with Calcium sup 2+, Calcium2+ efflux was measured after dilution into a medium containing various concentrations of propofol and 6 micro Meter Calcium sup 2+(filled circles) to activate ryanodine receptor (RYR)-mediated Calcium2+ efflux, or 10 mM EGTA, 10 mM MgCl2, 10 micro Meter ruthenium red (open circles) to inhibit RYR-mediated efflux. (Insets) Difference between Calcium2+ efflux in the presence of 6 micro Meter Calcium2+ and in the presence of RYR channel inhibitors. Points are means plus/minus SE of duplicate measurements from four MH- susceptible and four normal preparations.
Figure 1. Effect of propofol on the rate constant of Calcium2+ efflux from sarcoplasmic reticulum vesicles. Malignant hyperthermia (MH)-susceptible (A) and normal (B) vesicles were loaded with Calcium sup 2+, Calcium2+ efflux was measured after dilution into a medium containing various concentrations of propofol and 6 micro Meter Calcium sup 2+(filled circles) to activate ryanodine receptor (RYR)-mediated Calcium2+ efflux, or 10 mM EGTA, 10 mM MgCl2, 10 micro Meter ruthenium red (open circles) to inhibit RYR-mediated efflux. (Insets) Difference between Calcium2+ efflux in the presence of 6 micro Meter Calcium2+ and in the presence of RYR channel inhibitors. Points are means plus/minus SE of duplicate measurements from four MH- susceptible and four normal preparations.
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Figure 1. Effect of propofol on the rate constant of Calcium2+ efflux from sarcoplasmic reticulum vesicles. Malignant hyperthermia (MH)-susceptible (A) and normal (B) vesicles were loaded with Calcium sup 2+, Calcium2+ efflux was measured after dilution into a medium containing various concentrations of propofol and 6 micro Meter Calcium sup 2+(filled circles) to activate ryanodine receptor (RYR)-mediated Calcium2+ efflux, or 10 mM EGTA, 10 mM MgCl2, 10 micro Meter ruthenium red (open circles) to inhibit RYR-mediated efflux. (Insets) Difference between Calcium2+ efflux in the presence of 6 micro Meter Calcium2+ and in the presence of RYR channel inhibitors. Points are means plus/minus SE of duplicate measurements from four MH- susceptible and four normal preparations.
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RYR activity was directly monitored using [sup 3 Hydrogen]- ryanodine as a specific ligand for the open state of the RYR channel. [22 ] In the presence of 6 micro Meter Calcium2+, MH-susceptible SR vesicle preparations bound significantly more [sup 3 Hydrogen]ryanodine than did normal preparations (Figure 2). Consistent with our Calcium2+ efflux results, propofol concentrations of 1- 100 micro Meter had no effect on [sup 3 Hydrogen]ryanodine binding to MH-susceptible or normal muscle SR vesicles. Concentrations of propofol greater than 100 micro Meter had a small inhibitory effect on [sup 3 Hydrogen]ryanodine binding to MH-susceptible and normal membranes (p < 0.03, in the presence of 300 micro Meter propofol).
Figure 2. Effect of propofol on [sup 3 Hydrogen]ryanodine binding to sarcoplasmic reticulum vesicles. [sup 3 Hydrogen]Ryanodine binding to malignant hyperthermia (MH)-susceptible (filled circles) and normal (open circles) preparations was measured at 22 degrees Celsius, pH 7.0, in the presence of 6 micro Meter Calcium2+. Data are means plus/minus SE of duplicate measurements from four MH-susceptible and four normal preparations.
Figure 2. Effect of propofol on [sup 3 Hydrogen]ryanodine binding to sarcoplasmic reticulum vesicles. [sup 3 Hydrogen]Ryanodine binding to malignant hyperthermia (MH)-susceptible (filled circles) and normal (open circles) preparations was measured at 22 degrees Celsius, pH 7.0, in the presence of 6 micro Meter Calcium2+. Data are means plus/minus SE of duplicate measurements from four MH-susceptible and four normal preparations.
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Figure 2. Effect of propofol on [sup 3 Hydrogen]ryanodine binding to sarcoplasmic reticulum vesicles. [sup 3 Hydrogen]Ryanodine binding to malignant hyperthermia (MH)-susceptible (filled circles) and normal (open circles) preparations was measured at 22 degrees Celsius, pH 7.0, in the presence of 6 micro Meter Calcium2+. Data are means plus/minus SE of duplicate measurements from four MH-susceptible and four normal preparations.
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Effect of Propofol on Dihydropyridine Receptor Activity
To investigate the effect of propofol on DHPR function, the binding of the dihydropyridine Calcium2+ channel blocker PN 200- 110 to MH-susceptible and normal SR vesicle preparations was examined. [sup 3 Hydrogen]PN 200–110 binding was measured in the presence of 0.2 nM (Figure 3(A)) and 2.5 nM (Figure 3(B)) PN 200–110. These concentrations were chosen to approximate 1x and 10x the dissociation constant of PN200–110 binding to porcine skeletal muscle membranes [10 ] so that an effect of propofol on either the dissociation constant or maximal PN200–110 binding capacity of our membrane preparations would be detectable. At both of the PN200–110 concentrations examined, propofol inhibited [sup 3 Hydrogen]PN200–110 binding to MH-susceptible and normal preparations with a similar concentration dependence. The concentration of propofol producing 50% inhibition of [sup 3 Hydrogen]PN200–110 binding was 53 micro Meter in the presence of 0.2 mM PN200–110, and 100 micro Meter in the presence of 2.5 nM PN 200–110 (as calculated by Hill plots); 300 micro Meter propofol completely inhibited [sup 3 Hydrogen]PN200–110 binding to SR vesicles.
Figure 3. Effect of propofol on [sup 3 Hydrogen]PN200–110 binding to sarcoplasmic reticulum vesicles. [sup 3 Hydrogen]PN200–110 binding to malignant hyperthermia (MH)-susceptible (filled circles) and normal (open circles) preparations was determined at 22 degrees Celsius, pH 7.4, in the presence of 0.2 nM (A) or 2.5 nM (B) PN200–110. Measurements of control [sup 3 Hydrogen]PN200–110 binding in the absence of propofol were 3.09 plus/minus 0.19 pmol/mg (A) and 6.64 plus/minus 0.57 pmol/mg (B). Data are means plus/minus SE of duplicate measurements from three MH-susceptible and three normal preparations.
Figure 3. Effect of propofol on [sup 3 Hydrogen]PN200–110 binding to sarcoplasmic reticulum vesicles. [sup 3 Hydrogen]PN200–110 binding to malignant hyperthermia (MH)-susceptible (filled circles) and normal (open circles) preparations was determined at 22 degrees Celsius, pH 7.4, in the presence of 0.2 nM (A) or 2.5 nM (B) PN200–110. Measurements of control [sup 3 Hydrogen]PN200–110 binding in the absence of propofol were 3.09 plus/minus 0.19 pmol/mg (A) and 6.64 plus/minus 0.57 pmol/mg (B). Data are means plus/minus SE of duplicate measurements from three MH-susceptible and three normal preparations.
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Figure 3. Effect of propofol on [sup 3 Hydrogen]PN200–110 binding to sarcoplasmic reticulum vesicles. [sup 3 Hydrogen]PN200–110 binding to malignant hyperthermia (MH)-susceptible (filled circles) and normal (open circles) preparations was determined at 22 degrees Celsius, pH 7.4, in the presence of 0.2 nM (A) or 2.5 nM (B) PN200–110. Measurements of control [sup 3 Hydrogen]PN200–110 binding in the absence of propofol were 3.09 plus/minus 0.19 pmol/mg (A) and 6.64 plus/minus 0.57 pmol/mg (B). Data are means plus/minus SE of duplicate measurements from three MH-susceptible and three normal preparations.
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Effect of Propofol on Calcium sup 2+-Adenosine Triphosphatase Activity
The effect of propofol on the Calcium2+. ATPase activity of MH-susceptible and normal SR vesicle preparations was biphasic (Figure 4(A)). Calcium2+-ATPase activity was stimulated by 10- 100 micro Meter propofol with maximal stimulation at 50 micro Meter propofol. Stimulation of MH-susceptible and normal preparations did not differ (178 plus/minus 21% vs. 183 plus/minus 16%, respectively, in the presence of 50 micro Meter propofol). Stimulation of Calcium2+- ATPase activity was progressively decreased at propofol concentrations greater than 100 micro Meter, and in the presence of 300 micro Meter propofol was decreased to 75% of control activity in the absence of propofol.
Figure 4. (A) Effect of propofol on the Calcium2+-adenosine triphosphatase (Calcium2+-ATPase) activity of sarcoplasmic reticulum vesicles. The Calcium2+-ATPase activity of malignant hyperthermia (MH)-susceptible (filled circles) and normal (open circles) preparations was measured at 22 degrees Celsius, pH 7.0. (B) Effect of propofol on [45]Calcium2+ uptake by skeletal muscle membrane vesicles. ATP-dependent Calcium2+ uptake by MH-susceptible and normal preparations was measured at 22 degrees Celsius, pH 7.0, in the presence of 100 micro Meter ruthenium red. Control activities in the absence of propofol were 0.28 plus/minus 0.03 micro mol inorganic phosphate *symbol* mg protein1*symbol* min1(A) and 0.59 plus/minus 0.07 micro mol Calcium2+*symbol* mg protein1*symbol* min1(B). All points represent means plus/minus SE of duplicate measurements from three MH-susceptible and three normal preparations.
Figure 4. (A) Effect of propofol on the Calcium2+-adenosine triphosphatase (Calcium2+-ATPase) activity of sarcoplasmic reticulum vesicles. The Calcium2+-ATPase activity of malignant hyperthermia (MH)-susceptible (filled circles) and normal (open circles) preparations was measured at 22 degrees Celsius, pH 7.0. (B) Effect of propofol on [45]Calcium2+ uptake by skeletal muscle membrane vesicles. ATP-dependent Calcium2+ uptake by MH-susceptible and normal preparations was measured at 22 degrees Celsius, pH 7.0, in the presence of 100 micro Meter ruthenium red. Control activities in the absence of propofol were 0.28 plus/minus 0.03 micro mol inorganic phosphate *symbol* mg protein1*symbol* min1(A) and 0.59 plus/minus 0.07 micro mol Calcium2+*symbol* mg protein1*symbol* min1(B). All points represent means plus/minus SE of duplicate measurements from three MH-susceptible and three normal preparations.
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Figure 4. (A) Effect of propofol on the Calcium2+-adenosine triphosphatase (Calcium2+-ATPase) activity of sarcoplasmic reticulum vesicles. The Calcium2+-ATPase activity of malignant hyperthermia (MH)-susceptible (filled circles) and normal (open circles) preparations was measured at 22 degrees Celsius, pH 7.0. (B) Effect of propofol on [45]Calcium2+ uptake by skeletal muscle membrane vesicles. ATP-dependent Calcium2+ uptake by MH-susceptible and normal preparations was measured at 22 degrees Celsius, pH 7.0, in the presence of 100 micro Meter ruthenium red. Control activities in the absence of propofol were 0.28 plus/minus 0.03 micro mol inorganic phosphate *symbol* mg protein1*symbol* min1(A) and 0.59 plus/minus 0.07 micro mol Calcium2+*symbol* mg protein1*symbol* min1(B). All points represent means plus/minus SE of duplicate measurements from three MH-susceptible and three normal preparations.
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To determine whether the effects of propofol on Calcium2+-ATPase activity were reflected by similar effects on Calcium2+ accumulation, we also examined the effect of propofol on [45]Calcium sup 2+ uptake by MH-susceptible and normal SR vesicles. Consistent with the Calcium2+-ATPase results. [15 ] Calcium2+ uptake was maximally stimulated by 50 micro Meter propofol and inhibited by higher concentrations (Figure 4(B)).
Discussion
In the porcine model of MH used in this study, a single mutation in the skeletal muscle RYR (arginine 615 — cysteine 615) is associated with MH susceptibility. [4 ] MH susceptibility in some human families has also been linked to polymorphisms in and near the skeletal muscle RYR gene, [5,6 ] and to date RYR defects remain the only identified molecular cause of MH susceptibility. However, the arginine 615 — cysteine 615 mutation corresponding to the porcine mutation has been identified in only a small number of human families. [23,24 ] In other families, MH susceptibility shows no linkage to RYR markers. [25 ] The underlying genetic basis of human MH is thus more complex than in the porcine model. Nonetheless, investigations of anesthetic effects on skeletal muscle Calcium2+ homeostasis in the presence of this single defined mutation provide insights that will be important in understanding the pathogenesis of MH in families with other mutations as well.
Previous studies have implicated activation of RYR Calcium sup 2+ channels by inhalation anesthetics as a major factor in the pathogenesis of MH. Thus, the rate of Calcium2+ release from skeletal muscle SR vesicles was increased in the presence of clinical concentrations of MH-triggering inhalation anesthetics. [7,8 ] Similarly, inhalation anesthetics increased the open probability of RYR channels in planar lipid bilayers [26–28 ] and stimulated ryanodine binding to SR vesicles. [29 ] We hypothesized that if the triggering of MH is indeed a function of RYR activation by anesthesia, then a nontriggering anesthetic such as propofol should not activate RYR channels. Alternatively, effects on other membrane components controlling myoplasmic Calcium2+ may compensate for effects of this anesthetic on RYR function. We therefore investigated propofol's effects on MH-susceptible and normal RYR, DHPR, and Calcium2+-ATPase function.
Our results demonstrate that propofol did not activate RYR Calcium2+ channels. Thus, RYR-mediated Calcium2+ efflux from MH-susceptible and from normal SR vesicles was unaffected by 10–500 micro Meter propofol (Figure 1, insets). Similarly, ryanodine binding to MH-susceptible and normal SR was unaffected by 1–100 micro Meter propofol (Figure 2). At higher concentrations of propofol (300 micro Meter), ryanodine binding to MH-susceptible and normal SR vesicles was slightly inhibited. Because ryanodine interacts with the open state of RYR channels, [22 ] this inhibition of ryanodine binding by high concentrations of propofol might be taken as evidence of an inhibitory effect on RYR channel activity. However, as these same propofol concentrations had no significant effect on RYR-mediated Calcium2+ efflux (Figure 1, insets), it is unlikely this inhibition of ryanodine binding represents a direct inhibitory action on RYR channel activity. Furthermore, the concentrations of propofol required to inhibit ryanodine binding to our SR vesicle preparations are far higher than relevant clinical concentrations in vivo. For example, blood propofol concentrations necessary to produce unconsciousness in laboratory animals were reported to be in the range of 5–20 micro Meter [30 ]; free propofol concentrations have been estimated to be closer to 1 micro Meter, because a major fraction is bound by plasma proteins. [31 ] The concentration of propofol required to significantly inhibit SR vesicle ryanodine binding (300 micro Meter) is thus more than 100 times clinical concentrations, indicating that RYR channels are relatively insensitive to propofol. In contrast, gamma-aminobutyric acid A chloride channel activity is altered by propofol concentrations less than 1 micro Meter. [32 ].
Propofol (10–300 micro Meter) inhibited the binding of the DHPR ligand PN200–110 to MH-susceptible and normal SR vesicle preparations (Figure 3). The concentrations of propofol required to inhibit PN 200–110 binding (10–30 micro Meter) were an order of magnitude less than those required to inhibit ryanodine binding, indicating that DHPR channels are more sensitive to propofol than RYR channels. That inhibition of PN200–110 binding was similar at saturating and subsaturating PN200–110 concentrations suggests that the major effect of propofol was to reduce the maximal dihydropyridine binding capacity of our SR vesicle preparations. This effect of propofol on PN 200–110 binding is thus comparable to previously described effects of inhalation anesthetics. For example, we have previously shown that halothane, enflurane, and isoflurane (0.1–2.0 mM) also inhibited skeletal muscle membrane PN200–110 binding capacity. [11 ] Similarly. Lamb et al. reported corresponding reductions in DHPR-mediated charge movement and Calcium2+ current in the presence of halothane. [33 ] Although the concentrations of propofol required to significantly inhibit PN200–110 binding may be somewhat greater than clinical concentrations, [31 ] our findings suggest that propofol and the inhalation anesthetics have qualitatively similar effects on skeletal muscle DHPR function.
The Calcium2+-ATPase activity of skeletal muscle SR vesicle preparations was also affected by concentrations of propofol that had no effect on RYR activity. The threshold for stimulation of Calcium2+-ATPase activity was approximately 10 micro Meter propofol for MH-susceptible and for normal preparations, whereas propofol concentrations greater than 200 micro Meter inhibited Calcium sup 2+-ATPase activity (Figure 3). This biphasic effect of propofol on Calcium2+-ATPase activity is also analogous to the effects of inhalation anesthetics. [7,8 ] For example, under experimental conditions equivalent to those used in Figure 4(B), we previously reported that halothane, enflurane, and isoflurane (0.1–0.5 mM) each stimulated Calcium2+ uptake by SR vesicle preparations, whereas higher concentrations of these inhalation anesthetics inhibited Calcium sup 2+ uptake. [8 ] Considering the differences in the structures and physical properties of propofol and the inhalation anesthetics, it is perhaps remarkable that these anesthetics have similar effects on Calcium2+-ATPase and DHPR function. Of interest, a recent report suggests that the stimulation of Calcium2+-ATPase activity by various general anesthetics may reflect the dissociation of oligomeric Calcium2+-ATPase enzyme complexes in the SR membrane. [34 ].
A comparison of our results with those of previous investigations of the effects of propofol on cardiac muscle Calcium2+ regulation suggests that propofol may have similar effects on cardiac and skeletal muscle Calcium2+ channels. For example, Puttick and Terrar [35 ] and Takahashi et al. [36 ] reported that propofol inhibited DHPR-mediated (L-type) Calcium2+ currents in guinea pig ventricular myocytes. The concentrations of propofol that produced approximately 50% inhibition of L-type Calcium2+ currents (50–100 micro Meter), [45,30 ] are equivalent to concentrations that produced 50% inhibition of PN 200–110 binding to skeletal muscle membrane vesicles (Figure 3). These authors also indirectly examined the effect of propofol on cardiac SR function by analyzing Calcium2+- activated tail currents. Their observations indicated that propofol had little or no effect on Calcium2+ release from cardiac SR. [35,37 ] Cook and Housmans reached a similar conclusion based on their observation that exposure to ryanodine did not influence the negative inotropic effect of propofol on isolated ventricular myocardium. [38 ] Thus, cardiac and skeletal muscle RYR Calcium2+ channels may be similarly insensitive to propofol.
In conclusion, the current study demonstrates effects of propofol on Calcium2+ regulation by isolated MH-susceptible and normal muscle membrane preparations that are consistent with this agent's properties as a nontriggering general anesthetic. In particular, the absence of an effect of clinically relevant concentrations of propofol on RYR-mediated Calcium2+ efflux and ryanodine binding contrasts with the stimulatory effects of MH-triggering inhalation anesthetics on RYR channel activity. This difference in the way that propofol and inhalation anesthetics affect RYR channels further supports the view that an anesthetic's effect on RYR channels is a critical factor determining whether it is a trigger of MH. Although current data do not rule out the possibility that propofol may influence RYR channel function in MH patients with other mutations, this seems unlikely in that clinical concentrations of propofol also had no effect on normal RYR function. In addition, the apparent insensitivity of RYR channels to propofol suggests that activation of these intracellular Calcium2+ channels is not a fundamental property of all general anesthetics, but rather reflects a more specific interaction between inhalation anesthetics and RYR channels.
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Figure 1. Effect of propofol on the rate constant of Calcium2+ efflux from sarcoplasmic reticulum vesicles. Malignant hyperthermia (MH)-susceptible (A) and normal (B) vesicles were loaded with Calcium sup 2+, Calcium2+ efflux was measured after dilution into a medium containing various concentrations of propofol and 6 micro Meter Calcium sup 2+(filled circles) to activate ryanodine receptor (RYR)-mediated Calcium2+ efflux, or 10 mM EGTA, 10 mM MgCl2, 10 micro Meter ruthenium red (open circles) to inhibit RYR-mediated efflux. (Insets) Difference between Calcium2+ efflux in the presence of 6 micro Meter Calcium2+ and in the presence of RYR channel inhibitors. Points are means plus/minus SE of duplicate measurements from four MH- susceptible and four normal preparations.
Figure 1. Effect of propofol on the rate constant of Calcium2+ efflux from sarcoplasmic reticulum vesicles. Malignant hyperthermia (MH)-susceptible (A) and normal (B) vesicles were loaded with Calcium sup 2+, Calcium2+ efflux was measured after dilution into a medium containing various concentrations of propofol and 6 micro Meter Calcium sup 2+(filled circles) to activate ryanodine receptor (RYR)-mediated Calcium2+ efflux, or 10 mM EGTA, 10 mM MgCl2, 10 micro Meter ruthenium red (open circles) to inhibit RYR-mediated efflux. (Insets) Difference between Calcium2+ efflux in the presence of 6 micro Meter Calcium2+ and in the presence of RYR channel inhibitors. Points are means plus/minus SE of duplicate measurements from four MH- susceptible and four normal preparations.
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Figure 1. Effect of propofol on the rate constant of Calcium2+ efflux from sarcoplasmic reticulum vesicles. Malignant hyperthermia (MH)-susceptible (A) and normal (B) vesicles were loaded with Calcium sup 2+, Calcium2+ efflux was measured after dilution into a medium containing various concentrations of propofol and 6 micro Meter Calcium sup 2+(filled circles) to activate ryanodine receptor (RYR)-mediated Calcium2+ efflux, or 10 mM EGTA, 10 mM MgCl2, 10 micro Meter ruthenium red (open circles) to inhibit RYR-mediated efflux. (Insets) Difference between Calcium2+ efflux in the presence of 6 micro Meter Calcium2+ and in the presence of RYR channel inhibitors. Points are means plus/minus SE of duplicate measurements from four MH- susceptible and four normal preparations.
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Figure 2. Effect of propofol on [sup 3 Hydrogen]ryanodine binding to sarcoplasmic reticulum vesicles. [sup 3 Hydrogen]Ryanodine binding to malignant hyperthermia (MH)-susceptible (filled circles) and normal (open circles) preparations was measured at 22 degrees Celsius, pH 7.0, in the presence of 6 micro Meter Calcium2+. Data are means plus/minus SE of duplicate measurements from four MH-susceptible and four normal preparations.
Figure 2. Effect of propofol on [sup 3 Hydrogen]ryanodine binding to sarcoplasmic reticulum vesicles. [sup 3 Hydrogen]Ryanodine binding to malignant hyperthermia (MH)-susceptible (filled circles) and normal (open circles) preparations was measured at 22 degrees Celsius, pH 7.0, in the presence of 6 micro Meter Calcium2+. Data are means plus/minus SE of duplicate measurements from four MH-susceptible and four normal preparations.
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Figure 2. Effect of propofol on [sup 3 Hydrogen]ryanodine binding to sarcoplasmic reticulum vesicles. [sup 3 Hydrogen]Ryanodine binding to malignant hyperthermia (MH)-susceptible (filled circles) and normal (open circles) preparations was measured at 22 degrees Celsius, pH 7.0, in the presence of 6 micro Meter Calcium2+. Data are means plus/minus SE of duplicate measurements from four MH-susceptible and four normal preparations.
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Figure 3. Effect of propofol on [sup 3 Hydrogen]PN200–110 binding to sarcoplasmic reticulum vesicles. [sup 3 Hydrogen]PN200–110 binding to malignant hyperthermia (MH)-susceptible (filled circles) and normal (open circles) preparations was determined at 22 degrees Celsius, pH 7.4, in the presence of 0.2 nM (A) or 2.5 nM (B) PN200–110. Measurements of control [sup 3 Hydrogen]PN200–110 binding in the absence of propofol were 3.09 plus/minus 0.19 pmol/mg (A) and 6.64 plus/minus 0.57 pmol/mg (B). Data are means plus/minus SE of duplicate measurements from three MH-susceptible and three normal preparations.
Figure 3. Effect of propofol on [sup 3 Hydrogen]PN200–110 binding to sarcoplasmic reticulum vesicles. [sup 3 Hydrogen]PN200–110 binding to malignant hyperthermia (MH)-susceptible (filled circles) and normal (open circles) preparations was determined at 22 degrees Celsius, pH 7.4, in the presence of 0.2 nM (A) or 2.5 nM (B) PN200–110. Measurements of control [sup 3 Hydrogen]PN200–110 binding in the absence of propofol were 3.09 plus/minus 0.19 pmol/mg (A) and 6.64 plus/minus 0.57 pmol/mg (B). Data are means plus/minus SE of duplicate measurements from three MH-susceptible and three normal preparations.
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Figure 3. Effect of propofol on [sup 3 Hydrogen]PN200–110 binding to sarcoplasmic reticulum vesicles. [sup 3 Hydrogen]PN200–110 binding to malignant hyperthermia (MH)-susceptible (filled circles) and normal (open circles) preparations was determined at 22 degrees Celsius, pH 7.4, in the presence of 0.2 nM (A) or 2.5 nM (B) PN200–110. Measurements of control [sup 3 Hydrogen]PN200–110 binding in the absence of propofol were 3.09 plus/minus 0.19 pmol/mg (A) and 6.64 plus/minus 0.57 pmol/mg (B). Data are means plus/minus SE of duplicate measurements from three MH-susceptible and three normal preparations.
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Figure 4. (A) Effect of propofol on the Calcium2+-adenosine triphosphatase (Calcium2+-ATPase) activity of sarcoplasmic reticulum vesicles. The Calcium2+-ATPase activity of malignant hyperthermia (MH)-susceptible (filled circles) and normal (open circles) preparations was measured at 22 degrees Celsius, pH 7.0. (B) Effect of propofol on [45]Calcium2+ uptake by skeletal muscle membrane vesicles. ATP-dependent Calcium2+ uptake by MH-susceptible and normal preparations was measured at 22 degrees Celsius, pH 7.0, in the presence of 100 micro Meter ruthenium red. Control activities in the absence of propofol were 0.28 plus/minus 0.03 micro mol inorganic phosphate *symbol* mg protein1*symbol* min1(A) and 0.59 plus/minus 0.07 micro mol Calcium2+*symbol* mg protein1*symbol* min1(B). All points represent means plus/minus SE of duplicate measurements from three MH-susceptible and three normal preparations.
Figure 4. (A) Effect of propofol on the Calcium2+-adenosine triphosphatase (Calcium2+-ATPase) activity of sarcoplasmic reticulum vesicles. The Calcium2+-ATPase activity of malignant hyperthermia (MH)-susceptible (filled circles) and normal (open circles) preparations was measured at 22 degrees Celsius, pH 7.0. (B) Effect of propofol on [45]Calcium2+ uptake by skeletal muscle membrane vesicles. ATP-dependent Calcium2+ uptake by MH-susceptible and normal preparations was measured at 22 degrees Celsius, pH 7.0, in the presence of 100 micro Meter ruthenium red. Control activities in the absence of propofol were 0.28 plus/minus 0.03 micro mol inorganic phosphate *symbol* mg protein1*symbol* min1(A) and 0.59 plus/minus 0.07 micro mol Calcium2+*symbol* mg protein1*symbol* min1(B). All points represent means plus/minus SE of duplicate measurements from three MH-susceptible and three normal preparations.
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Figure 4. (A) Effect of propofol on the Calcium2+-adenosine triphosphatase (Calcium2+-ATPase) activity of sarcoplasmic reticulum vesicles. The Calcium2+-ATPase activity of malignant hyperthermia (MH)-susceptible (filled circles) and normal (open circles) preparations was measured at 22 degrees Celsius, pH 7.0. (B) Effect of propofol on [45]Calcium2+ uptake by skeletal muscle membrane vesicles. ATP-dependent Calcium2+ uptake by MH-susceptible and normal preparations was measured at 22 degrees Celsius, pH 7.0, in the presence of 100 micro Meter ruthenium red. Control activities in the absence of propofol were 0.28 plus/minus 0.03 micro mol inorganic phosphate *symbol* mg protein1*symbol* min1(A) and 0.59 plus/minus 0.07 micro mol Calcium2+*symbol* mg protein1*symbol* min1(B). All points represent means plus/minus SE of duplicate measurements from three MH-susceptible and three normal preparations.
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