Alcohol, Vol.9, pp. 513-517, 1992
0741-8329/92 $5.00 + .00 Copyright©1992PergamonPress Ltd.
Printed in the U.S.A. All rightsreserved.
Opposite Effects of Dimethyl Sulfoxide and Ethanol on Synaptic Membrane Fluidity R O B E R T E. M R A K
Department o f Pathology, John L. McClellan Memorial Veterans" Hospital and University o f Arkansas for Medical Sciences, Little Rock, A R 72205 Received 27 M a r c h 1992; A c c e p t e d 10 J u n e 1992
MRAK, R. E. Oppositeeffects of dimethyl sulfoxide and ethanol on synaptic membranefluidity. ALCOHOL 9(6) 513517, 1992.-Dimethyl sulfoxide (DMSO) is an organic solvent with myriad biological actions, including actions on synaptic membrane transport processes. In this study, fluorescence polarizations of the probes diphenylhexatriene (DPH: a probe of the hydrophobic membrane core), trimethylammonium-diphenylhexatriene (a probe of the superficial domain of the cytofacial synaptic membrane leaflet) and diphenylhexatriene propionic acid (a probe of the superficial domain of the exofacial synaptic plasma membrane leaflet) were measured in isolated rat cerebral synaptic plasma membranes. DMSO, added in vitro, increased fluorescence polarization of all of these intramembranous probes, an effect opposite that observed with the addition of ethanol. The fluorescence polarization increase appeared at lower concentrations of DMSO for the superficial membrane region probes (6~e vol/vol DMSO) than for the membrane core probe (10~e vol/vol DMSO). This is again in contrast to the effects of ethanol, which required lesser concentrations to decrease fluorescence polarization of DPH (50 mM ethanol) than that of the derivative probes (200 mM ethanol). The enhancement of DPH fluorescence polarization produced by DMSO was antagonized by the concomitant addition of ethanol. These results suggest an ordering effect of DMSO on synaptic plasma membranes, with greater effects in superficial membrane domains. Fluorescence polarization Synaptic plasma membranes Dimethyl sulfoxide Membrane fluidity Diphenylhexatriene Ethanol Trimethylammonium-diphenylhexatriene Diphenylhexatriene propionic acid
DIMETHYL sulfoxide is an organic solvent whose biological actions range from analgesic and anti-inflammatory actions (32) to induction of differentiation in cultured cells (20) to alterations of membrane transport activities (8,34). In mammalian brain extracts, dimethyl sulfoxide has been shown to inhibit synaptosomal uptake of choline and ~-aminobutyric acid (18) and to enhance (Na+,K+)-ATPase affinity for Na + and for K + (29-31). This latter result is in contrast to the effects of ethanol, which decrease (Na +,K+)-ATPase affinity for Na + and for K + (29,30). Scanning calorimetry investigations of artificial phospholipid bilayers have suggested a membrane site of action for dimethyl sulfoxide, and have further suggested an ordering, or "rigidifying" effect of this agent on such synthetic bilayers (11,12). This proposed action is opposite to the disordering, or "fluidizing" effects of ethanol in synaptic membranes (2,5), and suggests that these two agents might antagonize one another in vitro. There is, however, only one limited investigation of the biophysical effects o f dimethyl sulfoxide on biological membranes (4). This study examines the effects of dimethyl sulfoxide on fluorescence polarization of intramembranous probes in iso-
lated rat cerebral synaptic plasma membranes. The effects of dimethyl sulfoxide on diphenylhexatriene, a probe which preferentiaily localizes to the more-fluid membrane core domain (28), are compared with the effects on the derivative probes trimethylammonium-diphenylhexatriene and diphenylhexatriene propionic acid, probes which preferentially localize to the more superficial domains of the membrane bilayer (21,33). These probes show further specificity in that the cationic trimethylammonium-diphenylhexatriene has a greater affinity for the negatively charged inner (cytofacial) membrane leaflet, while the anionic diphenylhexatriene propionic acid shows greater affinity for the more neutral outer (exofacial) membrane leaflet (25). METHOD
Materials Dimethyl sulfoxide was obtained from Fisher Scientific (Pittsburgh, PA). Fluorescent probes were obtained from Molecular Probes, Inc. (Eugene, OR): 1,6-diphenyl-l,3,5hexatriene (diphenylhexatriene, Cat. No. D-202); 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluene-
Requests for reprints should be addressed to Laboratory Service (113), John L. McClellan Memorial Veterans' Hospital, 4300 West Seventh Street, Little Rock, AR 72205. 513
514
MRAK
sulfonate (trimethylammonium-diphenylhexatriene, Cat. No. T-204); and 3-(p-(6-phenyl)-l,3,5-hexatrienyl)phenylpropionic acid (diphenylhexatriene propionic acid, Cat. No. P-459).
Preparation of Synaptic Membranes Isolation of synaptic plasma membranes was performed as previously described (16). Briefly, Sprague-Dawley rats cerebral hemispheres (forebrains) were homogenized and a (1000g × 10 min) supernatant prepared. This was pelleted (11,000g × 20 rain) and washed twice by resuspension and recentrifugation. This washed crude synaptosomal pellet was lysed by hypo-osmotic shock, brought to 34070 wt/wt sucrose (390 g/l), layered under a solution of 28.5070 wt/wt sucrose (319 g/l), and centrifuged (49,000g x 16 h). Synaptic plasma membranes were recovered from the 28.5-340/0 interface and pelleted (87,000g x 2 h). A white outer rim of the pellet was recovered and resuspended in 0.32 M sucrose/2.5 mM Na Hepes (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid) (pH 7.4) at 10-20 mg membrane protein/ml. Preparations were assayed for protein (10,24) and for bound phosphorus (1,22), and then quick-frozen and stored in liquid nitrogen until used.
Fluorescence Polarization Fluorescence polarization studies were performed as previously described (14,15). Stock 1 mM solutions of fluorescence probes were prepared in tetrahydrofuran (diphenylhexatriene) or in 50°70 (vol/vol) aqueous tetrahydrofuran (trimethylammonium-diphenylhexatriene and diphenylhexatriene propionic acid). These were dispersed as 1,0/zM aqueous solutions in 0.1 M NaCl/10 mM Na Hepes buffer (pH 7.1). Equal volumes of dispersed probe and diluted synaptic plasma membranes (100/zg membrane protein, or 3.5 #g membrane bound phosphorus, per ml buffer) were mixed to pro-
duce final assay concentrations of 0.5 t~M probe and 50/~g membrane protein/ml, and a final probe:phospholipid molar ratio of approximately 1 : 110. Steady-state fluorescence polarization of the equilibrated samples was measured at 37°C using a Perkin-Elmer 650-40 fluorescence spectrophotometer, with excitation and emission wavelengths of 360 and 430 nm, and bandwidths of 3 and 20 nm respectively. Fluorescence polarization, P, was calculated as (Ill - I±)/(Iii + I±), where /ll and I l are the fluorescence intensities parallel to and perpendicular to (respectively) the polarization of the excitation beam.
Statistics Significance of observed differences were assessed using Student's t-test. A test for paired data was used throughout, except in comparing the control (zero additions) fluorescence polarizations values for the various probes in Table 1. RESULTS Polarization of fluorescence was observed for probes equilibrated with isolated synaptic plasma membranes (Table 1). This polarization was greater for trimethylammonium-diphenylhexatriene and for diphenylhexatriene propionic acid than for diphenylhexatriene itself. This is in accord with the presumed localization of diphenylhexatriene to the more-fluid membrane core (28), and the presumed localization of the derivative probes to the more ordered superficial bilayer domains (21,33). In addition, fluorescence polarization of trimethylammonium-diphenylhexatrienewas slightly, but significantly, greater than that of diphenylhexatriene propionic acid, in agreement with the presumed preference of these probes for the more-ordered cytofacial and less-ordered exofacial membrane leaflets, respectively (25). Ethanol, added in vitro to isolated synaptic plasma mem-
TABLE 1 EFFECTS OF ETHANOL AND DIMETHYL SULFOXIDEON FLUORESCENCEPOLARIZATION OF DIPHENYLHEXATRIENEPROBES IN ISOLATED SYNAPTIC PLASMA MEMBRANES* Probe Diphenylhexatriene
TrimethylammoniumDiphenylhexatriene
Diphenylhexatriene PropionicAcid
Fluorescence Polarization (P)
Ethanol nt 0mM 50 mM 200mM
6 0.272 + 0.001~ 0.270 + 0.0006§ 0.267 +_ 0.001¶
6 0.336 + 0.001 0.335 + 0.001 0.330 + 0.001¶
6 0.330 + 0.007 0.331 + 0.007 0.328 + 0.007#
Dimethyl sulfoxide n 0 mM 6070(vol/vol) 10% (vol/vol)
6 0.273 + 0.002 0.274 + 0.002 0.277 +_ 0.00211
6 0.338 + 0.002 0.342 + 0.002[] 0.343 + 0.0021]
5 0.335 + 0.001 0.337 + 0.001[[ 0.338 + 0.002§
*Assays conducted at 370C with 100/~g isolated synaptic plasma membrane protein and 1.0 nmol probe in 2.0 ml (estimated probe: phospholipid ratio of approximately 1:110mol/mol). tNumber of different synaptic plasma membrane preparations assayed. ~Values expressed as mean + SE. §,¶,#,[IValue is significantly different from corresponding value in the absence of ethanol or dimethyl sulfoxide: #p = 0.05, §p < 0.05, ILp < 0.01, ¶ < 0.005. In addition, the control (no additions) values for each probe are all significantly different from one another (p < 0.05 or better).
OPPOSITE EFFECTS OF DMSO AND ETHANOL
515
branes, decreased fluorescence polarization of all three probes used (Table 1). The effect was most pronounced with diphenylhexatriene, with a statistically significant decrease seen at 50 mM ethanol. This agrees with previous studies showing preferential ethanol fluidization of the hydrophobic core region of synaptic plasma membranes (5). For trimethylammonium-diphenylhexatriene and for diphenylhexatriene propionic acid, statistically significant decreases in fluorescence polarization appeared at 200 mM ethanol. Dimethyl sulfoxide, added in vitro to isolated synaptic plasma membranes, increased fluorescence polarization of all three probes used (Table 1). For diphenylhexatriene, fluorescence polarization showed a slight (nonsignificant) increase between 4070 and 8% (vol/vol) dimethyl sulfoxide, with statistical significance attained at 10°70 (vol/vol) dimethyl sulfoxide (Fig. 1). In contrast to the results obtained in the presence of ethanol, this effect of dimethyl sulfoxide was apparent at lower concentrations of dimethyl sulfoxide when the derivative probes, trimethylammonium-diphenylhexatriene and diphenylhexatriene propionic acid, were used; these showed statistically significant increases in fluorescence polarization in the presence of 6070 (vol/vol) dimethyl sulfoxide (Table 1). The dimethyl sulfoxide-induced increase in fluorescence polarization of diphenylhexatriene was antagonized by the addition of ethanol (Fig. 2). A significant 4.807o increase in fluorescence polarization observed in the presence of 16070 (vol/ vol) dimethyl sulfoxide was halved by the addition of 0.2 M ethanol. DISCUSSION Dimethyl sulfoxide, added in vitro to isolated synaptic plasma membranes, increased fluorescence polarization of the probes diphenylhexatriene, trimethylammonium-diphenylhexatriene, and diphenylhexatriene propionic acid. This effect was antagonized by ethanol added in vitro and was manifested over a concentration range of 4-1007o (vol/vol) dimethyl sulfoxide. These results suggest that dimethyl sulfoxide has an
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i
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I
!
i
I
I
i
!
i
i
i
0 I
4
8
12
m
16
DIMETHYL SULFOXIDE (%v/v) FIG. 1. Dimethyl sulfoxide enhancement of diphenylhexatriene fluorescence polarization in isolated rat cerebral synaptic plasma membranes, measured at 37°C. Data presented as mean percent change from control (zero dimethyl sulfoxide) fluorescence polarization :t= SE for four (0.4-1.6°70, 4070, 13070,and 16070vol/vol dimethyl sulfoxide), five (2.0070 vol/vol dimethyl sulfoxide), or six (6-10070 vol/vol dimethyl sulfoxide) different synaptic plasma membrane preparations. The mean control (zero dimethyl sulfoxide) fluorescence polarization value was 0.272 + 0.001 for seven different synaptic plasma membrane preparations. *Value is significantly different from control (zero dimethyl sulfoxide) value (p < 0.05).
4
0 NO
I
ADDITIONS
o
I
I
o.I 0.2 ETHANOL ( M )
I
0.3
FIG. 2. Ethanol antagonism of dimethyl sulfoxide enhancement of diphenyl-hexatriene fluorescence polarization in isolated rat cerebral synaptic plasma membranes at 37°C. Data presented as mean percent change from control (no additions) fluorescence polarization + SE for four different synaptic plasma membrane preparations. The mean control (no additions) fluorescence polarization value was 0.272 + 0.001. *Value is significantly different from control (no additions) value (p < 0.001). **Value is significantly different from that obtained in the presence of 16% vol/vol dimethyl sulfoxide and no ethanol (p < 0.05).
ordering, or "rigidifying" effect on synaptic plasma membranes. Furthermore, this rigidifying effect was seen at lower dimethyl sulfoxide concentrations for the derivative probes, trimethylammonium-diphenylhexatriene and diphenylhexatriene propionic acid [which report from the superficial domains of the membrane bilayer (21,33)], than for the parent molecule, diphenylhexatriene [which reports from the membrane core (28)]. These differences, although small, are opposite to the pattern obtained with ethanol, for which significant effects are first noted by diphenylhexatriene. These results thus suggest that the rigidifying effect of dimethyl sulfoxide is greater in the superficial portions of the membrane lipid bilayer rather than at the membrane core. This may reflect poor penetration of the polar dimethyl sulfoxide molecule into the membrane core. There is one previous report of dimethyl sulfoxide-induced changes in membrane order of isolated natural membranes. Gorvel et al. (4) used flow cytometry to measure fluorescence polarization of diphenylhexatriene in single-membrane vesicles derived from rabbit enterocytes. They reported a 25% increase in fluorescence polarization in basolateral enterocyte membranes incubated with 0.1 °7o dimethyl sulfoxide. This effect is more than 10 times the increase in diphenylhexatriene fluorescence polarization found in the present study (1-2%), at a dimethyl sulfoxide concentration one one-hundredth that employed in the present study (10070 vol/vol). In contrast to the results of Gorvel et al., the present study detected no diphenylhexatriene fluorescence polarization at 0.4070 (vol/ vol) dimethyl sulfoxide (Fig. 1), even using a probe concentration twice that employed in their study. While it is possible that enterocyte membranes are uniquely sensitive to dimethyl sulfoxide, this seems insufficient to explain the remarkable differences observed. There is no statistical analysis of results in the study of Gorvel et al., and thus the possibility that these results are spurious must be considered. Numerous compounds and experimental treatments have
516
MRAK
been shown to disorder, or "fluidize" natural bilayer membranes, including ethanol (2), barbiturates (6), most anesthetics (26), cholesterol depletion (17), and increasing temperature. Ordering, or "rigidifying" treatments, in contrast, are somewhat less commonly found, although this can be effected by alteration of membrane lipid composition or by decreasing temperature (27). Dimethyl sulfoxide is an organic solvent with myriad biological effects (3,9), including effects on the activities of membrane-bound enzymes (7,13) and transmembrane movements (19,23). Dimethyl sulfoxide has been shown to inhibit uptake of 3,-aminobutyric acid and of choline in isolated synaptosomes (18), and to increase K + and Na + affinity of (NA + ,K+) ATPase from brain (29-31). These effects are seen at dimethyl-sulfoxide concentrations similar to those employed in the present study (5-2007o). The dimethyl sulfoxide effect on (Na+,K+)-ATPase is in contrast to observed ethanol effects on this enzyme (29,30). An ordering effect of dimethyl sulfoxide on membranes was suggested by Lyman et al. (11,12) on the basis of a dimethyl sulfoxide-induced rise in transition temperatures of artificial phospholipid bilayers, detected using scanning calorimetry. This effect was antagonized by the addition of the
local anesthetic (and membrane-disordering agent), dibucaine hydrochloride (12). The concentration range of dimethyl sulfoxide examined by Lyman et al. (2-30070) is similar to that used in the present study. The present results confirm on ordering effect of dimethyl sulfoxide on lipid bilayer membranes, and extend the observations of Lyman et al. by employing natural biological membranes and a fluorescent probe technique. The present results further show a preferential effect of dimethyl sulfoxide in the superficial domains of the membrane bilayer. Both of these results are opposite to those produced by ethanol, which disorders ("fluidizes") biological membranes and acts preferentially at the membrane core. The observed antagonism between the superficial actions of dimethyl sulfoxide and the deep actions of ethanol suggests biophysical interactions between these different intramembrane lipid domains. ACKNOWLEDGEMENTS The author thanks Dr. Paula North for valuable discussions, and James Yarbrough and Virginia Fitzhugh for their excellent technical assistance. Supported in part by the Veterans Administration and in part by NIH Grant AA06915.
REFERENCES 1. Chen, P. S.; Toribara, T. Y.; Warner, H. Microdetermination of phosphorus. Anal. Chem. 28:1756-1758; 1956. 2. Chin, J. H.; Goldsteln, D. B. Effects of low concentrations of ethanol on the fluidity of spin-labelled erythrocytes and brain membranes. Molec. Pharmacol. 13:435-441; 1977. 3. David, N. A. The pharmacology of dimethyl sulfoxide. Ann. Rev. Pharmacol. 12:353-374; 1972. 4. Gorvel, J.-P.; Mawas, C.; Maroux, S.; Mishal, Z. Flow cytometry is an new method for the characterization of intestinal plasma membrane. Bioehem. J. 221:453-457; 1984. 5. Harris, R. A.; Schroeder, F. Ethanol and the physical properties of brain membranes; fluorescence studies. Molec. Pharmacol. 20: 128-137; 1981. 6. Harris, R. A.; Sehroeder, F. Effects of barbiturates and ethanol on the physical properties of brain membranes. J. Pharmacol. Exp. Ther. 223:424-431; 1982. 7. Hynie, S.; Klenerova, V. Effects of dimethyl sulfoxide and other dipolar aprotic solvents on rat hepatic adenylate cyclase. Potentiating effects on glucagon and guanylylimidodiphosphate stimulation. Nannyn-Schmiedeberg's Arch. Pharmacol. 310:231-236; 1980. 8. Jourdon, P.; Berwald-Netter, Y.; Dubois, J.-M. Effects of dimethy!-sulfoxide on membrane currents of neuroblastoma x glioma hybrid cell. Biochim. Biophys. Acta 856:399--402; 1986. 9. Leake, C. D., ed. Biological actions of dimethyl sulfoxide. Ann. NY Acad. Sci. 141:1-671; 1967. 10. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. F. Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193:265-275; 1951. 11. Lyman, G. H.; Preisler, H. D.; Papahadjopouios, D. Membrane action of DMSO and other chemical inducers of Friend leukaemic cell differentiation. Nature 262:360-363; 1976. 12. Lyman, G. H.; Papahadjopoulos, D.; Preisler, H. D. Phospholipid membrane stabilization by dimethylsulfoxide and other inducers of Friend leukemic cell differentiation. Biochim. Biophys. Acta 448:460-473; 1976. 13. Maynard, J. R.; Fintei, D. J.; Pitlick, F. A.; Nemerson, Y. Tissue factor in cultured cells. Pharmacologic effects. Lab. Invest. 35: 550-557; 1976. 14. Mrak, R. E. Isolation and characterization of transverse tubule from normal and dystrophic mice. Biochim. Biophys. Acta 774: 35-42; 1984. 15. Mrak, R. E.; Fleischer, S. Lipid composition of sarcoplasmic
16. 17. 18. 19.
20. 21.
22. 23. 24. 25. 26. 27. 28.
reticulum from mice with muscular dystrophy. Muscle Nerve 5: 439-446; 1982. Mrak, R. E.; North, P. E. Triphasic effects of short chain nalcohols on synaptic transport of choline and of -r-aminobutyric acid. Biochim. Biophys. Acta 984:97-103; 1989. North, P.; Fleischer, S. Alteration of synaptic membrane cholesterol/phospholipid ratio using a lipid transfer protein: Effect on 3'-aminobutyric acid uptake. J. Biol. Chem. 258:1242-1253; 1983. North, P. E.; Mrak, R. E. Synaptosomal uptake of choline and of gamma-aminobutyric acid: effects of ethanol and of dimethylsulfoxide. Neurotoxicology 10:569-576; 1989. Nygren, P.; Larsson, E.; Rastad, J.; Akerstr6m, G.; Gylfe, E. Dimethyl sulfoxide increases cytoplasmic Ca :+ concentration and inhibits parathyroid hormone release in normal bovine and pathological human parathyroid cells. Biochim. Biophys. Acta 928: 194-198; 1987. Preisler, H. D.; Lyman, G. Differentiation of erythroleukemia cells in vitro: properties of chemical inducers. Cell Differentiation 4:179-185; 1975. Prendergast, F. G.; Haugiand, R. P.; Callahan, P. J. l[4(Trimethylamino)-phenyl]-6-phenylhexa- 1,3,5-triene: Synthesis, fluorescence properties, and use as a fluorescence probe of lipid bilayers. Biochemistry 20:7333-7338; 1981. Rouser, G.; Fleischer, S. Isolation, characterization, and determination of polar lipids of mitochondria. Meth. Enzymol. 10:385406; 1967. Sandvig, K.; Madshus, I. H.; Olsnes, S. Dimethyl sulfoxide protects cells against polypeptide toxins and poliovirus. Biochem. J. 219:935-940; 1984. Schacterle, G. R.; Pollack, R. L. A simplified method for the quantitative assay of small amounts of protein in biological material. Anal. Biochem. 51:654-655; 1973. Schroeder, F.; Morrison, W. J.; Gorka, C.; Wood, W. G. Transbilayer effects of ethanol on fluidity of brain membrane leaflets. Biochim. Biophys. Acta 946:85-94; 1988. Seeman, P. The membrane actions of anesthetics and tranquilizers. Pharmacol. Rev. 24:583--655; 1972. Shiuitzky, M. Membrane fluidity and cellular functions. In: Shinitzky, M., ed. Physiology of membrane fluidity, vol. I. Boca Raton, FL: CRC Press; 1984:1-52. Shinitzky, M.; Barenholz, Y. Fluidity parameters of lipid regions determined by fluorescence polarization. Biochim. Biophys. Acta 515:367-394; 1978.
OPPOSITE EFFECTS OF DMSO AND ETHANOL 29. Swann, A. C. Brain 0Na+,K+)-ATPase. Opposite effects of ethanol and dimethyl sulfoxide on temperature dependence of enzyme conformation and univalent cation binding. J. Biol. Chem. 258: 11780-11786; 1983. 30. Swann, A. C. Inhibition of (Na +,K+)-ATPase by fluoride: Evidence for a membrane adaptation to ethanol. Alcohol 7:91-95; 1990. 31. Swann, A. C.; Albers, R. W. Sodium + potassium-activated ATPase of mammalian brain. Regulation of phosphatase activity. Biochim. Biophys. Acta 382:437-456; 1975. 32. Teigland, M. B.; Saurino, V. R. Clinical evaluation of dimethyl
517 sulfoxid¢ in equine applications. Ann. NY Acad. Sci. 141:471477; 1967. 33. Trotter, P. J.; Storch, J. 3-[p-(6-phenyl)-l,3,5-hexatrienyl]phenyl-propionic acid (PA-DPH): Characterization as a fluorescent membrane probe and binding to fatty acid binding proteins. Biochim. Biophys. Acta 982:131-139; 1989. 34. van Hock, A. N.; de Jong, M. D.; van Os, C. H. Effects of dimethyl-sulfoxide and mercurial suifhydryl reagents on water and solute permeability of rat kidney brush border membranes. Biochim. Biophys. Acta 1030:203-210; 1990.
0741-8329/92 $5.00 + .00 Copyright©1992PergamonPress Ltd.
Printed in the U.S.A. All rightsreserved.
Opposite Effects of Dimethyl Sulfoxide and Ethanol on Synaptic Membrane Fluidity R O B E R T E. M R A K
Department o f Pathology, John L. McClellan Memorial Veterans" Hospital and University o f Arkansas for Medical Sciences, Little Rock, A R 72205 Received 27 M a r c h 1992; A c c e p t e d 10 J u n e 1992
MRAK, R. E. Oppositeeffects of dimethyl sulfoxide and ethanol on synaptic membranefluidity. ALCOHOL 9(6) 513517, 1992.-Dimethyl sulfoxide (DMSO) is an organic solvent with myriad biological actions, including actions on synaptic membrane transport processes. In this study, fluorescence polarizations of the probes diphenylhexatriene (DPH: a probe of the hydrophobic membrane core), trimethylammonium-diphenylhexatriene (a probe of the superficial domain of the cytofacial synaptic membrane leaflet) and diphenylhexatriene propionic acid (a probe of the superficial domain of the exofacial synaptic plasma membrane leaflet) were measured in isolated rat cerebral synaptic plasma membranes. DMSO, added in vitro, increased fluorescence polarization of all of these intramembranous probes, an effect opposite that observed with the addition of ethanol. The fluorescence polarization increase appeared at lower concentrations of DMSO for the superficial membrane region probes (6~e vol/vol DMSO) than for the membrane core probe (10~e vol/vol DMSO). This is again in contrast to the effects of ethanol, which required lesser concentrations to decrease fluorescence polarization of DPH (50 mM ethanol) than that of the derivative probes (200 mM ethanol). The enhancement of DPH fluorescence polarization produced by DMSO was antagonized by the concomitant addition of ethanol. These results suggest an ordering effect of DMSO on synaptic plasma membranes, with greater effects in superficial membrane domains. Fluorescence polarization Synaptic plasma membranes Dimethyl sulfoxide Membrane fluidity Diphenylhexatriene Ethanol Trimethylammonium-diphenylhexatriene Diphenylhexatriene propionic acid
DIMETHYL sulfoxide is an organic solvent whose biological actions range from analgesic and anti-inflammatory actions (32) to induction of differentiation in cultured cells (20) to alterations of membrane transport activities (8,34). In mammalian brain extracts, dimethyl sulfoxide has been shown to inhibit synaptosomal uptake of choline and ~-aminobutyric acid (18) and to enhance (Na+,K+)-ATPase affinity for Na + and for K + (29-31). This latter result is in contrast to the effects of ethanol, which decrease (Na +,K+)-ATPase affinity for Na + and for K + (29,30). Scanning calorimetry investigations of artificial phospholipid bilayers have suggested a membrane site of action for dimethyl sulfoxide, and have further suggested an ordering, or "rigidifying" effect of this agent on such synthetic bilayers (11,12). This proposed action is opposite to the disordering, or "fluidizing" effects of ethanol in synaptic membranes (2,5), and suggests that these two agents might antagonize one another in vitro. There is, however, only one limited investigation of the biophysical effects o f dimethyl sulfoxide on biological membranes (4). This study examines the effects of dimethyl sulfoxide on fluorescence polarization of intramembranous probes in iso-
lated rat cerebral synaptic plasma membranes. The effects of dimethyl sulfoxide on diphenylhexatriene, a probe which preferentiaily localizes to the more-fluid membrane core domain (28), are compared with the effects on the derivative probes trimethylammonium-diphenylhexatriene and diphenylhexatriene propionic acid, probes which preferentially localize to the more superficial domains of the membrane bilayer (21,33). These probes show further specificity in that the cationic trimethylammonium-diphenylhexatriene has a greater affinity for the negatively charged inner (cytofacial) membrane leaflet, while the anionic diphenylhexatriene propionic acid shows greater affinity for the more neutral outer (exofacial) membrane leaflet (25). METHOD
Materials Dimethyl sulfoxide was obtained from Fisher Scientific (Pittsburgh, PA). Fluorescent probes were obtained from Molecular Probes, Inc. (Eugene, OR): 1,6-diphenyl-l,3,5hexatriene (diphenylhexatriene, Cat. No. D-202); 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluene-
Requests for reprints should be addressed to Laboratory Service (113), John L. McClellan Memorial Veterans' Hospital, 4300 West Seventh Street, Little Rock, AR 72205. 513
514
MRAK
sulfonate (trimethylammonium-diphenylhexatriene, Cat. No. T-204); and 3-(p-(6-phenyl)-l,3,5-hexatrienyl)phenylpropionic acid (diphenylhexatriene propionic acid, Cat. No. P-459).
Preparation of Synaptic Membranes Isolation of synaptic plasma membranes was performed as previously described (16). Briefly, Sprague-Dawley rats cerebral hemispheres (forebrains) were homogenized and a (1000g × 10 min) supernatant prepared. This was pelleted (11,000g × 20 rain) and washed twice by resuspension and recentrifugation. This washed crude synaptosomal pellet was lysed by hypo-osmotic shock, brought to 34070 wt/wt sucrose (390 g/l), layered under a solution of 28.5070 wt/wt sucrose (319 g/l), and centrifuged (49,000g x 16 h). Synaptic plasma membranes were recovered from the 28.5-340/0 interface and pelleted (87,000g x 2 h). A white outer rim of the pellet was recovered and resuspended in 0.32 M sucrose/2.5 mM Na Hepes (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid) (pH 7.4) at 10-20 mg membrane protein/ml. Preparations were assayed for protein (10,24) and for bound phosphorus (1,22), and then quick-frozen and stored in liquid nitrogen until used.
Fluorescence Polarization Fluorescence polarization studies were performed as previously described (14,15). Stock 1 mM solutions of fluorescence probes were prepared in tetrahydrofuran (diphenylhexatriene) or in 50°70 (vol/vol) aqueous tetrahydrofuran (trimethylammonium-diphenylhexatriene and diphenylhexatriene propionic acid). These were dispersed as 1,0/zM aqueous solutions in 0.1 M NaCl/10 mM Na Hepes buffer (pH 7.1). Equal volumes of dispersed probe and diluted synaptic plasma membranes (100/zg membrane protein, or 3.5 #g membrane bound phosphorus, per ml buffer) were mixed to pro-
duce final assay concentrations of 0.5 t~M probe and 50/~g membrane protein/ml, and a final probe:phospholipid molar ratio of approximately 1 : 110. Steady-state fluorescence polarization of the equilibrated samples was measured at 37°C using a Perkin-Elmer 650-40 fluorescence spectrophotometer, with excitation and emission wavelengths of 360 and 430 nm, and bandwidths of 3 and 20 nm respectively. Fluorescence polarization, P, was calculated as (Ill - I±)/(Iii + I±), where /ll and I l are the fluorescence intensities parallel to and perpendicular to (respectively) the polarization of the excitation beam.
Statistics Significance of observed differences were assessed using Student's t-test. A test for paired data was used throughout, except in comparing the control (zero additions) fluorescence polarizations values for the various probes in Table 1. RESULTS Polarization of fluorescence was observed for probes equilibrated with isolated synaptic plasma membranes (Table 1). This polarization was greater for trimethylammonium-diphenylhexatriene and for diphenylhexatriene propionic acid than for diphenylhexatriene itself. This is in accord with the presumed localization of diphenylhexatriene to the more-fluid membrane core (28), and the presumed localization of the derivative probes to the more ordered superficial bilayer domains (21,33). In addition, fluorescence polarization of trimethylammonium-diphenylhexatrienewas slightly, but significantly, greater than that of diphenylhexatriene propionic acid, in agreement with the presumed preference of these probes for the more-ordered cytofacial and less-ordered exofacial membrane leaflets, respectively (25). Ethanol, added in vitro to isolated synaptic plasma mem-
TABLE 1 EFFECTS OF ETHANOL AND DIMETHYL SULFOXIDEON FLUORESCENCEPOLARIZATION OF DIPHENYLHEXATRIENEPROBES IN ISOLATED SYNAPTIC PLASMA MEMBRANES* Probe Diphenylhexatriene
TrimethylammoniumDiphenylhexatriene
Diphenylhexatriene PropionicAcid
Fluorescence Polarization (P)
Ethanol nt 0mM 50 mM 200mM
6 0.272 + 0.001~ 0.270 + 0.0006§ 0.267 +_ 0.001¶
6 0.336 + 0.001 0.335 + 0.001 0.330 + 0.001¶
6 0.330 + 0.007 0.331 + 0.007 0.328 + 0.007#
Dimethyl sulfoxide n 0 mM 6070(vol/vol) 10% (vol/vol)
6 0.273 + 0.002 0.274 + 0.002 0.277 +_ 0.00211
6 0.338 + 0.002 0.342 + 0.002[] 0.343 + 0.0021]
5 0.335 + 0.001 0.337 + 0.001[[ 0.338 + 0.002§
*Assays conducted at 370C with 100/~g isolated synaptic plasma membrane protein and 1.0 nmol probe in 2.0 ml (estimated probe: phospholipid ratio of approximately 1:110mol/mol). tNumber of different synaptic plasma membrane preparations assayed. ~Values expressed as mean + SE. §,¶,#,[IValue is significantly different from corresponding value in the absence of ethanol or dimethyl sulfoxide: #p = 0.05, §p < 0.05, ILp < 0.01, ¶ < 0.005. In addition, the control (no additions) values for each probe are all significantly different from one another (p < 0.05 or better).
OPPOSITE EFFECTS OF DMSO AND ETHANOL
515
branes, decreased fluorescence polarization of all three probes used (Table 1). The effect was most pronounced with diphenylhexatriene, with a statistically significant decrease seen at 50 mM ethanol. This agrees with previous studies showing preferential ethanol fluidization of the hydrophobic core region of synaptic plasma membranes (5). For trimethylammonium-diphenylhexatriene and for diphenylhexatriene propionic acid, statistically significant decreases in fluorescence polarization appeared at 200 mM ethanol. Dimethyl sulfoxide, added in vitro to isolated synaptic plasma membranes, increased fluorescence polarization of all three probes used (Table 1). For diphenylhexatriene, fluorescence polarization showed a slight (nonsignificant) increase between 4070 and 8% (vol/vol) dimethyl sulfoxide, with statistical significance attained at 10°70 (vol/vol) dimethyl sulfoxide (Fig. 1). In contrast to the results obtained in the presence of ethanol, this effect of dimethyl sulfoxide was apparent at lower concentrations of dimethyl sulfoxide when the derivative probes, trimethylammonium-diphenylhexatriene and diphenylhexatriene propionic acid, were used; these showed statistically significant increases in fluorescence polarization in the presence of 6070 (vol/vol) dimethyl sulfoxide (Table 1). The dimethyl sulfoxide-induced increase in fluorescence polarization of diphenylhexatriene was antagonized by the addition of ethanol (Fig. 2). A significant 4.807o increase in fluorescence polarization observed in the presence of 16070 (vol/ vol) dimethyl sulfoxide was halved by the addition of 0.2 M ethanol. DISCUSSION Dimethyl sulfoxide, added in vitro to isolated synaptic plasma membranes, increased fluorescence polarization of the probes diphenylhexatriene, trimethylammonium-diphenylhexatriene, and diphenylhexatriene propionic acid. This effect was antagonized by ethanol added in vitro and was manifested over a concentration range of 4-1007o (vol/vol) dimethyl sulfoxide. These results suggest that dimethyl sulfoxide has an
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DIMETHYL SULFOXIDE (%v/v) FIG. 1. Dimethyl sulfoxide enhancement of diphenylhexatriene fluorescence polarization in isolated rat cerebral synaptic plasma membranes, measured at 37°C. Data presented as mean percent change from control (zero dimethyl sulfoxide) fluorescence polarization :t= SE for four (0.4-1.6°70, 4070, 13070,and 16070vol/vol dimethyl sulfoxide), five (2.0070 vol/vol dimethyl sulfoxide), or six (6-10070 vol/vol dimethyl sulfoxide) different synaptic plasma membrane preparations. The mean control (zero dimethyl sulfoxide) fluorescence polarization value was 0.272 + 0.001 for seven different synaptic plasma membrane preparations. *Value is significantly different from control (zero dimethyl sulfoxide) value (p < 0.05).
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o.I 0.2 ETHANOL ( M )
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FIG. 2. Ethanol antagonism of dimethyl sulfoxide enhancement of diphenyl-hexatriene fluorescence polarization in isolated rat cerebral synaptic plasma membranes at 37°C. Data presented as mean percent change from control (no additions) fluorescence polarization + SE for four different synaptic plasma membrane preparations. The mean control (no additions) fluorescence polarization value was 0.272 + 0.001. *Value is significantly different from control (no additions) value (p < 0.001). **Value is significantly different from that obtained in the presence of 16% vol/vol dimethyl sulfoxide and no ethanol (p < 0.05).
ordering, or "rigidifying" effect on synaptic plasma membranes. Furthermore, this rigidifying effect was seen at lower dimethyl sulfoxide concentrations for the derivative probes, trimethylammonium-diphenylhexatriene and diphenylhexatriene propionic acid [which report from the superficial domains of the membrane bilayer (21,33)], than for the parent molecule, diphenylhexatriene [which reports from the membrane core (28)]. These differences, although small, are opposite to the pattern obtained with ethanol, for which significant effects are first noted by diphenylhexatriene. These results thus suggest that the rigidifying effect of dimethyl sulfoxide is greater in the superficial portions of the membrane lipid bilayer rather than at the membrane core. This may reflect poor penetration of the polar dimethyl sulfoxide molecule into the membrane core. There is one previous report of dimethyl sulfoxide-induced changes in membrane order of isolated natural membranes. Gorvel et al. (4) used flow cytometry to measure fluorescence polarization of diphenylhexatriene in single-membrane vesicles derived from rabbit enterocytes. They reported a 25% increase in fluorescence polarization in basolateral enterocyte membranes incubated with 0.1 °7o dimethyl sulfoxide. This effect is more than 10 times the increase in diphenylhexatriene fluorescence polarization found in the present study (1-2%), at a dimethyl sulfoxide concentration one one-hundredth that employed in the present study (10070 vol/vol). In contrast to the results of Gorvel et al., the present study detected no diphenylhexatriene fluorescence polarization at 0.4070 (vol/ vol) dimethyl sulfoxide (Fig. 1), even using a probe concentration twice that employed in their study. While it is possible that enterocyte membranes are uniquely sensitive to dimethyl sulfoxide, this seems insufficient to explain the remarkable differences observed. There is no statistical analysis of results in the study of Gorvel et al., and thus the possibility that these results are spurious must be considered. Numerous compounds and experimental treatments have
516
MRAK
been shown to disorder, or "fluidize" natural bilayer membranes, including ethanol (2), barbiturates (6), most anesthetics (26), cholesterol depletion (17), and increasing temperature. Ordering, or "rigidifying" treatments, in contrast, are somewhat less commonly found, although this can be effected by alteration of membrane lipid composition or by decreasing temperature (27). Dimethyl sulfoxide is an organic solvent with myriad biological effects (3,9), including effects on the activities of membrane-bound enzymes (7,13) and transmembrane movements (19,23). Dimethyl sulfoxide has been shown to inhibit uptake of 3,-aminobutyric acid and of choline in isolated synaptosomes (18), and to increase K + and Na + affinity of (NA + ,K+) ATPase from brain (29-31). These effects are seen at dimethyl-sulfoxide concentrations similar to those employed in the present study (5-2007o). The dimethyl sulfoxide effect on (Na+,K+)-ATPase is in contrast to observed ethanol effects on this enzyme (29,30). An ordering effect of dimethyl sulfoxide on membranes was suggested by Lyman et al. (11,12) on the basis of a dimethyl sulfoxide-induced rise in transition temperatures of artificial phospholipid bilayers, detected using scanning calorimetry. This effect was antagonized by the addition of the
local anesthetic (and membrane-disordering agent), dibucaine hydrochloride (12). The concentration range of dimethyl sulfoxide examined by Lyman et al. (2-30070) is similar to that used in the present study. The present results confirm on ordering effect of dimethyl sulfoxide on lipid bilayer membranes, and extend the observations of Lyman et al. by employing natural biological membranes and a fluorescent probe technique. The present results further show a preferential effect of dimethyl sulfoxide in the superficial domains of the membrane bilayer. Both of these results are opposite to those produced by ethanol, which disorders ("fluidizes") biological membranes and acts preferentially at the membrane core. The observed antagonism between the superficial actions of dimethyl sulfoxide and the deep actions of ethanol suggests biophysical interactions between these different intramembrane lipid domains. ACKNOWLEDGEMENTS The author thanks Dr. Paula North for valuable discussions, and James Yarbrough and Virginia Fitzhugh for their excellent technical assistance. Supported in part by the Veterans Administration and in part by NIH Grant AA06915.
REFERENCES 1. Chen, P. S.; Toribara, T. Y.; Warner, H. Microdetermination of phosphorus. Anal. Chem. 28:1756-1758; 1956. 2. Chin, J. H.; Goldsteln, D. B. Effects of low concentrations of ethanol on the fluidity of spin-labelled erythrocytes and brain membranes. Molec. Pharmacol. 13:435-441; 1977. 3. David, N. A. The pharmacology of dimethyl sulfoxide. Ann. Rev. Pharmacol. 12:353-374; 1972. 4. Gorvel, J.-P.; Mawas, C.; Maroux, S.; Mishal, Z. Flow cytometry is an new method for the characterization of intestinal plasma membrane. Bioehem. J. 221:453-457; 1984. 5. Harris, R. A.; Schroeder, F. Ethanol and the physical properties of brain membranes; fluorescence studies. Molec. Pharmacol. 20: 128-137; 1981. 6. Harris, R. A.; Sehroeder, F. Effects of barbiturates and ethanol on the physical properties of brain membranes. J. Pharmacol. Exp. Ther. 223:424-431; 1982. 7. Hynie, S.; Klenerova, V. Effects of dimethyl sulfoxide and other dipolar aprotic solvents on rat hepatic adenylate cyclase. Potentiating effects on glucagon and guanylylimidodiphosphate stimulation. Nannyn-Schmiedeberg's Arch. Pharmacol. 310:231-236; 1980. 8. Jourdon, P.; Berwald-Netter, Y.; Dubois, J.-M. Effects of dimethy!-sulfoxide on membrane currents of neuroblastoma x glioma hybrid cell. Biochim. Biophys. Acta 856:399--402; 1986. 9. Leake, C. D., ed. Biological actions of dimethyl sulfoxide. Ann. NY Acad. Sci. 141:1-671; 1967. 10. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. F. Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193:265-275; 1951. 11. Lyman, G. H.; Preisler, H. D.; Papahadjopouios, D. Membrane action of DMSO and other chemical inducers of Friend leukaemic cell differentiation. Nature 262:360-363; 1976. 12. Lyman, G. H.; Papahadjopoulos, D.; Preisler, H. D. Phospholipid membrane stabilization by dimethylsulfoxide and other inducers of Friend leukemic cell differentiation. Biochim. Biophys. Acta 448:460-473; 1976. 13. Maynard, J. R.; Fintei, D. J.; Pitlick, F. A.; Nemerson, Y. Tissue factor in cultured cells. Pharmacologic effects. Lab. Invest. 35: 550-557; 1976. 14. Mrak, R. E. Isolation and characterization of transverse tubule from normal and dystrophic mice. Biochim. Biophys. Acta 774: 35-42; 1984. 15. Mrak, R. E.; Fleischer, S. Lipid composition of sarcoplasmic
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517 sulfoxid¢ in equine applications. Ann. NY Acad. Sci. 141:471477; 1967. 33. Trotter, P. J.; Storch, J. 3-[p-(6-phenyl)-l,3,5-hexatrienyl]phenyl-propionic acid (PA-DPH): Characterization as a fluorescent membrane probe and binding to fatty acid binding proteins. Biochim. Biophys. Acta 982:131-139; 1989. 34. van Hock, A. N.; de Jong, M. D.; van Os, C. H. Effects of dimethyl-sulfoxide and mercurial suifhydryl reagents on water and solute permeability of rat kidney brush border membranes. Biochim. Biophys. Acta 1030:203-210; 1990.
