ARCHIVES
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 281, No. 1, August 15, pp. 36-40,199O
Abnormal Fluidity State in Membranes Hyperthermia Pig Skeletal Muscle
of Malignant
Edmond Rock,*’ Mohammed Sidi Mammar,* Xavier Vignon,* Marie-Antoinette Thomas,? and Jacques Virett *INRA-Theix, Station de Recherches sur la viande, 63122 Ceyrat, France, and TCRSSA, Division Biophysique, B.P. 87,38702 La Tronche, France
Received January
5,1990, and in revised form April
16,199O
The fluidity state was analyzed on sarcoplasmic reticulum .membranes and phospholipid vesicles prepared from normal and malignant hyperthermia susceptible pig muscle. Electron spin resonance studies were performed to determine the fluidity state at the region near the polar headgroups and in the central core of the bilayer using 5-nitroxide (5-NS) and 16-nitroxide stearic acid (16-NS), respectively. With the S-NS label, no differences were found between normal and malignant hyperthermia sarcoplasmic reticulum (MH SR) membranes whereas with the 16-NS label, a significant increase of the activation energy was shown with MH membranes. Lower values of fluorescence anisotropy observed with DPH-labeled MH membranes as compared with normal ones, confirmed the higher abnormal fluidity state of these membranes. The fluidizing effect of halothane, a triggering agent of malignant hyperthermia syndrome, was also studied in these membranes. We show that a relatively low concentration of the drug destabilized not only the diseased sarcoplasmic reticulum membranes but also the vesicles made of total phospholipids extracted from MH skeletal muscle. Together, these findings strongly suggest that an overall increase in membrane fluidity may be implied in the MH disease, improving the general membrane defect hyo 1990 Academic press, IIIC. pothesis for this syndrome.
Porcine malignant hyperthermia (MH)2 is an inherited skeletal muscle disorder characterized by acidosis, rise in body temperature, and muscle contracture (1). r To whom correspondence should be addressed. * Abbreviations used: MH, malignant hyperthermia; SR, sarcoplasmic reticulum; DMSO, dimethyl sulfoxide; ESR, electron spin resonance; PL, phospholipids; DPH, 1,6-diphenyl-1,3,5-hexatriene; 5NS, 2-(3-carboxypropyl)-2-tridecyl-4,4-dimethyl-3-oxazolidinyloxyl; 16NS, 2-(l0-carboxydecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl. 36
This syndrome may be induced by halothane challenge, by a fluorinated anesthetic, or by physical stress such as high temperature, transport, or fighting. Malignant hyperthermia induces a substantial economic loss in the pig industry not only because of the death but also because of production of undesirable, pale, soft, and exudative meat (2). This pharmacogenetic disease has been shown to be the consequence of a sudden rise in myoplasmic free calcium ions (3), suggesting a defect in the regulation of calcium homeostasis and the involvement of muscle cell membranes (4). Among them, MH sarcoplasmic reticulum (SR) membranes were shown to be affected, particularly in the calcium-induced calcium release mechanism (5,6) and in the related calcium release channel (7). On the other hand, halothane is known to alter the physical behavior of synthetic or natural membranes (8, 9). This hydrophobic compound was shown to increase the fluidity state of the membranes and to perturb their permeability and their functional properties (10). In this report, we analyzed the fluidity state of SR membranes and of phospholipid vesicles prepared from normal and MH skeletal muscle, in the presence and in the absence of halothane. Our results indicate an abnormal fluidity state of MH membranes which additionally show a higher sensitivity toward halothane action at the lipid level. These findings strongly suggest that some metabolic actions of lipids can be involved in the MH syndrome and improve the general membrane defect hypothesis (11). MATERIALS
AND
METHODS
Animals. MH susceptibility was assessed by halothane challenge according to the procedure described by Ollivier et al. (12). The animals were slaughtered by electrical stunning and exsanguination. The trapezius muscles were removed and immediately placed in homogenizing cold buffer and the masseter muscles were dissected, frozen in liquid nitrogen, and stored at -80% until use. 0003-9861/90
$3.00
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction
in any form reserved.
MEMBRANE
FLUIDITY
AND
MALIGNANT
37
HYPERTHERMIA
For both spectroscopic studies, halothane (half-diluted in DMSO) was added so that the final concentration of DMSQ never exceeded 0.5% (v/v). The concentration of the anesthetic in the aqueous solution was determined by means of gas-liquid chromatography.
60 58 56 35.4 Y
52
RESULTS
ho
ESR Studies
48 46 AA ..
10
30 20 Temperature (“C)
35
FIG. 1. Effect of halothane on temperature-induced changes of outer hyperfine splitting of 5-NS spin probe in sarcoplasmic reticulum membranes isolated from normal (N) and malignant hyperthermic (MH) skeletal muscle. One hundred microliters of membrane suspension was mixed with 5-NS spin label in the presence or in the absence of 1.65 pmol halothane/mg protein and scanned as described under Materials and Methods. Means & SE for three different preparations.
Isolation of SR membranes. The membranes were prepared from carefully dissected trapezius muscles that were homogenized in 10 vol (w/v) of 20 mM Hepes, 300 mM Saccharose, 1 mM dithiothreitol, 1% fluoride (pH 7.0) bovine serum albumin, 500 pM phenylmethylsulfonyl for 2 x 15 s in a Waring Blendor set at low speed. The homogenate was centrifuged at 8000g for 15 min. The supernatant was filtered through eight layers of cheesecloth and centrifuged at 40,OOOg for 70 min. The pellet was resuspended in 10 mM Hepes, 300 mM Saccharose, (pH 6.8) and was treated with 0.6 M KC1 for 4 h. The suspension was then centrifuged at 40,OOOgfor 70 min and the pellet was washed again in the same buffer. The heavy SR membranes were finally suspended at 20-30 mg/ml in 10 mM Hepes, 150 mM KC1 (pH 6.8). Protein concentration was determined by the Lowry method (13). Lipid extraction. Total lipids were extracted from frozen masseter muscles by the method of Folch et al. (14). An antioxidant, butylated hydroxytoluene, was added at 5 pg/mi. The phospholipids were separated from neutral lipids by chromatography on a silicic acid column. The methanol extract was then evaporated, resuspended in chloroform, and stored at -20°C. Aliquots of phospholipids were dried under nitrogen and dispersed by vigorous shaking in a buffer containing 10 mM Hepes and 150 mM KC1 (pH 6.8). The phospholipid content of these liposomes was determined according to Bartlett (15) with KHzPOl as standard. The same procedure was used to determine the content of phosphore in SR membranes. Electron spin resonance studies. The rotational mobility of the lateral chains of phospholipids was measured using two fatty acid spin labels, N-oxyl-4’,4’-dimethyloxazolidine derivatives of stearic acid (5NS and 16-NS). These labels were added to membrane suspensions from a stock solution in dimethyl sulfoxide (DMSO) at a ratio of about 1 mol per 50 to 100 mol of phospholipids. Under these conditions, the contributions of unbound spin labels were negligible. From ESR spectra obtained with a Varian E-109 or E-3 spectrometer, we determined the outer extrema (2T//, which reflect the order parameter (16)) and the rotation frequency (v’) for 5-NS and 16-NS, respectively. In the latter case, an Arrhenius plot of Y+ allowed us to determine the activation energy (E,). Fluorescence measurements. A SEFAM Fluofluidimeter (INSERM-CNRS) with fixed excitation and emission polarization filter coupled to a microcomputer was used to measure the fluorescence anisotropy. Three microliters of l,&diphenyl-1,3,5-hexatriene (DPH) dissolved in tetrahydrofuran was added to 3 ml of 10 mM Hepes, 150 mM KC1 (pH 6.8) containing 0.2 mg/ml of membrane suspension or 0.1 mg/ml liposomes. The ratio is about 1 mol per 100 mol of phospholipids. This mixture was incubated at 25°C for 1 h and analyzed at the same temperature.
The fluidity state of the lipids was studied at two levels of the bilayer using 5-NS and 16-NS as probes with SR membranes derived from normal and MH pigs. 5-NS probes the membrane bilayer near its polar part. Figure 1 presents the variation of the outer hyperfine splitting (2T//) as a function of temperature. The normal and MH SR membranes were tested in the presence or in the absence of 1.65 pmol halothane/mg of protein. As expected, a rise in temperature induces a decrease of 2T//, indicating a disorganization of the orientation of the lipids resulting from a greater fluidity. However, no significant additional difference has been detected between normal and hyperthermic membranes either in the presence or in the absence of halothane. The hydrophobic domains of these membranes were labeled with the 16-NS spin probe. The rotation frequency (v’) determined in the presence or in the absence of 1.60 pmol halothane/mg protein is shown in Table I. Addition of the anesthetic in normal membranes significantly increases the value of the rotation frequency, indicating a greater mobility of the probe inside the membrane as a consequence of the well-known fluidization effect of halothane (17). With MH membranes the same order of u+ value was observed in the absence of halothane, demonstrating a high level of internal fluidity in these membranes. This fluidity state is not signifi-
TABLE
I
Analysis of Internal Fluidity of SR Membranes from Normal and Malignant Hyperthermic Pig Skeletal Muscle in the Presence and in the Absence of Halothane Parameter
Membrane
-Halothane
v+ (108.s-‘)
SR normal
4.72 kO.24 (1.2%) 7.34 k 1.10
(0.5%)
0.148 -t 0.005 (1.2%) 0.131 + 0.001
(1.8%)
(n=3) SR MH
+Halothane
(n.s.)
7.45 -t 0.29 (ns.) 8.72 ? 1.60
(n=5) Fluorescence anisotropy
SR normal
(n=5) SR MH (n=5)
(2.1%)
0,139 +- 0.005 (4.8%) 0.124 +- 0.003
Note. The rotation frequency (u’) was calculated from ESR spectra of 16-NS probed membranes and the fluorescence anisotropy was determined with DPH-labeled membranes. Experimental conditions were the same as those described in Figs. 1 and 2. Halothane concentration was 1.60 pmol/mg protein. Both parameters were determined at 25 + 1°C (*SE). Number in parenthesis refers to Student’s t statistical significancy (n.s., not significant).
38
ROCK
-0,007 I 0,o
1 , I I I 0,5 l,o 1,s 2,0 Halothane (pmoledmg protein)
1 2,s
FIG. 2. Effect of halothane on the fluorescence anisotropy of DPH in sarcoplasmic reticulum membranes isolated from normal (N) and malignant hyperthermic (MH) skeletal muscle. Experimental conditions: 150 mM KCl, 20 mM Hepes (pH 6.8), 2 MM DPH, 0.2 mg/ml of proteins, 25°C. Each point is the mean + SE for four and five normal and MH SR preparations, respectively.
cantly disturbed by the addition of halothane so that in its presence the MH membranes show the same physical state as halothane-treated normal ones. In the absence of halothane, the E, calculated from an Arrhenius plot of v+ was 20.1 + 1.8 and 26.8 _+ 1.8 kJ . mol-l. deg-’ for normal and malignant hyperthermic SR membranes. In the presence of the anesthetic, there were no significant differences between normal and MH membranes, 23.6 f 1.6 and 24.3 rt 2.0 kJ.mol-‘.deg-l, respectively. Fluorescence Studies Another probe which is known to be localized in the central core of the bilayer, i.e., DPH confirmed the surprising result obtained with the 16-NS probe in the absence of halothane. Indeed, as shown in Table I, the fluorescence anisotropy of DPH, determined in the absence of the drug, is significantly lower in MH membranes, suggesting a higher internal fluidity state. The addition of halothane decreases the fluorescence anisotropy of DPH in normal and MH membranes so that the disordering state of MH membranes is still increased in the presence of the drug. These discrepancies between ESR and fluorescence experiments could result from the better sensitivity of DPH to the detection of the disordering effects of halothane already noted with synthetic vesicles (18) and also from the widespread localization of the probes into the hydrophobic regions of the bilayer. Figure 2 represents the effect of increasing concentrations of halothane on the variation of the fluorescence anisotropy in DPH-labeled SR membranes isolated from normal and MH muscles. Here again, at least for concentrations lower than 1.5 pmol/mg protein, halothane induces a greater disordering effect with MH membranes as compared to normal ones. Moreover, for
ET AL.
the lowest concentration of halothane tested in this study, a paradoxal effect of the drug within these membranes exists. Indeed, MH membranes are already fluidized whereas the normal vesicles show higher anisotropy values than in the absence of halothane. We also observed that DMSO alone increased the fluorescence anisotropy of both kind of membranes. Thus, for 0.01% (v/ v), the maximum concentration of DMSO used in our study, the increase is about 0.004 f 0.001 and 0.002 + 0.002 unit for normal and MH SR membranes, respectively (n = 3). The higher ordering effect of DMSO in normal membranes may account for the increase of DPH fluorescence anisotropy with these membranes. The disordering effect of halothane was also tested with liposomes made of total polar lipids purified from normal and MH masseter muscles. In the absence of halothane, no differences have been detected between normal and MH vesicles (0.079 f 0.008 versus 0.080 _+0.004). However, the addition of increasing concentrations of halothane induces greater fluidity in MH liposomes than in normal ones (Fig. 3). This effect is more pronounced for concentrations above 1.5 pmol halothane/pmol phospholipids. The mean values obtained with lower concentrations of the anesthetic still demonstrate a higher potency of the drug to fluidize MH liposomes although these values may not be significantly different from those observed without any addition or in the presence of DMSO alone. Despite this concentration effect, the disordering occurred at a relatively high amount of the drug within the liposomes as compared with native membranes. Thus for 1 pmol halothane/mg protein, the molar ratio (Ri) is equal to 1.3 since the content of phospholipids was estimated at 0.756 +- 0.043 pmol phospholipid (n = 6). At this ratio, halothane already disturbed the SR membranes (Fig. 2) whereas the liposomes were only slightly affected (Fig. 3). From this stronger increase in the mobility of lipids in native mem-
2
2 B .B 3
MN -MH
0,000
8 -0,005 r 8 $4
g -0,010 B LT.
a -0,0151 0 1
Galothke
(lfi)
5
6
FIG. 3. Effect of halothane on the fluorescence anisotropy of DPH in phospholipid vesicles from normal (N) and malignant hyperthermic (MH) skeletal muscle. Experiments were done with 0.1 mg/ml of liposomes prepared from three different MH and normal pig skeletal muscle. Ri represents the molar ratio of halothane versus phospholipids. Each point is the mean f SE. Other conditions were the same as those in Fig. 2.
MEMBRANE
FLUIDITY
AND
MALIGNANT
HYPERTHERMIA
strongly dependent on the fluidity state and recently it was shown that the endogenous activity in rat liver membranes is increased in fluid membranes (32). Therefore, this observation may explain the increased amount of fatty acids in MH tissue (33). The binding of nitrendipine in MH pig T-tubules was shown to be lower than that of normal ones (30). At the same time, an increase DISCUSSION in temperature from 22 to 37°C known to increase the Halothane is the main triggering agent of MH syn- fluidity state of the membranes, decreased the binding drome in susceptible individuals. However, its action on level of normal membranes with only a slight effect on the cellular membranes is poorly understood. In the MH membranes. In agreement with authors’ interpretapresent study, the use of ESR probes clearly shows that tion, the lower value observed with MH membranes may halothane mainly perturbs the hydrophobic core of the result from a possible defect in the lipid environment of membrane, suggesting that it is confined to the interface the receptor as an increase in its fluidity as depicted in of the two leaflets of the bilayer. The higher fluidity state our study. Last, many studies have described a defect of of this region in native membranes from MH muscles the calcium-induced calcium release mechanism in MH suggests that this area is organized differently then are SR membranes. Particularly, higher rate constants have normal ones. This could be the result of a different com- been shown in hyperthermic membranes (31). For the transport processes, the rate constant is inversely proposition of lipids such as the content of phosphatidylinoportional to lipid microviscosity (for a review see Ref. sit01 or the content of arachidonic acid in phosphatidylserine and phosphatidylethanolamine that have been (34)). Once more, the higher rate constant for MH memshown to be reduced in MH longissimus dorsi muscles branes is in agreement with the higher fluidity state of these membranes. Beside these instances, such a physi(21), but other study shows almost no differences in total lipid contents of the same muscle (22) or of its MH SR cal default in lipid behavior may also account for the membranes (23). These controversial findings may come findings of abnormalities in other cell membrane activifrom the procedures used to extract the lipids and also ties, i.e., the erythrocytes (35) or the lymphocytes (36) from the physiological state of the pig and of the muscle. from MH susceptible individuals, improving the general In our case, postmortem removing of muscles may also membrane defect hypothesis for MH syndrome (11). affect the lipolytic activity leading to a differential lipid Other investigations are being carried out to further composition between MH and normal muscles. How- characterize the event(s) at the origin of this physical ever, studies on cholesterol distribution in muscle sam- anomaly of MH membranes. ples kept in Krebs solution strongly suggested that MH membranes were already affected in situ (data not shown). Thus, other factors affecting the physical pa- REFERENCES 1. Gronert, G. A. (1980) Anesthesiology 63,395-423. rameters of biological membranes, such as lipid peroxidation (24) or the content of gangliosides (25), must be 2. Sybesma, W., and Eikelenboom, G. (1969) Neth. J. Vet. Sci. 2, 155-160. investigated to clarify this point. Whatever the origin of 3. Lopez, J. R., Allen, P. D., Alamo, L., Jones, D., and Sreter, F. this high level of fluidity in MH membranes, it affects (1988) Muscle Nerve 11,82-88. mainly the lipid-protein interactions since liposomes 4. Ellis, F. R., and Heffron, J. J. A. (1985) in Recent Advances in from normal and MH muscle did not have different beAnaesthesia and Analgesia (Atkinson, R. S., and Adams, A. P., haviors in the absence of halothane. However, the Eds.), pp. 173-207, Churchill, London. greater response of MH liposomes to halothane action 5. Nelson, T. E., and Sweo, T. (1988) Anesthesiology 69,571-577. suggests that a default at the lipid-lipid or lipid-water 6. Toshio, O., Endo, M., Nakano, T., Morohoshi, Y., Wakinawa, K., (20) interactions may also exist. and Ohga, A. (1989) Amer. J. Physiol. 266, C358-C367. The fluidity state of biological membranes may modu7. Mickelson, J. R., Gallant, E. M., Litterer, L. A., Johnson, K. M., late the activities of enzymes such as adenylate cyclase Rempel, W. E., and Louis, C. F. (1988) J. Biol. Ch.em. 263,9310in erythrocytes (26) or the Ca’+-ATPase in SR mem9315. branes (27). Recently, it was also shown that disordering 8. Roth, S. H. (1980) Fed. Proc. 39,1595-1599. toward the center of the membrane bilayer was benefi9. Diamond, E. M., and Berman, M. C. (1980) Biochem. Pharmacol. cial on transport activity (28). Related to studies on MH 29,375-381. syndrome, the high level of internal fluidity in MH mem- 10. LaBella, F. S. (1981) Canad. J. Physiol. Phurmacol. 59,432-442. branes observed in this study could account for at least 11. Denborough, M. A. (1980) Pharmucol. Z’her. 9,357-365. three abnormal activities described for these diseased 12. Ollivier, L., Sellier, P., and Monin, G. (1978) Ann. Genet. Sel. membranes. These are PLase A2 activity (29), the dihyAnim. 10,191-208. dropyridine receptor (30), and the calcium-induced cal- 13. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. cium release mechanism (31). The PLase A2 activity is (1951) J. Biol. Chem. 193,265-275.
branes, it was deduced that halothane weakens the lipidprotein interaction (19). Other studies on the interaction of halothane with dipalmitoylphosphatidylcholine liposomes suggest that the anesthetic weakens the water-lipid interaction (20).
40
ROCK ET AL.
14. Folch, J., Lees, M., and Sloane-Stanley, G. H. (1957) J. Biol. Chem. 226,497-509. 15. Bartlett, G. R. (1959) J. Biol. Chem. 234,466-468. 16. Thomas, M. A., Lamas, E., Daveloose, D., Viret, J., Fardeau, M., and Leterrier, F. (1987) Clin. Chim. Acta 164.83-91. 17. Trudell, J. R., Hubbell, W. L., and Cohen, E. N. (1973) B&him. Biophys. Acta 291,321-327. 18. Pang, K. Y., Chang, T. L., andMiller, K. W. (1979) Mol. Phurmncol. 15,729-738. 19. Lenaz, G., Curatola, G., Mazzanti, L., Parenti-Castelli, G., and Bertoli, E. (1978) B&hem. Pharmacol. 27,2835-2844. 20. Ueda, I., Hirakawa, M., Arakawa, K., and Kamaya, H. (1986) Anesthesiology 64,67-71. 21. Eichenger, H. M., Seewald, M. J., and Iaizzo, P. A. (1989) in Proceedings, 35th International Congress of Meat Science and Technology (Copenhagen), pp. 1119-1123. 22. Fletcher, J. E., Rosenberg, H., Michaux, K., Cheah, K. S., and Cheah, A. M. (1988) B&hem. Cell Bid. 66,917-921. 23. Mickelson, J. R., Ross, J. A., Reed, B. K., and Louis, C. F. (1986) B&him. Biophys. Acta 862,318328. 24. Duthie, G. G., Arthur, J. R., Bremner, P., Kikuchi, Y., and Nicvol, F. (1989) Amer. J. Vet. Res. 50,84-87. 25. Harris, R. A., and Groh, G. I. (1985) Anesthesiology 62,115-119.
26. Salesse, R., Garnier, J., Leterrier, F., Daveloose, D., and Viret, J. (1982) Biochemistry21,1581-1586. 21. Bigelow, D. J., and Thomas, D. D. (1987) J. Biol. Chem. 262, 13,449-13,456. 28. Philipson, K. D., and Ward, R. (1987) B&him. Biophys. Actu 987,152-158. 29. Cheah, K. S., and Cheah, A. M. (1984) in Biomembranes (Kates, M., and Monson, L. A. Eds.), Vol. 12, pp. 661-687, Plenum, New York/London. 30. Ervasti, J. M., Claessens, M. T., Mickelson, J. R., and Louis, C. F. (1989) J. Biol. Chem. 264,2711-271’7. 31. Kim, D. H., Sreter, F. A., Ohnishi, S. T., Ryan, J. F., Roberts, J., Allen, P. D., Meszaros, L. G., Antoniu, B., and Ikemoto, N. (1984) Biochim. Biophys. Acta 775,320-327. 32. Koumanov, K. S., and Momchilova-Pankova, A. M. (1989) in Biomembranes et Nutrition (Leger, C. L., and Bereziat, G. Eds.), Vol. 195, pp. 229-237, INSERM, Paris. 33. Fletcher, J. E., and Rosenberg, H. (1986) Brit. J. Anuesth. 68, 1433-1439. 34. Shinitzky, M. (1984) in Physiology of Membrane Fluidity (Shinitzky, M., Ed.), Vol. 1, pp. 39-51, CRC Press, Boca Raton, FL. 35. O’Brien, P. J., Rooney, M. T., Reik, T. R., et al. (1985) Amer. J. Vet. Res. 46,1451-1456. 36. Klip, A., Ramlal, T., Walker, D., et al. (1987) Anesth. An&. 66, 381-385.
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 281, No. 1, August 15, pp. 36-40,199O
Abnormal Fluidity State in Membranes Hyperthermia Pig Skeletal Muscle
of Malignant
Edmond Rock,*’ Mohammed Sidi Mammar,* Xavier Vignon,* Marie-Antoinette Thomas,? and Jacques Virett *INRA-Theix, Station de Recherches sur la viande, 63122 Ceyrat, France, and TCRSSA, Division Biophysique, B.P. 87,38702 La Tronche, France
Received January
5,1990, and in revised form April
16,199O
The fluidity state was analyzed on sarcoplasmic reticulum .membranes and phospholipid vesicles prepared from normal and malignant hyperthermia susceptible pig muscle. Electron spin resonance studies were performed to determine the fluidity state at the region near the polar headgroups and in the central core of the bilayer using 5-nitroxide (5-NS) and 16-nitroxide stearic acid (16-NS), respectively. With the S-NS label, no differences were found between normal and malignant hyperthermia sarcoplasmic reticulum (MH SR) membranes whereas with the 16-NS label, a significant increase of the activation energy was shown with MH membranes. Lower values of fluorescence anisotropy observed with DPH-labeled MH membranes as compared with normal ones, confirmed the higher abnormal fluidity state of these membranes. The fluidizing effect of halothane, a triggering agent of malignant hyperthermia syndrome, was also studied in these membranes. We show that a relatively low concentration of the drug destabilized not only the diseased sarcoplasmic reticulum membranes but also the vesicles made of total phospholipids extracted from MH skeletal muscle. Together, these findings strongly suggest that an overall increase in membrane fluidity may be implied in the MH disease, improving the general membrane defect hyo 1990 Academic press, IIIC. pothesis for this syndrome.
Porcine malignant hyperthermia (MH)2 is an inherited skeletal muscle disorder characterized by acidosis, rise in body temperature, and muscle contracture (1). r To whom correspondence should be addressed. * Abbreviations used: MH, malignant hyperthermia; SR, sarcoplasmic reticulum; DMSO, dimethyl sulfoxide; ESR, electron spin resonance; PL, phospholipids; DPH, 1,6-diphenyl-1,3,5-hexatriene; 5NS, 2-(3-carboxypropyl)-2-tridecyl-4,4-dimethyl-3-oxazolidinyloxyl; 16NS, 2-(l0-carboxydecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl. 36
This syndrome may be induced by halothane challenge, by a fluorinated anesthetic, or by physical stress such as high temperature, transport, or fighting. Malignant hyperthermia induces a substantial economic loss in the pig industry not only because of the death but also because of production of undesirable, pale, soft, and exudative meat (2). This pharmacogenetic disease has been shown to be the consequence of a sudden rise in myoplasmic free calcium ions (3), suggesting a defect in the regulation of calcium homeostasis and the involvement of muscle cell membranes (4). Among them, MH sarcoplasmic reticulum (SR) membranes were shown to be affected, particularly in the calcium-induced calcium release mechanism (5,6) and in the related calcium release channel (7). On the other hand, halothane is known to alter the physical behavior of synthetic or natural membranes (8, 9). This hydrophobic compound was shown to increase the fluidity state of the membranes and to perturb their permeability and their functional properties (10). In this report, we analyzed the fluidity state of SR membranes and of phospholipid vesicles prepared from normal and MH skeletal muscle, in the presence and in the absence of halothane. Our results indicate an abnormal fluidity state of MH membranes which additionally show a higher sensitivity toward halothane action at the lipid level. These findings strongly suggest that some metabolic actions of lipids can be involved in the MH syndrome and improve the general membrane defect hypothesis (11). MATERIALS
AND
METHODS
Animals. MH susceptibility was assessed by halothane challenge according to the procedure described by Ollivier et al. (12). The animals were slaughtered by electrical stunning and exsanguination. The trapezius muscles were removed and immediately placed in homogenizing cold buffer and the masseter muscles were dissected, frozen in liquid nitrogen, and stored at -80% until use. 0003-9861/90
$3.00
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction
in any form reserved.
MEMBRANE
FLUIDITY
AND
MALIGNANT
37
HYPERTHERMIA
For both spectroscopic studies, halothane (half-diluted in DMSO) was added so that the final concentration of DMSQ never exceeded 0.5% (v/v). The concentration of the anesthetic in the aqueous solution was determined by means of gas-liquid chromatography.
60 58 56 35.4 Y
52
RESULTS
ho
ESR Studies
48 46 AA ..
10
30 20 Temperature (“C)
35
FIG. 1. Effect of halothane on temperature-induced changes of outer hyperfine splitting of 5-NS spin probe in sarcoplasmic reticulum membranes isolated from normal (N) and malignant hyperthermic (MH) skeletal muscle. One hundred microliters of membrane suspension was mixed with 5-NS spin label in the presence or in the absence of 1.65 pmol halothane/mg protein and scanned as described under Materials and Methods. Means & SE for three different preparations.
Isolation of SR membranes. The membranes were prepared from carefully dissected trapezius muscles that were homogenized in 10 vol (w/v) of 20 mM Hepes, 300 mM Saccharose, 1 mM dithiothreitol, 1% fluoride (pH 7.0) bovine serum albumin, 500 pM phenylmethylsulfonyl for 2 x 15 s in a Waring Blendor set at low speed. The homogenate was centrifuged at 8000g for 15 min. The supernatant was filtered through eight layers of cheesecloth and centrifuged at 40,OOOg for 70 min. The pellet was resuspended in 10 mM Hepes, 300 mM Saccharose, (pH 6.8) and was treated with 0.6 M KC1 for 4 h. The suspension was then centrifuged at 40,OOOgfor 70 min and the pellet was washed again in the same buffer. The heavy SR membranes were finally suspended at 20-30 mg/ml in 10 mM Hepes, 150 mM KC1 (pH 6.8). Protein concentration was determined by the Lowry method (13). Lipid extraction. Total lipids were extracted from frozen masseter muscles by the method of Folch et al. (14). An antioxidant, butylated hydroxytoluene, was added at 5 pg/mi. The phospholipids were separated from neutral lipids by chromatography on a silicic acid column. The methanol extract was then evaporated, resuspended in chloroform, and stored at -20°C. Aliquots of phospholipids were dried under nitrogen and dispersed by vigorous shaking in a buffer containing 10 mM Hepes and 150 mM KC1 (pH 6.8). The phospholipid content of these liposomes was determined according to Bartlett (15) with KHzPOl as standard. The same procedure was used to determine the content of phosphore in SR membranes. Electron spin resonance studies. The rotational mobility of the lateral chains of phospholipids was measured using two fatty acid spin labels, N-oxyl-4’,4’-dimethyloxazolidine derivatives of stearic acid (5NS and 16-NS). These labels were added to membrane suspensions from a stock solution in dimethyl sulfoxide (DMSO) at a ratio of about 1 mol per 50 to 100 mol of phospholipids. Under these conditions, the contributions of unbound spin labels were negligible. From ESR spectra obtained with a Varian E-109 or E-3 spectrometer, we determined the outer extrema (2T//, which reflect the order parameter (16)) and the rotation frequency (v’) for 5-NS and 16-NS, respectively. In the latter case, an Arrhenius plot of Y+ allowed us to determine the activation energy (E,). Fluorescence measurements. A SEFAM Fluofluidimeter (INSERM-CNRS) with fixed excitation and emission polarization filter coupled to a microcomputer was used to measure the fluorescence anisotropy. Three microliters of l,&diphenyl-1,3,5-hexatriene (DPH) dissolved in tetrahydrofuran was added to 3 ml of 10 mM Hepes, 150 mM KC1 (pH 6.8) containing 0.2 mg/ml of membrane suspension or 0.1 mg/ml liposomes. The ratio is about 1 mol per 100 mol of phospholipids. This mixture was incubated at 25°C for 1 h and analyzed at the same temperature.
The fluidity state of the lipids was studied at two levels of the bilayer using 5-NS and 16-NS as probes with SR membranes derived from normal and MH pigs. 5-NS probes the membrane bilayer near its polar part. Figure 1 presents the variation of the outer hyperfine splitting (2T//) as a function of temperature. The normal and MH SR membranes were tested in the presence or in the absence of 1.65 pmol halothane/mg of protein. As expected, a rise in temperature induces a decrease of 2T//, indicating a disorganization of the orientation of the lipids resulting from a greater fluidity. However, no significant additional difference has been detected between normal and hyperthermic membranes either in the presence or in the absence of halothane. The hydrophobic domains of these membranes were labeled with the 16-NS spin probe. The rotation frequency (v’) determined in the presence or in the absence of 1.60 pmol halothane/mg protein is shown in Table I. Addition of the anesthetic in normal membranes significantly increases the value of the rotation frequency, indicating a greater mobility of the probe inside the membrane as a consequence of the well-known fluidization effect of halothane (17). With MH membranes the same order of u+ value was observed in the absence of halothane, demonstrating a high level of internal fluidity in these membranes. This fluidity state is not signifi-
TABLE
I
Analysis of Internal Fluidity of SR Membranes from Normal and Malignant Hyperthermic Pig Skeletal Muscle in the Presence and in the Absence of Halothane Parameter
Membrane
-Halothane
v+ (108.s-‘)
SR normal
4.72 kO.24 (1.2%) 7.34 k 1.10
(0.5%)
0.148 -t 0.005 (1.2%) 0.131 + 0.001
(1.8%)
(n=3) SR MH
+Halothane
(n.s.)
7.45 -t 0.29 (ns.) 8.72 ? 1.60
(n=5) Fluorescence anisotropy
SR normal
(n=5) SR MH (n=5)
(2.1%)
0,139 +- 0.005 (4.8%) 0.124 +- 0.003
Note. The rotation frequency (u’) was calculated from ESR spectra of 16-NS probed membranes and the fluorescence anisotropy was determined with DPH-labeled membranes. Experimental conditions were the same as those described in Figs. 1 and 2. Halothane concentration was 1.60 pmol/mg protein. Both parameters were determined at 25 + 1°C (*SE). Number in parenthesis refers to Student’s t statistical significancy (n.s., not significant).
38
ROCK
-0,007 I 0,o
1 , I I I 0,5 l,o 1,s 2,0 Halothane (pmoledmg protein)
1 2,s
FIG. 2. Effect of halothane on the fluorescence anisotropy of DPH in sarcoplasmic reticulum membranes isolated from normal (N) and malignant hyperthermic (MH) skeletal muscle. Experimental conditions: 150 mM KCl, 20 mM Hepes (pH 6.8), 2 MM DPH, 0.2 mg/ml of proteins, 25°C. Each point is the mean + SE for four and five normal and MH SR preparations, respectively.
cantly disturbed by the addition of halothane so that in its presence the MH membranes show the same physical state as halothane-treated normal ones. In the absence of halothane, the E, calculated from an Arrhenius plot of v+ was 20.1 + 1.8 and 26.8 _+ 1.8 kJ . mol-l. deg-’ for normal and malignant hyperthermic SR membranes. In the presence of the anesthetic, there were no significant differences between normal and MH membranes, 23.6 f 1.6 and 24.3 rt 2.0 kJ.mol-‘.deg-l, respectively. Fluorescence Studies Another probe which is known to be localized in the central core of the bilayer, i.e., DPH confirmed the surprising result obtained with the 16-NS probe in the absence of halothane. Indeed, as shown in Table I, the fluorescence anisotropy of DPH, determined in the absence of the drug, is significantly lower in MH membranes, suggesting a higher internal fluidity state. The addition of halothane decreases the fluorescence anisotropy of DPH in normal and MH membranes so that the disordering state of MH membranes is still increased in the presence of the drug. These discrepancies between ESR and fluorescence experiments could result from the better sensitivity of DPH to the detection of the disordering effects of halothane already noted with synthetic vesicles (18) and also from the widespread localization of the probes into the hydrophobic regions of the bilayer. Figure 2 represents the effect of increasing concentrations of halothane on the variation of the fluorescence anisotropy in DPH-labeled SR membranes isolated from normal and MH muscles. Here again, at least for concentrations lower than 1.5 pmol/mg protein, halothane induces a greater disordering effect with MH membranes as compared to normal ones. Moreover, for
ET AL.
the lowest concentration of halothane tested in this study, a paradoxal effect of the drug within these membranes exists. Indeed, MH membranes are already fluidized whereas the normal vesicles show higher anisotropy values than in the absence of halothane. We also observed that DMSO alone increased the fluorescence anisotropy of both kind of membranes. Thus, for 0.01% (v/ v), the maximum concentration of DMSO used in our study, the increase is about 0.004 f 0.001 and 0.002 + 0.002 unit for normal and MH SR membranes, respectively (n = 3). The higher ordering effect of DMSO in normal membranes may account for the increase of DPH fluorescence anisotropy with these membranes. The disordering effect of halothane was also tested with liposomes made of total polar lipids purified from normal and MH masseter muscles. In the absence of halothane, no differences have been detected between normal and MH vesicles (0.079 f 0.008 versus 0.080 _+0.004). However, the addition of increasing concentrations of halothane induces greater fluidity in MH liposomes than in normal ones (Fig. 3). This effect is more pronounced for concentrations above 1.5 pmol halothane/pmol phospholipids. The mean values obtained with lower concentrations of the anesthetic still demonstrate a higher potency of the drug to fluidize MH liposomes although these values may not be significantly different from those observed without any addition or in the presence of DMSO alone. Despite this concentration effect, the disordering occurred at a relatively high amount of the drug within the liposomes as compared with native membranes. Thus for 1 pmol halothane/mg protein, the molar ratio (Ri) is equal to 1.3 since the content of phospholipids was estimated at 0.756 +- 0.043 pmol phospholipid (n = 6). At this ratio, halothane already disturbed the SR membranes (Fig. 2) whereas the liposomes were only slightly affected (Fig. 3). From this stronger increase in the mobility of lipids in native mem-
2
2 B .B 3
MN -MH
0,000
8 -0,005 r 8 $4
g -0,010 B LT.
a -0,0151 0 1
Galothke
(lfi)
5
6
FIG. 3. Effect of halothane on the fluorescence anisotropy of DPH in phospholipid vesicles from normal (N) and malignant hyperthermic (MH) skeletal muscle. Experiments were done with 0.1 mg/ml of liposomes prepared from three different MH and normal pig skeletal muscle. Ri represents the molar ratio of halothane versus phospholipids. Each point is the mean f SE. Other conditions were the same as those in Fig. 2.
MEMBRANE
FLUIDITY
AND
MALIGNANT
HYPERTHERMIA
strongly dependent on the fluidity state and recently it was shown that the endogenous activity in rat liver membranes is increased in fluid membranes (32). Therefore, this observation may explain the increased amount of fatty acids in MH tissue (33). The binding of nitrendipine in MH pig T-tubules was shown to be lower than that of normal ones (30). At the same time, an increase DISCUSSION in temperature from 22 to 37°C known to increase the Halothane is the main triggering agent of MH syn- fluidity state of the membranes, decreased the binding drome in susceptible individuals. However, its action on level of normal membranes with only a slight effect on the cellular membranes is poorly understood. In the MH membranes. In agreement with authors’ interpretapresent study, the use of ESR probes clearly shows that tion, the lower value observed with MH membranes may halothane mainly perturbs the hydrophobic core of the result from a possible defect in the lipid environment of membrane, suggesting that it is confined to the interface the receptor as an increase in its fluidity as depicted in of the two leaflets of the bilayer. The higher fluidity state our study. Last, many studies have described a defect of of this region in native membranes from MH muscles the calcium-induced calcium release mechanism in MH suggests that this area is organized differently then are SR membranes. Particularly, higher rate constants have normal ones. This could be the result of a different com- been shown in hyperthermic membranes (31). For the transport processes, the rate constant is inversely proposition of lipids such as the content of phosphatidylinoportional to lipid microviscosity (for a review see Ref. sit01 or the content of arachidonic acid in phosphatidylserine and phosphatidylethanolamine that have been (34)). Once more, the higher rate constant for MH memshown to be reduced in MH longissimus dorsi muscles branes is in agreement with the higher fluidity state of these membranes. Beside these instances, such a physi(21), but other study shows almost no differences in total lipid contents of the same muscle (22) or of its MH SR cal default in lipid behavior may also account for the membranes (23). These controversial findings may come findings of abnormalities in other cell membrane activifrom the procedures used to extract the lipids and also ties, i.e., the erythrocytes (35) or the lymphocytes (36) from the physiological state of the pig and of the muscle. from MH susceptible individuals, improving the general In our case, postmortem removing of muscles may also membrane defect hypothesis for MH syndrome (11). affect the lipolytic activity leading to a differential lipid Other investigations are being carried out to further composition between MH and normal muscles. How- characterize the event(s) at the origin of this physical ever, studies on cholesterol distribution in muscle sam- anomaly of MH membranes. ples kept in Krebs solution strongly suggested that MH membranes were already affected in situ (data not shown). Thus, other factors affecting the physical pa- REFERENCES 1. Gronert, G. A. (1980) Anesthesiology 63,395-423. rameters of biological membranes, such as lipid peroxidation (24) or the content of gangliosides (25), must be 2. Sybesma, W., and Eikelenboom, G. (1969) Neth. J. Vet. Sci. 2, 155-160. investigated to clarify this point. Whatever the origin of 3. Lopez, J. R., Allen, P. D., Alamo, L., Jones, D., and Sreter, F. this high level of fluidity in MH membranes, it affects (1988) Muscle Nerve 11,82-88. mainly the lipid-protein interactions since liposomes 4. Ellis, F. R., and Heffron, J. J. A. (1985) in Recent Advances in from normal and MH muscle did not have different beAnaesthesia and Analgesia (Atkinson, R. S., and Adams, A. P., haviors in the absence of halothane. However, the Eds.), pp. 173-207, Churchill, London. greater response of MH liposomes to halothane action 5. Nelson, T. E., and Sweo, T. (1988) Anesthesiology 69,571-577. suggests that a default at the lipid-lipid or lipid-water 6. Toshio, O., Endo, M., Nakano, T., Morohoshi, Y., Wakinawa, K., (20) interactions may also exist. and Ohga, A. (1989) Amer. J. Physiol. 266, C358-C367. The fluidity state of biological membranes may modu7. Mickelson, J. R., Gallant, E. M., Litterer, L. A., Johnson, K. M., late the activities of enzymes such as adenylate cyclase Rempel, W. E., and Louis, C. F. (1988) J. Biol. Ch.em. 263,9310in erythrocytes (26) or the Ca’+-ATPase in SR mem9315. branes (27). Recently, it was also shown that disordering 8. Roth, S. H. (1980) Fed. Proc. 39,1595-1599. toward the center of the membrane bilayer was benefi9. Diamond, E. M., and Berman, M. C. (1980) Biochem. Pharmacol. cial on transport activity (28). Related to studies on MH 29,375-381. syndrome, the high level of internal fluidity in MH mem- 10. LaBella, F. S. (1981) Canad. J. Physiol. Phurmacol. 59,432-442. branes observed in this study could account for at least 11. Denborough, M. A. (1980) Pharmucol. 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40
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26. Salesse, R., Garnier, J., Leterrier, F., Daveloose, D., and Viret, J. (1982) Biochemistry21,1581-1586. 21. Bigelow, D. J., and Thomas, D. D. (1987) J. Biol. Chem. 262, 13,449-13,456. 28. Philipson, K. D., and Ward, R. (1987) B&him. Biophys. Actu 987,152-158. 29. Cheah, K. S., and Cheah, A. M. (1984) in Biomembranes (Kates, M., and Monson, L. A. Eds.), Vol. 12, pp. 661-687, Plenum, New York/London. 30. Ervasti, J. M., Claessens, M. T., Mickelson, J. R., and Louis, C. F. (1989) J. Biol. Chem. 264,2711-271’7. 31. Kim, D. H., Sreter, F. A., Ohnishi, S. T., Ryan, J. F., Roberts, J., Allen, P. D., Meszaros, L. G., Antoniu, B., and Ikemoto, N. (1984) Biochim. Biophys. Acta 775,320-327. 32. Koumanov, K. S., and Momchilova-Pankova, A. M. (1989) in Biomembranes et Nutrition (Leger, C. L., and Bereziat, G. Eds.), Vol. 195, pp. 229-237, INSERM, Paris. 33. Fletcher, J. E., and Rosenberg, H. (1986) Brit. J. Anuesth. 68, 1433-1439. 34. Shinitzky, M. (1984) in Physiology of Membrane Fluidity (Shinitzky, M., Ed.), Vol. 1, pp. 39-51, CRC Press, Boca Raton, FL. 35. O’Brien, P. J., Rooney, M. T., Reik, T. R., et al. (1985) Amer. J. Vet. Res. 46,1451-1456. 36. Klip, A., Ramlal, T., Walker, D., et al. (1987) Anesth. An&. 66, 381-385.
